The selected nucleic acid molecule, a chimeric gene, recombinant vector, strain of bacteria, toxin, insecticidal composition, method of producing toxin, the method of obtaining insect-resistant plants and a method of combating insects
The invention relates to biotechnology, in particular to biotechnology crops. Produce nucleotide sequences from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus lumintecens, the expression of which is the formation of new insecticidal toxins. Transform the cell-hosana chimeric gene comprising one selected from the nucleotide sequences. Get composition and formulation containing insecticidal toxins, suitable for insect pests. The invention improves plant resistance to insect pests, and thus increase the yield of agricultural products. 8 N., and 15 C.p. f-crystals.
The invention relates to new toxins from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus lumintecens, to nucleotide sequences, the expression of which toxins are formed, and a process for the production and usage of toxins and the corresponding nucleotide sequences for controlling insects.
Insect pests are a major cause of crop losses. Only in the US, the annual yield losses due to lesions of different insect species are evaluated pressurecialis also makes it difficult for the growers and blogowogo, manufacturers of decorative colors, and they are a source of trouble for gardeners and homeowners.
In the main insect pests are struggling through intensive use of chemical insecticides possessing activity against inhibition of insect growth, prevent food or insect, or leading to their death. This method can effectively deal with insects, but such chemicals struggle sometimes also affect other beneficial insect species. Another problem arising from the widespread use of chemical pesticides is the emergence of resistant strains of insects. This problem is partially solved using different strategies for sustainability management, however, there is increasing need for the creation of alternative agents for pest control. Also used with satisfactory result of biological agents against insects, such as strains of Bacillus thuringiensis expressing insecticidal toxins such as endotoxins, and they represent an alternative or Supplement to chemical insecticides. Recently been selected genes encoding some of these endotoxins, and was fitted the ohmic perspective insect pests. In particular, expression of transgenic insecticidal toxins such as endotoxins of Bacillus thuringiensis provides effective protection against certain insect pests, and transgenic plants expressing such toxins are commercially available that allows farmers to reduce the number of used chemical agents against insects. However, even in this case, there is a possibility of development of resistance and may be limited to the number of only a small number of certain types of insect pests. Thus, it remains a long-conscious but not satisfied the need for the creation of new and effective agents to combat insects that would bring farmers economic benefits and be acceptable from the point of view of impact on the environment.
The present invention is directed to solving long-standing problems of creation of new agents to combat insects. First of all the necessary agents, which are aimed at combating important from an economic point of view insect pests and allow you to effectively deal with the strains of insects that are resistant to existing agents for combating insects. In addition, I preferred the hentov for combating insects of special interest are certain classes of nematodes of the genera Heterorhabdus and Steinernema, due to their insecticidal properties. They kill insect larvae and their offspring feed on the dead larvae. In fact insecticidal activity due to symbiotic bacteria living in nematodes. Such symbiotic bacteria Photorhabdus are in the case of Heterorhabdus and Xenorhabdus in the case of Steinernema. The present invention relates to nucleotide sequences isolated from Xenorhabdus nematophilus, to nucleotide sequences, almost similar, the expression of which leads to the production of insecticidal toxins that are toxic to important from an economic point of view, pests, primarily pests of plants. In addition, the invention relates to insecticidal toxin resulting from the expression of the nucleotide sequence, and to compositions and preparative forms, including insectopedia toxin, which have the ability to inhibit (suppress) the survival of insect pests, their growth or reproductive ability, or limit caused by insect damage or loss of crops of cultivated plants. In addition, the invention relates to a method for production of the toxin and the methods of application n the deposits to impart resistance to insects, and method of application of the toxin, and compositions and preparative forms, including toxin, for example, by treatment with toxin composition or preparative form of infected insects space, or for prophylactic treatment of susceptible to insects or plants that are potential targets, to protect or create resistance against harmful insects.
A new toxin has a high insecticidal activity against Plutella xylostella (cabbage moth), important from an economic point of view of the insect pest. The toxin can be used in numerous strategies for combating insects, leading to the achievement of maximum efficiency combined with minimal impact on the environment.
One of the objects of the present invention is selected nucleic acid molecule, comprising: (a) a nucleotide sequence that is almost identical to the nucleotide sequence selected from the group comprising: the nucleotide 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14; or (b) the nucleotide sequence, isooctanol nucleotide sequence of the criminal code of the e of one toxin, possessing activity against insects. In one of the embodiments of the invention the nucleotide sequence is isooctanol nucleotide sequence that is almost identical to nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. Preferably the nucleotide sequence is almost identical to nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. More preferably, the nucleotide sequence encodes the amino acid sequence selected from the group comprising SEQ ID NO.:2, 3, 5, 7, 9, 11, 13 and 15. Most preferably, the nucleotide sequence comprises nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. In another embodiment, the nucleotide sequence comprises the DNA fragment length of approximately 3.0 T. p. N., contained in pCIB9369 (NRRL B-21883).
According to a preferred variant implementation of the toxins resulting from the expression of the nucleic acid molecules according to the invention possess activity against Plutella xylostella.
Other objektom, 45, or 50 (preferably 20) base pairs a part of the nucleotide sequence is identical to the corresponding consisting of 20, 25, 30, 35, 40, 45 or 50 (preferably 20) consecutive base pairs of a part of the nucleotide sequence selected from the group comprising: the nucleotide 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, moreover, expression of the nucleic acid molecule leads to the formation of at least one toxin with activity against insects.
The object of the present invention is a chimeric gene containing a heterologous promoter sequence functionally linked with the nucleic acid molecule according to the invention. In addition, an object of the present invention is a recombinant vector containing this chimeric gene. Another object of the present invention is a host cell containing such chimeric gene. A host cell according to this object of the present invention may be a bacterial cell, a yeast cell or a plant cell, preferably plant cell. And another object of the present invention is a plant containing such rastitel is retene are toxins, the resulting expression of the DNA molecules of the present invention. According to a preferred variant implementation of the toxins of the present invention possess activity against Plutella xylostella.
In one embodiment, the implementation of the toxins produced by E. coli strain having registration number NRRL B-21883.
In another embodiment, the toxin according to the invention contains an amino acid sequence selected from the group comprising SEQ ID NO.:2, 3, 5, 7, 9, 11, 13 and 15.
It is also an object of the invention is a composition comprising effective as an insecticide quantity of toxin according to the invention. Another object of the present invention is a method of producing a toxin that has activity against insects, providing: (a) obtaining a host cell containing a chimeric gene that comprises a heterologous promoter sequence functionally linked with the nucleic acid molecule according to the invention; and (b) the expression of the nucleic acid molecule in the cell, which leads to the formation of at least one toxin with activity against insects.
Another object of the present invention suitable for all kinds of the nucleic acids according to the invention, moreover, the nucleic acid molecule is able to be expressed in the plant in a quantity effective against insects. According to a preferred variant implementation of insects is Plutella xylostella.
And another object of the present invention is a method of combating insects, introducing the insect effective amount of a toxin of the present invention. According to a preferred variant implementation of the insect is a Plutella xylostella. Preferably, the toxin enters the body of an insect oral route.
Another object of the present invention is a method of implementation of the mutation of the nucleic acid molecules of the present invention, according to which a molecule of nucleic acid digested with obtaining a population of random double-stranded fragments of the desired length and perform the following steps:
a) adding to the resulting population of random double-stranded fragments one or more single - or double-stranded oligonucleotides, each of the oligonucleotides contains the identity and scope of heterological against double-stranded matrix polynucleate is obtaining single-stranded fragments;
C) incubation of the resulting population of single-stranded fragments with a polymerase under conditions in which there is resaturate these single-stranded fragments at the areas of identity with the formation of pairs denaturirovannykh fragments, and these areas of identity are sufficient for one member of the pair has primeaval replication of the other, leading to the formation of double-stranded polynucleotide containing the mutation; and
g) repeating the second and third stages at least for two more cycles, and the resulting mixture in the second stage loop contains a double-stranded polynucleotide with mutation obtained in the third stage of the previous cycle, resulting in further cycles lead to the formation of additional quantities of double-stranded polynucleotide containing the mutation.
Other objects and advantages of the present invention will be obvious to experts in the field based on the study of the description and examples, not limiting its scope.
The concept of "activity" in relation to toxins according to the invention means that the effect of the toxins as agents for combating n who have the ability to stop or disrupt food insect that may cause or may cause death of the insect. If the toxin according to the invention enters the body of an insect, the result usually is the death of an insect or insect ceases to be powered from a source, from which the toxin is delivered to the insect.
The term "coupled with/functionally associated with" refers to two nucleotide sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is associated with" a DNA sequence that encodes a RNA or a protein if the two sequences are functionally related or located so that the regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.
The term "chimeric gene" refers to a recombinant nucleotide sequence in which a promoter or regulatory nucleotide sequence is functionally connected or connected with a nucleotide sequence that encodes an mRNA or which is expressed in the form of protein, so that the regulatory nucleotide sequence is able to regulate transcription or expression of the tie is the norm is not functionally related to connected with it, nucleotide sequence, as it occurs in natural conditions.
The term "coding sequence" means a nucleotide sequence that is transcribed with the formation of RNA, such as mRNA, rRNA, tRNA, snPHK (M. I.RNA, sense RNA or antisense RNA. Preferably then the RNA is translated in the body with the formation of a protein.
The concept of a "fight" with insects means to inhibit the toxic action of the ability of insect pests to survive, to grow, to feed and/or reproduce, or to restrict associated with insect damage or loss of crops of cultivated plants. When fighting insects can kill or not to kill insects, although preferably this concept means to kill insects.
The notion of "introduction (intake)" toxin means that the toxin comes in contact with insects, which leads to the toxic effects and the fight against insects. The toxin can enter the body in many known ways, for example, orally at eating insects, or in contact with insects as a result of expression of the toxin transgenic plant, using preparative(s) form-based protein composition (s) for opisina.
The term "expression cassette" in the context of the present description refers to a nucleotide sequence capable of expression of a particular nucleotide sequence in an appropriate cell host, which includes the promoter functionally linked with interest nucleotide sequence that is functionally linked to termination signals. It is normal also contains sequences required for proper translation of the nucleotide sequence. The expression cassette containing the interest nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. The expression cassette may also be a tape, which occurs in natural conditions, but which was obtained in recombinant form, suitable for heterologous expression. However, as a rule, the expression cassette is heterologous in relation to the owner, i.e. a certain DNA sequence of the expression cassette does not occur under natural conditions in the cell the owner and must be Introduzione in the expression cassette may be under the control of a constitutive promoter or inducible promoter, which initiates transcription only when the cell host process a particular external stimulus. In the case of a multicellular organism such as a plant, the promoter can also be specific to a particular tissue or organ or stage of development.
The term "gene" refers to a particular region within the genome, which in addition to the above coding sequence contains other, primarily regulatory nucleotide sequences responsible for the control of expression, i.e. transcription and translation of the coding region. A gene may also include other 5’- and 3’-noncoding sequence and the termination sequence. Other elements that may also be present in the gene are, for example, introns.
The term "gene of interest" refers to any gene that is carried into the plant gives the plant the required properties, such as resistance to antibiotics, resistance to viruses, resistant to insects, disease resistance, or resistance to other pests, resistance to herbicides, improved nutritional value, improved performance from the standpoint of industrial plumage is carried in plants for the production of important from an economic point of view, enzymes or metabolites in plants.
"Heterologous" nucleic acid sequence refers to a nucleotide sequence, which under natural conditions is not associated with the host-cell into which it is injected, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequences.
"Homologous" nucleic acid sequence refers to a nucleotide sequence, which in natural conditions associated with the host-cell into which it is injected.
The term "homologous recombination" refers to the mutual exchange of fragments of the nucleic acid between homologous molecules of nucleic acids.
The term "insecticide" refers to toxic biological activity, through which it is possible to deal with insects, it is preferable to kill them.
The nucleotide sequence is "izoldovna" nucleotide sequence with which comparison is made, if the nucleotide sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by a nucleotide sequence which is soleirolii acid or an enzyme, thanks man exist outside of their natural environment and, therefore, are not natural products. The selected nucleic acid molecule or enzyme may exist in a purified form or may exist in an unnatural environment, such as, for example, a recombinant cell host.
"Nucleic acid molecule" or "nucleotide sequence" refers to a linear segment of a single - or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention the nucleic acid molecule preferably denotes a segment of DNA. The term "ORF" refers to the open reading frame.
The term "plant" means any plant at any stage of development, in particular seed plants.
The term "plant cell" refers to a structural and physiological unit of a plant, including protoplast and cellular membranes. The plant cell may be in the form of separate cells or cultured cells, or be a part of highly organized units, such as, for example, plant tissue, an organ of the plant or the whole plant.
The concept of "the cult of the emer, the protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo bags, eggs and embryos at different stages of development.
The term "plant material" refers to the leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, otocam, cell cultures or tissues, or any other part or product of a plant.
The concept of "organ of a plant" means a separate and structured differentiated part of a plant such as root, stem, leaf, leaf Bud or Bud.
The term "plant tissue" in the context of the present description refers to a group of plant cells organized into structural or functional unit. Means any plant tissue in planta or in culture. This term includes whole plants, plant organs, plant seeds, tissue culture, and any group of plant cells organized into structural and/or functional units, but are not limited to them. The application of this concept in combination with a specific type of plant tissue, as defined above, or for some other signs fall under this definition, or outside depending the concept of "promoter" means a noncoding DNA sequence, located against the course of transcription of the coding region, which contains the binding site of RNA polymerase II initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.
The concept of "protoplast" indicates the selected plant cell without cell membrane or only part of the cell membrane.
The concept of "regulatory elements" refers to sequences involved in the control of expression of the nucleotide sequence. Regulatory elements include a promoter functionally linked with interest nucleotide sequence, and signals termination. They also usually include sequences required for proper translation of the nucleotide sequence.
In its broadest sense, the notion of "almost the same" when it is used in the present description with reference to the nucleotide sequence, means a nucleotide sequence corresponding to the nucleotide sequence, with which comparison is made, and consider the sequence encodes a polypeptide having almost the performed comparison, for example, a polypeptide having only the replacement of amino acids that do not affect the function of the polypeptide. Preferably almost the same as the nucleotide sequence encodes a polypeptide encoded by the nucleotide sequence, with which comparison is made. Preferably, the percentage identity is almost the same as the nucleotide sequence and the nucleotide sequence, with which comparison is made, constituted at least 80%, more preferably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%. The nucleotide sequence, "almost the same" nucleotide sequence with which comparison is made, as a rule, hybridizes with the nucleic acid molecule with which a comparison is made in the following conditions: 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1 mm add at 50°C; washing twice SSC (2xSSC), 0,1% - ordinator at 50°C, more preferably in 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1 mm add at 50°C; shaded single SSC (1xSSC), 0,1% - ordinator at 50°C, more preferably in 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP44, 1 mm add at 50°C; washing in 0.1 xSSC, 0.1% of th-ordinator at 50°C, more preferably 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1 mm add at 50°C; washing in 0.1 xSSC, 0.1% of th-ordinator at 65°C.
The term "synthetic" refers to nucleotide sequences, including structural features that are not present in the naturally occurring sequence. For example, a synthetic sequence is called artificial sequence characterized by the lowest G+C content and the normal distribution of codons, genes characteristic of dicots and/or monocotyledonous plants.
The term "transformation" means the introduction of a heterologous nucleotide sequence in a cell of the host or in the body. In particular, the "transformation" refers to a stable integration of the DNA molecule into the genome of interest of the body.
The term "transformed/transgenic/recombinant" refers to the body of the host, such as a bacterium or plant, which introduced the heterologous molecule is a nucleic acid. The nucleic acid molecule can be stably integrated into the host genome or molecule nucleic cyclospasmol or infect other programs. It should be understood that the concepts of transformed cells, tissues or plants include not only the final product of the transformation process, but also transgenic progeny. The concept of "normal", "nereshennye" or "non-recombinant" refers to the wild-type organism, such as bacteria or plant that does not contain the heterologous nucleic acid molecule.
The nucleotides are indicated by their bases by the following standard abbreviations: adenine (a), cytosine (C), thymine (T) and guanine (G). Similarly, the amino acid is indicated by the following standard abbreviations: alanine (l; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Il; I), leucine (Leu; L), lysine (Lys; K), methionine (Met, M), phenylalanine (Phe, F); Proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp; W), tyrosine (Tight; Y) and valine (Val; V). In addition, (XAA; X stands for any amino acid.
A brief description of the sequences presented in the sequence listing
SEQ ID NO:1 is a sequence of DNA fragment length of approximately 3.0 T. p. N., contained in clone Xenorhabdus nematophilus pCIB is a sequence of protein with molecular weight ~15 kDa, encoded by orf1 clone pCIB9369.
SEQ ID NO:3 is a sequence similar to protein esterase juvenile hormone with a molecular mass of ~47,7 kDa encoded by orf2 clone RSV.
SEQ ID NO:4 represents the DNA sequence of orf1 clone Xenorhabdus nematophilus pCIB9381.
SEQ ID NO:5 is the sequence of the protein encoded by orf1 of the clone pCIB9381.
SEQ ID NO:6 represents the DNA sequence of orf2 clone Xenorhabdus nematophilus pCIB9381.
SEQ ID NO:7 is a sequence similar to protein of juvenile hormone esterase encoded by orf2 clone pCIB9381.
SEQ ID NO:8 represents the DNA sequence of orf1 clone Xenorhabdus poinarii pCIB9354.
SEQ ID NO:9 is the sequence of the protein encoded by orf1 of the clone pGIB9354.
SEQ ID NO:10 represents the DNA sequence of orf2 clone Xenorhabdus poinarii pCIB9354.
SEQ ID NO:11 is a sequence similar to protein of juvenile hormone esterase encoded by orf2 clone pCIB9354.
SEQ ID NO:12 represents the DNA sequence of orf1 clone Photorhabdus luminescens pCIB9383-21.
SEQ ID NO:13 is the sequence of the protein encoded by orf1 of the clone pCIB9383-21.
The following products were deposited in the Collection of patented crops the agricultural research service (Agricultural Research Service, Patent Culture Collection (NRRL), 1815 Northern University Street, Peoria, Illinois 61604, USA) in accordance with the Budapest Treaty on the international recognition of the Deposit of microorganisms for the purposes of patenting procedures. All restrictions on the availability of deposited products should be permanently eliminated after issuance of the patent.
New nucleotide sequence, the expression of which leads to formation of insecticidal toxins
The present invention relates to nucleotide sequences, the expression of which leads to the formation of new toxins, and to the obtaining and use of toxins for insect pests. Nucleotide sequence isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus luminescens, which representatives of the family Enterobacteriaceae. Bacteria of the genus Xenorhabdus are symbiotic bacteria nematodes of the genus Steinernema. Bacteria of the genus Photorhabdus are symbiotic bacteria nematodes of the genus Heterorhabditis. Nematodes Colo is CIGNA activity due to symbiotic bacteria R. R. Xenorhabdus and Photorhabdus. Applicants were the first who managed to highlight the nucleotide sequence of the present invention. The expression of the nucleotide sequences of the present invention leads to the formation of toxins, which can be used against lepidopteran insects, such as Plutella xylostella (cabbage moth).
The nucleotide sequence of the present invention contained in the clone RSV, characterized by the presence of DNA fragment length of approximately 3.0, etc., N. that is deposited according to the Budapest Treaty on the Deposit for patenting under registration number NRRL B-21883. The sequence of this DNA fragment is presented in SEQ ID NO:1. In SEQ ID NO:1 contains two open reading frames (ORF) (nucleotides 569-979 and nucleotides 1045-2334 respectively) encoding proteins with predicted molecular mass of 15 kDa and 47.7 kDa (sequence SEQ ID NO:2 and SEQ ID NO:3, respectively). Two ORF organized in an operon-like structure. Search for known sequences with homology to each ORF, using the UWGCG programs Blast and Gap did not reveal sequence with any significant homology with RF No. 1, and has revealed is not considered significant. Gap-analysis of the protein encoded by ORF No. 2 RSV conducted using the Blast program revealed 30,6% amino acid sequence identity and 44.1% amino acid sequence similarity with the sequence of the protein associated with juvenile hormone esterase (GenBank registration number 2921553, Henikoff and others, PNAS USA 89: 10915-10919 (1992)). The nucleotide sequence of the present invention was also compared with known sequences Xenorhabdus nematophilus, encoding the insecticidal toxin tox4 (WO 95/00647), however, significant homology was found. The DNA fragment length 3,0, etc., ad also was compared with the nucleotide sequence published in WO 98/08388. Using the UWGCG program Gap compared twenty-two sequence of 60 nucleotides each (60-measures) of DNA fragment length 38,2, etc., ad, described in WO 98/08388, with a DNA fragment with a length of 3.0, etc., AD. according to the present invention. The nucleotide sequence of the first 60-measure starts with the base 1 DNA fragment length 38,2, etc., ad, and the remaining 60-measures are located at intervals of approximately 2.0, etc., N. Analyzed each of the 22 sequences, complementary to sequence them. The highest percentage of identity that b is 3%, what is not considered significant homology. In addition, by the method of southern blotting five different fragments of the DNA sequence by the length of 38.2, etc., ad tested against hybridization with a DNA fragment with a length of 3.0, etc., AD. according to the present invention. Neither one of them was found positive hybridization signal.
In nucleotide sequences of each clone RSV, RSV and RSV-21 were also found two open reading frames. The nucleotide sequences of the two ORFS in each of RSV and RSV-21 had a high degree of homology with the sequences of the ORF RSV. Therefore, RF No. 2 proteins RSV and RSV-21 have almost the same homology with protein associated with juvenile hormone esterase that RF No. 2 protein RSV. The nucleotide sequence RF No. 1 RSV 77% identical to the nucleotide sequence of the ORF No. 1 RSV, the nucleotide sequence RF No. 2 RCW 79% identical to the nucleotide sequence RF No. 2 pCIB9369. ORF No. 2 protein RSV also has homology to proteins that are associated with juvenile hormone esterase (29,2% identity of amino acid sequences, 42,2% similarity of amino acid sequences).
The nucleotide sequences according to the invention can be, recycling in the sequence listing as the basis for design of primers for PCR. For example, oligonucleotides having sequences comprising, respectively, first and last 20-25 consecutive nucleotides of the sequence of orf1 SEQ ID NO:1 (e.g., nucleotides 569-588 and 957-976 SEQ ID NO:1) can be used as primers for PCR to amplify the coding sequence of orf1 (nucleotides 569-976 SEQ ID NO:1) directly from strain-source (strain of Xenorhabdus nematophilus ATCC 19061). In a similar manner from the corresponding strains with PCR using the ends of coding sequences listed in the sequence listing as the basis for primers for PCR can be amplified by other sequences of genes according to the invention.
In another preferred embodiment, the insecticidal toxins include at least one polypeptide encoded by a nucleotide sequence according to the invention. Molecular weight insecticidal toxin of the present invention according to experiments with fractionation by size is more than 6000 Da. After treatment with proteinase K in the bioanalysis on insects nab the toxins virtually resistant to treatment with proteinase K. Insecticidal toxins retain their insecticidal activity after storage for 2 weeks at 22°C or at 4°C. They also retain their insecticidal activity after freeze drying and storage for 2 weeks at 22°C. Insecticidal toxins also have activity after incubation for 5 min at 60°With, however, they lose their insecticidal activity after incubation for 5 min at 100°s or 80°C.
According to other variants of the implementation of the nucleotide sequence of the present invention can be modified by the inclusion of non-specific (random) mutations using a method known as recombination in vitro or rearrangement of DNA. This method is described by Stemmer and other, Nature 370: 389-391 (1994) and in U.S. patent 5605793, which is incorporated into this description by reference. On the basis of the nucleotide sequence presented in the present description, and its variants with improved properties, such as increased insecticidal activity, increased stability or different specificity or a broader spectrum of insect pests, which targets receive millions of copies of nucleotide sequences with Muta the excitation of double-stranded polynucleotide, which includes the nucleotide sequence of the present invention, and represents the matrix of the double-stranded polynucleotide split into two double-stranded random fragment of the desired size, and provides the stage adding to the resulting population of double-stranded random fragments one or more single - or double-stranded oligonucleotides, and these oligonucleotides comprise an area of identity and an area of heterological relatively representing a matrix of double-stranded polynucleotide; denaturation mixture of double-stranded random fragments and oligonucleotides with obtaining single-stranded fragments; incubating the resulting population of single-stranded fragments with a polymerase under conditions which result in renaturation of these single-stranded fragments in these areas of identity with obtaining pairs denaturirovannykh fragments, moreover, these areas of identity sufficient to premirovany replication of one of the members of the other pair, resulting in the double-stranded polynucleotide with the mutation; and repeating the second and third stages at least for two cycles, and obtained in the second stage the stage the previous cycle, and with optional cycle get additional amount of double-stranded polynucleotide with mutation. According to a preferred variant implementation of the concentration of certain types of double-stranded random fragments in the population of double-stranded random fragments is less than 1% in recalculation on weight of total DNA. According to another preferred variant implementation representing a matrix of double-stranded nucleotide comprises at least about 100 species of polynucleotides. According to another preferred variant of implementation, the size of double-stranded random fragments is from about 5 base pairs to 5 T. p. N. And according to another preferred variant implementation of the fourth stage of the method comprises repeating the second and third stages for at least 10 cycles.
Expression of nucleotide sequences in heterologous microorganisms-hosts
Insecticidal toxins, representing biological agents for combating insects, produced by expression of the nucleotide sequences in heterologous cells masters, able to Express the nucleotide consequently is eficacia at least one nucleotide sequence according to the present invention in place of its location in the chromosome. Such modifications include mutations or deletions of existing regulatory elements, leading to changes in the expression of the nucleotide sequence, or the inclusion of new regulatory elements controlling the expression of the nucleotide sequence. In another embodiment, the cells Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus luminescens introduce additional copies of one or more nucleotide sequences or by integration into the chromosome, or by the introduction can replicate outside of the chromosome molecules that contain nucleotide sequences.
In another embodiment, at measure one nucleotide sequence according to the invention is inserted into a suitable expression cassette containing a promoter and termination signals. The expression of the nucleotide sequence may be constitutive or for initiation of translation can be used inducible promoter that responds to different types of stimuli. In a preferred embodiment, the cage in which is expressed the toxin, is a microorganism such as a virus, bacterium or fungus. In a preferred embodiment, the virus, such as baculovirus is as relevant insecticidal toxin after infection respective eukaryotic cells, suitable for virus replication and expression of the nucleotide sequence. Produced thus insecticidal toxin is used as an insecticidal agent. Alternatively, use baculoviruses, are designed so that they include the nucleotide sequence, in order to infect insects in vivo and kill them or as a result of expression of the insecticidal toxin, or by a combination of viral infection and expression of insecticidal toxin.
Bacterial cells can also serve as hosts for expression of the nucleotide sequences according to the invention. In a preferred embodiment, use of non-pathogenic commensal bacteria that can live and multiply in the tissues of plants, the so-called endophyte, or non-pathogenic commensal bacteria, which are able to colonize phyllosphere or the rhizosphere, the so-called epiphytes. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flawobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi such as Trichoderma and Gliocladium are possible hosts for expression of the nucleotide sequence is s in this area, and they are specific in relation to various suitable hosts. For example, for the expression of heterologous genes in E. coli can be used in expression vectors ALAC.223-3 and ALAC.223-2, built in the form of a transcriptional or translational fusion under the control of the promoter tac or trc. For the expression of operons that encode multiple ORF, the most simple method consists in embedding operon in the form of transcriptional merge in such a vector as ALAC.223-3 that allows the use of related binding site of the ribosome heterologous genes. In this area are also known methods of implementation overexpression in gram-positive species, such as Bacillus, and they can be used in the context of the present invention (Quax, etc., Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz and others (EDS)), American Society for Microbiology, Washington (1993)). Alternative systems to ensure overexpression based on, for example, yeast vectors, and they include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman and Skatrud (ed-ry), American Society for Microbiology, Washington (1993)); Deguin and Barre, Biotechnology 12: 173-177 (1994); van den Berg and others, Biotechnology 8: 135-139 (1990)).
In another preferred embodiment, at least one is in published patent application EU 0472494 and in WO 94/01561), which has the characteristics required for biological control. In another preferred embodiment, the nucleotide sequence according to the invention is transferred into a strain of Pseudomonas auerofaciens 30-84 that also has the characteristics required for biological control. Expression in heterologous strains intended for biological control requires the selection of vectors suitable for replication in the selected host, and the appropriate selection of the promoter. In this area are well known methods for expression in gram-negative and gram-positive bacteria and fungi.
Expression of nucleotide sequences in plant tissue
In the most preferred embodiment, at least one of the insecticidal toxins of the invention Express a higher organism, e.g. a plant. In this case, transgenic plants expressing an effective amount of toxins to protect themselves from insect pests. If an insect eats such a transgenic plant, it also absorbs downregulation of toxins. This may deter the insect from further eating plant tissues or even the eye is obreteniyu inserted into the expression cassette, which preferably stably integrate into the genome of plants. In another preferred embodiment, the nucleotide sequence include non-pathogenic sampleservice virus. Plants transformed according to the present invention can provide a monocotyledonous or dicotyledonous plants, such as corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, pumpkin large Pepo pumpkin, hemp, zucchini, Apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, BlackBerry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thalaiana and woody plants such as coniferous and deciduous trees, but not limited to.
After desired nucleotide sequence transformed certain types of plants, it may be propagated in that species or moved into other varieties of the same species, primarily in commercial importance with which retenu preferably Express in transgenic plants, thus leading to the biosynthesis of the corresponding toxin in transgenic plants. In this way receive a transgenic plant having increased resistance to insects. For their expression in transgenic plants DNA molecules may require modification and optimization. Although in many cases the genes of microorganisms can be expressed with high levels in plants without modification, low expression in transgenic plants may be due to the fact that the nucleotide sequence of microorganisms have codons that are preferred for plants. In this area it is known that all organisms have specific preferences regarding the most frequent codon and the codons in the nucleotide sequence, which comprises the DNA molecule of the present invention, can be replaced in order to meet the specific preferences of the plants, preserving the amino acid sequence that is encoded by them. In addition, the high level of expression in plants is most easily achieved with the use of the coding sequences in which the GC content is at least about 35%, the preference is more positive than approximately 60%. The nucleotide sequences of microorganisms with low GC content, may not be expressed due to the presence of motifs ATTA, which can destabilize transcripts, and motives of AATAAA, which can cause inadequate polyadenylation. Although the preferred sequence of genes can accordingly be expressed as in monocotyledonous and dicotyledonous plant species, sequences can be modified to suit the specific preferences of monocotyledonous or dicotyledonous plant codon and GC content, since, as has been established, these preferences is different (Murray and others, Nucl. Acid. Res. 17: 477-498 (1989)). In addition, the nucleotide sequence is subjected to screening for the presence of illegal splicing sites, which may lead to shortening of the transcripts. All necessary changes in nucleotide sequences, such as the changes described above, is performed using well known methods siteprovides mutagenesis, PCR and construction of synthetic genes using the methods described in the published patent applications EP 0385962 (in the name of the Monsanto company), EP 0359472 the change necessary to modify the sequence, adjacent to the site of initiation methionine. For example, they can be modified by inclusion of sequences for which it is known that they are effective in plants. Joshi suggested suitable for plant consensus sequence (NAR 15: 6643-6653 (1987)), and the company Clontech proposed additional consensus initiator broadcast (1993/1994 catalog, p. 210). These consensus sequences can be used with nucleotide sequences according to this invention. These sequences include constructs that contain the nucleotide sequence, up to ATG inclusive (leaving the second amino acid unmodified) or in the alternative to following the ATG codon GTC inclusive (in this case, allow for the possibility of modification of the second amino acid of the transgene).
In transgenic plants, the expression of nucleotide sequences is under the control of the promoter, which has the ability to function in plants. The choice of promoter may vary depending on the temporal and spatial pattern of expression, and depending on the target species. So it is preferable expression nucleot panicles, the rods corn cobs, and so on), in the roots and/or leaves. However, in many cases you are required to have protection from more than one type of pest insects, so it is advisable to expression occurred in several types of tissue. Although it is established that many promoters of dicotyledonous plants can function in monocots and Vice versa, ideally for expression in dicotyledonous choose the bipartite promoters, and for expression in monocots choose the promoters monocots. However, there are no restrictions on the source of the selected promoters; it is sufficient that they were functionally active in controlling the expression of DNA molecules in the target cell.
Preferred promoters that are constitutively expressed, include the promoters from the genes encoding actin or ubiquitin, and the 35S promoter and CaMV 19S. The nucleotide sequence of the present invention can also be expressed under the control of a chemically regulated promoters. This allows to synthesize insecticidal toxins only when cultivated plants treated with inducing chemical compounds. The preferred method of chemical induction of gene expression is automatic inducible promoter is the promoter of PR-1a tobacco.
The preferred category promoters are induced by wounding promoters. Described by many promoters which ensure expression in areas of injury, and infection by the pathogen. Ideally, such a promoter should be active only locally at the site of infection, and in this case insecticidal toxins accumulate only in those cells in which it is necessary to synthesize insecticidal toxins to kill the attacking insect pests. Preferred promoters of this type include promoters, described by Stanford and others, Mol. Gen. Genet. 215: 200-208 (1989), Xu and other Plant Molec. Biol. 22: 573-588 (1993), Logemann and other Plant Cell 1: 151-161 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek and other Plant Molec. Biol. 22: 129-142 (1993) and Warner and other Plant J. 3: 191-201 (1993)).
Preferred tissue-specific schema expression includes expression, specific for green tissue-specific root-specific stem and specific flower. Promoters suitable for expression in green tissues, include many promoters that regulate expression of genes involved in photosynthesis, and many of them originating from both monocotyledonous and dicotyledonous plants, were cloned. Preferably repectfully promoter for specific root expression is the promoter, described by de Framond (FEBS 290: 103-106 (1991); EP 0452269 on the name of the company Ciba-Geigy). Preferred specific to stem the promoter is the promoter described in U.S. patent 5625136 (in the name of the company Ciba-Geigy), which controls the expression of a gene trpA corn.
In preferred variants of the invention, the transgenic plants Express a nucleotide sequence according to the invention-specific root. In other preferred embodiments, the implementation of the transgenic plants Express a nucleotide sequence under the control of a promoter induced by injury or induced by infection by a pathogen.
In addition to the selection of an appropriate promoter, in structures designed for expression of proteins in plants requires a terminator of transcription, which must be attached in the course of transcription relative to the heterologous nucleotide sequence. In this area are known and can be used several of these terminators (e.g., tm1 CaMV, rbcS E9). According to the present invention can be applied to any terminator for which he is known to function in plants.
In the expression cassette, predstaviteljnosti, have the ability to enhance expression such as intron sequences (e.g. genes Adh1 and bronze1) and viral leader sequences (e.g., tobacco mosaic virus (TMV), virus chlorotic leaf spot of maize (MCMV), and alfalfa mosaic virus (AMV)).
It may be preferable to direct the expression of DNA molecules in various cellular compartments of the plant. In some cases, may require localization in the cytosol, whereas in other cases it may be preferred localization in any of the subcellular organelle. Subcellular localization of the encoded by a transgene of the enzyme can be carried out using well-known in the field of methods. Generally, DNA encoding guide the peptide of known gene product, involved in the directed migration in a specific organelle, process and merge against the course of transcription relative to the nucleotide sequence. For chloroplast known a number of such guides transfer sequences and found that they function in heterologous constructs. The expression of the nucleotide sequences of the present invention is also directed to e is e to achieve this.
Vectors suitable for transformation of plants below in the present description. For indirect Adrobacterium transformation can be applied to binary vectors or vectors carrying at least one border sequence of T-DNA, however, for direct gene transfer any suitable vector and may be preferred linear DNA containing only interest design. In the case of direct gene transfer can be used transformation of certain types of DNA or cotransformation (Schocher, etc., Biotechnology 4: 1093-1096 (1986)). As for direct gene transfer and indirect Adrobacterium transfer usually (but not necessarily) apply the transformation using breeding marker, which can impart resistance to an antibiotic (kanamycin, hygromycin or metotrexat) or herbicide (Basta). Examples of such markers are neomycinphosphotransferase, GigabitEthernet, dihydrotetrazolo, phosphonomethylglycine, dehalogenase 2,2-dichloropropionic acid synthase of Ecotoxical, 5-enolpyruvylshikimate-paspatinath, gallerynitrous, protoporphyrinogen, acetyl-COA-carboxylase, dihydropteridine, chlorophenylacetone.
The above-described recombinant DNA molecule according to the invention can be introduced into the plant cell numerous known in the field methods. Professionals in this field should be obvious that the choice of method may depend on the type of plants intended for transformation. Suitable methods of transforming plant cells include microinjection (Crossway, etc., BioTechniques 4: 320-334 (1986)), electroporation (Riggs and others, Proc. Natl. Acad. Sci. USA 83: 5602-5606 (1986); transformation mediated by Agrobacterium (Hinchee and others, Biotechnology 6: 915-921 (1988); (concerning transformation of maize plants, see Ishida and others, Nature Biotechnology 14: 745-750 (June 1996)), direct gene transfer (Paszkowski and others, EMBO J. 3: 2717-2722 (1984)), ballistic introduction of particles using devices supplied by firms Agracetus, Inc., Madison, Wisconsin and Dupont, Inc. Wilmington, Delaware (see, for example, Sanford and others, U.S. patent 4945050; and McCabe and others, Biotechnology 6: 923-926 (1988)). Cm. also Weissinger and others, Annual Rev. Genet. 22: 421-477 (1988); Sanford and others, Particulate Science and Technology 5: 27-37 (1987) (bow); Svab and others, Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou and other, Plant Physiol. 87: 671-674 (1988) (soy); McCab etc., Bio/Technology 6: 923-926 (1988) (soybean); McCabe and others, Bio/Technology 6: 923-926 (1988) (soy); Klein and others, Proc. Natl. Acad. Sci. USA, 85: 4305-4309 (1988) (maize); Klein and others, Bio/Technology 6: 559-563 (1988) (maize); Klein the Biotechnology 11: 194-200 (1993) (maize); Shimamoto and other, Nature 338: 274-277 (1989) (rice); Christou and others, Biotechnology 9: 957-962 (rice); Datta and others, Bio/Technology 8: 736-740 (1990) (rice); the application for the European patent EP 0332581 (cocksfoot and other Pooideae); Vasil and others, Biotechnology 11: 1553-1558 (1993) (wheat); Weeks, etc., Plant Physiol. 102: 1077-1084 (1993) (wheat); Wan et al., Plant Physiol. 104; 37 to 48 (1994) (barley); Jahne and others, Theor. Appl. Genet. 89: 525-533 (1994) (barley); Umbeck, etc., Bio/Technology 5: 263-266 (1987) (cotton); Casas and others, Proc. Natl. Acad. Sci. USA, 90: 11212-11216 (December 1993) (sorghum); Somers and others, Bio/Technology 10: 1589-1594 (December 1992) (oat); Torbert and others, Plant Cell Reports 14: 635-640 (1995) (oat); Weeks, etc., Plant Physiol. 102: 1077-1084 (1993) (wheat); Chang and others, WO 94/13822 (wheat) and Nehra and others, The Plant Journal 5: 285-297 (1994) (wheat). The most preferred number of options for implementing the introduction of recombinant DNA molecules in maize using bombardment microkeratome can be found at Koziel, etc., Biotechnology 11: 194-200 (1993), Hill and others, Euphytica 85: 119-123 (1995) and Koziel, etc., Annals of the New York Academy of Sciences 792: 164-171 (1996). Additional preferred embodiment is a method of transformation of protoplast corn, described in EP 0292435. Transformation can be carried out with the use of certain types of DNA or more types of DNA (i.e., cotransformation), and both of these methods are suitable for use in applying the sequence, kodiruyushchemu according to the present invention is directly transformed plastid genome. The main advantage of plastid transformation is that plastids, as a rule, able to Express bacterial genes without significant modification and that plastids are able to Express multiple open reading frames under the control of a single promoter. The method of transformation of plastids are described in detail in patents US 5451513, 4545817 and 5545818; in the international patent application WO 95/16783; and McBride and others, Proc. Natl. Acad. Sci. USA 91: 7301-7306 (1994). The main method of chloroplast transformation involves introducing suitable the target tissue sites cloned plastid DNA flanking breeding token together with the gene of interest, for example, using the method booballistics transfer or transformation protoplast (for example, transformation mediated by calcium chloride or PEG). Flanking region of length 1-1,5, etc., ad, called sequences, providing directional migration, facilitate homologous recombination with the plastid genome and, thus, enable you to replace or modify specific region of the plastome. Initially as breeding markers for transformation used point mutations in the chloroplast 16S genes-pPHK and rps12 USA 87: 8526-8530 (1990); Staub J. M. and Maliga P., Plant Cell 4: 39-45 (1992). This results in stable homoplasmic of transformants at a frequency of approximately 1 transformant per 100 bombing leaves target. The presence of cloning sites between these markers allows you to create a vector for the introduction of alien genes directional migration in the plastid (Staub J. M. and Maliga P., EMBO J. 12: 601-606 (1993)). A significant increase in the frequency of transformation get by replacing the genes are recessive rRNA or R-protein, contributing to antibiotic resistance, the dominant breeding marker, the bacterial aadA gene coding for pectinmethylesterase the enzyme aminoglycoside-3’-adinistrator (Svab, Z. and Maliga, P. Proc. Natl. Acad. Sci. USA 90: 913-917 (1993)). Earlier this token was successfully used to occur with high frequency transformation of the plastid genome of the green Alga Chlamydomonas reinhardtii (Goldschmidt-Clermont M., Nucl. Acids Res. 19: 4083-4089 (1991)). In this area other known breeding markers suitable for the transformation of plastids, and they fall under the scope of the invention. Typically, to achieve homoplastic state after the transformation takes approximately 15-20 cycles of cell division. Expression in plastids, in which genes integrated ludaday plant cell, has the advantage consisting in the fact that a huge number of copies exceeds the number of copies of expressed genes in the nuclear DNA, helps to ensure the levels of expression, which can easily exceed 10% of the total soluble protein of the plant. In a preferred embodiment, the nucleotide sequence of the present invention is inserted into the vector, providing directional migration in the plastid, and transform them plastid genome of the respective host plant. Plants receive, homoplasies in respect of plastid genomes, which contain the nucleotide sequence of the present invention and which preferably is capable of the expression of the nucleotide sequence with a high level.
Preparation preparative forms containing insecticidal composition
Under the scope of the invention also includes compositions comprising at least one of the insecticidal toxins of the present invention. For effective control of insect pests such compositions should preferably contain a sufficient amount of toxin. These amounts vary depending on subject to the protection of culture, the. the preferred embodiment of the composition, including insecticidal toxins contain cell-hosts expressing toxins, not subjected to additional cleaning. In another preferred embodiment, cells expressing insecticidal toxins, is subjected to lyophilization prior to their use as insecticidal agent. In another embodiment, creating such insecticidal toxins that are secreted by cells of the host. In cases where it is desired cleaning toxins from host cells in which they are expressed, can be achieved in various degrees of purification of insecticidal toxins.
Under the scope of the present invention also falls within the method of preparation of compositions comprising at least one insecticidal toxin of the present invention, mixed until smooth with one or more compounds or groups of compounds listed in this description. The present invention also relates to a method of processing plant capable of handling plants insecticidal toxins or compositions containing insecticidal toxins. Insecticidal toxins may be made in the form cvetelina with other biologically active compounds. These compounds can be represented as fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be characterised by the selective action of herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or a mixture of several of these preparations, if necessary together with other carriers, surfactants or conducive to the application of adjuvants commonly used in the manufacture of preparative forms. Suitable carriers and adjuvants can be solid or liquid, and they are substances commonly used in the manufacture of preparative forms, i.e. suitable or regenerated mineral substances, solvents, dispersing agents, wetting agents, adhesives, binders, or udobreniya.
The preferred method of application of the insecticidal toxins of the present invention is the spraying of the habitat of the insect pest, such as soil, surface water or the leaves of plants. The number of treatments and application rates depend on the type and intensity of infection insect-pest. Insecticidal macrostructure plants with a liquid composition or in the soil of compounds in solid form, for example in granular form (soil application). Insecticidal toxins can also be applied to seeds (coating) either by impregnating the seeds with a liquid composition containing insecticidal toxin or coated solid composition. In some cases you can also apply other types of processing, such as selective processing of trunks or buds of plants. Insecticidal toxins can also be used as baits placed on the soil surface or beneath the surface.
Insecticidal toxins are used in unmodified form or preferably together with the adjuvants commonly used in the field of machinery manufacturing preparative forms, and, therefore, they can be prepared in the usual way in the form of emulsion concentrates, covering pastes, ready-to-use spray or dilutable solutions, diluted emulsions, wettable powders, soluble powders, Farrukh Dustov, granules, and also in encapsulated form, for example in polymeric substances. Depending on the nature of the compositions in accordance with the objectives and the prevailing circumstances choose processing methods, such as spraying the flies or drugs, containing insecticidal toxins, and optionally a solid or liquid adjuvant, get in a known manner, for example, by homogeneous mixing and/or grinding the active substances with extenders, for example with solvents, solid carriers and, if necessary, surface active substances (detergents).
Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8-12 carbon atoms, for example, xiaowei mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctylphthalate, aliphatic hydrocarbons, such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, onomatology or monotropy ethers of ethylene glycol, ketones, such as cyclohexanone, highly polar solvents such as N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxydecane vegetable oils, such as epoxydecane coconut oil or soybean oil, or water.
The solid carriers used, for example, Farrukh Dustov and dispersible powders, are usually natural mineral fillers such as calcite, talc, kolinapin silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are such porous types, for example pumice, broken brick, thick or bentonite; and suitable nenormiruemym carriers are materials such as calcite or sand. In addition, there may be used a large number of pre-granulated materials of inorganic or organic origin, for example, particularly preferred dolomite or crushed into powder residues.
Suitable surface-active compounds are nonionic, cationogenic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. Under the concept of "surface active substances" also includes mixtures of surface-active substances. Suitable anionic surfactants can be a water-soluble Soaps and water-soluble synthetic surface-active substances.
Suitable Soaps are the alkali metal salts, salts of alkaline earth metal or unsubstituted or substituted ammonium salts and higher fatty acids having chains containing 10-22 carbon atoms), such as sodium and is obtained, for example, from coconut or tallow oil. In addition, can also be used methyltaurine salts of fatty acids.
More often, however, used the so-called synthetic surfactants, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
The fatty sulfonates or sulfates are usually applied in the form of salts of alkali metals, salts of alkaline earth metal or unsubstituted or substituted ammonium salts, and they usually have an alkyl radical containing 8-22 carbon atoms, which also includes the alkyl fragment of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a mixture of sulfates of fatty alcohols obtained from natural fatty acids. These compounds also include salts of esters of sulfuric acid, and adducts of sulfonic acids of fatty alcohol/ethylene oxide. Sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid group and one radical of a fatty acid containing from 8 to 22 carbon atoms. Examples of alkylarylsulfonates are sodium, calcium or triethanolamine salt modellen is islote/formaldehyde. Also suitable are corresponding phosphates, e.g. salts of ether phosphoric acid and adduct para-Nonylphenol with 4 to 14 by moles of ethylene oxide.
Nonionic surfactants preferably are derived polyglycolic ether and aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and ALKYLPHENOLS, these derivatives contain 3-30 glycolic ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon fragment and 6-18 carbon atoms in the alkyl fragment of ALKYLPHENOLS.
In addition, suitable nonionic surfactants are the water-soluble adducts of polyethylene oxide and polypropylenglycol, ethylenediaminetetra and alkylpiperidines containing 1-10 carbon atoms in the alkyl chain, and the adducts contain 20 to 250 groups etilenglikolevykh ether and 10-100 groups of propylene glycol ether. These compounds usually contain 1 to 5 fragments of ethylene glycol on a fragment of propylene glycol.
Typical examples of nonionic surfactants are nonylphenolethoxylates, ethers, castor oil and polyglycol, adducts polypropyle fatty acids and polyoxyethylenesorbitan, such as triolein polyoxide-lincolniana, are also suitable nonionic surface-active substances.
Cationogenic surfactants preferably are salts of Quaternary ammonium compounds, which have, as N-substituent at least one8-C22alkyl radical and, in addition, as further substituents, unsubstituted or halogenated lower alkyl, benzyl or hydroxy-(lower) alkyl radicals. Preferred salts in the form of halides, methylsulfate or ethylsulfate, for example the chloride of stearylamine or bromide, benilde(2-chloroethyl)ethylamine.
Surfactants commonly used in the field of preparation preparative forms described e.g. in "McCutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp. Ridgwood, New Jersey, 1979; and Sisley and Wood, "Encyclopedia of Surface Active Agents", Chemical Publoshing Co., Inc. New York, 1980.
The invention is described hereinafter with reference to the following examples. These examples are only for illustration purposes and are not intended to limit the invention, unless otherwise indicated. Used standard methods of recombinant DNA and molekulyarnoj); in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory, Cold Spring Harbor, NY (1989); and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Ezperiments with Gene Fusions, Cold Spring Laboratory, Cold Spring Harbor, NY (1984).
A. Selection of nucleotide sequences, the expression of which leads to the formation of toxins with activity against insects of the order Lepidoptera
Example 1. Cultivation of strains of Xenorhabdus and Photorhabdus
For analyses of biological activity against insects of the following strains are cultivated in a nutrient medium, recommended ATS, at 25°C for 3 days. For DNA extraction of the culture grown in the same conditions within 24 hours
The strain of Xenorhabdus nematophilus ATCC 19061.
The strain of Xenorhabdus nematophilus Pst1, isolate the USDA.
The strain of Xenorhabdus poinarii ATCC 49122.
Strain Photorhabdus luminescens Ps5, isolate the USDA.
Analyses of biological activity against Plutella xylostella (Px) is conducted by introducing aliquot 50 ál of each culture E. coli on solid synthetic medium for P. xylostella (Biever and Boldt, Annals of Entomological Society of America, 1971; Shelton and others, J. Ent. Sci. 26: 17). 5 newly hatched larvae of P. xylostella from the laboratory colony, adapted to stern, is placed in each Cup containing to aliroot with 10 caterpillars. Trays with cups placed in a thermostat at 72°F for 3 days with a light cycle light:dark 14:10 (hours). Record the number of live larvae in each Cup.
Example 3. The results of the analysis of biological activity
In the analysis of biological activity according to the method described in example 2, using broth containing strain of Xenorhabdus nematophilus ATCC 19061, leads to 100% mortality of Plutella xylostella (Px). Similarly, in the analysis of biological activity according to the method described in example 2 using each of the broths containing strain of Xenorhabdus nematophilus Pst1, the strain of Xenorhabdus poinarii ATCC 49122 and strains of Photorhabdus luminescens Ps5, leads to 100% mortality of Plutella xylostella (Px).
Example 4. Design kosmidou library
Full DNA isolated from the strain of Xenorhabdus nematophilus ATCC 19061 by processing svezhevykrashennyh cells, resuspending in 100 mm Tris, pH 8, 10 mm etc, for 30 min at 37°To secrete lysozyme, taken at a concentration of 2 mg/ml Add proteinase K at a final concentration of 100 μg/ml in 0.5% LTOs and incubated at 45°C. the Solution becomes clear and very viscous. The concentration of LTOs increase to 1% and add 300 mm NaCl and an equal amount of the mixture of phenol-chloroform-isoamyl alcohol. Sample ouu phase is mixed with 0.7 volumes of isopropanol and centrifuged. Debris DNA is washed three times with 70% ethanol and gently resuspending 0.5 xTe (agitator). 6 μg DNA treated with 0.3% Sau3/µg DNA at 37°C for 3.5 min in a volume of 100 μl. Then the sample was incubated for 30 min at 65°C to inactivate the enzyme and then incubated for 30 min at 37°C with 2 units of alkaline phosphatase from calf intestine. The sample is mixed with an equal volume of a mixture of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed and mixed with 0.7 volumes of isopropanol and centrifuged. Debris resuspending 0.5 xTe with a concentration of 100 ng/ml.
Comedy vector SuperCos (firm Stratagene, La JOLLA, California) receive according to the supplier's instructions with your use of the site cloning BamHI. The resulting vector SuperCos at a concentration of 100 ng/ml are ligated over night at 6°C in a volume of 5 μl in a 2:1 ratio with the DNA of X. nematophilus, previously cleaved with Sau3A. The resulting ligation mix is Packed with a set of Gigapack III XL (firm Stratagene) according to the supplier's instructions. Packaged phages infect cells of E. coli strain XL-1MR (firm Stratagene) according to the supplier's instructions. Kosmidou library plated on L-agar containing 50 μg/ml konamis a density of 50 colonies/plate. Cells are washed with L-broth and mixed with 20% glycerol and frozen at -80°C.
For the genome of X. nematophilus size of 4.2 M. p. N. 450 clones with an average size of 40, etc., ad correspond to a 4-fold overlap of the genome. Therefore, when screening 450 clones there is a 99% probability of detecting any gene.
Cosignee library strain of Xenorhabdus nematophilus Pst1, Xenorhabdus strain poinarii ATCC 49122 and strains of Photorhabdus luminescens Ps5 design in the same way.
Example 5. The results of the biological analysis of cosmid and identification of clones possessing insecticidal activity
For analysis of biological activity against insects subjected to screening 400 E. coli clones from each kosmidou libraries to identify clones with activity against Plutella xylostella. Cloned kosmidou strain of Xenorhabdus nematophilus ATCC 19061 having insecticidal activity, is designated as RSV. Possessing insecticidal activity of a cloned kosmidou length 42, etc., N. from the strain of Xenorhabdus nematophilus Pst1 designated as RSV. Possessing insecticidal activity of a cloned kosmidou length 42, etc., N. from Xenorhabdus strain poinarii ATCC 49122 designated as RSV. Possessing insecticidal activity of the cloned KOs is having insecticidal activity of Clone RSV digested with SacII and produce a DNA fragment of length 9, etc., N. This fragment is inserted into the Bluescript vector cleaved with SacII. The resulting ligation mixture to transform cells of E. coli strain DH5 according to the method described in Molecular Cloning, 2nd edition (Sambrook and others). The resulting transformation mixture is plated on L-agar containing 100 μg/ml ampicillin, and incubated overnight at 37°C. Selected colonies grown in L-broth containing 100 μg/ml ampicillin, and plasmid DNA secrete using alkaline miniprep according to the method described in Molecular Cloning, 2nd edition. Allocated using SacII clone length 9, etc., N. denote as RSV-3, and it has insecticidal activity, causing 100% mortality of Plutella xylostella. There are 3 µg RSV-3, cleaved with the help of 0.3 units of Sau3A 1 µg DNA for 4, 6 and 8 min at 37°C and maintained at 75°C for 15 minutes, the Samples are arranged into groups and inserted into the plasmid pUC19 previously cleaved with BamHI and treated with alkaline phosphatase intestine of the calf. The resulting ligation product transform cells of E. coli strain DH5, plated on L-agar containing XgaI/Amp, according to the method described in Molecular Cloning, and grown overnight at 37°C. Colony of white color is selected and VeriSign digested with EcoRI/HindIII and the new resulting restriction fragments are subjected to sequencing. Primers for sequencing receive from the company Genosys Biotechnologies (the woodland, TX). Sequencing carried out according to dideoxy method on the basis of breaking the circuit and carry out using automated DNA sequencing machine model 377 firms Applied Biosystems Inc. (Forster city, Calif.). Sequence unite using the program Sequencher 3.0 firms Gene Code Corporation, Ann arbor, Michigan).
After identifying sites of restriction and possible ORF plasmid RSV-3 were cleaved with ClaI and fragment length 3,0, etc., N. exhale, clone in the Bluescript vector and transform them cells of E. coli strain DH5. Selected colonies are grown according to the method described above, and plasmid DNA secrete the method of alkaline lysis. Subclan identify as RSV and it causes 100% mortality of Plutella xylostella in the experiments for the study of biological activity. Strain RSV deposited in culture Collections for the purposes of patenting USDA ARS 12 November 1997 under registration number NRRL B-21883.
The fragment length of 20 T. p. N., designated as RSV, subcloning of Comedy RSV using cleavage with NotI.
Example 7. The results of the analysis of biological activity
Analyses of biological act is>P CLASS="ptx2">+ - significant inhibition of growth
+ + - the death of >40% but less than 100%,
+ + + - 100% death.
These results indicate that insecticidal toxin resulting from the expression in pCIB9369 nucleotide sequence with a length of 3.0 T. p. N., has a high activity against Plutella xylostella.
Analyses of the biological activity of strains RSV, RSV and RSV-21 have shown that these strains also possess high insecticidal activity against Plutella xylostella.
Example 8. Determination of particle size, causing insecticidal activity
Comedy clone Xenorhabdus nematophilus pCIB9369 and the vector BlueScript company Stratagene in the E. coli strain DH5 grown in a medium consisting of 50% of Terrific broth and 50% broth, Luria, supplemented with 50 μg/ml ampicillin. Cultures grown in shake flasks (300 ml flasks with baffles 1000 ml) at 250 rpm overnight at 37°C. the Culture of each strain centrifuged at 7000 rpm in a centrifuge type Sorvall GS at 4°C. Pelletierine cells resuspended in 30 ml of 50 mm NaCI, 25 mm Tris-buffer, pH 7.0. The concentrated cells were destroyed by exposure to ultrasound, using devices like Branson Model 450 Sonicator in the leak. The resulting ultrasonic irradiation, the product is centrifuged in a centrifuge type Sorvall SS34 at 6000 rpm for 10 min at 4°C. the Resulting supernatant filtered through a filter with a pore size of 0.2 μm. Debris spin-on products resulting from exposure to ultrasound, resuspended in 30 ml of 50 mm NaCl, 25 mm Tris-buffer, pH 7.0.
Fractions of the effluent volume to 3 ml contribute in a column of type Bio-Rad Econo-Pac 10DG, pre-equilibrated with 10 ml of 50 mm NaCl, 25 mm Tris-buffer, pH 7.0. Discard the flow collected during the loading of the samples. Samples fractionary by successive addition of two portions of 4 ml each equilibrating buffer NaCl Tris. The first three fractions are selected for analysis. The first fraction should contain all products having a molecular weight above about 6000 Da. These fractions should contain products with a molecular mass of less than 6000 Yes.
The sample is subjected to ultrasonic irradiation of the filtrate and resuspending debris after ultrasonic irradiation test along with the three fractions obtained using a 10DG columns, their activity against cvezhevypavshie larvae of P. xylostella method kontaktira, the first fraction of the sample ID 9369 obtained from the column, was highly effective against P. xylostella. The second and third fractions of the sample ID 9369 was inactive. None of the samples obtained from cultures of strain DH5 plasmid BlueScript, had no activity. These results indicate that the insecticidal activity inherent in the clone X. nematophilus pCIB9369, as well as its homologues RSV, RSV and RSV-21 caused by the fragment having a molecular weight greater than 6000 Yes.
Example 9. The stability of the insecticidal activity
300 ml of the broth Luria, supplemented with 100 μg/ml ampicillin, inoculant the plasmid pCIB9369 and grown over night at 37°C.
The sample is placed in a sterile test tubes with screw caps volume of 15 ml and stored at 22°C and 4°C. One sample is centrifuged, the supernatant removed, dried by freezing and stored at 22°C. These samples are kept in these conditions for 2 weeks and then analyze their biological activity against P. xylostella. The product obtained by drying, freezing, resuspended in equal volume as described previously for sample, freeze dried. All samples resuspended with shaking. Another arr>the example 10. Inactivation insecticidal activity when heated to Determine the stability of the toxin when heated. Culture of the strain E. coli RSV (strain-host E. coli DH5 carrying the DNA fragment Xenorhabdus nematophilus length 3,0, etc., ad) is grown in a mixture (50:50) broth, Luria and Terrific-broth. The culture is grown at 37°C in test tubes on a rotary shaker. Samples of each culture volume of 1 ml was placed in an Eppendorf tube of 1.5 ml and incubated at 60°s and 80°C. After 5 min, the samples are removed from thermostat and allow to cool to room temperature. The sample along with the untreated portion of the culture analyzed in relation to the activity to P. xylostella. 50 μl of the sample is applied by spraying on food, allowed to dry and placed on the surface cvezhevypavshie caterpillars of P. xylostella. The samples are incubated for 5 days at room temperature.
The untreated sample and the sample treated by heating to 60°C, caused 100% mortality. The sample treated by heating to 80°C and a control sample representing only the food did not cause visible destruction.
Example 11. Influenced by processing insecticidal activity
Comedy clone Xenorhabdus nematophilus pCIB9369 and the vector BlueScript company Stratagene in W is of picillin. Cultures grown in shake flasks (300 ml flasks with baffles 1000 ml) at 250 rpm overnight at 37°C. the Culture of each strain centrifuged at 7000 rpm in a centrifuge type Sorvall GS at 4°C. Pelletierine cells resuspended in 30 ml of 50 mm NaCl, 25 mm Tris-buffer, pH 7.0. Concentrated destroy cells by exposure to ultrasound, using devices like Branson Model 450 Sonicator for about eight 10-second cycles with the use of cooling on ice between cycles. The resulting ultrasonic irradiation, the product is centrifuged in a centrifuge type Sorvall SS34 at 6000 rpm for 10 min at 4°C. the Resulting supernatant filtered through a filter with a pore size of 0.2 μm. The concentration of CA++samples of the supernatant volume of 1 ml was adjusted to 5 mm by adding l2. Add protease K (firm Gibco BRL; Gaithersburg, MD) to a concentration of 500 µg/ml Samples incubated at 37°C for 2 and 24 hours Prepared and incubated control samples, in which type of CA++but do not add the protease.
The filtrate is irradiated with ultrasound, obtained from samples containing pCIB9369, and samples containing pCIB9369, incubated in when the, inkubirovanie in the presence of protease K (with the addition of CA++) caused less destruction of caterpillars of Plutella constituting approximately 90%).
Example 12. Comparative sequence analysis and protein sequence Shua and the sequence of the protein of juvenile hormone esterase
The nucleotide sequence pCIB9369 (SEQ ID NO:1) has two open reading frames (ORFS) in the provisions of nucleotides 569-976 and 1045-2334. According to the results of the search using the UWGCG programs Blast and Gap ORF No. 1 has no homology with any one of the sequences from GenBank. Gap-analysis of the protein encoded by ORF No. 2 RSV conducted using the Blast program revealed insignificant homology (21% identity) with the protein of Bacillus thuringensis Shua. When this Gap-analysis of the protein encoded by ORF No. 2 RSV conducted using the Blast program revealed a certain degree of homology (30,6% identity of amino acid sequences and 44.1% similarity of amino acid sequences) with protein, similar to juvenile hormone esterase (GenBank registration number 2921553; Henikoff and others, PNAS USA 89: 10915-10919 (1992).
Nucleotide sequence pCIB9381, pCIB9354 and RSV-21 also have two open reading frames cadaverine, ORF No. 2 proteins pCIB9381 and RSV-21 has practically the same homology with the associated with juvenile hormone esterase protein that ORF No. 2 protein RSV. The nucleotide sequence of ORF No. 1 protein pCIB9354 identical to 77% nucleotide sequence of the ORF number 1 protein pCIB9369, a nucleotide sequence of ORF No. 2 protein pCIB9354 identical to 79% nucleotide sequence of the ORF No. 2 protein pCIB9369. ORF No. 2 protein pCIB9354 also has homology with the associated with juvenile hormone esterase protein (amino acid sequence identity 29,2%, the similarity of amino acid sequences 42,2%).
Example 13. Comparison of the sequence pCIB9369 with the sequences described in WO 98/08388
Of DNA fragment length 38,2 T. p. H., the nucleotide sequence described in WO 98/08388, allocate twenty-two sequence of 60 nucleotides each (60-measures) and compared with the nucleotide sequence RSV-3, including pCIB9369. The first 60-Mer starts with the base 1 DNA fragment length 38,2, etc., ad, and the rest are located on the DNA fragment at intervals of approximately 2, etc., ad Their position on the DNA fragment length 38,2, etc., ad are listed below:
1-60; 2041-2100; 4021-4080; 60; 8041-38100; 38161-38220.
Sequences were compared using the UWGCG program Gap and analyzed how each of the 22 60-dimensional sequence and complementary sequence them. The results of this comparative analysis suggests that the highest percentage of identity is 53%, which in this area is not considered a significant degree of homology.
Example 14. Analysis by the method of southern blotting using probes derived from the sequences of WO 98/08388
For amplification of DNA fragments of DNA fragment length 38,2, etc., ad, described in WO 98/08388, design pairs of oligonucleotides. Oligonucleotides receive from the company Genosys Biotechnologies (The Woodlands, Texas) and their position on the DNA fragment length 38,2, etc. ad below. Also indicated their sizes and the size of amplified using PCR fragments:
VK1046: position 20-40,
VK1047: position 2078-2100.
The size of the fragment amplified by PCR using primers VK1046 and VK1047: 2080 base pairs,
VK1048: position 11221-11241,
VK1049: position 13360-13380.
The size of the fragment amplified by PCR using the CLASS="ptx2">The size of the fragment amplified by PCR using primers VK1050 and VK1051: 1979 base pairs,
VK1052: position 18901-18921,
VK1053: position 20321-20340.
The size of the fragment amplified by PCR using primers VK1052 and VK1053: 1439 base pairs.
VK1054: position 34261-34281,
VK1055: position 35320-35340 base pairs,
The size of the fragment amplified by PCR using primers VK1054 and VK1055: 1079 base pairs.
PCR is carried out in thermoacetica type Perkin-Elmer 9600 using the following conditions: 94°C, 2 min; then 30 cycles at 94°C, 30 s; 54°C, 30 s; 72°C, 4 min Samples containing 800 ng DNA Xenorhabdus nematophilus, of 0.1-0.5 μm of each pair of oligonucleotides, 250 μm dNTP, 5 units of Taq polymerase and a single (1x) buffer (firm Perkin-Elmer) in a final volume of 100 μl. The resulting reaction mixture, precipitated with ethanol, resuspended in THOSE and bring in 1% TBE-gel SeaPlaque, FMC, Rocklend, Maine). After electrophoresis and staining with ethidium bromide fragments cut out from the gel and visualize under UV rays. Slices of the gel, melted at 65°C and aliquots of 10 μl mixed with 10 μl of distilled water, boiled for 5 min and placed on ice. Then mixed with 15 μl of buffer dltf32P and 1 μl polymerase maple. Response the introduction of labels carried out for 60 min at room temperature. Samples of purified on columns of type Nick (firm Pharmacia Biotech) according to the recommendations of the supplier. Probes boiled for 5 min and placed on ice.
Analysis by the method of southern blotting is carried out by splitting the total DNA Xenorhabdus nematophilus, DNA isolated from cosmid RSV and RSV (Comedy have overlapping segment with a length of 25 T. p. N. and they both contain the DNA fragment RSV; RSV used to sublimirovanny), DNA isolated from subclones RSV-3 (SacII fragment length 9, etc., ad) and pCIB9369 (ClaI-fragment length 2,96, etc., ad), cleaved with ClaI, SacII or HindIII. The products resulting from cleavage, put on a 0.75% agarose TBE gel and dispersed throughout the night. Do the pictures and gel process according to the instructions of the company Bio-Rad, concerning blotting using a membrane for hybridization with a Zeta-probe. After blotting the membrane was kept at 80°C for 30 minutes and Then the membrane is placed in a 7% LTOs containing 250 mm sodium phosphate, pH to 7.2, and incubated at 67°C for 30 minutes Add fresh solution after equilibration at 67°add the above radioactive probes, and the p is m-ordinator for 30 min at 67°C. The membrane exhibited on film for 1 h and 3 h Film show, and the results suggest that probes for PCR based on the sequence described in WO 98/08388, not hybridize with DNA of cosmid or DNA subclones described in this application. However, for DNA X. nematophilus there is a strong hybridization signal.
These findings support the results of the comparative analysis of sequences and indicate that the nucleotide sequence of clone RSV different from the nucleotide sequence described in WO 98/08388.
B. Expression of the nucleotide sequences according to the invention in heterologous microorganisms-hosts
Microorganisms suitable for heterologous expression of nucleotide sequences according to the invention, can be any microorganisms that are able to colonize plants or the rhizosphere. To do this, they must be brought into contact with insect pests. These include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, gram-positive microorganism Bacillus and fungi Trichoderma and Gliocladium, and the yeast Saccharomyces cerevisiae. Preferred heterologous hosts are Pderma viride, Trichoderma harzianum, Gliocladium virens and Saccharomyces cerevisiae.
Example 15. Expression of nucleotide sequences in E. coil and other gram-negative bacteria
Many of the genes expressed in gram-negative bacteria heterologous way. Expression vector RK-3 (catalog number of the firm Pharmacia No. 27-4935-01) allows expression in E. coli. This vector has a strong tac promoter (Brosius J., and others, Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG (isopropyl--D-thiogalactoside). For use in E. coli developed a large number of other expression systems. Induced by heating the expression vector PPL(catalogue number of the firm Pharmacia No. 27-4946-01) contains a strictly regulated promoter of the bacteriophage , allowing expression of proteins with a high level. The lac promoter is another ensuring the expression of the promoter, however, it is not possible to achieve such high levels of expression, as the tac promoter. Adding in these expression vector system replicons with a wide range of hosts becomes possible expression of the nucleotide sequences in closely related gram-negative bacteria, such as Pseudomonas, Enterobacter, Serratia and Ageev, enabling replication in many species of gram-negative bacteria.
For expression in E. coli under control of the tac promoter (i.e., trp-lac) requires induction with IPTG. If this promoter (e.g., in a plasmid with a wide range of hosts pLRKD211) embed in Pseudomonas, he will have constitutive activity that does not require induction with IPTG. This promoter trp-lac can be placed before any gene or operon of interest for expression in Pseudomonas or any other closely related bacteria to implement the constitutive expression of this gene. Thus, the nucleotide sequence, the expression of which leads to formation of insecticidal toxin, may be under the control of a strong constitutive promoter and transferred into a bacterium with the ability to colonize plants or the rhizosphere, which makes this organism in insecticidal agent. Other suitable promoters can be used to implement the constitutive expression of a nucleotide sequence in gram-negative bacteria. They include, for example, the promoter regulatory genes gafA and lemA (WO 94/01561) from Pseudomonas and the promoter of the operon IAA Pseudomonas savastanoi (Gaf is entrusted bacteria
Heterologous expression of nucleotide sequences in gram-positive bacteria is another tool producing insecticidal toxins. The most fully characterized expression system for Bacillus and Streptomyces. It is established that the promoter of the gene causing resistance to erythromycin (ermR) from Streptococcus pneumoniae has activity in gram-positive aerobic and anaerobic microorganisms, as well as in E. coli (Trieu-Cuot and others, Nucl. Acids Res. 18: 3660 (1990)). Another promoter of the gene, which imparts resistance to the antibiotic thiostrepton used in cloning vectors for Streptomyces (Bibb, Mol. Gen. Genet. 199: 26-36 (1985)). For expression in Bacillus also suitable Shuttle vector RNT (Lereclus, FEMS Environ. Lett. 60: 211-218 (1989)). The big advantage of this approach is that many gram-positive bacteria form spores that can be used in the compositions, which allows you to create insecticidal agents with extensive shelf life. Species of Bacillus and Streptomyces are active colonizers of the soil.
Example 17. Expression of nucleotide sequences in fungi
Found that Trichoderma harzianum and Gliocladium virens allow you to reach different levels of biological control in Palestina sequence, expression of which leads to formation of insecticidal toxin, can also be expressed in fungi. This can be accomplished by various methods, well known in this field. One of them is the transformation of protoplasts of the fungus, PEG-mediated, or carried out by the method of electroporation. An alternative for this transformation of protoplasts or other cells of the fungus with the ability to develop in the regenerated Mature patterns, can be used particle bombardment. Vector pAN7-1, originally created for transformation of Aspergillus and currently widely used for the transformation of fungi (Curragh and others, Mycol. Res. 97(3): 313-317 (1992); Tooley and others, Curr. Genet. 21: 55-60 (1992); Punt and others, Gene 56: 117-124 (1987)), are manufactured so that it contained the nucleotide sequence. This plasmid contains a gene causing resistance to hygromycin, from E. coli, flanked by the gpd promoter and trpC terminator of Aspergillus nidulans (Punt and others, Gene 56: 117-124 (1987)).
In a preferred embodiment, the nucleotide sequence according to the invention Express in yeast Saccharomyces cerevisiae. For example, each of the two ORF RSV, RSV, RSV or RSV clone in a separate vectors together with inducible the likon size of 2 μm. The vectors preferably differ only in their tokens growth in yeast. S. cerevisiae transformed with the constructs separately and in combination with each other. ORF expressed together and they are tested against the expression of the protein and the presence of insecticidal activity.
C. Formulation containing insecticidal toxin
Insecticidal formulation prepared with the use of active substances, which include either a dedicated toxin, or alternatively, suspension or concentrates cells that produce it, and those described in the examples. For example, to control insect pests can be used E. coli cells expressing the insecticidal toxin. The following describes the Preparative form that can be prepared in liquid or solid form.
Example 18. Liquid formulations of insecticidal compositions
The active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier and then the solvent is evaporated under vacuum.
By carefully mixing the carriers with the active ingredient get ready-to-use dusty.
Example 19. Solid is the ingredients in the compositions are given in wt.%.
The active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, receiving wettable powders which can be diluted with water to obtain suspensions of the desired concentration.
Emulsions of the desired concentration is obtained from this concentrate by dilution with water.
Ready-to-use dusty obtained by mixing the active ingredient with the carriers and grinding the mixture in a suitable mill.
The active substance is mixed and milled with the adjuvants and the mixture is then moistened with water. The mixture ekstragiruyut and then dried in the air stream.
Finely ground active ingredient is uniformly fed into the mixer, to the kaolin moistened with polyethylene glycol. In this way we obtain a dust free granules coated.
Finely ground active ingredient is thoroughly mixed with the adjuvants, receiving a suspension concentrate from which suspensions of the desired concentration can be obtained by dilution with water.
The above insecticidal preparative forms of process plants Sagami pests with insecticidal toxin.
, The Expression of nucleotide sequences in transgenic plants
The nucleotide sequence presented in the present description, can be introduced into the plant cells using conventional methods of recombinant DNA. As a rule, they use the embedding coding sequence according to the invention in an expression system to which the coding sequence is heterologous (i.e., not present in normal condition) using standard cloning techniques known in this field. The vector contains the necessary elements for the transcription and translation of the built-in protein coding sequences. Can be applied in numerous well-known in this field vector system, such as plasmids, bacteriophages and other modified viruses. Suitable vectors include viral vectors, such as systems based on lambda vectors gtl1, gt10 and Charon 4; plasmid vectors, such as l121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems, but are not limited to them. The components of the expression system can also be modified to increase the expression level. what can be found in the present description the expression system can be used to transform virtually any cells cultivated plants in appropriate conditions. Transformed cells can be regenerated to obtain whole plants, while the nucleotide sequence according to the invention gives transgenic plants resistant to insects.
Example 22. Modification of the coding sequences and related sequences
DNA molecules presented in this description can be modified for expression in transgenic host plants. The host-plant that expresses the nucleotide sequence and which produces in their cells insecticidal toxins, has a high resistance to attack by insects and, thus, better able to withstand losses associated with such an attack.
Transgenic expression of genes in plants originating from microorganisms, may require modification of these genes in order to achieve and optimize their expression in plants. In particular, bacterial ORF encoding various enzymes, but which in the native microorganism are encoded by the same transcript is expressed in the plant better if they are encoded by different transcripts. To achieve this, each ORF originating from microorganis’-end of the ORF and terminator of transcription plants at the 3’end of the ORF. The selected ORF sequence preferably includes the initiating ATG codon and the termination stop codon, but it may contain additional sequence in addition to the initiating codon ATG and stop codon. In addition, the ORF may be shortened, but still maintain a desired activity; in the case of particularly long ORF shortened version that retains the activity may be preferred for expression in transgenic organisms. It is understood that the term "promoter plants and terminator of transcription plants" refers to promoters and transcription terminators, functioning in the plant. They also include promoters and transcription terminators, which can be isolated from sources which are not plants, such as viruses (e.g., cauliflower mosaic virus (CaMV)).
In some cases, modification of the coding ORF sequences and related sequences may not be required. Simply select the fragment containing interest ORF, and embed it in the course of transcription relative to the promoter of a plant. For example, Gaffney and others, (Science 261: 754-756 (1993)) has successfully completed the nahG gene expression in Pseudomonas transgenic plants under the control of a promotion the nas was still attached to ORF nahG against the course of transcription relative to the ATG codon and base pairs was still attached to it in the course of transcription relative to the stop codon. Preferably, a small contiguous sequence from a microorganism remained attached against the course of transcription relative to the ATG codon and downstream of transcription relative to the stop codon. In practice, the possibility of creating such a design may depend on the availability of restriction sites.
In other cases, the expression of genes selected from sources representing the microorganisms that can cause problems. These problems are well-known in this field and they are common to genes isolated from certain sources, such as Bacillus. These problems can occur for the nucleotide sequences of the present invention, and to overcome them can be carried out modification of these genes using the well-known in the field of methods. May encounter the following problems:
1. The most frequent codons
In plants, the most frequent codons different from the most frequent codons in some types of microorganisms. A comparison of the most frequent codons in the cloned ORF of the microorganism most frequently occurring codons of the genes of plants (and officeroute. As a rule, the result of the evolution of monocotyledonous plants plants have a pronounced preference in respect of the nucleotides C and G in the third position nucleotide sequence, while in dicotyledonous plants in this position, the most frequent nucleotides or So by modifying gene by incorporating of the most common codon for a specific transgenic species target many of the following issues relating to the content of GC/AT and illegal splicing sites can be overcome.
2. The content of GC/AT
As a rule, the plants have a GC content of more than 35%. The use of ORF sequences having a high content of nucleotides a and T, can present serious problems for plants. First, apparently, motives ATTA cause destabilization of transcripts, and they are detected at the 3’-end of many short-lived mRNAs. Secondly, the presence of polyadenylation signals, such as AATAAA in inappropriate positions in the transcript, apparently, causes premature termination of transcription. In addition, in monocotyledonous plants sequence with a high content of AT can be interpreted as sites of splicing (see below).
Before initiating codon ATG in the sequence 14 genes in maize:
Such analysis can be performed for the target species of plants, which embed the nucleotide sequence and the sequence adjacent to the ATG can be modified to include the preferred nucleotides.
4. Remove illegal sites splicing
Genes cloned from sources not related to plants, and is not optimized for expression in plants, can also contain motifs that can be recognized in plants as 5’- or 3’-splicing sites, and they can be cleaved, resulting in shortened form or with deletions transcripts. Such sites can be removed using known in the field of methods.
Methods of modification of coding sequences and related sequences are well known in this field. In those cases, when the original level of expression of the DNA molecule of the present invention is low and appears to be desirable to carry out the but according to methods well known in this field. These methods are described, for example, in published patent applications EP 0385962 (in the name of the Monsanto company), EP 0359472 (in the name of the company Lubrizol) and WO 93/07278 (to the name of the company Ciba-Geygi), all of them are included in the present description by reference. In many cases it is preferable to analyze the expression level of gene structures using protocols of short-term tests (which are well known in this area) before their transfer into transgenic plants.
Example 23. Construction of plant expression cassettes
Coding sequences intended for expression in transgenic plants, first unite in expression cassettes behind a suitable promoter which is able to be expressed in the plant. The expression cassette may also include any other sequences necessary or selected for the expression of the transgene. Such sequences include transcription terminators, alien sequence, intended to increase expression, such as introns, vital sequence and sequence intended for directed gene transfer product in specific organelles and cellular to the global transforming the vectors according to the method described below. Below is a description of the various components of a typical cassette expression.
The choice of promoter used in expression cassettes, defines the spatial and temporal pattern of expression of the transgene in the transgenic plant. Selected promoters allow for expression of transgenes in specific cell types (such as the epidermal cells of leaves, mesophyll cells, cells of the root cortex) or in specific tissues or organs (e.g. roots, leaves or flowers), and this choice depends on the desired place of accumulation of the gene product. Alternatively, the selected promoter can control the expression of a gene under different conditions of induction. Promoters can vary in its strength, i.e. the ability to stimulate transcription. Depending on the system host cell may be any of numerous suitable promoters, including the native promoter of the gene. Examples of promoters that can be used in expression cassettes, not limiting the scope of the invention.
A. Constitutive expression: the promoter ubiquitin
Ubiquitin represents a different gene product, investmente in transgenic plants (for example, in the sunflower - Binet and other, Plant Science 79: 87-94 (1991), in corn - Christensen and others, Plant Molec. Biol. 12: 619-632 (1989); and Arabidopsis - Norris and others, Plant Mol. Biol. 21: 895-906 (1993)). The promoter of the gene ubiquitin maize was investigated in transgenic systems monocotyledonous plants and its sequence and vectors designed for transforming monocots, is described in the published patent application EP 0342926 (in the name of the company Lubrizol), included in the present description by reference. In addition, Taylor and others (Plant Cell Rep. 12: 491-495 (1993) describes the vector (RANS), which contains the promoter ubiquitin corn and first intron, and noted its high activity in cell suspensions of numerous monocotyledonous plants in the introduction using bombardment microkeratome. The promoter ubiquitin Arabidopsis ideal for use with nucleotide sequences of the present invention. The promoter ubiquitin suitable for gene expression in transgenic plants, both in monocots and dicots. Suitable vectors are derived from vector runs or any transforming vectors presented in the present description, modified by embedding the corresponding promoter ubiquitin and/or intron sequences.
B. Constitutively Express the emer 23), which is incorporated into this description by reference. Plasmid pCGN1761 contains the "double 35S promoter and terminator of transcription tml with a unique EcoRI site between the promoter and the terminator and has a frame Fig. Construct a plasmid, which is a derivative of pCGN1761, which is a modified polylinker, including sites NotI and XhoI in addition to the existing EcoRI site. This derivative plasmid is designated as pCGN1761ENX. pCGN1761ENX can be used for cloning of cDNA sequences or gene sequences (including sequences of microbial ORF) within its polylinker for their expression in transgenic plants under the control of the 35S promoter. Full cassette of this design, including the 35S promoter-coding sequence-tml terminator, can be derived in sites HindIII, SphI, SalI, and XbaI at the 5’end of the promoter and XbaI sites, BamHI and BglI at the 3’end of the terminator for transfer to transformation vectors such as those described above. In addition, a fragment of the double 35S promoter can be removed by 5’-cleavage with HindIII, SphI, SalI, XbaI or > PST, 3’-end in any site restriction of polylinker (EcoRI, NotI or XhoI) to be replaced by another promoter. If necessary, around the cloning site can be made Timeline in cases when you want overexpression. For example, pCGN1761ENX can be modified by optimization of iniziali broadcast according to the method described in example 37 patent US 5639949, which is included in the present description by reference.
C. Constitutive expression: the promoter of actin
There are several isoforms of actin, expressed in most cell types, and therefore, the actin promoter is a good choice as a constitutive promoter. In particular, was cloned and characterized the promoter of the gene of rice Act1 (McElroy and others, Plant Cell 2: 163-171 (1990)). It is established that the promoter fragment with a length of 1.3 T. p. N. contains all regulatory elements necessary for expression in rice protoplasts. In addition, they constructed numerous expression vectors comprising the Act1 promoter, specifically for use in monocotyledonous plants (McElroy and others, Mol.Gen. Genet. 231: 150-160 (1991)). They include intron 1 Act1, 5’-flanking sequence of the Adh1 and intron 1 Adh1 (alcohol dehydrogenase gene of maize) and the sequence of the promoter CaMV 35S. Who are the most expression vectors are represented merge 35S and Act1 intron or 5’-flanking sequence of the Adh1 and Act1 intron. Optimization of the Promoter expression cassettes, described in (McElroy and others, Mol.Gen. Genet. 231: 150-160 (1991)) can be easily modified for expression of the gene of the present invention and it is particularly suitable for use in monocotyledonous host plants. For example, fragments containing the promoter, may be removed from structures McElroy and used to replace the double 35S promoter in pCGN1761ENX, which then becomes suitable for embedding specific gene sequences. Fused genes, designed, can then be moved into the appropriate transforming vectors. In another research report, it is reported that the promoter of the rice Act1 with its first intron also leads to high level expression in cultured cells of barley (Chibbar, etc., Plant Cell Rep. 12: 506-509 (1993)).
, Inducible expression, the promoter of PR-1a
The double 35S promoter in pCGN1761ENX can be replaced by any other selected promoter, which provides a high level of expression. For example, the double 35S promoter can be replaced by one of the chemically regulated promoters, which are described in U.S. patent 5614395. The selected promoter is preferably otscheplaut from its source using restricted, but alternatively it can be ampie. After PCR amplification of the promoter must be resequencer to check the error amplification after cloning of the amplified promoter in the vector-target. The chemically/pathogen-regulated promoter of the PR-1a tobacco, isolated from the plasmid RSV (concerning the construction, see example 21 the patent application EP 0332104 included in the present description by reference) and transferred to plasmid pCGN1761ENX (Ukness and others, 1992). Plasmid RSV cleaved with NcoI and the resulting 3’-protruding end of the linearized fragment "dull" by treating the DNA polymerase of phage T4. Then the fragment digested with HindIII and the resulting fragment containing the promoter of PR-la, purified using gel and clone in pCGN1761ENX, removed the double 35S promoter. This is carried out by cleavage with XhoI and "break" the end using the polymerase of phage T4, followed by cleavage with HindIII and the allocation of a larger fragment containing the vector-terminator, in which clone the fragment of the promoter RSV. This allows you to get the vector, which is derived pCGN1761ENX, which contains the promoter of PR-1a and the tml terminator, and a built-in polylinker with unique sites EcoRI and NotI. Selected coor) can then be moved to any selected transformation vector, including vectors below. Various chemical controls can be used to induce the expression of the coding sequence in plants transformed according to the present invention, including derivatives benzothiadiazole, isonicotinic acid and salicylic acid, are described in patents US 5523311 and 5614395.
D. Inducible expression of the promoter. induced by ethanol
To ensure the inducible expression of the coding sequence of the present invention can also be applied promoter induced by certain alcohols or ketones, for example, ethanol. This promoter is, for example, the promoter alcA gene of Aspergillus nidulans (Caddick and others, Nat. Biotechnol. 16: 177-180 (1998)). In A. nidulans alcA gene encodes alcoholdehydrogenase I, the expression of which is regulated by the AlcR transcription factors in the presence of the chemical inducer. For the purposes of the present invention coding sequence CAT plasmid Rasa:SAT, comprising the promoter sequence of the gene l, merged with the minimal 35S promoter (Caddick and others, Nat. Biotechnol. 16: 177-180 (1998)), replace the coding sequence of the present invention, receiving the expression cassette containing the coding posmestny in this region methods.
that is, Inducible expression, promoter induced by glucocorticoid
As can be seen, the induction of expression of the nucleotide sequence of the present invention using systems based on steroid hormones. For example, you can use the induction system, mediated by glucocorticoid (Aoyama and Chua, The Plant Journal 11: 605-612 (1997)), and to induce gene expression by treatment with a glucocorticoid, for example, a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration in the range from 0.1 to 1 mm, more preferably in the range from 10 to 100 mm. For the purposes of the present invention the sequence of the gene luciferase replace nucleotide sequence according to the invention, by constructing an expression cassette containing the nucleotide sequence according to the invention, under the control of six copies of the GAL4 located against the course of transcription relative to the activating sequences fused with the minimal promoter 35S. This is carried out using methods well known in the field. Transactivity factor includes the DNA binding domain of GAL4 (Keegan and others, Science 231: 699-704 (1986)), merged with transactivator glucocorticoid rats (Picard and others, Cell 54: 1073-1080 (1988)). The expression of the fused protein is controlled by any known in this field promoter suitable for expression in plants. This expression cassette containing the nucleotide sequence according to the invention, merged with the design 6GL4/minimal promoter is inserted into the plant. Thus ensure the specificity of the fused protein in relation to the tissue or organ, resulting in the inducible expression of insecticidal toxin specific in relation to the tissue or organ.
W. Specific to the root of the expression
Another scheme of gene expression is an expression in the root. Suitable promoter for expression in the root is the promoter, described de Framound (FEBS 290: 103-106 (1991), and in published patent application EP 0452269, which is incorporated into this description by reference. This promoter is transferred into a suitable vector, such as pCGN1761ENX, for embedding the selected gene and the subsequent transfer of the full cassette promoter-gene-terminator in the interest of transforming the vector.
C. Promoters induced by wounding
Induced by wounding promoters can also be used for gene expression. Described by many takia), Firek and other Plant Molec. Biol. 22: 129-142 (1993), Warner and other Plant J. 3: 191-201 (1993), and they are all suitable for use in accordance with the present invention. Logemann with co-authors described sequence located against the course of transcription relative to the 5’-end of the gene wun1 dicotyledonous plants - potatoes. Hee al. found that induced by wounding the promoter of dicotyledonous plants of potato (pin2) has activity in monocotyledonous plant is rice. In addition, Rohrmeier and Lehle described the cloning of a cDNA Wip1 corn, which is induced by wounding and which can be used to highlight related promoter using standard techniques. Similarly Firek with co-workers and Warmer with co-authors described induced injury of genes from monocotyledonous plants Asparagus officinalis, which is expressed locally at the site of injury and in places of introduction of the pathogen. Using cloning techniques well known in the field, these promoters can be transferred into suitable vectors, merged with genes and used for gene expression in injured plants wound.
I. Preferred for the core expression
In the patent application WO 93/07278, included in the present description by reference, describes vitalnet gene and promoter located from the start of transcription to the nucleotide - 1726. Using standard methods of molecular biology of the promoter or its part can be moved in a vector, as pCGN1761, where it can replace the 35S promoter and used for expression of a foreign gene is preferred for the core. In essence fragments containing preferred for core promoter or part thereof, can be transferred to any vector and modified for use in transgenic plants.
K. Specific leaf expression
The gene of maize encodes phosphoenolpyruvate (RERS) described Hudspeth and Grula (Plant Molec. Biol. 12: 579-589 (1989)). Using standard methods of molecular biology the promoter of this gene can be used to ensure the expression of any gene in transgenic plants specific to leaves.
HP-Specific pollen expression
In the patent application WO 93/07278 described the selection gene Kalnyshevsky protein kinase (CDPK) corn, which is expressed in cells of pollen. The sequence of the gene and promoter extends to the position of 1400 base pairs from the site of transcription initiation. With whom in the vector, such as pCGN1761, where they can replace the 35S promoter, and it can be used to provide expression of the nucleotide sequence of the present invention is specific in relation to pollen.
2. The transcription terminators
For use in the expression cassettes suitable wide variety of transcription terminators. They are responsible for the termination of transcription beyond the transgene and for its proper polyadenylation. The corresponding transcription terminators are terminators, for which it is known that they possess the ability to function in plants, and they include the terminator 35S CaMV, the tml terminator, the terminator napaliensis, terminator a pea rbcS. They can be used both in monocots and dicots. In addition, there may be used the native terminator of transcription of the genes.
3. Sequences for amplification or expression regulation
Numerous sequences that enhance expression of a unit of transcription of the gene, and these sequences may be used in combination with the genes of the present invention to enhance their expression in transgenic plants.
Ustanovlenny. For example, it was found that the introns of the gene of maize Adhl when introduced into cells of maize significantly enhance the expression of the gene of the wild type under the control of the cognate promoter. Found that intron 1 is particularly effective and enhances the expression of hybrid structures at the confluence with the gene chloramphenicol-acetyltransferase (Callis and others, Genes Develop. 1: 1183-1200 (1987)). In this same experimental system intron of the gene bronzel corn shows a similar effect in relation to increased expression (Callis, etc. above). The sequence of intron include conventional method of plant transformation vectors, usually consisting untranslated leader sequence.
We also know a large number of non-translated leader sequences derived from viruses that enhance the expression, and which are particularly effective in the cells of dicotyledonous plants. In particular, it is established that the leader sequence from tobacco mosaic virus (TMV, the "sequence"), from virus chlorotic leaf spot of maize (MCMV) and alfalfa mosaic virus (AMV) effectively enhance the expression (see, for example, Galli and others, Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski, etc., Plant Molec. Biol. 15: 65-79 (1990)).
4. Her gene transfer products and sequences that control the functioning of these mechanisms, described in great detail. For example, the directed transfer of the gene products to the chloroplast is controlled by the signal sequence found in the N-end different protein, which is cleaved during chloroplast import to, resulting in a Mature protein (see, for example, Comai, etc., J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused with a heterologous gene products for efficient import of heterologous products in the chloroplast (van den Broeck and others, Nature 313: 358-363 1985)). DNA encoding appropriate signal sequence, can be isolated from the 5’-end of cDNA encoding the RUBISCO protein, protein CAB, the enzyme EPSP synthase, protein GS2 and a number of other proteins, for which it is known that they are localized in the chloroplast. Cm. also the section entitled "Expression by directional migration in the chloroplast in example 37 patent US 5639949).
Other gene products are localized in other organelles, such as mitochondria and peroxisome (see, for example, Unger and others, Plant Molec. Biol. 13: 411-418 (1989)). cDNA encoding these products can also be subjected to impacts on the of sledovatelnot are encoded by the nuclear DNA ATPase and specific for mitochondrial isoform of aspartate aminotransferase. Directional migration in cellular protein bullock was described by Rogers and others (Proc. Natl. Acad Sci. USA 82: 6512-6516 (1985)).
In addition, were characterized by sequence, leading to directed gene transfer products to other cellular compartments. N-terminal sequences responsible for directional transport in the endoplasmic reticulum (ER), the apoplast and extracellular secretion of aleurone cells (Koehler and But, Plant Cell 2: 769-783 (1990)). In addition, N-terminal sequences in combination with C-terminal sequences responsible for directed gene transfer products in vacuoles (Shinshi and other, Plant Molec. Biol. 14: 357-368 (1990)).
By merging the above-described respective guides of the sequences of interest transgenic sequences can be sent transgenic product in any organelle or any compartment of the cell. For the directional migration, for example, in the chloroplast signal sequence of the chloroplast RUBISCO gene, CAB gene, gene, EPSP synthase gene GS2 merge in reading frame with the N-terminal ATG codon of the transgene. The selected signal sequence must include a prominent site of cleavage, and when designing hybrids cladophora cases this requirement can be satisfied by adding a small number of amino acids between the cleavage site and the ATG of the transgene, or alternatively, by substitution of several amino acids within the sequence of the transgene. Hybrids, designed for import into the chloroplast, can be assessed in relation to the efficiency of absorption by the chloroplast through the translation of in vitro transcribed in vitro structures with the subsequent absorption in vitro chloroplast using methods described by Bartlett and others in: Edelmann and others (ed) Methods in Chloroplast Molecular Biology, Elsevier. PP 1081-1091 (1982)); Wasmann, etc. (Mol. Gen. Genet. 205: 446-453 (1986)). These design methods are well known in this field and equally suitable for both mitochondria and peroxisome.
The above mechanisms of directional transport in the cell can be used not only in conjunction with their cognate promoters, but also with heterologous promoters in order to implement specific target directional migration by regulation of transcription by the promoter, which has a pattern of expression that differs from the schema of the expression of the promoter, from which comes the directional signal transfer.
Example 24. Construction of plant transformation vectors
Specialists in this area known for numerous transformation vectors m of such vectors. The choice of vector depends on the preferred method of transformation and the types of targets that are intended for transformation. For certain types of preferred targets can be different markers for breeding on the basis of resistance to the antibiotic or herbicide. Typically used for transformation of breeding markers include the nptll gene, which causes resistance to kanamycin and related antibiotics (Messing and Essen, Gene, 19: 259-268 (1982); Bevan and others, Nature 304: 184-187 (1983)), the bar gene causing resistance to the herbicide phosphinotricin (White and others, Nucl. Acids Res. 18: 1062 (1990), Spencer and others, Theor. Appl. Genet. 79: 625-631 (1990)), the hpH gene causing resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol. Cell Biol, 4: 2929-2931 (1984)) and the dhfr gene, which causes resistance to methotrexate (Bourouis and others, EMBO J., 2: 1099-1104 (1983)) and the gene EPSPS protein, causing resistance to glyphosate (patent US 4940935 and 5188642).
1. Construction of vectors suitable for transformation using Agrobacterium
For transformation using Agrobacterium tumefaciens numerous suitable vectors. They usually have at least one border sequence of T-DNA and include such vectors as pBIN19 (Bevan, Nucl. Acids Res., (1984)) and pXYZ. Described below constru:
Binary vectors RSV and RSV used to construct recombinant vectors designed for use with Agrobacterium and their design is as follows. Create pTJS75kan by splitting pTJS75 (Schmidhauser and Helinski, J. Bacteriol, 164: 446-455 (1985)) with NarI, which allows you to cut out the gene of resistance to tetracycline, with subsequent embedding of the fragment As from plasmid pUC4K carrying NPTII (Messing and Essen, Gene, 19: 259-268 (1982)); Bevan and others, Nature, 304: 184-187 (1983); McBridge and other, Plant Molecular Biology, 14: 266-276, (1990)). XhoI linkers are ligated with EcoRV fragment RSV containing the left and right border sequences of T-DNA in plant breeding chimeric gene nos/nptII and polylinker pUC (Rothstein and others, Gene, 53: 153-161 (1987)), and clone cleaved with XhoI fragment of plasmid pTJS75 cleaved with SalI, creating RSV (see also EP 0332104, example 19 ). Vector RSV contains polylinker the following unique sites recognized by restrictase (restriction sites): EcoRI, SstI, kpni restriction sites, BglII, XbaI and SalI. Vector RSV is derived RSV and it is created by inserting polylinker additional restriction sites. Unique restriction sites in polylinker vector RSV are EcoRI, SstI, kpni restriction sites, BglII, XbaI, SalI, luI, BclI, AvrII, ApaI, HpaI, and StuI. In addition to t the kanamycin plants and bacteria, the left and right border sequences of T-DNA in Agrobacterium mediated transformation, the trfA function which is derived from RK2, for mobilization between E. coli and other hosts, and functions OriT and OriV, also derived from RK2. Polylinker RSV suitable for the cloning of plant expression cassettes containing their own regulatory signals.
B. Vector pCIB10 and its derivatives, selected on the basis of resistance to hygromycin
The binary vector pCIB10 contains a gene causing resistance to kanamycin in plants, the right and left border sequences of the T-DNA contains sequences from plasmids pRK252 with a wide range of hosts, which allows its replication both in E. coli and in Agrobacterium. Its design is described by Rothstein and others, Gene, 53: 153-161, (1987)). The Gritz and others (Gene, 25: 179-188, (1983)) describes the various derivatives designed pCIB10, which include gene phosphotransfer-times, causing resistance to hygromycin Century. These derivatives allow the selection of cells of transgenic plants or only on the basis of resistance to hygromycin (RSV), or on the basis of resistance to hygromycin and kanamycin (RSV, RSV).
2. Age is erium tumefaciens allows you to bypass the requirement for sequences of T-DNA in the selected transforming the vector and therefore, the vectors lacking these sequences may be used in addition to vectors, such as described above, which contain sequences of T-DNA. Methods of transformation that are not based on the use of Agrobacterium include transformation using particle bombardment, the absorption protoplasts (for example, using PEG and electroporation and microinjection. The choice of vector depends largely on the selection of the preferred species to be transformed. Below are some typical design vectors.
RSV is a vector, which is derived from Fig suitable methods for direct gene transfer in combination with selection in relation to herbicide basta (or phosphinotricin). Plasmid RSV includes the promoter CaMV 35S, functionally associated with the GUS gene of E. coli, and the transcription terminator 35S CaMV, and it is described in published PCT application WO 93/07278. The 35S promoter of this vector contains two ATG sequence at the 5’-end of the site of initiation. These sites are subjected to mutation using standard PCR methods in such a way as to remove both ATG and form the SspI restriction enzymes cut sites and PvuII. New restriction sites negotating site of initiation. The resulting derived plasmids RSV identified as RSV. Then GUS gene cut from RSV by cleavage with Sall and SacI, all dull and re are ligated, receiving plasmid RSV. Plasmid JI82 obtained from the John Innes Center, Norwich, and cut out the SmaI fragment length 400 base pairs, containing the bar gene from Streptomyces viridochromogenes, and inserted into the HpaI site of plasmid RSV (Thompson and others, EMBO J., 6: 2519-2523, (1987)). This allows to obtain plasmid RSV containing the bar gene under the control of the promoter of CaMV 35S and terminator for breeding on the basis of resistance to the herbicide, the gene causing resistance to ampicillin (for selection in E. coli) and polylinker with a unique SphI sites, PST, HindIII and BamHI. This vector suitable for cloning of plant expression cassettes containing their own regulatory signals.
B. pSOG19 and pSOG35:
pSOG35 is a transforming vector, in which as a breeding marker gene is present digidrofolatreduktazy (DHFR) E. coli causing resistance to methotrexate. To amplify the 35S promoter (~800 base pairs), intron 6 of the gene of maize Adh1 (~550 base pairs) and fragment length 18 base pairs netransliruemoi leader sequence GUS from the li, also amplified by PCR and these two PCR fragments combine with SacI fragment- > PST from RV (Clontech), which contains a basis of the vector pUC19 and terminator nepalensis. The Assembly of these fragments allows to obtain plasmid pSOG19 containing the 35S promoter to merge with the sequence of intron 6, leader GUS, DHFR gene and the terminator nepalensis. Replacement leader GUS in pSOG19 on the leader sequence of the virus chlorotic mosaic corn allows to obtain the vector pSOG35. pSOG19 and pSOG35 carry the gene Fig causing resistance to ampicillin, and have sites HindIII, SphI, PST and EcoRI, suitable for the cloning of foreign sequences.
3. Vector suitable for transformation of the chloroplast
For expression of the nucleotide sequence of the present invention in plastids of plants using plastid transformation vector rn (WO 97/32011, example 36). The nucleotide sequence is inserted into rn, replacing her coding sequence PROTOX (protoporphyrinogen). Then this vector is used for plastid transformation and selection of transformants on the basis of resistance to spectinomycin. In an alternative embodiment, the nucleotide sequence is inserted into RRN tutoral PROTOX.
Example 25. Transformation
After the nucleotide sequence cloned in an expression system, it will transform the plant cell. Methods for transformation and regeneration of plants are well known in this field. For example, the introduction of foreign DNA, as well as direct DNA absorption, introduction via liposomes, electroporation, microinjection and using micronarrative, use Ti-plasmid. In addition, for the transformation of plant cells can be used bacteria of the genus Agrobacterium. Described below are representative methods of transformation of both dicotyledonous and monocotyledonous plants, as well as a representative method of transformation of plastids.
1. Transformation of dicotyledonous plants
Methods for transformation of dicotyledonous plants is well known in this field and include methods based on the use of Agrobacterium, and methods that do not require the use of Agrobacterium. Methods that do not require Agrobacterium include the uptake of exogenous genetic material directly protoplasts or cells. This can be accomplished by absorption, indirect PEG or electroporation, injection with bombardi the Mol. Gen. Genet. 199: 169-177 (1985), Reich, etc., Biotechnology 4: 1001-1004 (1986) and Klein et al., Nature 327: 70-73 (1987). In each case, the transformed cells to regenerate whole plants using standard techniques known in this field.
Transformation mediated by Agrobacterium, is the preferred method of transformation of dicotyledons because of the high efficiency of transformation using this method and its wide applicability to a range of different species. Transformation using Agrobacterium usually includes the transfer of the binary vector carrying interest of foreign DNA (e.g., RSV or RSV), in a suitable Agrobacterium strain which may depend on the complement of vir genes of Agrobacterium strain-master, and which may be located either on coresidential Ti-plasmid or on the chromosome (for example, strain CIB542 for RSV and pCIB2001 (Uknes and other Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium produced by technovillage mating using E. coli carrying the recombinant binary vector, strain-helper E. coli carrying a plasmid, such as pRK2031, and which is able to mobilize the recombinant binary vector to directed migration in strain-target Agrobacterium. In tzer, Nucl. Acids Res. 16:9877 (1988)).
Transformation of plant species to target with recombinant Agrobacterium usually includes joint cultivation of Agrobacterium with explants of plants and is performed according to protocols well known in this field. Transformed tissue regenerate on breeding environment containing a marker for selection on the basis of resistance to the antibiotic or herbicide, located between the border sequences of T-DNA binary plasmids.
Another method of transforming cells of a plant genome based on the introduction of inert or has biological activity of particles in tissues or cells of plants. This method is described in U.S. patent 4945050, 5036006 and 5100792 (all in the name of Sanford and others). Typically, this method consists in the introduction of inert or has biological activity of particles in cells under conditions that allow them to penetrate the outer membrane of the cells and to carry out embedding inside her. If you are using inert particles, the vector can be introduced into the cell by coating on particles of the vector containing the target gene. In an alternative embodiment, the cell-target may be surrounded by a vector so that the vector is transferred into the glue is active particles (e.g., dried yeast, dried bacteria or bacteriophages, each (th), which contains DNA, which is designed for injection).
2. The transformation of monocotyledonous plants
The transformation of most monocot plant species at the present time has also become a tradition. Preferred methods include direct gene transfer into protoplasts using methods based on PEG or electroporation, and particle bombardment of callus tissue. Transformation can be carried out in a single species of DNA or with multiple types of DNA (i.e., cotransformation), and both of these methods are suitable for use in the present invention. The advantage of cotransformation may be that it does not require a full vector design and gives the opportunity to obtain transgenic plants with non-linked loci for the gene of interest and breeding token that allows you to remove breeding marker in subsequent generations, if it is deemed necessary. However, the disadvantage of using the method of cotransformation is that the frequency with which different types of DNA integrated into the genome, is less than 100% (Schocher and other Biotechnology 4: 1093-1096 (1986)).
In applications of Nauruz, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm and others, Plant Cell 2: 603-618 (1990) and Fromm and others, in Biotechnology 8: 833-839 (1990)) have described methods of transformation of maize line derived from line A, using particle bombardment. Methods of transformation of elite inbred lines of maize by particle bombardment is also described in WO 93/07278 and Koziel with co-authors (Biotechnology 11: 194-200 (1993)). This method includes the use of immature embryos of maize in length, 1.5-2.5 mm, cut from the cob of corn after 14-15 days after pollination, and devices for bombing type PDS-l000 He Biolistics.
Transformation of rice can also be carried out using methods of direct gene transfer using protoplasts or particle bombardment. Mediated by protoplast transformation described for Japanese and Indian types of rice (Zhang and others, Plant Cell Rep. 7: 379-384 (1988); Shimamoto and other, Nature 338: 274-277 (1989); Datta and others, Biotechnology 8: 736-740 (1990)). Both types of rice are also transformed by standard methods using particle bombardment (Christou and others, Biotechnology 9: 957-962 (1991)). In addition, WP 93/21335 described methods of transformation of rice using electroporation.®when the pressure of the punch ~1000 pounds per square inch and the standard screen with a pore size of 80 mesh. After bombardment, the embryos are placed back into the darkness for a recovery period of approximately 24 h (still in osmoticum). After 24 h, the embryos are removed from osmoticum and then placed back in the environment for induction, where they remain for approximately one month prior to regeneration. Approximately one month later the embryo explants with developing embryogenic the callus is transferred into the environment for regeneration (MS + 1 mg/l NICK, 5 mg/l Ledger (gibellina acid) containing, in addition, the corresponding selectivity agent (10 mg/l Basta if RSV and 2 mg/l of methotrexate in the case of pSOG35). After approximately one month developed shoots are transferred into larger sterile containers, known as "GA7s" containing MS with half fortress, 2% sucrose and the same concentration of SESM. WP 94/00977 and U.S. patent 5591616, which are both included in the present description by reference).
3. Transformation of plastids
Seeds of Nicotiana tabacum cultivar Xanthi nc ' are planted at the rate of 7 pieces in a Cup on a circle with a diameter of 1 inch on T agar medium and 12-14 days after sowing the bombardment particles of tungsten size of 1 μm (M10, the company Biorad, Hercules, CA) coated with DNA plasmids rn and rn, mainly according to the method described in Svab z and Maliga P., PNAS 90, 913-917 (1993). Been bombarded seedlings are incubated on T medium for two days, then cut off the leaves and put abaxial side up in bright light (350-500 mcmole photons/m2/s) on plates with RMOP medium (Svab, Z., Hajdukiewicz P, and Maliga P. PNAS 87, 8526-8530 (1990)) containing 500 μg/ml spectinomycin dihydrochloride (firm Sigma, St.Louis, MO). Resistant seedlings, which in 3-8 weeks after the bombing appear discolored bottom leaves, subcloning on the same selective medium, allow to form callus and secondary seedlings allocate and subcloning. Complete separation of copies of the transformed plastid genome (homoplasmic state) in the independent subclones determined using standard methods southern blotting DNA (Mettler I. J. Plant Mol. Biol. Reporter 5, 346-349 (1987)) are divided into 1-percentage Tris-borate (TBE) agarose gels, transferred to nylon membranes (firm Amersham) and probe labeled with32P premirovanii random DNA sequences corresponding to the BamHI/HindIII-DNA fragment with a length of 0.7 T. p. N. from RS, which contains part of the sequence of the rps7/12, providing directional migration in the plastid. Homoplasmic shoots are planted in aseptic conditions on containing spectinomycin environment MS/IBA (McBride, K. E. and other PNAS 91, 7301-7305 (1994)) and transferred to the greenhouse.
D. Breeding and seed production
Plants obtained by transformation of the nucleotide sequence of the present invention, can refer to any of a wide variety of species of plants, including monocotyledonous and dicotyledonous plants; however, preferably the plants used in the method according to the invention, chosen from the list above are important from an agricultural point of view of the target cultures. The ability to expression of the gene of the present invention in combination with other characteristics that are important from the point of view of productivity and quality, can be given to lines of plants through breeding. Principles and NY (1981); Crop Breeding, Wood D. R. (editor) American Society of Agronomy, Madison, Wisconsin (1983); Mayo O. theory of Plant Breeding, 2-e Izd., Clarendon Press, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co.,, Berlin (1986)).
Genetic traits, constructed in the above-described transgenic seeds and plants, are transmitted through sexual reproduction or vegetative growth and, thus, can be preserved and passed by inheritance to the seed plants. Usually specified retention and transfer to the offspring carried out using well-known agricultural techniques developed for specific purposes, such as tillage, sowing or harvesting. Can also apply specialized methods such as hydroponics or indoor gardening. Because cultivated plants susceptible to attack and damage caused by insects or infections, and also subject to competition from weeds, to improve crop conduct activities to control weeds, plant diseases, insects, nematodes and other harmful factors. They include mechanical methods such as tillage or remove Sorna city, the nematicides, growth regulators, agents that promote maturation, and insecticides.
In addition, the advantage of genetic characteristics of transgenic plants and seeds according to the invention can be used in plant breeding to create plants with improved properties, such as tolerance to pests, herbicides, or stress, with improved nutritional value, increased productivity or improved structure to help reduce losses from lodging or shedding. For different stages of the breeding is characterized by deliberate human intervention, such as the selection of lines to be crossing, direct the pollen parent lines or the selection of an appropriate progeny plants. Depending on the desired properties choose different methods of selection. Appropriate methods well known in the field and include hybridization, inbreeding, return crossing, multi-line crossing, mixing varieties, interspecific hybridization, methods aneuploidy and so on, but not limited to. Methods of hybridization also include sterilization plants with the purpose of obtaining a male or female sterile plants by mechanical, chemical, or the range, that the genome is male sterile, but fertile female plants will be likewise acquiring the properties of both parental lines. Thus, transgenic seeds and plants according to the present invention can be used for selection of improved lines of plants, for example, that allows to increase the effectiveness of conventional methods such as treatment with herbicide or pesticide, or gives you the ability to do without these methods, thanks to the modified genetic characteristics of these plants. In addition, there can be obtained new plants with improved tolerance to stress through genetic optimization "device" that allows you to harvest the highest quality products compared to products derived from plants that do not have the ability to resist similar hazardous conditions in the course of their development.
Example 27. Seed production
In the production of seed quality, germination and uniformity of seed are important characteristics of the product, while the quality of germination and uniformity of seed collected and sold by the farmer, are not important. Since it is difficult to grow a cultivated plant out of contact with SNIA seeds with good germination manufacturers, growers, conditioning and sale of clean seed, developed quite extensive and targeted methods of seed production. So, usually the farmer is practicing the purchase of certified seed material that meets certain quality standards, instead use the seeds from his own culture. Breeding material, which is used as seed material, usually treated with a protective coating, including herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Commonly used protective coatings include such compounds as Captan, carboxin, thiram (TMTD®), metalaxyl (Apron®and pirimiphosmethyl (Actelinc®). If necessary, these compounds are produced in the form of preparative forms together with further carriers, surfactants or conducive to the application of adjuvants commonly used in the field of preparative forms to protect from damage caused by bacteria, fungi or insect pests. The protective coating can be applied by soaking the material for propagation of the liquid composition or by the processing of the kidneys or of the fruit.
Another subject of the invention are new agricultural methods, such as the above methods are characterized by the use of transgenic plants, transgenic plant material or transgenic seed according to the present invention.
Seeds can be placed in a bag, container or vessel, made of suitable packing material, the bag or container must be able to close for the safety of the seeds. Bag, container or vessel can be designed either for a short or long term storage of seeds, or for both. Examples of suitable packaging materials include paper, such as Kraft paper, rigid or flexible plastic or other polymeric material, glass or metal. Preferably, bag, container or vessel consisted of several layers of packaging materials of the same type or of different types. In one of the embodiments bag, container or vessel made in such a way as to exclude or limit the contact of the seeds with water or moisture. In one example, the bag, the container or vessel is sealed, for example, sealed with heat, so they put water absorbent materials. In another embodiment, bag, container, or vessel, or the packing material of which it consists, is treated in order to restrict, suppress or prevent infection by disease, pollution or other hazards arising from the storage or transport seeds.
An example of such treatment is sterilization, for example, by chemical means or by irradiation. Under the scope of the present invention falls within the bag designed for commercial use, containing the seeds of transgenic plants carrying the gene of the present invention, which is expressed in the transformed plant with a higher level than in the wild type plant, together with a suitable carrier, and instructions for use to ensure the sustainability of plants to a wide range of diseases.
The above-described embodiments of the above to illustrate. This description of the invention enables the person skilled in the art to make numerous variations of the invention. It should be understood that all such obvious and predictable variations fall under the scope of the attached claims.
1. The selected nucleic acid molecule comprising a nucleotide sequence that encodes at least one toxin active against Plutella xylostella, where the nucleotide sequence of (a) hybridized with nucleotide sequence selected from the group comprising nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14 under the following conditions: 7%hydrated sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1mm add at 50°C With washing in 0.1 x SSC, 0.01 to x-ordinator at 65°, or (b) the nucleotide sequence, isooctanol nucleotide sequence specified in subparagraph (a).
2. The selected nucleic acid molecule under item 1, characterized in that the nucleotide sequence is isooctanol nucleotide sequence that hybridizes with nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14 under the following conditions: 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1mm add at 50°C With washing in 0.1 x SSC, 0.01 to x-ordinator at 65°C.
3. Selected molecule of nucleic acid is cleotide 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14 under the following conditions: 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1mm add at 50°C With washing in 0.1 x SSC, 0.01 to x-ordinator at 65°C.
4. The selected nucleic acid molecule under item 1, wherein the nucleotide sequence encodes the amino acid sequence selected from the group comprising SEQ ID NO.:2, 3, 5, 7, 9, 11, 13 and 15.
5. The selected nucleic acid molecule under item 1, characterized in that the nucleotide sequence includes nucleotides 569-979 SEQ ID NO:1, nucleotides 1045-2334 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
6. The selected nucleic acid molecule under item 1, characterized in that the nucleotide sequence hybridizes with the nucleotides 569-979 SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:12 under the following conditions: 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1mm add at 50°C With washing in 0.1 x SSC, 0.01 to x-ordinator at 65°C.
7. The selected nucleic acid molecule under item 1, wherein the nucleotide sequence encodes the amino acid sequence represented in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:13.
8. The selected nucleic acid molecule SEQ ID NO:10 or SEQ ID NO:14 under the following conditions: 7% sodium dodecyl sulphate (LTOs), 0.5 M NaP4, 1mm add at 50°C With washing in 0.1 x SSC, 0.01 to x-ordinator at 65°C.
9. The selected nucleic acid molecule under item 1, wherein the nucleotide sequence encodes the amino acid sequence represented in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:15.
10. The selected nucleic acid molecule under item 1, characterized in that the nucleotide sequence is a DNA fragment of approximately 3.0, etc. acting, which is included in the plasmid pCIB9369 (NRRL B-21883).
11. Chimeric gene comprising a heterologous promoter sequence functionally linked with the nucleic acid molecule under item 1, where this chimeric gene is able to be expressed in microbial or plant cell host.
12. Recombinant plasmid comprising a chimeric gene under item 11.
13. Bacterial strain E. coli NRRL B-21883 producing toxin active against Plutella xylostella.
14. The toxin, resulting from expression of the DNA molecule under item 1, and this toxin is active against Plutella xylostella.
15. The toxin on p. 14, where the toxin is produced by a strain of E. coli, with registration number NRRL B-21883.
16. The toxin on p. 14, where the toxin in the 17. The toxin on p. 16, where the toxin comprises the amino acid sequence selected from the group comprising SEQ ID NO:2, 5, 9 and 13.
18. The toxin on p. 16, where the toxin comprises the amino acid sequence selected from the group comprising SEQ ID NO:3, 7, 11 and 15.
19. Insecticidal composition comprising possessing insecticidal effective amount of a toxin on p. 14 and agricultural acceptable carrier.
20. A method of producing a toxin that has activity against Plutella xylostella, providing for (a) the transformation strain host cells a nucleic acid molecule according to any one of paragraphs.1-9 and (b) the expression of the nucleic acid molecules in these cells, hosts, which leads to the formation of at least one toxin with activity against insects.
21. The method of obtaining the insect-resistant plants, introducing into the plant the nucleic acid molecule under item 1, and the nucleic acid molecule is able to be expressed in the plant in a quantity effective against Plutella xylostella.
22. The way to deal with Plutella xylostella, introducing the insect effective amount of the toxin under item 14.
23. The method according to p. 22, g is