Recombinant stable occluded nuclear polyhedrosis virus (options), insecticidal composition and method for controlling insects on the site
(57) Abstract:The present invention relates to baculovirus insecticides. Proposed stable alladinian recombinant nuclear polyhedrosis virus, which infected cells of their insect Express the toxin Scorpion or spider Segestria. The toxin is secreted from insect cells. These recombinant viruses can be used to combat insects. Proposed insecticidal composition comprising an inert carrier and stable occluded recombinant virus. Also a method of combating insects at the site, providing on site an effective amount of stable occluded recombinant virus. 10 S. p. F.-ly, 19 ill., table 4. The present invention relates to baculovirus insecticides.Specific to insect baculoviruses are an alternative to the use of chemical agents for insect control in agriculture. They have been used for these purposes for many years. Baculoviruses are advantages of selectivity with respect to Milenium organisms and those that do not pollute the environment. Believe that they are safe for the fact, that they are relatively slow kill micheneau insect, which prevents the rapid termination of damage in the food insects. Development of techniques for genetic constructions in the field of baculoviruses has given the opportunity to suggest that the efficacy of such agents can be enhanced by expression in a host-specific insect toxins, hormones or enzymes, thereby reducing the time required for killing insect, or at least, preventing eating their plants.Still modications efficiency of baculovirus insecticides had limited success. Corbonell et al. (1988) expressed specific insect toxin Scorpion from Buthus eupeus, using Autographa californica nuclear polyhedrosis virus (AcMNPV), but was unable to determine the biological activity of recombinant toxin. Maeda (1989) included gene diuretic hormone from Manduca sexta in Bombyx mori MNPV, and reduces the time required for killing the host about 20%. Hammock et al. (1990) expressed the juvenile hormone esterase in Trichoplusia ni larvae using poliakin-negative AcMNPV, and identified a significant reduction in weight gain at higher doses of virus. And finally, Merryweather et al. (1990) and Martens et al. (1990) included the l toxic to insect larvae per se, not modified the efficiency of the virus.We currently have included synthetic gene specific to the insect toxin under the control of a baculovirus promoter in recombinant AcMNPV. We found that there is a significant increase in pathogenicity to the insect, if the sequence encoding the toxin, to merge with a sequence that encodes a signal peptide, which causes the secretion of the protein.The invention relates to stable occluded recombinant virus nuclear palindrone suitable for use as an insecticide, and the virus contains one of the following constructs:
- construct vector pAcST3 shown in Fig. 4B,
- construct vector pAcJL2ST shown in Fig. 9,
- construct vector pAcjHEST shown in Fig. 10,
- construct vector pAcSTST shown in Fig. 11,
- construct vector pAcCP2ST shown in Fig. 12,
- construct vector pAcOXYST shown in Fig. 13,
- construct vector pAcBPST shown in Fig. 14,
- construct vector pAcSefJT shown in Fig. 19.In the present invention a method of combating insects at the site, which includes decome digests of recombinant baculoviruses, viral DNA multiplicities inside the insect, and the toxin is expressed and secreted, destroying insect. The death of the insect is faster than when using recombinant baculoviruses, which are not provided with a sequence of secretory signal. This provides an important advantage, because it reduces the number of plants damaged by insects to their death.The present invention can be applied to any suitable baculovirus family Baculoviridae, for example, type Baculovirus. Baculoviruses can be a nuclear polyhedrosis viruses (NPV), for example, AcMNPV, Bombyx mori NPV, Heliothis zea NPV and Buzura suppressuria NPV. The genome of the recombinant NPV according to the method of the present invention includes a functional polyhedrin gene. Therefore, the expression proidennogo protein usually manages j natural baculovirus polyhedrin. Because recombinant baculoviruses are functional in the environment, these viruses must be sustainable and must possess the ability to create a protective shell.The genome of recombinant baculoviruses also includes chimeric gene, encoding an insecticidal toxin, or its functional derivative, the signal peptide which promo gene recombinant baculoviruses may consist of wild-type genome, which contains the chimeric gene and the elements controlling its transcription and translation. Usually on the genetic mechanism of wild-type genome is not affected by the addition of a chimeric gene with its control sequences. Expression of proteins encoded natural wild-type genome, remains intact. So, get a viable, stable, infectious recombinant baculovirus.The toxin can be a neurotoxin, usually a neurotoxin, affecting cellular ion channels. A suitable toxin is a toxin that is secreted by the cells that normally produce the toxin. The toxin is usually a polypeptide toxin a poisonous animal or microorganism. Suitable toxin after purification to homogeneity is toxic to the insect in the introduction. Preferably, the toxin acted selectively against insects and had no action on mammals. Recombinant baculoviruses can expressionate and secrete two or more toxin.A preferred class of toxins are polypeptides that are secreted from the cells in which they are synthesized inside the animal in the sack of poison, from which the toxin R>1. The Scorpion toxin-specific operating on insects. (type Arthropoda. The Class Arachnida. Squad Scorpionida) Buthus eupeus (Central Asian Scorpion) (see sequence 1 at the end of text).Sequence I3A and I4A related I1A. Androctonus australis Hector (AaH IT): see Fig. 1. The other two are specific to the insect toxin with related amino acid sequences were isolated from this species AaH IT1 and AaH IT2 (Loret, 1990) (see sequence 2 and 3).It was found that this toxin has two isoforms l containing Ile at positions 15 and 2 Val. (Lester, D., Lazarovici, P., Pelhate, M., Ziotkin, E. , Biochim. Biophys. Acta701, 370, 1982) (see sequence 4 and 5).2. The spider toxin Segesdria florentina (Sef IT) can also be used (see sequence 6).Signal peptide provides for secretion of the toxin from the cells in which it is produced. The signal sequence may be a sequence capable of secretion of the protein from mammalian cells or insect, so it can be a sequence of a mammal, as a secretory signal sequence for interleukin 2 (IL-2), or such a sequence of the insect, as the secretory signal sequence of the virus signal sequence, for example, such as a secretory signal sequence gp 67 protein.Suitable signal sequences can therefore include (see last-minute 7-12).References (see end of text).The signal peptide can be chipped off from the toxin, and the toxin can be secreted. Therefore, the signal peptide can be condensed with the N-terminal or C-terminal amino acid residue of the toxin, either directly or through linker sequences. The linker sequence may contain from 1 to 10 amino acid residues, for example, from 1 to 5 residues.Recombinant baculovirus of the present invention can be obtained by:
(i) cloning chimerical gene encoding a foreign protein containing the toxin or its functional derivative, and a signal peptide, a vector of baculovirus transfer by restriction site in the forward direction from the promoter, capable of driving the expression of foreign proteins in insect cells; and
(ii) cotransfection cells susceptible to baculovirus infection with the recombinant vector transfer from stage (ii) and the intact DNA of wild-type baculovirus.Occurring homologous resiny with the promoter. Chimerical gene is usually singled out for use at the stage of cloning (i). Recombinant baculoviruses can also contain expressed by the polyhedrin gene. This can be achieved using on stage (i) wild-type baculovirus, which includes this gene. In another embodiment, the vector transfer can be used in stage (ii), which, in addition, contains a sequence encoding poliakin, operable associated with the promoter poliakine. In this case, at the stage (ii) you can use wild-type baculovirus without expressed by the polyhedrin gene. Preferably, therefore, to recombinant baculovirus contains natural polyhedrin gene. In other words, the sequence encoding poliakin and polyhedrin promoter, are natural for recombinant baculoviruses.Chimeric gene contains the coding sequence for the toxin. This coding sequence or the full chimerical gene can be synthesized. Oligonucleotides, which generally correspond to the target sequence, are synthesized and annealed. For this purpose, preferably adapt the codon preference for specific baculovirus into which you want to enable encoding polnum 3'-terminal codon for glycine, or equivalent sequence, after which broadcast can be modified to - NH2. Provides translational stop codon. Gene toxin can be cloned using known in the art methods. If a gene toxin synthesized separately, it are ligated with an appropriate secretion signal coding sequence.The secretory signal sequence is present, in order to ensure secretion of the toxin from insect cells infected with recombinant baculovirus of the present invention. The estimated signal sequence can be tested by constructing a recombinant baculoviruses infecting their Spodoptera frugiperda cells and exploring the supernatant of cultures of infected cells S. frugiperda for secreted toxin.In another embodiment, the hemolymph can be distinguished from insects infected with recombinant baculovirus, to enter sensitive to them insects and to assess toxicity. The toxin can enter both the larvae and adult individuals. For toxin active against Diptera, test insects can serve as M. domestica. Other suitable insects can be selected as a test species for others the transcription and translation, including the P10 promoter. The promoter controls the expression of the protein in insect cells infected with recombinant baculovirus.Cells susceptible to baculovirus infection, transfection recombinant vector transfer, containing chimerical gene, baculovirus to infect wild type at stage (ii) specified earlier. Such susceptible cells are typically cells cell lines insects, usually cells of Spodoptera frugiperda. After going homologous recombination, we can distinguish the recombinant baculovirus according to the method of the present invention.Recombinant baculovirus is stable. In other words, recombinant baculovirus stable enough for practical purposes. Poliakin negative NPV is not stable enough for practical purposes. Recombinant baculovirus is also infectious. Recombinant baculovirus can infect any susceptible him the insect. Another protein containing the toxin Express in cells of the insect and it is secreted from them. The toxin causes the death of the insect.Therefore, the recombinant baculovirus is used to control insect pests. Effective to the MP for pests-insects, and specific cultural plants that suffer from contamination by pests.In another embodiment, the recombinant baculoviruses can be applied to insects, for example, contained and grown in the laboratory insects, which then transferred the plot to infect subsequent generations. The dose of the recombinant virus is from 108up to 1013PIB per acre. Suitable baculovirus selected depending on the insect, subject to eradication. Recombinant baculovirus is usually applied by spraying, but it can be applied in the form of bait or dust. Recombinant baculovirus is digested by the larvae of insects, so it should be on the appropriate area at the time when it is expected hatching of the eggs of the insect. This period usually lasts from two to four weeks. The virus can be used alone or in combination with other biological control agents or chemical insecticides.Depending on the selected baculovirus this way of dealing can be used against insect the following groups:
Trichoptera, and especially
Lepidoptera.Due to the selection of cultural plants and forests; the insects include the following:
Heliothis armigera, zea, virescens
Spodoptera littoralis, exigua, litura, exempta,
Recombinant baculovirus of the present invention can themselves be consistently used as insecticides in the usual way, in which you can use unmodified native viruses, for example, introducing an effective amount to the area where the insects are susceptible to infection with this virus. It was found that recombinant baculoviruses viable or competent for such purposes due to the fact that contain native DNA sequence for reproduction, lethal infection and expression of the polypeptide toxin. Recombinant viruses can be constructed so that they are expressed and secretively two or more of the toxins.Insecticidal compositions containing the recombinant baculoviruses in insecticide effective amount typically include such inert carriers, such as clay, lactose and proteinogram materialistically, which are food attractants, and which can improve the stability contained in the composition of baculoviruses. Such compositions preferably stored at low temperatures, for example, when 14 - 20oC to ensure the safety of viruses.The composition proposed in the present invention, can be crushed compositions in the sense that they can consist of many small particles, in which the baculoviruses are stored in clay and/or protein matrix. The term "saved" is used here in the sense that such particles cover included in the matrix part of baculoviruses exposed on the surface of particles, and also include a full encapsulation of baculoviruses in the matrix, the latter is preferable, and is mainly for particles of the present invention. The size of the particles in the composition preferably should not exceed 150 microns, and the next distinctive feature of the present invention are satisfactory composition, with the sizes of particles from 5 to 100, preferably from 5 to 50 μm, and especially from 5 to 25 microns, which is quite easy to get.The matrix material is from 65 to 99.9% by weight of the total weight BNO use in the present compositions, can be any convenient treated clay with fine powder particles, such as kaolin, attapulgite and bentonite compounds, preventing clays. The preferred clays are olancha and attapulgite. Vegetable protein, which is used as a material for matrices, can also be any of a wide range of protein sources, which are finely ground form. These materials are preferably degreased, or in another embodiment, they contain virtually no fat. Vegetable sources of protein that can be specified as an illustration, include soybeans, cotton seeds, sunflower seeds, and extracts of various yeast. Preferred vegetable proteinsathome materials are proteins of soybean and cotton seeds, preferably from skimmed source, more preferably low-fat soybeans. Representative animal proteins are skim milk, casein and egg albumin. Proteincalorie materials derived from natural sources may contain significant amounts of deproteinised materials, and the terms "protein" and "proteincalorie materials" is used here to refer to materials that contain even so maknae vegetable proteins, usually contain from 40 to 75% real protein.Although the individual particles and the composition may contain relatively low concentrations of baculovirus, preferably, from the viewpoint of minimal practical use, so that the composition contained the active equivalent LD50at least 1.0 microgram/ml Efficacy of the composition can occur at doses in the range of from 0.001 μg/ml up to 1.0 microgram/ml, and typically in the range of from about 0,003 mg/ml to 0.4 mg/ml Determination of insecticidal activity or effectiveness of that use and referenced, based on the determination of quantities LD50expressed in μg/ml of the power required to achieve the level of dose for 50% growth of larvae of the first stage at a temperature of 30oC. This method is described in detail in J. Insect Pathology; 6,737-45 (1965) in relation to assessing the effectiveness of Trichopusia NPV.Powdered compositions according to the method of the present invention demonstrate the required number of insecticidal effectiveness, and usually are characterized by high resistance to the efficiency of decomposition due to photoinactivation and thermodenaturation. Other properties of the compositions, especially the physical characteristics that vary with different faces clay, usually characterized by good wettability, but tend to be less slidenote, and may in some cases be composed of softer particles. On the other hand, the composition in which the matrix consists only of vegetable protein, can be in General characterized by good adhesion and hardness, but have poorer wettability. Compositions containing large amounts of animal protein, have good adhesion but poor wettability and hardness. Accordingly, the preferred matrix materials are selected from the group consisting of vegetable protein, clay and mixtures thereof. It was also found that compositions in which the matrix is composed of a homogeneous mixture of vegetable protein and clay has the advantages of good wettability, slidenote and stiffness, as well as other desirable characteristics. Accordingly, the most preferred compositions of the present invention are matrix consisting of a mixture of plant proteins and clay. The weight ratio of vegetable protein to the clay may be in the range of from 0.1 to 10 weight. parts of the protein by weight. part clay and preferably in the range from 0.3 to 4 weight. parts of plant proteinase of the invention can be encapsulated in a biodegradable polymer, for example, the alginate Culigel (trade mark). The polymer may include a screen for ultraviolet light and/or feeding stimulant. Powdered sunscreen agent, for example, carbon-based stains, carbon, aluminiumoxid, titanium dioxide, clay, flour or fluorescent materials may be present in the polymer.The following examples illustrate the invention. In accordance with the attached drawings:
In Fig. 1 presents AcMNPV gp67 signal sequence and AaHIT coding sequence, gp67 signal peptide is highlighted two potential codon that initiates translation in bold.In Fig. 2 presents the sequence of the synthetic oligonucleotides used in the construction AaHIT gene. The nucleotide sequence represented by 5'-3'-direction (a1, b3, c5, d7) and in the direction of 3'-5' (a2, b4, c6 and d8). Sites of restriction enzymes that are critical for Assembly of the gene of toxin presented above sequence. The initiation codon of translation is underlined and the stop codon of translation (TAA) are marked with an asterisk above the second nucleotide. "Stuffer" is the area between the BamH1 and Acc1 sites included to facilitate double parivara these two sites, neophocaena between Acc1 and Xba1 sites and pUC18 (Site Acc18 designed to fracture during insertion), forming pSTD. c5/c6 include between BamH1 and Xba1 sites pSTD for education pSTDC. It is digested by BamH1 and Acc1 for the release of "Stuffer" section, and a1/a2 clone these sites to create pSTDCA. This digest Acc1 and insert b3/b4 to create a pLS-ST.In Fig. 3A shows the construction of pAcST-1. Synthetic oligonucleotides encoding AaHIT, were collected in pUC18, as shown in Fig. 2, to obtain the pLS-ST. Then full AaHIT encoding section, removed from the pLS-ST, and inserted into the Bgl II site pAcUW2B, vector transfer baculovirus containing a copy of the p10 promoter, located in the forward direction full AcMNPV polyhedrin gene. The resulting vector transfer pAcST-1 contains a complete AaHIT coding sequence without signal peptide.In Fig. 3b presents the design pAcST-3. Synthetic AaHIT removed from the pLS-ST and insert BamH I site pAcATM1 to get pAcATV-1-ST. Mutagenesis under the action of customers carry out communication between the gp67 secretion signal and AaHIT coding section to obtain favorable proteasome site of cleavage in a modified plasmid pAcST-2. Modified gp67-Aunt encoding section removed from pAcST-2, and insert in pAcUW2B to get pAcST-3.In Fig. 4A presents the nucleotide posledovatelno (360-569). The site of initiation of transcription of the p10 promoter (position 167-170; TAAG) is indicated in bold. The relationship between the signal peptide and toxin shown to mutagenesis. Nucleotides were deleted, as shown below.In Fig. 4b presents the nucleotide sequence of AcMNPV p10 gene promoter in accordance with the gp67 signal peptide coded plot and AaHIT coding section. Above the sequence presents restriction map of the plasmid vector transfer (pAcST3), which were used to produce recombinant virus.In Fig. 5 shows the expression of proteins in cells infected with the virus S. frigiperda (Sf). These cells were infected with AcST-3 or the wild-type virus, and pulse-state of35S-cysteine for 1 hour at various times after infection as described in methods. Proteins were isolated by 10-30% polyacrylamide gradient gel before exposure of film to x-rays. Specified position AaHIT and polyhedrin protein.In Fig. 6 presents the results of immunoblotting analysis VIH (HPLC) fractions AcH full of toxin and AcST-3 infected cells environment. Fractions 25-28 from WICH were selected by 10-30% polyacrylamide gradient gel and were SS="ptx2">In Fig. 7 presents full raw AaH venom or the environment from Sf cells infected with AcST-3, which were fractionated and used in VIH (see example 1). Analysis of selected fractions was carried out by introducing 4 ál in M. domestica and watching paralysis or death.In Fig. 8 presents a superficial defeat leaves the larva infected AcST-3 and AcMNPV (wild type).In Fig. 9 presents the nucleotide sequence of AcMNPV p10 gene promoter in Association with the site coding for the signal peptide of interleukin 2, and AaHIT coding section. Over this sequence depicts a restriction map of the plasmid vector transfer (pAcI12ST) used to produce recombinant virus.In Fig. 10 shows the nucleotide sequence of AcMNPV p10 gene promoter in Association with the site coding for the signal peptide of juvenile hormone esterase and AaHIT coding section. Over this sequence presents a restriction map of the plasmid vector transfer (pAcJHEST) used to produce recombinant virus.In Fig. 15 shows the synthesis of proteins in virus-infected Sf cells, as shown in Fig. 5 (a) ACI12ST; (b) AcJHEST; (c) AcBPST.initial peptide selected two potential initiation codons broadcast in bold.In Fig. 17 shows the sequence of the synthetic oligonucleotides used in the construction SefIT gene. The nucleotides presented in the direction 5'-3' (OS3711) and in the direction of 3'-5' (OS3712). Sites of restriction enzymes used to sublimirovanny pairs of oligonucleotides in plasmids, are presented below. The initiation codon of translation is underlined and the stop codon of translation (TAG) is indicated by an asterisk above the second oligonucleotide. After annealing the oligonucleotides include pDH7, between the Sph I and BamH I sites. pDH7 is derived pEMBL19, which has a secretion signal AcNPV gp67 between the unique Bgl II site located instead of the Hind III site of polylinker, and Sph I.In Fig. 18 presents the nucleotide sequence of AcMNPV h10 promoter (1 to 240) in Association with the gp67 area coding for the signal peptide (246-359). The rest of the sequence is a fragment of the coding SefIT. The sequence presents after mutagenesis communication between gp67 plot and SefIT plot.In Fig. 19 shows the nucleotide sequence of AcMNPV p10 gene promoter in Association with the site coding gp67 signal peptide, and SefIT coded plot. Above the sequence presented re the ASS="ptx2">Example
1. Materials and methods
(A) Viruses and cells
AcMNPV C6 (Possee, 1986) and all recombinant viruses are cultivated in Spodoptera frugiperda (IPLB-Sf-21) cells (Vaughn et al., 1977) in the 28oC in TC100 with the addition of 5% or 10% serum fetal calf (FCS) in the presence of penicillin and streptomycin. The infective ability of the virus was assessed using analysis of belascoaran according to the method of Brown and Faulkner (1977).Purification of the virus: the Polyhedron purified from infected cells or insects on the way ish et al. (1977), but with the addition of 0.1% (weight/volume) SDS for all solutions. SDS was removed by washing the polyhedron water at the end of the cleaning process. The number of polyhedra estimate using the method of dry account Overlying (1976).(B) Constructing a synthetic AaHIT and SefIT coding sequences
Protein sequence AaHIT turn in the nucleotide sequence, using the offset preferred codon AcMNPV polyhedrin (Hooft van Iddenkinge et al, 1983) and p10 (Kuzio et al, 1984) genes. The expected DNA sequence analyzed on a VAX computer using the tools of molecular biology Studena to identify sites of restriction enzyme. They are used to construct four complementary pairs of synthetic legkim way to four nucleotides before the ATG codon corresponded to nucleotides AcMNPV polyhedrin gene, preserving, thus, the integrity of the microenvironment of the initiation of translation; the codon the end of the broadcast include at the 3' end of the coding area. The corresponding restriction sites of the enzyme include to facilitate the inclusion of synthetic oligonucleotides into pUC18 and then cut in whole AaHIT coding section BamHI and Bgl II for further manipulation. Oligonucleotides are collected in full AaHIT coding sequence using pUC18 (Yanisch-Perron et al., 1985), using standard protocols (Sambrook et al., 1989). Further details of this procedure previously described in relation to Fig. 2, 3A and 3b. AaHIT coding section in the final construction of the plasmid pLS-ST, is sequenced using terminators dideoxynucleotide circuits (Sanger et al., 1977) to confirm their authenticity.Similarly protein sequence Sef IT turned into nucleotide sequence information. Synthesize two complementary oligonucleotide, OS3711 and OS3712 (Fig. 17). After annealing these oligonucleotides include plasmid pDH7, between sites Sph I and BamH I. pDH7 is derived pEMBL19 (Dente et al., 1983), which sod is ef IT encodes the plot in the construction of plasmids, pDH7-Sef IT is sequenced in the way described earlier to confirm its authenticity.(C) Construction of transfer vectors and baculovirus
(i) pAcST-1 (comparison): To obtain practical virus insecticide necessary to AaHIT coding sequence was included in baculovirus, which retains a functional polyhedrin gene. Vector transfer pAcUW2B (Weyer et al. , 1990) satisfies this criterion. It is based on AcMNPV EcoR I-I fragment, which includes the polyhedrin gene. He has a complete copy of the polyhedrin gene, a copy of the p10 promoter and a cloning site Bgl II with SV 40 sequence of the transcription has been completed, included in the position 90 nucleotides in the opposite direction from the polyhedrin ATG codon and in the opposite orientation. AaHIT coding section excised from pLS-ST, using BamH I and Bgl II, purified after fractionation in the gel, gliroides at a low temperature, and include in the Bgl II site of the vector transfer cUW2B (Weyer et al., 1990) to obtain pAcST-I (Fig. 3a).(ii) pAcST-2: To obtain the viruses that have the potential to secrete AaHIT coding the site, include pAcATMI, vector transfer with a copy of the gp67 signal peptide sequence (Whitford et al., 1989) after the polyhedrin promoter. Patch, Cody. 3b). pAcATMI is derived pAcCL29 (Livingstone and Jones, 1989), the vector of transfer on the basis of the polyhedrin promoter containing M13 intergeneric plot, which makes it easier to obtain single-stranded DNA through the use of M13 helper phage (Livingstone and Jones, 1989). The relationship between the gp67 signal sequence and AaHIT modify using M13 oligonucleotide directed mutagenesis (Kunkel, 1985), using GP-67-SC. Tox, is shown in Fig. 4A to obtain the corresponding DNA sequence that encodes a site of recognition suitable protease: at this stage also removes the source of the initiation codon ATG broadcast from AaHIT. Changes made in gp67 the connection shown in Fig. 4A along with a sequence of p10 promoter.(iii) pAcST-3 (invention): gp67 signal sequence and AaHIT removed from pAcST-2, digesting Xho II, and putting into a Bgl II site in pAcUW2B to get pAcST-3 (Fig. 3b).(iv) pAcl12ST (invention): AaHIT synthetic coding section include both BamH I / Bgl II digested DNA fragment into the BamH I site with polylinker site of BLUESCRIPT M13 phagemid (Stratagene). The orientation of the coding of the site confirm restriction enzymatic Cartaromana. The sequence encoding the signal peptide of IL-2 (Taniguchi et al., 1963), includes m is Obedinenie between the sequences encoding the signal peptide and toxin change, using oligonucleotide-directed mutagenesis (Kunkel, 1985) to obtain the optimal site recognition protease for cleavage of secretory peptide. The sequence confirmed using the methods of the termination circuit. The DNA fragment containing parts, encoding the signal peptide and toxin excised using Xho II and include Bgl II site of the vector baculovirus transfer pAcUW21 to get pAcI12ST. AcUW21 is derived pAcUW2B and has shortened viral sequences flanking the gene polyhedrin and p10 gene promoter; it also contains the appropriate regulatory sequence to ensure the production of single-stranded DNA in the bacteria, after superantisypware due M13 KO7. pAcUW21 design, issaka nucleotide DNA fragment 5408 from pAcUW2B and including it in pEMBL19, which was digested Hind III and EcoR I, and the ends of the DNA recovered DNA polymerase and treated before generowania phosphatase intestine of the calf. In Fig. 9 shows the sequence of the plot, the coding I1-2 signal peptide, in Association with AaHIT coding sequence.(v) pAcJHEST (invention): Synthetic DNA encoding JHE signal peptide design, including two pairs coR I sites. Then, this plasmid digested by BamH I (within 3' of the site JHE sequence), the section that encodes the toxin include as BamHI/Bgl II-digested DNA fragment. The relationship between JHE signal peptide and areas encoding the toxin change, using oligonucleotide-directed mutagenesis as described previously. Then these sequences produce Bgl II perwara and include in pAcUW21 by Bgl II site to obtain pAcJHEST. In Fig. 10 presents a plot that encodes a signal peptide JHE in Association with AaHIT coding sequence.(vi) pAcSTST (invention): For a copy of the AaHIT gene with a signal peptide sequence for natural AaHIT obtained by polymerase chain reaction Bougis R. E., et al., 1989) on the 5' end and Bgl II at both ends. 5' primer (see at the end of the sequence 13).3' primer:
TATATAAGATCT TTA GTT AAT AAT AGT AGT GTC
Then digested Bgl II fragment inserted into pAcUW21 unique Bgl II site. This vector indicate pAcSTST. In Fig. 11 presents AaHIT signal peptide and AaHIT coding section.(vii) pAcCP2ST (invention): This vector are given as and for pAcSTST, except that the add signal peptide sequence for CP2 from Drosophila melongaster (Syder, M. et al., 1982) in which iMER is:
TATATAAGATCT TTA GTT AAT AAT AGT AGT GCT. In Fig. 12 presents the sequence of the signal peptide and AaHIT coding of the site.(viii) pAcOXYST (invention): This vector get as pAcSTST, excluding the signal peptide sequence for oxytocin from rats (IvelI, R. and Tichter, D., 1984) is added instead of natural AaHIT signal peptide. the 5' primer has the form (sequence 15)
the 3' primer has the form:
TATATAAGATCT TTA GTT AAT AAT AGT AGT GTC. The sequence of oxytocin signal peptide and AaHIT coding section shown in Fig. 13.(ix) pAcUW21 BP (invention): the Promoter of the basic protein gene excised from pAcMPI (Hill-Perkins et al., 1990) with ASp 718 and BamH I and inserted between the same sites in pDH7 to get pDH7BP. The polymerase chain reaction is used then to obtain copies of the promoter core protein gene, and add Xba I site at 5' end of the promoter fragment. Then this fragment is inserted instead of the p10 gene promoter in pAcUW21 and denote pAcUW21BP. The DNA sequence containing the gp67 signal peptide, and a plot that encodes a toxin, excised from pAcST-3, using Xho II and include Bgl II site pAcUW21BP to get pAcBPST. The sequence of the basic protein and p10 gannage): gp67 encoding section is placed in a frame with SefIT coded plot (pDH7-SefIT), using site directed mutagenesis as described previously. The change is confirmed by sequencing. Full encoding section (gp67 and SefIT) excised from pDH7-SefIT with Bgl II and BamH I and include Bgl II site pAcUW21 to get pAcSefIT. The sequence SefIT the coding section in Association with the gp67 signal peptide site and p10 gene promoter is shown in Fig. 18 and 19.(D) Obtaining recombinant viruses
(i) AcST-1 (comparison): Spodoptera frugiperda cells were jointly transfection with pAcST-1 and poliakin hegative AcRP-6-SC (Kitts et al., 1990) infectious viral DNA using the calcium phosphate procedure (Smith et al. , 1983). Recombinant viruses is chosen as poliakin-positive phenotype in the standard analysis Belkoopsoyuz.(ii) AcST-3 (invention): pAcST-3 together transfection with AcRP6-SC, using the method lipofectin (Felgner et al., 1987, Groebe et al., 1990), and viruses sceneroot as for the previous cotransfection.(iii) AcI12ST, AcJHEST, AcBPST, AcSTST, AcCP2ST, AcOXYST, AcSefIT (all inventions): all described as for AcST-3.Recombinant viruses are cleaned five successive analyses belascoaran determine their authenticity southern hybridization EcoR I peravurani DNA from infected cells using the ratinov infected cells or fractions VIH (high performance liquid chromatography)
Protein extracts of unlabeled virus infected cells (see E previous or next G) was isolated in 10-30% denaturing polyacrylamide gels, and transferred to nitrocellulose filters using the apparatus of electroblotting Bio-Rad for two hours at 100 In 25 mm Tris, 5,SM-glycine and 20% methanol as a buffer transfer. The filter is incubated in PBS containing 0.05% tween-20 (PBST) with 3-5% fat powder milk as a blocking agent. All this gently shaken for 1 hour at room temperature, then incubated for 2 hours with serum of Guinea pigs against synthetic AaHIT peptide, diluted 1/100 in PBST (titer more than 1: 10000 in indirect ELISA against the homologous peptide). The filter is then washed several times in PBST to remove unbound antibodies. The bound antibodies detected using artimarca mumps lgG-alkaline fosfatazy conjugate.(G) WJH AaH full of toxin and infected cell supernatant fluids and in vivo analysis in Musca domestica: a test for toxicity when injected
Full raw AaH venom obtained from Sigma in the form of frozen-dried sample (1 mg), resuspended in 20 μl of DMSO, then diluted to 100 μm in H2O. Precipitated in the sludge Mat is utilized VIH, using LCD system with detector Spectroflow 280 nm. For division use Spherisord, C8with a particle diameter of 5 μm, 25 cm column. As A solvent used is 0.15 M monitorial. pH 2.7 and the solvent is acetonitrile. The lead elution with a linear gradient from 15 to 40% in A 40 minutes at a flow rate of 1 ml/min, and collect 40 fractions of 1 ml Fractions are dried by freezing to remove acetonitrile and ammonium formate, then resuspended in 200 ál of 0.1 mg/ml BSA in water. 0.5 µl of each fraction injected with 10 adults of M. domestica (males and females) using a needle syringe, 25 gauge.Analysis of the supernatant from infected cells is done using the supernatant liquid with 2 cups of cells infected S. frugiperda as source material (i.e., 2 ml). This concentrate in 4 times, using the filtering system Method (HCC) by centrifugation at 500 g for 3 hours in the centrifuge Sorvall RC5. 100 μl of the concentrated material is loaded into a column VGH, and are treated the same way as full of poison. After processing the sample on VGH, fractions are dried by freezing and resuspended in 100 μl of 0.1 mg/ml BSA. 4 μl of each fraction used for the introduction of M. domestica.(H) Receiving viruses in insects
T. ni vyd is that once missed by insects. For the initial implementation, the third stage of T. ni larvae were given feed that contained the crude extract of infected cells, after which the larvae transferred to fresh diet. Immediately after the death of the insects they collect and process. The polyhedron purified and used for subsequent analyses.(1) the Biological activity of haemolymph extracted from infected cells
The biological activity of the toxin appreciate, imposing on 4 µl of serial dilutions of the specific sample in the dorso lateral area of the abdomen of M. domestica, between the sclera. The fractions that contain the toxin derived from VGH for aAH full of poison, used to create standards for assessing the amount of a toxin isolated from the haemolymph. Fractions are dried by freezing and again suspended to a concentration of 90 ng/100 μl water. This is again diluted in PBS to facilitate the introduction of 4 µl, containing 18, 9, 4,5, or 1.25 to 2.25 ng toxin insects (dose 18 ng was excessive for LD50for toxin).(J) Determining LD50< / BR>In the second stage larvae of T. ni (approximately 0.6-0.7 mg) separately fed five serial diluted with a known titer of the virus on small pieces of artificial feed in microtiter plates. Maximum dose of s strains of AcMNPV satisfy these conditions, is 120, 60, 30, 15 and 7.5 PIB on the larva. Those larvae that had consumed the dose for 24 hours, and those who did not use were excluded from the analysis. The rest were transferred to individual containers with artificial feed and kept for 24 hours in a dark incubator. Larvae were daily examined and cadaver was removed, placed on a glass slide, and the cause of death was determined under a microscope. The results were analyzed using probit analysis to estimate LD50(Finney, 1971).(K) Determining ST50
To obtain the newborn (neonate) larvae, which are used in such studies, adults are placed in cells containing filter paper for oviposition. Filters with testicles surface sterilize, and store in plastic containers. After hatching, the hatchlings do not give food for 3-6 hours before give drip food with different suspensions of viruses. Suspension tint 5% blue food dye to visualize feed. Small droplets of this suspension are placed in Petri dishes concentric rings. Larvae placed in the center of these concentric circles, then move through drops, taking a small amount of Gulati, remain in the center of the Cup, which allows simultaneous feed and select healthy individuals. In preliminary studies it was shown that in this type of experiments, the larvae of T. ni consumes 0,0087 ( 0,0023) ál (Hughes and Wood, 1981. Hughes et al., 1986) and in these ST50 analyses suspension viruses contained 1106polyhedra/ml After feeding, the larvae were kept in separate containers with artificial food. After 24 hours, the larvae inspect all dead larvae removed, as their death is associated with manipulation; after 48 hours the larvae again examined, and then the intervals between inspections is reduced. All dead larvae were removed and the cause of death is determined by appearance and examination under microscope. Assessment ST50 spend on the program Vistat (Boyce Thompson Institute, Ithaca. New York).(L) reducing the damage dealt when feeding insects infected AcST-3
Third stage larvae (T. ni) are fed separately during the night pieces of artificial feed inoculated with 104bodies include polyhedra (PIB). Such a high dose leads to 100% mortality. After feeding, the larvae are divided into groups of 5, on the cabbage leaves that were grown under controlled conditions. Before feeding the sheets are immersed overnight in water to ensure the tx2">2. Results
(A) Synthesis of proteins by recombinant viruses
AaHIT has a protein sequence containing 70 amino acids (including methionine) with a predicted molecular weight of 8000. Analysis of extracts from cells of S. frugiperda cells infected AcST-1 (AaHIT without secretory signal) or AcST-3 (AaHIT with secretory signal) under denaturing 10-30% polyacrylamide gel, which is not defined additional proteins of the expected size, or Comassie staining Blue or silver. Then virus-infected cells have been labelled with [35S] cysteine at various times after infection (Fig. 5). The results show an additional protein, which migrates between the expected position of a virus p10 protein (p10 proteins without cysteine residues). Synthesis alleged AaHIT maximum between 18 and 36 hours after infection. The identity of the protein is confirmed using the Western blot testing analysis AcST-1 or AcST-3 infected cell extracts with anticorodal brought to synthetic AaHIT.(B) VIH AaH full of poison and synthetic AaHIT
On the chromatograms VIH visible peaks with an optical density at 280 nm around fractions 27/28, which are present in the full poison, icing analysis of fractions between 25 and 28 indicates the presence AaHIT in fractions 27 and 28 of the crude venom, and 26 27 supernatant of cells infected AcST-3 (Fig. 6).Injection of M. domestica small amounts of each of these fractions confirms that for these fractions is observed paralytic and lethal activity. Immobilization due to toxin observed one hour after injection, and the loss becomes evident 24 hours after injection (Fig. 7).(C) Biological activity of haemolymph extracted from infected larvae
Secretion AaHIT from AcST-3 infected cells in vivo estimate, highlighting the hemolymph of infected T. ni larvae and injecting it in M. domestica. Larvae (stage 4) T. ni infect 105PIB AcST-3. After 72 hours after infection make the incision at the end of prolegs and select the hemolymph (20 µl). To limit mechanisatie, pipette was used to collect the hemolymph of all samples immersed in a solution of phenyltoloxamine. 4 μl of this haemolymph used for injection of the test larvae of M. domestica and assess toxicity. The reaction compared with standard curves obtained using the AaHIT full of poison purified on VGH, and the results obtained are presented in table 1. The reaction of M. domestica indicates the concentration of toxin in the larvae of T. ni around 1.5-2.0 ng/ál of hemali the spine of recombinant viruses appreciate determining the magnitude of LD50. Infect larvae of T. ni second stage. Re individuals administered doses of 7, 15, 30, 60, or 120 PIB. After probit analysis LD50values for unmodified AcMNPV was 44 (95% confidence limit of 35 and 55), AcST-1 38 (95% limits of confidence 31 and 46) and AcST-3 (invention) - 31 (95% limits of trust 27 and 37). LD50values for AcMNPV and AcST-3 is markedly different for a = 0,05. The difference in LD50relatively small, and the increase is 1,4191 with 95% limits of confidence 1,026216-1,9846.ST50 appreciate after infection of newborns. However, show a very marked decrease in the time required for killing larvae. ST50 for AcMNPV was 113,1 (95% confidence limits 112-115), for AcST-1 - 109,2 (95% limits of confidence 107-110) and AcST-3 (invention) amounted to 85.8 hours (95% limits of confidence 85-87 hours). Result for AcST-3 is markedly different from the values for the unmodified virus AcST-1, which contains the toxin coding sequence without the secretion signal = 0,0001). This means that AcST-3 virus kills T. ni larvae, at least 25% faster than AcMNPV. The results obtained are summarized in table 2.(E) the Pathology of infection
And what of ICIEC, infected AcST-1 or AcMNPV. Infection AcST-1 or AcMNPV leads to massive caused by a virus damage, causing liquefaction of the larvae, accompanied by white color. When the infection AcST-3 larvae retain their structural integrity and remain green.(F) the Reduction of damage caused by feeding of larvae infected with AcST-3
Efficiency AcST-3 recombinant virus by reducing the damage caused to cultivated plants of T. ni larvae, shown in Fig.4.(G) Synthesis of proteins by recombinant viruses with an alternative signal peptides
Gene expression of toxin included in each of the recombinant viruses, testing, infecting Spodoptera frugiperda cells and introducing RFID tags in a separate culture plates with the same time intervals in 1 hour. The obtained results are presented in Fig. 15a, b. AcJHEST produce a certain amount of toxin after 12 hours after infection, with a maximum of synthesis between 18-36 hours mi after infection; after which the rate of production of protein is reduced. Comparable results to see I12ST-infected cells, although the release of the toxin from the cells infected with the virus, the torus
In Fig. 15c presents also the kinetics of production of toxin Scorpion in AcBPST-infected cells. Obviously, the virus initiates the production of toxin in large quantities after 12 hours after infection. The output is much higher than from any other cells infected with recombinant viruses containing the toxin gene under the control of one p10 gene promoter. The production of toxin in AcBPST-infected cells continues until 48 hours after infection.(I) the Biological activity of the toxin, secreted in the medium from virus-infected cells
Table 3 provides biological activity AaHIT, secreted into the culture medium that supports the growth of virus-infected Sf cells. Wednesday investigate, using M. domestica, as described in (C). Obviously, AcST-1 does not emit the active toxin in the environment.(J) In vivo activity of recombinant viruses with alternative signals secretion or hybrid gene promoter
The magnitude of LD50for each recombinant virus assessed as previously described (D). Found that they are similar. St50evaluate after infection of newborns. ST50for AcST-3 accounts for 71.4 coctavlyaet 87.2 hours (95% limits of confidence 86,1-86,3), for AcBPST is 73.1 hours (95% limits of confidence 71,8-74,4).Table 4 shows LD50and ST50for recombinant baculovirus containing AaHIT.E. coli XZ-blue containing pAcATM-1, E. coli HB101 containing pAcUW2B and E. coli SURETM containing pAcST-3, were deposited in the National Collection of Industrial and Marine Bacteria, Aberdeen, UK, 22 March 1991 under registration numbers NCIMB 40393, NCIMB 40394 and NCIMB 40395, respectively. 1. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene toxin Scorpion DNA sequence that encodes a signal peptide gp67, P10 promoter, polyhedrin gene with its own promoter, and containing 2 restriction site XbaI, 1 restriction site BglII, 1 the restriction site HindIII and 1 restriction site kpni restriction sites.2. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene Scorpion toxin, DNA sequences encoding the XbaI.3. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene toxin Scorpion DNA sequence that encodes a signal sequence of the juvenile hormone esterase, P10 promoter, polyhedrin gene with its own promoter, and containing 2 restriction site XbaI.4. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene toxin Scorpion DNA sequence that encodes a signal sequence AaHIT, P10 promoter, polyhedrin gene with its own promoter, and containing 2 restriction site XbaI, 1 the restriction site BamHI, 1 the restriction site HindIII, 1 restriction site kpni restriction sites.5. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene Scorpion toxin, DNA sequences coderush the AIT enzyme XbaI, 1 the restriction site BamHI, 1 the restriction site HindIII and 1 restriction site kpni restriction sites.6. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene toxin Scorpion DNA sequence that encodes a signal sequence of oxytocin, P10 promoter, polyhedrin gene with its own promoter, and containing 2 restriction site XbaI, 1 the restriction site BamHI, 1 the restriction site HindIII and 1 restriction site kpni restriction sites.7. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following consecutive items: gene toxin Scorpion DNA sequence that encodes a signal sequence gp 67, P10 promoter, promoter of the gene of the main protein of AcMNPV, polyhedrin gene with its own promoter, and containing the restriction site XbaI.8. Stable occluded recombinant nuclear polyhedrosis virus for use as an insecticide, and the virus contains DNA fragment consisting of the following placentas is sledovatelnot gp 67, the P10 promoter, polyhedrin gene with its own promoter, and containing 1 BgIII restriction site, 1 the restriction site XbaI, 1 the restriction site BamHI, 1 the restriction site HindIII, 1 restriction site kpni restriction sites.9. Insecticidal composition comprising an inert carrier and an active component, characterized in that as the active component contains stable occluded recombinant virus in PP.1 - 8 in an effective amount.10. A method of combating insects at the site, providing on a plot of effective number insecticide-active viral drug, wherein as a virus use stable occluding the recombinant virus according to PP.1 - 8.
FIELD: plant production.
SUBSTANCE: method includes spraying of vegetative solanaceous plants with Steinermena feltiae suspension in combination as antidesiccant with agent obtained from biomass of Mortierella jenkinii micromycete according to claimed technology.
EFFECT: insect pest control with improved effect.
FIELD: plant production.
SUBSTANCE: method includes spraying of vegetative solanaceous plants with Steinermena feltiae suspension in combination as antidesiccant with agent obtained from biomass of Mortierella marburgansis micromycete according to claimed technology.
EFFECT: insect pest control with improved effect.