Gene providing plant disease resistance

FIELD: biotechnology, in particular isolated DNA molecule providing plant disease resistance and method for providing of disease resistance to plants.

SUBSTANCE: DNA molecular containing N1M1 is isolated. Recombinant vector including active in plant promoter functionally bonded with said DNA is constructed and plant is transformed by this vector.

EFFECT: decreased technological charges and increased land productivity.

9 cl, 37 dwg, 18 tbl, 10 ex

 

The present invention relates to the resistance of plants to diseases and to the identification and selection of plants resistant to diseases. In particular, the present invention relates to the identification, isolation and characterization of the gene causing the resistance of plants to a wide range of diseases.

Plants are constantly exposed to a wide spectrum of pathogenic organisms, including viruses, bacteria, fungi and nematodes. Cultivated plants are especially vulnerable because they are usually grown as genetically uniform monocultures, while losses in the defeat of the disease can be serious.

However, most plants have their own innate defense mechanisms against pathogenic organisms. Natural variability for the trait resistance to phytopathogens was viallinen plant breeders and phytopathologists and used for breeding of many cultivated plants. These natural resistance genes to diseases often cause high-level resistance or immunity against pathogens.

Many plant species the initial inoculation with the pathogen, causing paralysis, can immunize plants against subsequent infection. This acquired resistance to disease first described in 1901 and probably plays an important ro the ü in the preservation of plants in nature. Especially fully described in the examples of plant immunity is the phenomenon of systemic acquired resistance (SAR). systemic acquired resistance and induced resistance in plants such as tobacco, Arabidopsis and cucumber. In these systems inoculation with the pathogen, causing necrosis, leading to systemic protection against subsequent infections by this pathogen, as well as a number of other important from an agricultural point of view, bacteria, fungi, and viral pathogens.

Systemic acquired resistance may also be called chemical immunizing compounds, certain chemicals, which cause the immune response in plants. Such compounds may be of natural origin, for example, salicylic acid (CK), or they can be synthesized by chemical agents such as 2,6-dichloroaniline acid (INC.) and S-methyl ether benzo(1,2,3)thiadiazole-7-karbaminovoi acid (BTK). Treatment with pathogen or immunizing compound induces the expression of at least nine sets of genes in tobacco, the most well characterized species. Other plants can be expressed with different number and types of genes. The level of induction associated with SAR genes induced by immunizing compounds, more than 10,000 times greater than the primary level. In cha is in the surrounding area, SAR is characterized by the expression of SAR genes, including genes associated with pathogenesis (PR). pathogenesis-related).

SAR-genes are indicated after infection by a pathogen. Some of these genes play a role in making the plant systemic acquired resistance. These plant proteins are induced in significant amounts in response to infection by various pathogens, including viruses, bacteria and fungi. PR-proteins were originally discovered in tobacco plants (Nicotiana tabacum), characterized by hypersensitivity to infection with tobacco mosaic virus (TMV). Then PR-proteins have been found in many plant species (see Redolfi and others (1983), Neth j Plant Pathol 89: 245-254; Van Loon (1985), Plant Mol. Biol. 4: 111-116; Uknes and others, (1992) Plant Cell 4: 645-656). Such proteins are probably the overall protective system response of plants to infection by pathogens.

Associated with the pathogenesis proteins include proteins SAR8.2a and SAR8.2b, acidic and basic forms of proteins of tobacco PR-1a, PR-1b and PR-1c, basic proteins PR-1’, PR-2, PR-2’, PR-2 and PR-N, PR-O, PR-O’, PR-4, PR-P, PR-Q, PR-S and PR-R, peroxidase cucumber, basic cucumber peroxidase, chitinase, which is the master copy of PR-P or PR-Q, beta-1,3-glucanase (glucan-endo-1,3-beta-glucosidase, EC 3.2.1.39), which is the master copy of PR-2, PR-N or PR-O, and induced by the pathogen of cucumber chitinase. These PR-proteins are described, for example, Uknes and others (1992) The Plant Cell 4:645-656, and specified in this article references.

SAR or SAR-like genes are expressed in all types of plants with systemic acquired resistance. The expression of these genes may be detected by sensing using the known DNA sequence of the gene SAR, as described, for example, Lawton and others (1992), Proceedings of the Second European Federation of Plant Pathology (1983), In: Mechanisms of Defence Responses in Plants, edited by .Fritig and .Legrand, Kluwer Academic Publishers, Dordrecht, str-420; Uknes and others (1992), The Plant Cell 4: 645-656; Ward and others, (1991) The Plant Cell 3: 1085-1094. Methods of hybridization and cloning are well known in this field and are described, for example, in Molecular Cloning, A Laboratory Manual, 2nd ed., volumes 1-3, edited by Sambrook and others, Cold Spring Harbor Laboratory Press (1989), and referred to in this work references.

An alternative to this SAR or SAR-like genes can be obnarujeny other methods, such as screening of protein, ± screening (differential screening), etc., as described, for example, Liang and Pardee (1992), Science 257: 967-971; St. John and Davis (1979)Cell 16: 443.

Despite numerous studies and the use of complex and extensive activities aimed at crop protection, including genetic transformation of plants, losses from the disease annually continue to make millions of dollars. Genes for disease resistance cloned previously, however, a transgenic plant transformed with such genes, as a rule, possessed resistance to some of the States the mom of certain types of pathogens. Despite attempts to clone the genes related to SAR, the gene controlling resistance to a wide range of diseases has not been previously isolated and characterized.

Some independent evidence suggests that the resulting endogenous salicylic acid (SA) is involved in the pathway of signal transduction, combining perception of infection by the pathogen with the development of systemic acquired resistance. Mutants that retain the ability to accumulate SA in response to infection by the pathogen, but have already lost the ability to induce SAR genes or resistance after processing IC or INC., were described by Delaney and others, Proc. Natl. Acad. Sci. 92: 6602-6606 (1995) and in the application WO 94/16077 fully included in the present description by reference.

It was found that these mutants contain the mutant gene, and this gene in its wild form controls gene expression SAR and the phenomenon SAR. According to the invention it was found that the mutant gene gives mutant plants are sensitive to a wide range of diseases and causes the lack of ability to the induction by pathogens and chemical inducers.

The present invention relates to the identification, isolation and characterization of the gene of the wild type (NIM1), i.e. the gene that gives the plant the ability to activate SAR and SAR gene expression in response to Biologicheskie chemical inducers.

The mutant gene was identified subjected to mutagenesis of the Arabidopsis plants. It was found that these plants are defective in comparison with their normal reaction to infection by the pathogen in the sense that they do not Express genes associated with systemic acquired resistance (SAR) and are not able to show such stability. These mutants contain the defective gene, which is designated as nim1 (the gene responsible for maindocument immunity).

The present invention also relates to the use of the cloned gene NIM1 and its variants for producing transgenic plants resistant to a wide range of diseases, and to the thus obtained transgenic plants. The invention also relates to the use of the cloned gene NIM1 and its variants for screening to identify compounds able to induce in plants resistance to a wide range of diseases.

Brief description of drawings

Figure 1 shows the effect of chemical inducers on the induction of PR gene expression in wild-type plants and in nim1 plants.

Figure 2 shows the gene expression of PR-1 in infected with a pathogen Ws-O-plants and nim1 plants 6 days after the onset of infection.

Figure 3 shows the levels of accumulation of SA in Ws-About-plants and nim1 plants infected .syringae.

Figure 4 shows the genetic map of the NIM1-about the Asti, obtained using the methods PDF and PDP.

Figure 5 shows the physical map of the NIM1-area obtained by analysis of YAC clones.

Figure 6 shows the physical map of the extended set of sequence fragments (contig) P1/.

Figure 7 shows the physical map showing the position of the P1 clones and flanking YOU on PDAF markers and YAC.

On Fig given physical map of one of the elongated set of sequence fragments P1/YOU, containing the gene NIM1.

Figure 9 shows a combined genetic and subtle physical card NIM-field.

Figure 10 shows a map NIM-field.

Figure 11 shows the combined map NIM-region, including new PDF-tokens.

On Fig presents a schematic representation of recombinant D169 and S.

On Fig presents a General map of the chromosomal region around NIM1 identified recombinants, including YOU, YAC and Comedy in NIM1-field.

On Fig presents a sequence region of length 9,9 TPN clone YOU-04-containing gene NIM1.

(Fig shows the nucleotide sequence of the gene NIM1 and amino acid sequence of the gene product NI1, including changes in different alleles.

On Fig shows the expression of NIM1 induced INC., BTK SA and pathogen alleles in wild-type and mutant alleles nim1.

On Fig p the cauldron expression of PR-1 in plants having a mutation nim1, and in the wild type.

On Fig shows the level of resistance to diseases in different nim1 mutants.

On Fig the above amino acid sequences, including expressed sequence Tag-regions of the NIM1 protein and cDNA protein products of sequences of 4 genes of rice (see SEQ ID NO: 3).

Definition

AK:amino acid
PDF:polymorphism of the lengths of the amplified fragments
avrRpt2:avirulent gene Rpt2 isolated from Pseudomonas syringae
YOU:bacterial artificial chromosome
BTK:S-methyl ether benzo(1,2,3)thiadiazole-7-karbaminovoi acid
Col:the Arabidopsis ecotype Columbia
KF:combinations of enzymes
INC.:2,6-dichloroaniline acid
Ler:the Arabidopsis ecotype Landsberg erecta
NIM1:gene wild-type, causing the resistance of plants to diseases

nim:mutant allele NIM1 determining the sensitivity of plants to diseases
nim1:Mut is ntna line plants
LFS:the open reading frame
KP:combinations of primers
BCC:salicylic acid
SAR:systemic acquired resistance
PDP:the length polymorphism of simple sequence
Ws-O:ecopet Arabidopsis Wassilewskija
YAC:artificial chromosome yeast

Gene NIM1 was cloned methods of mapping and "walks" along the chromosome, which showed that the gene is in a region of length 105 TPN (see Fig and table 16). This area is limited L84.6b-marker on the left and L84.T2-marker on the right. Only three overlapping of Comedy obtained from wild type DNA from a region of length 105 TPN, restore (complementary) mutant phenotype; nim1 (Fig and table 16). The three Comedy overlap only in the length of 9.9 TPN indicated by the left end kosmidou clone D7 and the right end kosmidou clone D5, as shown in Fig. Many of the other Comedy constructed based on other plots area length 105 TPN, do not restore the phenotype nim1 (Fig and table 16). Almost full-size cDNA clone of the gene NIM1 allows you to identify intron-exon boundary sequences and to determine the amino acid placentas is the activity of the gene product. Only NIM1-region of the gene within the complementary region of length 9,9 t.ii.il has changes in the sequence of the different mutant alleles nim1 (table 18). Discovered that three other potential region of the gene do not have sequence changes that are associated with the phenotype nim1. Changes to sequences found in NIM1-region of the gene, consistent with altered function or loss of function of the gene product. The severity of the changes in NIM1-region of a gene in a mutant allele approximately correlates with the observed physiological role of this nim1-allele. Only NIM1-region of the gene had detectable RNA (transcription), and this RNA is detected numerous changes that are consistent with the physiological role of NIM1 in the pathogenesis (table 18 and Fig).

The present invention relates to selected gene fragment, gene NIM1, which is a key component of the metabolic pathways of systemic acquired resistance (SAR) in plants. Gene NIM1 is associated with activation of SAR chemical and biological inducers and in combination with such inductors required for the SAR and the expression of SAR genes.

Localization of the gene NIM determined using analysis methods molecular biology of the genome of mutant plants, for which it is known that they carry the mutant gene nim1, which gives the plant owners is very high the th sensitivity to a wide range of pathogens and weakens their ability to respond to pathogens and chemical SAR inducers. nim1 Mutants suitable as universal susceptible to plant diseases" (UCB)/because of their sensitivity to many strains and pathotypes pathogens, the host plant, as well as pathogens that normally do not infect the host plant, but which infect other hosts. They can be obtained by treatment of seeds or other biological material mutagenic agents with subsequent selection of progeny plants against UCB-phenotype by processing plants offspring known chemical inducers (e.g., INC.) systemic acquired response and subsequent infection of plants known pathogen. In such circumstances, the mutants with pendulums sustainability develop serious symptoms of the disease, while at namutoni plants chemical compound induces systemic acquired resistance. nim1 Mutants can be selected as mutant populations resulting from chemical or radiation mutagenesis and populations formed using indels (insertions) T-DNA and transposoninduced mutagenesis.

Methods of producing mutant lines of plants are well known in this field. min-Phenotype of plants used as a tool to identify a selection gene, which gives the plant the ability of the EC is to preservati resistance to a wide range of diseases.

The present invention relates to the selected DNA molecule comprising a mutant gene NI1, which represents the nim1 gene.

When using the nim1 mutant or plants to highlight the NIM1 gene wild type required for constitutive expression of SAR genes, signs of resilience in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Approaches and breeding methods known in this field and are described, for example, in the following publications: Welsh J.R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, edited by D.R. Wood, American Society of Agronomy Madison, Wisconsin (1983); Mayo, O., theory of Plant Breeding, 2nd ed., Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

Another object of the invention is a chimeric gene comprising active in the plant promoter functionally linked to a heterologous DNA molecule that encodes the amino acid sequence of the NIM1 gene product and its variants according to the invention.

Methodology construction of plant expression cassettes, as well as the introduction of foreign DNA into plants generally described in the art. As a rule, for introduction into plants of foreign DNA used Ti-plasmid vectors for delivery of foreign DNA. DL is such delivery was also used direct injection of DNA, liposomes, electroporation, microinjection and microarray. Such methods are described in this area, for example, in the following publications: Bilang and others (1991), Gene 100: 247-250; Scheid and others, (1991) Mol. Gen. Genet. 228: 104-112; Guerche and others, (1987) Plant Science 52: 111-116; Neuhause and others, (1987) Theor. Appl. Genet. 75: 30-36; Klein and others, (1987) Nature 327: 70-73; Howell and others, (1980) Science 208: 1265; Horsch and others, (1985) Science 227: 1229-1231; DeBlock and others, (1989) Plant discrimination 91: 694-701; Methods for Plant Molecular Biology (edited by Weissbach and Weissbach) Academic Press, Inc. (1988); Methods in Plant Molecular Biology (edited by Schuler and Zielinski) Academic Press, Inc. (1989), as well as in the application for U.S. patent 08/438666, filed may 10, 1995, and in the application WO 93/07278, both of these publications in full included in the present description by reference. It should be noted that the transformation method should depend on the plant cell to be transformed.

It is also clear that the components of the expression cassette can be modified to enhance expression. For example, can be used truncated sequence, nucleotide substitutions or other modifications. Transformed plant cells with modified expression systems must continue to take the overexpression or constitutive expression of SAR genes, which is necessary for the activation of SAR.

The DNA molecule or fragment of a gene, causing the plants resistance to disease due to the fact that they provide gene expression SAR) can be in the turned off in plant or bacterial cells using conventional recombinant DNA method. Typically, this technique involves embedding (insertion) of the DNA molecule in an expression system to which the DNA molecule is heterologous (i.e., in which they do not normally present). Heterologous DNA molecule is inserted into the expression system or vector in the proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the built-in sequences encoding the proteins. Can be used a large number of vector systems known in this field, such as plasmids, viral bacteriophages and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as vector system lambda Igti1, Igt10 and Charon 4, and plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, series pAR. pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII, and other similar systems. The DNA sequence can be cloned into the vector using standard in this field of cloning techniques described in Maniatis, etc. in Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory, Cold Spring Harbor, New York (1982).

Another object of the invention is a recombinant vector comprising the chimeric gene according to the invention.

Order to obtain efficient expression of the gene or gene fragment of the present invention the expression vector of the debtor is to be a promoter. RNA polymerase in the norm associated with the promoter and initiates transcription of the gene. Promoters vary in strength, i.e. the ability to provide transcription. Depending on the system host cell may be any of the many acceptable promoters. Acceptable promoters include the promoter ubiquitin, nos-promoter, the gene promoter of the small subunit of ribulosebisphosphate, the promoter of the small subunit binds chlorophyll a/b-polypeptide, the promoter 35S of cauliflower mosaic virus and promoters isolated from plant genes, it can be called, for example, article Vallejos and others, "Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to theRRNA (45S), the major chlorophyllA/BBinding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes," Genetics 112: 93-105 (1986), which describes the composition of the small subunits. The nos promoter and the 35S promoter of cauliflower mosaic virus is well known in this field.

When gene disease resistance according to the present invention cloned into the expression system, it is easy to transform the plant cell. Tissue of plants suitable for transformation include leaf tissue, root tissue, meristem and protoplasts.

For transformation of plant cells can be used by bacteria of the genus Agrobacterium. Suitable species of this bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Especially C is LeSabre to use the kind of Agrobacterium tumefaciens (e.g., strains LBA4404 or ENA) due to its well known ability to transform plants.

Another approach to transforming plant cells genome comprises introducing inert or biologically active particles in plant tissues and cells. This method is described in US 4945050, US 5036006 and US 5100792, issued in the name of Sanford and others In General, this method consists in the application of inert or biologically active particles at the cell under conditions that ensure their passage through the membrane of the cells and penetration into the cell. When inert particles, the vector may introductionthese into the cell by coating the particles with the vector containing the desired gene. In an alternative embodiment, the cell-target may be surrounded by a vector so that the vector into the cell followed by the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophage, each of which contains DNA, which is supposed to introductionat) can also be introduced into plant tissue cells.

The selection of the gene of the present invention can be used to impart disease resistance to a wide range of plant cells, including cells of gymnosperms, monocots and dicots plants. Although the gene can be integrated into any plant cell, which is included in these broad the forms of plants, especially suitable is the embedding in cells cultivated plants such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, pumpkin large ordinary pumpkin, zucchini, cucumber, Apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, BlackBerry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugar cane.

In suitable conditions, the expression system of the present invention can be used to transform cells of almost any crop. Transformed cells can regenerates in whole plants, resulting in gene confers resistance to diseases whole transgenic plants. As indicated above, the expression system may be modified so that the gene for disease resistance is expressed continuously or expression is constitutive.

Transformation

This system can be used in any plant that can be transformed and regenerated. Such methods of transformation and regeneration are well known in this field. In addition to the above publications can be called as follows: AnG., Watson, B.D. and S. Chiang, Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system, Plant Physiol. 81: 301 to 305, 1986; Fry, J., Barnason, A. and Horsch R.B., Transformation of Brassica napus with Agrobacterium tumefaciens based vectors, PI. Cell Rep. 6: 321-325, 1987; M.D. Block, Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens, Theor. appl. genet. 76: 767-774, 1988; Deblock, M., Brouwer D.D. and Tenning, P., Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the Expression of the bar and neo genes in the transgenic plants, Plant Physiol. 91: 694-701, 1989; Baribault T.J., Skene K.G.M., Cain P.A. and Scott N.S., Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase, PI. Cell Rep. 41: 1045-1049, 1990; Hinchee M.A.W., C.A. Newell, ConnorWard DV, T.A. Armstrong, Deaton W.R., Sato S.S. and Rozman R.J., Transformation and regeneration of non-solanaceous crop plants, Stadler. Genet. Symp. 203212.203-212, 1990; Barfield D.G. and E.C. Pua, Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation, PI. Cell Rep. 10: 308-314, 1991; Cousins Y.L., Lyon, B.R. and Llewellyn D.J., Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering, Aust. J. Plant Physiol. 18: 481-494, 1991; Chee P.P. and Slightom J.L., Transformation of Cucumber Tissues by Microprojectile Bombardment Identification of Plants Containing Functional and Nonfunctional Transferred Genes, GENE 118: 255-260, 1992; Christou, P., Ford T.L. and M. Kofron, The development of a variety-independent gene-transfer method for rice. Trends. Biotechnol. 10: 239-246, 1992; D Halluin K., Bossut M., Bonne E., Mazur C., Leemans J. and J. Botterman, Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants, Bio/Technol. 10: 309-314. 1992; Dhir S.K., S. Dhir, Savka M.A., F. Belanger, A.L. Kriz, Farrand S.K. and J.M. Widholm, Regeneration of Transgenic Soybean (Glycine Max) Plants from Electroporated Protoplasts, PLANT PHYSIOL 99: 81-88, 1992; S.B., Wu F.S. and Thorne BECAUSE, Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts, PI. Cell Rep. 11: 601-604, 1992; Blechi A.E., Genetic ransformation The New Tool for Wheat Improvement 78th Annual Meeting Keynote Address, CEREAL FOOD WORLD 38: 846-847, 1993; Casas, A.M., Kononowicz AK, Zehr U.B., Tomes D.T., J.D. Axtell, L.G. Butler, R.A. Bressan and P.M. Hasegawa, Transgenic Sorghum Plants via Microprojectile Bombardment, PROC NAT ACAD SCI USA 90: 11212-11216, 1993; Christou P., Philosophy and Practice of Variety of Independent Gene Transfer into Recalcitrant Crops, IN VITRO CELL DEV BIOL-PLANT 29P: 119-124, 1993; P. Damiani, Nenz E., F. Paolocci and Arcioni, S., Introduction of Hygromycin Resistance in Lotus spp Through Agrobacterium Rhizogenes Transformation, TRANSGENIC RES 2: 330-335, 1993; Davies D.R., Hamilton and J. Mullineaux P., Transformation of Peas, PI. Cell Rep. 12: 180-183, 1993; J.Z. Dong and Mchughen, A., Transgenic Flax Plants from Agrobacterium Mediated Transformation Incidence of Chimeric Regenerants and Inheritance of Transgenic Plants, PLANT SCI 91: 139-148, 1993; Fitch, M.M.M., R. Manshardt, Gonsalves D. and J.L. Slightom, Transgenic Papaya Plants from Agrobacterium Mediated Transformation of Somatic Embryos, PI. Cell Rep. 12: 245-249, 1993; Franklin and C.I. Trieu T.N., Transformation of the Forage Grass Caucasian Bluestem via Biolistic Bombardment Mediated DNA Transfer, PLANT PHYSIOL 102: 167, 1993; Golovkin, M.V., Abraham M., Morocz, S., Bottka, S., Feher, A. and D. Dudits, Production of Transgenic Maize Plants by Direct DNA Uptake into Embryogenic Protoplasts, PLANT SCI 90: 41 to 52, 1993; Guo G.Q., Z.H. Xu, Wei and Chen Z.M. H.M., Transgenic Plants Obtained from Wheat Protoplasts Transformed by Peg Mediated Direct Gene Transfer, CHIN SCI BULL 38: 2072-2078. 1993; Asano Y. and Ugaki M., Transgenic plants of Agrostis alba obtained by electroporationmediated direct gene transfer into protoplasts, PI. Cell Rep. 13, 1994; Ayres N.M. and Park, W.D., Genetic Transformation of Rice, CRIT REV PLANT SCI 13: 219-239, 1994; P. Barcelo, C. Hagel, D. Becker, Martin A. and H. Lorz, Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue, PLANT J 5: 583-592, 1994; Becker D., Brettschneider, R. and H. Lorz Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue, PLANT J 5: 299-307, 1994; Biswas G.C.G., Iglesias VA, S.K. Datta and Potrykus I., Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts, J BIOTECHNOL 32: 1-10, 1994; M. Borkowska, Kleczkowski K., Klos is., Jakubiec J. and Wielgat Century, the Transformation of Diploid Potato with an Agrobacterium Tumefaciens Binary Vector System.1. Methodological Approach, ACTA PHYSIOL PLANT 16: 225-230, 1994;

G.S. Brar, Cohen B.A., Vick C.L. and Johnson G.W., Recovery of Transgenic Peanut (Arachis Hypogaea L) Plants from Elite Cultivars Utilizing Accell(R) Technology, PLANT J 5: 745-753, 1994; Christou P., Genetic Engineering of Crop Legumes and Cereals Current Status and Recent Advances, AGRO FOOD IND HI TECH 5: 17-27, 1994; Chupeau M.C., Pautot, V. and Y. Chupeau, Recovery of Transgenic Trees After Electroporation of Poplar Protoplasts, TRANSGENIC RES 3: 13-19, 1994; S. Eapen and George L., Agrobacterium Tumefaciens Mediated Gene Transfer in Peanut (Arachis Hypogaea L), PI. Cell Rep. 13: 582-586, 1994; Hartman C.L., Lee L., Day, P.R. and Turner, N.E., Herbicide Resistant Turfgrass (Agrostis Palustris Huds) by Biolistic Transformation, BID-TECHNOLOGY 12: 919-923, 1994; Howe G.T., Goldfarb V. and Strauss S.H., Agrobacterium Mediated Transformation of Hybrid Poplar Suspension Cultures and Regeneration of Transformed Plants, Plant Cell Tissue &Organ Culture 36: 59-71, 1994; Konwar B.K., Agrobacterium Tumefaciens Mediated Genetic Transformation of Sugar Beet (Beta Vulgaris L), J PLANTBIOCHEM BIOTECHNOL 3: 37-41, 1994; Ritala, A., Aspegren, K., Kurten U., Salmenkalliomarttila M., L. Mannonen, Hannus R., Kauppinen V, Teeri so-CALLED. and Enari M., Fertile Transgenic Barley by Particle Bombardment of Immature Embryos, PLANT MOL BIOL 24: 317-325, 1994; Scorza R., Cordts J.M., D.W. Ramming and Emershad R.L., Transformation of Grape (Vitis Vinifera L) Somatic Embryos and Regeneration of Transgenic Plants, J CELL BIOCHEM: 102, 1994; Shimamoto K., Gene Expression in Transgenic proposed classification CURR OPINBIOTECHNOL 5: 158-162, 1994; Spangenberg G., Wang Z.Y., Nagel, J. and Potrykus I., Protoplast Culture and Generation of Transgenic Plants in Red Fescue (Festuca Rubra L.), PLANT SCI 97: 83-94, 1994; Spangenberg G., Wang Z.Y., Nagel, J. and Potrykus I., Gene Transfer and Regeneration of Transgenic Plants in Forage Grasses, J CELL BIOCHEM: 102, 1994; Y.C. Wan and Lemaux P.G., Generation of Large Numbers of Independently Transformed Fertile Barley Plants, PLANT PHYSIOL 104: 3748, 1994; Weeks J.T., Anderson O.D. and Blechi A.E., Stable Transformation of Wheat (Triticum Aestivum L) by Microprojectile Bombardment,J CELL BIOCHEM: 104, 1994; Ye, X.J., Brown S.K., Scorza R., Cordts J. and J.C. Sanford, Genetic Transformation of Peach Tissues by Particle Bombardment, JAMER SOCHORTSCI 119: 367-373, 1994; Spangenberg G., Wang Z.Y., Nagel, J. and Potrykus I.,

PROTOPLAST CULTURE AND GENERATION OF TRANSGENIC PLANTS IN RED FESCUE (FESTUCA RUBRA L.), Plant Science 1994 97: 83-94, 1995.

As nim1 plants as host plants may also have sensitivity to pathogens, in contrast to the usual range of hosts, from which they, obviously, fall, these plants also have great practical importance for molecular, genetic and biological study of the relationship between plant-pathogen. In addition, UCB-phenotype nim1 plants also allows you to use them for screening of fungicides. nim1 Mutants selected for a particular host, have great practical importance for the screening of fungicides with this host and pathogens of the host. The advantage is UCB-mutant phenotype that allows you to solve problems that arise due to the fact that the hosts have different sensitivity to different pathogens and pathotypes or even resistance to certain pathogens or pathotypes.

To pathogens according to the invention include, but are not limited to, viruses or viroids, for example, the tobacco mosaic virus or cucumber virus circular spots or necrosis virus, the virus of curly leaf pelargonium, virus craptastic red clover, dwarf virus Bush tomato and under the by viruses, mushrooms, for example, Phythophthora parasitica and Peronospora. tabacina, bacteria, for example Pseudomonas syringae and Pseudomonas tabaci, insects, such as aphids, for example, Myzus persicae, and Lepidoptera, for example, Heliothus spp., and nematodes, for example, Meloidogyne incognita. Methods according to the invention are suitable for numerous organisms that cause diseases of corn, including the causative agents of downy mildew, such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis, rust, such as Puccinia sorphi, Puccinia polysora and Physopella zeae, other fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exserohilum twcicum, Kabatiellu zeae and Bipolaris maydis, and bacteria, such as Erwinia stewartii, but are not limited to them.

Description listed sequences

SEQ ID NO: 1: genomic sequence length 9919 base pairs, is shown in Fig.

SEQ ID NO: 2: genomic sequence length 5655 base pairs, is shown in Fig.

SEQ ID NO: 3: amino acid (AA) sequence NIM-wild-type protein encoded/coding sequence (cds) SEQ ID NO: 2.

SEQ ID NO: 4: AK sequence (33-155) rice Rice-1 shown in Fig.

SEQ ID NO: 5: AK sequence (215-328) rice Rice-1 shown in Fig.

SEQ ID NO: 6: AK sequence (33-155) rice Rice-2, shown in Fig.

SEQ ID NO: 7: AK sequence (208-288) rice Rice-2, shown in Fig.

SEQ ID NO: 9: AK sequence (208-288) rice Rice-3, shown in Fig.

SEQ ID NO: 10: AK sequence (33-155) rice Rice-4, shown in Fig.

SEQ ID NO: 11: AK sequence (215-271) rice Rice-4, shown in Fig.

Pick up

The following vector molecules were deposited in the American type culture collection (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A.) in the following terms:

plasmid YOU-04 was deposited in ATSS may 8, 1996 under ATS 97543;

plasmid P1-18 was deposited in ATSC 13 June 1996 under ATS 97606;

cosmid D7 was deposited in ATSC September 25, 1996 under number PBX 97736.

Examples

Example 1: identification of the NIM1-clones using clone-based mapping: genetic mapping and high resolution physical mapping of NIM1 in Arabidopsis

1. Plant material and isolation of nim1 mutants

The nim1 mutants were isolated from two populations of Arabidopsis ecotype Ws-O according to the method described by Delaney and others (1995), PNAS 92, 6602-6606. One mutant population was in the form M 2 libraries originating from seeds that were subjected to induction mutagenese using ethylmethanesulfonate (EMC) (obtained from the company Lehle, Round Rock, TX), and the second was in the form of a population of T-DNA originating from seeds obtained from the Ohio State University Arabidopsis Biological Resource Center (Coumbus, OH).

The basis for screening of mutants with pendulumed immunity (nim1) was the search using subject induced mutagenesis populatiei plants that have resistance to a virulent pathogen couldn coil INC (2,6-dichlorophenylamino acid; Metraux and others, 1991, in Advances in Molecular Genetics of Plant-Microbe Interactions, volume 1, 432-439, Ed. by Hennecke and Verma; Kessmann and others, 1993, in the Mode of action of agrochemicals, Ed. by Honma Y; Vernooij and others, 1995, Molec. PI. Microbe Interaction 8, 228-234).

Plants of the mutant populations were grown to high density in large pallets marketed mixtures to grow. When the plants reach 2 weeks of age, the trays were sprayed with 0.25 mg/ml INC. After 4 days the plants were sprayed with spore suspension Peronospora. parasitica, isolate EmWa (EmWa) with a concentration of from 5×104up to 1×105spores/ml. Normally this fungus virulent against the Arabidopsis ecotype Ws-O, if resistance was not originally induced in these plants using INC or similar connection.

After incubation under conditions of high humidity were identified plants with visible symptoms of the disease, as a rule, on the 7th day after infection. These plants did not show resistance to fungi, despite the processing of inducing resistance chemical compound, and therefore, they were considered as a potential mutant plants that do not have the immunity is, nim-mutants (non-immunity). From 360,000 plants identified 75 potential nim mutants.

These potential mutant plants were removed from the box for seedlings, were placed in conditions with low humidity and allowed to give seeds. Plants obtained from this seed were subjected to screening in the same way as carried out the screening for susceptibility to isolate fungi EmWa, and in this case also after pre-processing INC. Progeny plants that showed symptoms of infection, was defined as nim mutants. Thus it was revealed six nim mutants. One line (nim1) was isolated from a population of T-DNA and five of the population, EMS treated.

2. Evaluation of plant responses to INC and other chemical inducers of resistance to diseases

I. Phenotypic analysis nim1

Salicylic acid (SA) and S-methyl ether benzo(1,2,3)thiadiazole-7-karbaminovoi acid (BTK) are two chemical compounds that are similarly INC induce the wild-type resistance to a wide spectrum of diseases, which is called systemic acquired resistance (SAR). Because, INC. does not induce resistance in nim1 plants, these plants also assessed the occurrence of disease resistance in response to the pre-processing IC and BTK as it is, in particular, described by Delaney and others, 1995, PNAS 92, 6602-6606).

RA the plants were sprayed with 1, 5 or 15 mm SC or 0.25 mg/ml BTK and 5 days later were infected with inoculum EmWa (as described above in example 1). As IC and BTK could not defend nim1 plants from damage by fungus, which can be seen by the presence of symptoms and growth of the fungus on these plants. Thus, nim1 plants insensitive to any of inducing SAR chemical compounds, suggesting that the mutation is located below during transcription of the period (EC) to the entry of these chemicals into the process of induction of resistance.

nim1 Plants were also evaluated for their susceptibility to disease when the infection two incompatible inoculum of P. parasitica Wela and Noco (i.e. those strains of the fungus did not cause diseases in plants Ws-O wild-type), nim1 Plants were sprayed with a suspension of conidia containing 5-10×104spores/ml of the strain Wela or Noco, and incubated at high humidity for 7 days. Unlike wild-type plants at nim1 plants developed disease symptoms in response to infection and Wela and Noco. Symptoms consisted of necrotic spots and flexion to the ground, with some of sporulation. After staining lactophenol blue hyphae of the fungus was clearly visible on the leaves nim1 plants. Thus, nim1 plants are sensitive to incompatible OK isolates .parasitica. These results indicate that nim1-R is stenia not only defective in relation to induced disease resistance, but they are also defective in respect of natural resistance to microorganisms that normally are not pathogenic.

II. Biochemical analysis nim1

IC, INC. and BTK induce Arabidopsis SAR and SAR gene expression, which include associated with pathogenesis genes (Pathogenesis Related genes) PR-1, PR-2 and PR-5. Since these compounds do not induce disease resistance in nim1, which in norm they are incompatible (as described in example 1.2, see above), this mutant line were analyzed in terms of SAR gene expression after treatment of SK, INC. or BTK.

After processing nim1 plants SK, INC. or BTK plant tissues were collected and analyzed for the accumulation of RNA genes PR-1, PR-2 and PR-5. Then total RNA was isolated from treated tissue was subjected to electrophoresis on agarose gel. Received repeated three times, the blots gel and each of them hybridized with a probe for one of these three genes SAR according to the method described by Delaney and others, 1995, PNAS 92, 6602-6606. Unlike what happens in the case of wild-type plants, chemical compounds did not ndesirable the accumulation of RNA in any of these three genes SAR in nim1 plants, as shown in figure 1. The totality of the results obtained suggests that the chemical compounds do not induce any SAR or SAR gene expression in nim1 plants.

Because chemical compounds are not induced SAR, neither the expression of SAR genes in nim1 plants, was of interest to investigate whether infection with a pathogen to induce the expression of SAR genes in these plants, as occurs in wild-type plants. Ws-O - and nim1 plants were sprayed with spores EmWa, as described previously, and after a certain period of time took the tissue for analysis of RNA. Infection by the pathogen (EmWa) Ws-O-wild type induced gene expression of PR-1 through 4 days after infection, as seen in figure 2. However, the nim1 plants, the gene expression of PR-1 was not induced within 6 days after infection, and its level was lower compared to the level in wild-type plants at this time. Thus, after infection by a pathogen gene expression of PR-1 in nim1 plants slows down and decreases compared to wild type.

Infection of wild-type plants by pathogens that cause necrotic reaction, leads to the accumulation of SA in infected tissues. It was shown that endogenous SA is required for signal transduction in SAR-path, i.e. the decomposition of endogenous SC leads to reduced resistance to disease. This suggests that the accumulation of SC is a marker for SAR-path (Gaffney and others, 1993, Science 261, 754-756).

Evaluated nim1 plants accumulate SA after pathogen infection. Virulent to tomato strain of Pseudomonas syringae DC 3000 bearing GE is avrRpt2, infected leaves of 4-week nim1 plants. After 2 days, the leaves were collected for analysis of IC according to the method described Delaney and others, 1995, PNAS 92, 6602-6606. This analysis showed that nim1 plants accumulate high levels of SA in infected leaves, as seen in figure 3. In uninfected leaves also, there is an accumulation of SC, but not to such a level as in the infected leaves, and just as it happened in Arabidopsis wild-type. This suggests that the nim-mutation on the genetic map is located below in the course of transcription relative to the marker, SC in the path of signal transduction. It seemed probable, because, INC. and BTK (inactive in nim1 plants) is known for the ability to stimulate a component in SAR-way below in the course of transcription from IC (Vernooij and others, 1995, Molec. PI. Microbe Interaction 8, 228-234; Friedrich and others, 1996, The Plant Journal 9, in press; Lawton and others, 1996, The Plant Journal 9, in press). In addition, as described in example 1.2, exogenous made IC does not protect nim1 plants from infection EmWa.

3. Genetic analysis nim1

Carried out backcross nim1 plants with plants of wild-type Ws-O and their F1 progeny after pre-processing, INC. tested against resistance EmWa according to the method described above in example 1.1. None of the F1 plants pretreated INC., no symptoms of infection, in EMA as in the control nim1 plants infected found. It follows that nim1-mutation is recessive.

The population of the second generation F2, obtained by crossing Ws-O × nim1, after pre-processing INC analysed in relation to disease resistance. In this population, approximately 1/4 (32 of 130 plants) after infection EmWa detected symptoms in plants, pre-sprayed INC., and 3/4 of plants not identified diseases. These results suggest that the nim-mutation is located in a separate genetic locus, and confirmed using the F1 data indicating the recessive nature of the mutation.

4. Identification of markers and genetic mapping of a locus NIM

For conventional cloning of the gene NIM-based mapping must be identified markers that are genetically closely related to mutation. This study was conducted in 2 stages. First nim1 plants were crossed with another genotype of Arabidopsis - Landsberg erecta (Ler) and revealed F2 plants resulting from the crossing, which had the phenotype nim1 (i.e. plants that are homozygous nim/nim locus NIM). Next, using molecular analysis of these plants revealed the plants which had the genotype Ler, located directly next to a DNA marker. In accordance with the criteria identified, these plants are PE is the query result of recombination between the marker and the locus of NIM. The frequency of recombinants indicates the distance on the genetic map between the marker and the locus NIM.

The second prerequisite for cloning on the basis of mapping is to identify markers that are genetically very close to the locus NIM, i.e. markers, which found very few recombinants. If the identified genetic markers that are very close, they can be used to isolate clones of genomic DNA, which is close to the locus NIM. Locus NIM can then be cloned by the method of "walking along the chromosome, if it is not already present on the cloned DNA. "Walk along the chromosome can be initiated from both sides of the gene. This method is based on obtaining overlapping clones, which are positioned successively closer and closer to the gene of interest. When a single DNA marker get the "walk"started, for example, with the Northern end, and reveal the absence of recombinants between this marker and the gene of interest, this marker is very close to the gene. However, if token found (s) recombinant(s) from the southern end of the clone from which the token was received, must make the cross over from the genome. By this definition, clone gene of interest. It must be localized between the bullet and the last token is m, located on the Northern end, which is determined by the smallest number of recombinants with the Northern end.

In the first stage, a large number of recombinants obtained when genetic crossing. In the second stage, the recombinants that are close to NIM gene, isolated using molecular markers. Many of the markers described in the literature and there are several methods of obtaining additional markers. Applied in this invention, the approach is based on the number of marker systems, including PDP and PDF (see below).

I. Genetic crossing

To map the chromosomal position of the gene NIM relatively PDP and PDF markers nim1 were crossed with Ler, creating Charterhouse population. Grown F2 plants resulting from the crossing, and leaves were collected for further DNA extraction. Then F2 plants were evaluated in relation to the nim1-phenotype according to the method described above in example 1.1. Also grew F3 populations derived from individual F2 plants, and evaluated in relation to the nim1-phenotype. DNA was extracted from preserved tissues of plants of F2 and F3, with the phenotype nim1, method b, as described by Rogers and Bendich, 1988, Plant Molecular Biology Manual, A6, 1-10. This DNA was used for mapping the gene NIM, as described below.

II. Markers length polymorphism simple sequence Markers polymorphism of the lengths of the simple placentas is Teleostei (PDP) ATHGENEA and nga111 have been described previously (Bell and Ecker, 1994, Genomics 19, 137-144). The primers used to detect these PDP markers listed in table 1.

Table 1

PDP-primernye sequence
The primer setThe sequence of primer (5’→3’)
ATHGENEA (1)ACC ATG CAT AGC TTA AAC TTC TTG ACA TAA CCA CAA ATA GGG GTG
ATHGENEA (2)ACC ATG CAT AGC TTA AAC TTC TTG CCA AAT GTC AAA ATA CTC GTC
nga111 (1)CTC CAG TTG GAA GCT AAA GGG TGT TTT TTA GGA CAA ATG GCG
nga111 (1)CTC CAG TTG GAA GCT AAA G TGT TTT TTA GGA CAA ATG G

Genetic mapping of the gene NIM relatively token ATHGENEA:

When using primers ATHGENEA (1) for PCR amplification of genomic DNA Ler, was expecting to receive a strip corresponding to 205 base pairs (BP), and when using genomic DNA Ws-O was expecting to receive a strip 211 BP (Bell and Ecker, 1994, Genomics 19, 137-144). The resulting amplification products are difficult to separate by conventional agarose gels. So have developed two different methods for separation and detection of these PCR fragments.

When ispolzovanie first method, a set of primers ATHGENEA (1) (table 1) was used for amplification of genomic DNA in the presence of labeled using 6-carboxyrhodamine UTP (dUTP-R110 obtained from the firm ABI), getting the sword is by using rhodamine PCR fragments. PCR reactions were analyzed on DNA sequencing machine, the resolution of which in respect of the DNA fragments is one nucleotide.

Used the following specific reagents: a single PCR buffer (1×PCR buffer, 2 mm MgCl2, 200 mm of each dNTP, 2 mm dUTP-R110, primers ATHGENEA (1) at a concentration of 0.75 mm, 10 ng DNA and 0.75 units of Taq polymerase in a 20 ml volume of the reaction mixture. The conditions of amplification were as follows: 3 min at 94°C, then 35 cycles of 15 s at 94°C, 15 s at 55°s and 30 s at 72°C. These samples were analyzed on DNA sequencing machine type ABI 377 DNA Sequencer, which is capable of detecting fluorescencebased DNA fragments with a resolution of one nucleotide (NT.). Using this method we obtained the following data on the genotype of the plant samples: the DNA fragment length 205 nucleotides were obtained from DNA Ler, and the band corresponding to 211 nucleotides were obtained from DNA Ws-O. Thus, can be easily separated DNA fragments having a length differing by 6 nucleotides, allowing you to easily specify the following genotype samples per locus ATHGENEA: homozygous Ws-O, homozygous Ler and heterozygous Ws-O/Ler.

To increase the performance of this system used a multiplex scheme. Some DNA samples were subjected to PCR amplification as described above, the primer set ATHGENEA (1), a other way the s was analyzed using primer set ATHGENEA (2) (shown in table 2), in each case in the presence of labeled using 6-carboxyrhodamine Dutt. The set of primers ATHGENEA (2) created on the basis of published sequence ATHGENEA (Simoens and others, 1988, Gene 67, 1-11). This set of primers is provided amplification of DNA fragment length 139 base pairs of DNA Ler and the band corresponding to 145 base pairs of DNA Ws-O. the reaction Conditions of amplification for a set of primers ATHGENEA (2) were identical to those described above for the primer set ATHGENEA (1).

Separate the reaction mixture obtained using the primer set ATHGENEA (1), and separate the reaction mixture obtained using the primer set ATHGENEA (2), were mixed together before electrophoresis in DNA sequencing machine type ABI 377 DNA Sequencer. This multiplex approach made it possible to carry out the sequencing machine genotyping of 2 samples in one track sequencing machine, one in the provisions 145/139 HT., and other provisions 211/205 NT.

In another method, the PCR fragments were labeled primer labeled with fluorescent dye FRAMES-6 (6-carboxyfluorescein) (Integrated DNA Technologies, Inc.). ATHGENEA primers for synthesis along the chain, in sets of primers ATHGENEA (1) and (2) are identical in their sequences (table 1). This primer was labeled with FAM-6 and used for PCR amplification using the following reagents (firm Perkin Elmer): lxXL-buffer, 1 mm MgCl2by 200 m is each dNTP, 0,50 mm of each primer (primer for synthesis along the chain, labeled with FAM-6), 10 ng genomic DNA and 0.5 units of Taq polymerase XL 20 ml volume of the reaction mixture. Conditions cycles were as follows: 3 min at 94°C, then 35 cycles of 15 s at 94°C, 15 s at 59°s and 30 s at 72°C. And in this case, a separate reaction mixture, obtained using the primer set ATHGENEA (1), and separate the reaction mixture, obtained using the primer set ATHGENEA (2), were mixed together before electrophoresis in DNA sequencing machine type ABI 377 DNA Sequencer. This multiplex approach made it possible to carry out the sequencing machine genotyping of 2 samples in one track sequencing machine, one in the provisions 145/139 HT., and other provisions 211/205 NT.

All samples from generations F2 and F3 plants with a phenotype nim1 evaluated with respect to their genotype at the locus ATHGENEA according to the above-described method. All samples that were heterozygous at this locus corresponded to plants with recombination between locus NIM1 and locus ATHGENEA. In a population consisting of 1144 of F2 plants with a phenotype nim1, and F3 populations with nim1-phenotype, which has been evaluated thus, 98 were heterozygous at the locus ATHGENEA, allowing to establish that the genetic distance between these PDP-locus and locus NIM1 is 4.3 cm (centimorgan). It follows that NIM1 is located on chromosome 1 near the marker ATHGENEA.

Genetic mapping of the gene NIM1 relatively token nga111:

Two sets of primers for PDP marker nga111 (described by Bell and Ecker, 1994, Genomics 19, 137-144) was used for amplification of genomic DNA plant generations F2 and F3 nim1-phenotype and control plants epitopes Ws-O and Ler. The set of primers nga111 (1) (described by Bell and Ecker, 1994, Genomics 19, 137-144 and are presented in table 1) was applied in the following conditions: 1×PCR buffer, 2 mm MgCl2, 200 mm of each dNTP, 0.75 mm of each primer, 10 ng of genomic DNA and 0.75 units of Taq polymerase in a 20 ml volume of the reaction mixture. The set of primers nga111 (2) (shown in table 1 and which is derived from a set of primers ngalll (D) used in other conditions: 1×PCR buffer, 1.5 mm MgCl2, 200 mm of each dNTP, 1 mm each primer, 10 ng of DNA and 1 unit of Taq polymerase in a 20 ml volume of the reaction mixture. Both reaction mixture was amplified by incubation at 94°C for 1 min, then 40 cycles of 15 s at 94°C, 15 s at 55°s and 30 s at 72°C.

Samples were analyzed on a 3-5-percentage agarose gels. The band received after DNA amplification Ws-O with any set of primers corresponded to 146 BP, and amplification of DNA Ler gave the band 162 BP Samples of plants heterozygous for the locus nga111 comply with the plants that had recombination between this PDP-marker loci NIM. In a population consisting of 1144 is astani F2 with phenotype nim1, and F3 populations with nim1-phenotype, 239 plants were heterozygous at the locus nga111 marker, allowing to establish that the genetic distance between these PDP-marker and the locus NIM1 is 10.4 cm. It follows that the locus NIM1 is located on chromosome 1. Because there are several plants with nim1-phenotype, which are heterozygous and ATHGENEA, and nga111, it was found that the locus NIM1 is located between the two markers, and ATHGENEA localized closer to the Northern end from the gene NIM1, a nga111 localized closer to the southern end from the NIM1 gene. This allows you to have NIM1 gene at a distance of approximately 10 cm closer to the Northern end from nga111, about position 85 on chromosome 1 (Lister and Dean, 1993, Plant J. 4, 745-750; Bell and Ecker, 1994, Genomics 19, 137-144).

III. Markers polymorphism of the lengths of the amplified fragments

For based on the mapping of the cloned gene NIM1 need to identify molecular markers that are as close as possible to this gene. For this purpose, the received tokens of length polymorphism of amplified fragments using the method of selective amplification of restriction fragments by the method described by Zabeau and Vos (1993, EP 0534858) and Vos and others (1995, Nucleic Acid Research 23, 4407-4414).

Description PDF-technology

Application PDF technology mapping based on the selective amplification of a series of DNA bands in two genetically different the x samples. The discovery of the fact that any resulting bands are different in the two genotypes, allows to identify these bands as markers for a given genotype. If for markers characteristic of the high frequency cosegregation with the gene insert (mutation), then the token is a close genetic locus.

Selective amplification of a small set of DNA fragments in complex with DNA sample obtained using the two-stage process. First get the DNA fragments by DNA cleavage by enzymes restrictable with subsequent legirovaniem adapters to the ends. In the second stage using the primers consisting of the sequence complementary to the adapters, plus the extension on the 3’-end (usually 0-3 nucleotide), to amplify only the DNA fragments, the ends of which are complementary to these primers. If applied elongation in a single nucleotide, theoretically each primer will "match" approximately 1/4 of all the fragments, and 1/16 of all fragments will have the primers "relevant" to both ends. Thus, these primers are amplification of a limited set of DNA fragments. By subsequent labeling with a radioactive isotope of one primer can be obtained even smaller sunamori visible bands.

PDF-analysis

For PDF analysis of DNA samples of 50 ng DNA Ross plaly with appropriate enzymes (usually EcoRI and Msel; see below) and adapters (listed in table 2, below) ligated with fragments obtained by treatment with restrictase (usually EcoRI and Msel). Sequences of primers and YAC clones, P1 and YOU are described in detail below. For amplification reactions used the matrix (approximately 0.5 ng DNA per reaction) using primers complementary to the adapters, with a short 3’-extensions (2 or 3 nucleotides; the sequences of the primers below). Because one of the primers is radioactiveman (usually EcoRI-primer), for separation of bands use only subnavbar amplified fragments visible when autoradiographical analysis of gel.

Conditions for amplification of cloned DNA (YAC, P1, cosmid) were as follows: 36 cycles of 30 s at 94°C (denaturation), annealing 30 s, and elongation for 1 min at 72°C. the annealing Temperature in the first cycle was 65°and it was lowered by 0.7°With each cycle over the next 12 cycles and then maintained at 56°C. For genomic DNA Arabidopsis amplification was performed in 2 stages: in the first stage (pre-amplification) DNA amplified with primers that have an elongation of 1 nucleotide (none of the primers were not marked). The reaction conditions for this reaction amplification were as follows: 20 cycles of 30 s denaturation (94°C), ATI is 1 min (56° C), and elongation for 1 min (72°). In the second stage reaction mixture for amplification were first diluted 10 times and then they were re-amplification for 36 cycles with primers containing full extension (using a single labeled primer) under the following conditions: 30 s at 94°C (denaturation), annealing 30 s, and elongation for 1 min at 72°C. the annealing Temperature in the first cycle was 65°and it was lowered by 0.7°With each cycle over the next 12 cycles and then maintained at level 56°C. the Final reaction products were separated on polyacrylamide gel and the gel was exposed to film, which allowed to visualize radioactiveman PCR strips. When this analysis was applied simultaneously to the DNA of the two genotypes were identified PDAF-bands, which were characterized as a diagnostic characteristic to one or the other genotype. Such informative band called PDF-bands or PDF-markers. Table 2 lists the adapters that are used for PDF analysis.

Table 2
EnzymeAdapter
EcoRI5’-CTCGTAGACTGCGTACC-3’

3’-CATCTGACGCATGGTTAA-5’
HindIII5’-CTCGTAGACTGCGTACC-3’

3’-CATCTGACGCATGGTCGA-5’
> PST 5 is the-CTCGTAGACTGCGTACATGCA-3

3’-CATCTGACGCATGT-5’
MseI5’-GACGATGAGTCCTGAG-3’

3’-TACTCAGGACTCAT-5’

Getting PDAF markers and precise mapping of the locus NIM1

A population of recombinant inbred lines derived from crosses of such Agapitov Arabidopsis, as Landsberg erecta (Ler) and Columbia (Col) (Lister and Dean, 1993, Plant J. 4, 745-750), used for screening PDAF markers. For PDF-screening used the following primers:

EcoRI-primers: 5’-GACTGCGTACCAATTCWN-3’

MseI-primers: 5’-GATGAGTCCTGAGTAAXWN-3’

The symbol "N" in the primers indicates that this area is variable (a, C, G or T), "W" stands for a or T, and "X" denotes With. Used all 8 possible primers for EcoRI and Msel-primer. This gives a total of 64 (8×8) combinations of primers (KP), which was used for DNA amplification of recombinant inbred lines and the parental genotypes Ler and Col, as described above. Amplification reaction was performed under denaturing polyacrilamide the gel in order to separate PDAF fragments by size and the gel was exposed to film. The film was analyzed from the point of view of all the bands that are present only in one genotype, i.e. analyzed in relation PDAF markers.

PDF markers, i.e. the DNA fragments polymorphic for both parents recombinant inbred lines were used to create a genetic map of the population of recombinant inbre the Noi line. In the below example 1.51 describes the mapping of the NIM1 gene on chromosome 1 of Arabidopsis approximately 85 position. Those PDAF markers that were mapped (using recombinant inbred lines) to a position between 81 and 88 chromosome 1 of Arabidopsis, were selected for analysis of recombinant plants for the presence of these PDF markers and, thus, for more accurate mapping of the gene NIM1. It was found that seven PDAF markers from this region are informative; they were polymorphic for both parents when crossing nim1 x Ler. Six PDAF markers were Ler-specific, i.e. these PDAF markers were absent in Ws and Col). One PDF marker was Ws-specific, i.e. Col-specific PDF-marker (not in Ler), which was also present in the Ws. These PDAF markers represent L81.1, L81.2, W83.1, L84, L85, L87 and L88 (L-a token is a specific ecotype Ler, a W-marker is specific for both ecotypes Col and Ws; numbers indicate the position on the map). These PDAF markers used for analysis of recombinant plants and breeding of nim1 × Ler (see below). In addition, PDF token S (specific to Col marker derived from recombinant inbred lines) were used for isolation of DNA clones (see below). Table 3 shows the sequences of the primers that were used to obtain these PDAF markers.

In that the face 3 shows the combinations of primers PDF markers obtained from a population of recombinant inbred lines.

"EcoRI-" denotes the sequence 5’-GACTGCGTACCAATTC-3’ and

"MseI-" denotes the sequence 5’-GATGAGTCCTGAGTAA-3’.

Table 3
PDF-markerThe appropriate combination of primers
L81.1EcoRI-CAMseI-CCG
L81.2EcoRI-AAMseI-CAA
W83.1EcoRI-CAMseI-CTC
L84EcoRI-AATMseI-CAA
L85EcoRI-CAMseI-CCT
L87EcoRI-CAMseI-CTT
L88EcoRI-AGMseI-CTA
SEcoRI-AGMseI-CCT

Detailed genetic map of the region was created using the above PDF markers, by typing recombinants. Only 337 of recombinant plants were obtained from 1144 plants of the second generation F2 with phenotype nim1. These recombinant plants were first subjected to screening using flanking the Northern end PDAF markers L81.2 and ATHGENEA and flanking the southern end markers L88 and nga111. Forty-eight plants were homozygous for the nim1/nim1 and heterozygous for ATHGENEA and L81.2, and 21 R is stenie were homozygous for the nim1/nim1 and heterozygous for nga111 and L88. These recombinant plants were then analyzed using nine PDF markers in NIM-region, including four PDF-token, which is obtained during the mapping population of recombinant inbred lines (W83.1, L84, L85 and L87), and five PDAF markers, obtained from the analysis of YAC clones (W83.3/W84.1, W84.2, W85.1, W86.1 and L86, see below).

Based on this analysis of the genetic map NIM1 is shown in figure 4. It is seen that 27 recombinants were found between marker W84.2 and NIM1, and 14 recombinants were found between W85.1 and NIM1. Marker L85 is located close to the NIM1, but this marker could not be mapped to the YAC clones, YOU or P1 (see below) and, therefore, could not be used to identify gene NIM1.

5. Physical mapping of NIM1-area

I. Selection of YAC clones using PDAF markers located close to NIM1

The CIC library, the YAC library of Arabidopsis ecotype Columbia (Bouchez and others 1995, 6th Int. Conf. on Arabidopsis Research (proceedings of the 6th International conference on Arabidopsis research, Madison, PCs Wisconsin) were subjected to screening against YAC clones in NIM-region. This library includes approximately 2.5 equivalent nuclear genome and has an average insert 450 TPN YAC Library was subjected to screening using two PDF markers: W83.1 and S. W83.1 is the most close to the Northern end of the NIM1 PDF-token received from recombin nteu inbred lines, and S is PDF-marker derived from recombinant inbred lines specific to Col (not in Ler and Ws). C86 mapped on the southern end of the NIM1 gene on the map the population of recombinant inbred lines. This is specific to Col PDF-marker is used instead of the close-specific Ler PDF markers (figure 4), since the latter PDAF markers revealed only ecotype Landsberg erecta and therefore could not be used for screening libraries YAC ecotype Columbia.

The YAC library was subjected to screening in two stages. First, cells YAC clones each plate twelve 96-well plates for micrometrology were United in a group (pool plates) and used for DNA extraction according to the method described by Ross and others (1991, Nucleic Acids Res. 19, 6053). The pools were subjected to screening with both PDAF-markers. Then from each positive pool plates DNA samples from each series (pool of 8 clones) and each column in a pool of 12 clones) were subjected to screening with PDAF-marker for which a pool of plates was positive. Using this method can be identified separate positive YAC clones. The screening led to the isolation of only four YAC clones: YAC 12F04 and YAC 12H07 were selected using Northern PDAF marker W83.1, a YAC 10G07 and YAC 7E03 - using southern AFLP marker C86 (item YAC clones use adopted for CIC n is merely). Received "fingerprints"

YAC using PDAF, which gave YAC-specific PDF fragments. Fingerprints YAC were compared and used to assess overlaps between YAC (see also tables 5 and 6). On the basis of obtained using PDF fingerprints found that the clone A largely overlaps clone 10G07 (see also table 5), and clone N similarly largely overlaps clone 12F04 (see also table 6).

III. Getting PDAF markers of clones UAS

As described above PDF markers were genetically relatively far from the gene NIM1 (see figure 3), additional PDAF markers were created with the purpose of finding markers that had been closer to gene NIM.

Screening additional originating from YAC PDF markers was performed using the following DNA samples: DNA from the YAC clones as described previously, we identified 4 YAC clone, clone yeast without YAC and three ecotypes of Arabidopsis, as Col, Ler, and Ws. Using this screening fragments specific YAC clones (missing in strain of yeast present in the ecotype Col), could be assessed in relation to polymorphism in plants Ler and Ws (parents recombinant plants identified in example 1.5, see below). Thus, all of the identified polymorphic fragments can serve as an additional PDF-markers. When PE is the first screening PDAF used a combination of enzymes (EC) EcoRI/MseI. When this screening examined two YAC clone 10G07 and ES (found using PDAF marker S, see below), the strain of yeast without YAC and three Arabidopsis ecotype Col, Ler, and Ws. Applied combinations of primers with selective movements can be subdivided into three groups, and they are presented in table 4. All were subjected to screening 256 (64+96+96) combinations of primers.

In the following table 4 shows the sequences of primers used in PDF-screening two YAC clones, 10G07 and E, strain of yeast, nestorgames YAC, and three ecotypes of Arabidopsis Col, Ler, and Ws. Used three combinations of primers. The symbol "N" in the primers suggests that this part was variable (a, C, G or T), "S" stands for C or G, "W" stands for a or T, and "Y" stands for C or T.

Table 4

EcoRI - primers:

5’-GACTGCGTACCAATTCGW-3’

5’- GACTGCGTACCAATTCTS-3’

MseI-primers:

5’- GATGAGTCCTGAGTAAAAS-3’

5’- GATGAGTCCTGAGTAAASA-3’

5’-GATGAGTCCTGAGTAAATN-3’

5’-GATGAGTCCTGAGTAACAN-3’

5’-GATGAGTCCTGAGTAACTN-3’

EcoRI-primers:

5’-GACTGCGTACCAATTCAN-3’

5’-GACTGCGTACCAATTCCW-3’

5’-GACTGCGTACCAATTCTW-3’

MseI-primers:

5’- GATGAGTCCTGAGTAAAAS-3’

5’- GATGAGTCCTGAGTAAASA-3’

5’-GATGAGTCCTGAGTAAGAY-3’

5’-GATGAGTCCTGAGTAAGTW-3’

5’-GATGAGTCCTGAGTAATCG-3’

5’-GATGAGTCCTGAGTAATCT-3’

5’-GATGAGTCCTGAGTAATGW-3’

EcoRI-primers:

5’-GACTGCGTACCAATTCGW-3’

5’-GACTGCGTACCAATTCTN-3’

MseI-primers:

5’-GATGAGTCCTGAGTAAGAW-3’

5’-GATGAGTCCTGAGTAAGCW-3’

5’-GATGAGTCCTGAGTAAGTW-3’

<> 5’-GATGAGTCCTGAGTAATAN-3’

5’-GATGAGTCCTGAGTAATCW-3’

5’-GATGAGTCCTGAGTAATGW-3’

5’-GATGAGTCCTGAGTAATTS-3’

In total, there were 83 Col-specific fragment, of which 62 were common to both YAC clones. Three fragments represented PDF marker polymorphism between Ws and Ler, two of which were PDAF-markers Ws (Col-fragment is also present in Ws and Ler), and one was PDAF token Ler (Col-fragment is also present in Ler and no Ws). These results are presented below in table 5.

Table 5 shows the number of shared and unique PDAF fragments found in the YAC clones 10G07 and E, and the number of informative PDF markers among these fragments in the genotypes Ws and Ler.

Table 5
 PDF fragments in the YAC clonesPDF-marker
10G07EWsLer
total626221
unique21000

Thus, PDAF-analysis allowed us to obtain three new PDF marker (see figure 4 and below). Their position relative to each other and relative to PDAF markers, recip is the R of recombinant inbred lines, were determined using analysis of recombinants with these PDF-markers.

The second screening PDAF markers was performed by analyzing all four identified YAC clone (see below) and using a combination of enzymes

> PST /MseI. Used the following primers:

> PST -primers:

5’-GACTGCGTACATGCAGAN-3’

5’-GACTGCGTACATGCAGCW-3’

5’-GACTGCGTACATGCAGGW-3’

5’-GACTGCGTACATGCAGTN-3’

MseI-primers:

5’-GATGAGTCCTGAGTAAAN-3’

5’-GATGAGTCCTGAGTAACW-3’

5’-GATGAGTCCTGAGTAAGW-3’

5’-GATGAGTCCTGAGTAATN-3’

The symbol "N" in the primers indicates the variable part of the sequence (a, C, G or T), and "W" in the primer means And or So Just used 144 (12×12) combinations of primers for screening all four selected YAC clones: 12F04, N, 10G07 and E, strain of yeast, nestorgames YAC, and three ecotypes of Arabidopsis Col, Ler, and Ws. They received a total of 219 PDF fragments, of which 144 were present in the YAC clones 12F04 and N (72 appeared to be unique to the clone 12F04 and 72 were common to both YAC clones) and of which 75 were present in the YAC clones 10G07 and E (33 appeared to be unique to the clone 10G07 and 42 are common to both YAC clones). Three fragments, derived from a set of YAC clones were polymorphic (PDAF markers Ws). These results are presented below in table 6.

Table 6 shows the number of shared and unique PDAF fragments identified in the YAC, and from these fragments, the number of informative PDF markers DL the genotypes Ws and Ler.

Table 6
 The number PDAF fragments in the YAC clonesPDF-markers
12F04N10G07EWsLer
total72720010
unique7200020
total00424200
unique0about33000

The results indicate that the YAC clone 12H07 is part of a larger YAC clone 12F04, and the YAC clone 7E03 is part of a larger YAC clone 10G07. These data suggest that larger YAC clones 12F04 and 10G07 do not overlap. The data obtained do not allow to determine the position of the NIM1 gene in any of these YAC clones. Full screening, including 400 combinations of primers, which gave 302 PDF-fragment in NIM-region, allowed us to obtain five suitable PDAF markers, four to the x appeared Ws-specific, one - Ler-specific. The location on the map these 5 additional PDAF markers was determined using analysis of recombinant plants (see figure 4 and below), and they are marked as W84.1 (a.k.a. W83.3), W84.2, W85.1, W86.1 and L86.

Table 7 summarizes the sequences of the primers that were used to obtain these PDAF markers. These 5 additional PDAF markers increased the total number PDAF markers to 12 in the area from L81.1 to L88 (see figure 4 and below).

Table 7 summarizes the combinations of primers PDF markers derived from YAC clones.

"EcoRI-" refers to the sequence 5’-GACTGCGTACCAATTC-3’,

"MseI-" refers to the sequence 5’-GATGAGTCCTGAGTAA-3’ and

">PST -" refers to the sequence 5’-GACTGCGTACATGCAG-3’.

Table 7
PDF-markerThe combination of primers with selective movements
W84.1 > PST -ATIMseI-TT
W84.2 > PST -AAMseI-TT
W85.1EcoRI-CTMseI-GTA
W86.1EcoRI-GTMseI-CTT
L86EcoRI-GTMseI-CTT

This information was used to create physical maps of the area, as shown in figure 5, with an approximate to the provisions of the YAC clones on the genetic map. The map shows that the area containing the locus NIM1 between markers W83.1 and W85.1, partly covered by a three YAC clones: 12F04 and 10G07/7E03.

III. The design of a set of sequence fragments P1/YOU, containing the gene NIM1

In previous sections described how the allocated PDF markers associated with NIM1-region, and how they identified and mapped these markers YAC clones. The results obtained with the localization of the NIM1 gene on the chromosome fragment, not allowed to determine the specific segment of DNA containing the gene NIM1: flanking PDF markers mapped different YAC clones that do not overlap. Therefore, it was impossible to determine the exact physical location of the gene NIM1; he could be localized either on either of the two YAC, or in the gaps between YAC. To close the physical gap between the flanking markers was selected alternative approach: the P1 library and YOU used to overlap the gaps between the flanking PDF-markers.

Used to close gaps library was a library of Arabidopsis ecotype Columbia PI, described by Liu and others (The Plant J. 7, 351-358, 1995) and the library of ecotype Columbia YOU described Choi and others (Internet: http://genome-www.stanford.edu/Arabidopsis/ww/Vol2/choi.html). Pl-library consists of approximately 10,000 clones with an average insert 80 TPN, and YOU-the library consists of approximately and the 4000 clones with an average insert 100 TPN Theoretically, these libraries represent approximately 10 equivalents of nuclear gene ohms (assuming that the size of the haploid genome of Arabidopsis is 120 MPN).

IV. Identification of clones P1 corresponding to the flanking markers For screening groups of P1 clones was used by flanking markers Ws84.2 and Ws85.1 using a strategy previously described for screening of YAC library (see example 1.51). Selected clones P1 with marker fragments, and provided "plasmid DNA. Received the DNA fingerprints of different P1-clones, using KF EcoRI/MseI and HindIII/MseI and primers without selective nucleotides. By comparing PDF-fingerprints created a physical map, i.e. a map that shows the size and overlap of clones. The number PDAF fragments that are unique, and the number PDAF fragments common to the clones, characterizes the degree of overlap. Map is shown in Fig.6. Getting PDF-fingerprints showed that it was constructed two sets of non-overlapping sequences P1, each of which contained one of the following flanking markers: P1-1 and P1-2, including the token Ws84.2, and P1-3 and P1-4, including the token W85.1. Consequently, the gap between the flanking markers has not been closed (6).

The position of the set of sequences of P1 with respect to the set of YAC sequences were determined, recip what I PDAF-fingerprints of clones YAC and P1 with the above-mentioned number of YAC-specific KP. Clones P1 P1-1 and P1-2, probably completely obscured by the YAC clone CIC12F04, but only partially YAC clone CIC12H07. Thus, the last clones P1 may be located on a set of sequences YAC CIC12H07/12F04 (6). The PI clones P1-3 and P1-4 is completely overlaps both YAC clone CIC7E03 and CIC10G07 and, it is likely that PDF token W86.1 and W85.1 mapped in the same set of sequences P1 (6).

Next token L85 was used to identify the corresponding P1 clones and YOU. L85 is a specific ecotype Landsberg marker, and therefore, was carried out by colony hybridization radioactiveman DNA L85 with P1 - YOU-filters. Was not identified no single clone P1 or YOU, for hybridization with L85. This confirms earlier evidence that the sequence L85 is absent in the genome of Arabidopsis ecotype Columbia, which is the most likely explanation for what had not been identified corresponding clones.

V. Extension flanking NIM1 sets of sequences P1

To extend flanking sets of sequences P1 used different approaches.

PDF-YAC fragments that are specific regarding the southern end of the YAC clone CIC12F04 (unique for CIC12F04, not in CIC 12H07), was used to identify clones P1 using PDAF-group screening libraries.

1. PDF-fragments of YAC YAC clone 10G07 that overlap P1-4, application is whether to identify clones P1 using PDF-screening set of libraries PL

2. EcoRI-restriction fragments from clone P1-6 (obtained from the stage 1 screening libraries P1, based on the use of PDAF) were used as probes for hybridization with the library YOU on the filters.

As a result of this screening were obtained from different clones P1 YOU and for all of them received PDAF-fingerprints using KF EcoRI/MseI and HindIII/MseI and primers without selective nucleotides. Created a new map, as described above, which is shown in Fig.7. Table 8 presents various PDF-KP, with PDAF fragments that mapped to the flanking YAC and used for screening libraries P1 in relation to the corresponding P1-clones.

Table 8 presents various PDF-KP, used for screening libraries P1. In the upper part of the table shows the CP, specific to Northern ends of the YAC, and in the lower part of the table shows the CP, specific to the southern ends of the YAC. Also indicated clones YAC and P1, are experiencing PDAF fragments.

Received sets of sequences P1/YOU length of approximately 250 TPN, covering the southern end of the YAC clone CIC12F04 (which did not go beyond this YAC clone) and that contained the marker W84.2. Got a set of sequences P1 length of approximately 150 TPN containing markers W85.1 and W86.1; this pic set is egovernance is fully enclosed by the YAC clone CIC7E03.

Analysis using PDF-marker designs, including a set of sequences P1/YOU covering gene NIM1, recombinants with markers from the southern end of the Northern set of sequences P1/YOU (WL84.4 and WL84.5, see further in the text and in table 11) showed that the earlier the stage of "walking on chromosome fails to construct a set of sequences containing the gene NIM1 (see next section). Therefore, the existing Northern-set sequences P1/YOU was extended to the southern end to "walk" across the NIM1 gene that would allow to identify and distinguish a specific segment of DNA containing the gene NIM1. Next approach was employed using hybridization, in which the P1 clones or YOU localized at the southern end of the Northern set of sequences PP1/YOU used to identify clones located closer to NIM1 (southern border sequence). New clones obtained by stages "walk", mapped in relation to the existing sets of sequences obtained using PDF-fingerprints using KF EcoRI/MseI and HindIII/MseI, as described above. Apparently, for "crossing" gene NIM1 need just 5 successive stages of the "walk". Table 9 shows the clones obtained from different stages of the "walk".

In table 9 for p is slichnih stages "walk" shows the hybridization probes, which was used for screening libraries P1 and YOU, and the selected clones, and hybridization with probes and extension sequence towards the southern end.

Table 9
StageProbeNew clones, extending the southern end
1P1-7YOU-02
2YOU-02P1-16, YOU-03
3YOU-03P1-17, P1-18
4P1-18P1-21, P1-20, YOU-04
5YOU-04P1-22, P1-23, P1-24, YOU-06, YOU-05

Physical map of different clones obtained from these stages "walks"that are listed on Fig. In General, it was analyzed distance of approximately 600 TBN, starting from the starting point of the "walk" token W84.2. The southern end of the set of sequences shown in Fig probably contains a gene NIM1 (see next section). A set of sequences, which stretches over 300 tpd to the southern end from the YAC clone CIC12F04 and probably not overlapping YAC clones CIC10G07 and CIC7E03 suggests that the gene NIM1 is located in the gap between the flanking YAC sets of sequences and that this gap has a size of at least 300 TPN

VI. The creation of a generalized genetic and physical maps NIM1-area

In the previous sections, we described how allocated PDAF markers associated with NIM1-area, how was revealed YAC clones corresponding to the flanking markers, and how has designed a set of sequences P1/YOU, reaching approximately 550 TPN to the southern end from the nearest to the Northern end flanking PDF marker W84.2. This section describes obtaining new PDAF markers from a set of sequences P1/YOU, the physical mapping of these markers in this set of sequences and genetic mapping of these markers using the available recombinants.

1. Getting new PDAF markers from a set of sequences P1/YOU

As described in the previous section, the set of sequences obtained by elongation of the P1 clones and YOU characterized using obtained on the basis of PDAF fingerprints using KF EcoRI/MseI and HindIII/MseI. This method allows to accurately determine the length of the overlapping areas of different clones P1 and YOU, and also get a number PDAF fragments that are specific for these clones. PDF primers without selective nucleotides were used to obtain fingerprints of purified plasmid DNA of the clones P1 or YOU. Selective nucleotides, however, I had are needed to make possible the application of these P1 - or YOU-specific PDF fragments for detection in Arabidopsis. By defining the end sequences of the amplified restriction fragments can be designed PDF primers with appropriate selective basis, for amplification P1 - or YOU-specific PDF-fragment in Arabidopsis. All PDF fragments originate from ecotype Columbia (Col) and, therefore, can also be identified, if PDAF markers Columbia are informative recombinants NIM1, which are the result of crossing ecotypes Landsberg erecta (Ler) and nim1 mutant ecotype Wassilewskija (Ws-nim). In principle there are 4 types PDAF fragments, two of which are suitable markers, as shown below in table 10.

Table 10 shows the types found PDAF markers. (+) or (-) indicates the presence or absence of PDAF-fragment.

Table 10
ColLerWs-nimThe token type
+++uninformative
++-Ler-marker
+-+Ws-token
+--uninformative

In General, obtaining fingerprints of clones P1 and YOU gave 30-40 EcoRI/MseI-PDF fragments and 60-80 HindIII/MseI-PDF fragments for each individual clone. Terminal sequences of individual fragments was determined by standard sequencing methods. Then tested sets specific PDF-primers with selective movements, consisting of three nucleotides, for both EcoRI or HindIII primers and Msel-primer using the following DNA:

1. DNA of clone P1/YOU, that was PDAF token;

2A. Yeast DNA;

2B. DNA YAC clone CIC12F04 (only for PDF fragment from P1-7);

2B. DNA YAC clone CIC10G07;

3A. DNA Col, which is a source libraries P1 and YOU;

3b. DNA Ler, parent 1 recombinants nim;

3V. DNA Ws-nim, parent 2 recombinants nim.

We selected 6 clones for sequence analysis of their EcoRI/MseI and HindIII/MseI-PDF fragments: YOU-01/P1-7, P1-17/P1-18, YOU-04/YOU-06. All PDF-fragments of clone P1-7 were detected in YAC CIC12F04, suggesting that this clone contained entirely within this YAC. None of P1/YOU-specific PDF fragments was not detected in the YAC clone CIC10G07, indicating that the set of sequences P1/YOU is not a bridge for the gap between the two flanking YAC sets of sequences. PDF markers selected for analysis of recombinants nim, are shown in table 11.

Table 11 shows selected PDF markers of PDF-KP specific to different clones P1 and YOU. "WL"-the token is a token that is obtained from the same KP and corresponds to two PDF-markers, i.e. Ws and Ler-marker, which probably fully connected in the phase repulsion in the analysis of recombinants NIM.

Table 11
OriginThe name of the markerThe combination PDF-primer
P1-7WL84.4EcoRI-AGC MseI-ACT
P1-7WL84.5HindIII-CTC MseI-TTC

OriginThe name of the markerThe combination PDF-primer
P1-17/P1-18Ler84.6aHmdIII-CGT MseI-ATT
P1-17/P1-18Ler84.6bHindIII-ATT MseI-CAT
P1-18Ler84.6cHindIII-TCT MseI-TAT
P1-18Ler84.7EcoRI-AAA MseI-AGA
YOU 04/06Ler84.8EcoRI-TTC MseI-AGT
YOU-06Ler84.9aEcoRI-AAA MseI-TGT
YOU-06Ler84.9bEcoRI-ATC MseI-TCC
YOU-06Ler84.9cEcoRI-ATG MseI-GTA

2. Physical the mapping of new PDAF markers

The above PDF markers mapped on the physical map by detecting their presence in different clones P1 and YOU. The results are shown in figures 9-11.

3. Genetic mapping of new PDAF markers All PDF markers analyzed in the selected set of recombinants. The results obtained are summarized in tables 12A, 12B and 12B.

PDF markers Ler84.8, Ler84.9a, Ler84.9b and Ler84.9c visible on the map on the southern side of the NIM1. Were detected recombinants, which are phenotypically manifested as nim1 (homozygous, genotype Ws-nim1/Ws-nim1) and heterozygous for these PDF-markers was detected Ler-specific PDF-marker genotype Ws-nim1/Ler). PDF token Ler84.8 was nearest to the NIM1: only one recombinant (S-105) was classified as heterozygous Ws-nim1/Ler and homozygous Ws-nim1/Ws-nim1. PDF markers Ler84.7 and Ler84.6c was completely cosegregating with NIM1: all recombinants had an identical genotype NIM1 and PDF token. Located closer to the Northern end from the NIM1 marker L84.6b was nearest to the NIM1: detected three recombinant plants with a phenotype nim1, C-074, D-169, E-103 (table 12B), which are heterozygous Ws-nim1/Ler on this m is rcaro. Using kosmidou set of sequences obtained from P1-18, YOU-04 and YOU-06, AFLP-markers Ler84.6b and Ler84.8 were mapped in P1-18 and YOU-04, respectively, and found that the physical distance between them is approximately 110 TPN This allows us to establish that nim1 must be localized on the DNA segment, whose length is 110 TPN From this analysis revealed that the gene NIM1 contained in clone YOU-04 or P1-18. Clones YOU-04 and P1-18 have been deposited in ATSS and assigned registration number of ATSS 97543 and ATSS 97606, respectively.

VII. Genetic and physical fine mapping of the gene NIM1

The previous section describes how the segment of DNA containing the gene NIM, was determined using the physical mapping flanking PDF markers (Ler84.6b and Ler84.8) on the set of sequences P1/YOU. Flanking markers are visible on the map in two overlapping clones P1-18 and YOU-04. This section describes the way more YOU-04-specific and R 1-18-specific PDF markers to increase the resolution genetic and physical mapping in the region containing the gene NIM1.

VIII. Get more PDEF markers of kosmidou set of sequences

Order more PDF markers for fine mapping NIM1 were selected 4 KF: > PST /MseI, XbaI/MseI, BstYI/MsI and TaqI/MseI. > PST /MseI - and XbaI/MseI-PDF fragments were obtained from clones P1-18 and YOU-04 and determined selective sequence required to identify Arabidopsis. Similarly PDF fragments and selective sequence was determined for BstYI/MseI and TaqI/MseI, but in this case the procedure was performed using komenich DNA: A11, C7, E1 and E8 for BstYI/MseI (full NIM1-region) and D7, E8 and E6 for TaqI/MseI (southern side NIM1-region). Informative PDF markers selected for further genetic and physical mapping, are shown in table 13. Additional adapters, which are used in this work are shown in table 14.

Table 13 shows PDAF markers were used for genetic and physical fine mapping of NIM1. "BstYI(T)" indicates the restriction site and the corresponding primer represented either AGATCT or GGATCT.

Table 13
MarkerKF/KP
Ler84.Y1BstYICD-GCTMseI-AAC
Ws84.Y2BstYI(T)-TCTMseI-GCA
Ler84.Y3BstYI(T)-AAGMseI-TAT
Ler84.Y4BstYI(T)-GTTMseI-AGA
Ws84.T1TaqI-TACMseI-GGA
Ler84.T2TaqI-TTG MseI-GGA

Table 14 shows the same with additional adapters, which are used to identify new PDAF markers.

IX. Physical mapping of new PDAF markers in komenich sets of sequences

Performed physical mapping of the above markers in the set of cosmid, determining their presence in various komenich clones (11).

1. Genetic mapping of new PDAF markers

For new PDAF markers was performed genetic mapping using PDF-analysis of immediate Northern and southern recombinants. Mapped closest to the Northern end (recombinant D169) and to the southern end (recombinant S) point recombination (see table 15). PDF-analysis showed that the recombinant D169 had southern recombination marker L84.Y1, but Northern recombination marker W84.Y2. Point recombination in recombinant S mapped between markers L84.T2 and L84.8. Using the available set of recombinants were able to further define the chromosomal interval containing NIM1; the distance between the flanking points recombinations, apparently, is 60-90 TPN (Fig).

2. Design kosmidou set of sequences

For complementaly plant phenotype nim1 neo the natural transformation nim1 plants NIM1 gene of the wild type. This can be done by transformation of these plants kosmidou containing this gene. For this purpose designed Kemeny set of sequences NIM1-region. Because of Arabidopsis transformed with Agrobacterium used Kemeny vector was represented by a binary vector.

DNA was extracted from YOU-04, YOU-06 and P1-18 and was carried out by partial cleavage with restrictase SauSAI. Fragments of length 20-25 TPN allocated in the sucrose gradient were pooled and filled with dATP and DSTF. The binary vector (04541) were digested with XhoI and filled dCTP and dTTP. Then the fragments ligated with the vector. Legirovannoi the mixture was Packed and transducible in E.coli.

Conducted screening kosmidou library clones YOU-04, YOU-06 and P1-18, and provided positive clones. These cosmid then determined PDAF-fingerprints and were formed from them a set of sequences of overlapping clones covering the NIM1-area. To determine the size of komenich inserts and performed limited mapping using restricted. The results are shown in figure 10.

Example 2: Identification of a clone containing the gene NIM1

1. The complementation using stable transformation

Comedy obtained from the above clones that covered NIM1-region (described above) was introduced into Agrobacterium by technovillage crossing. Comedy and then is used to transform the nim1-Arabidopsis by vacuum infiltration (Mindrinos, and others, 1994, Cell 78, 1089-1099) or by standard transformation of the root. The seeds of these plants were collected and germinated on agar plates with kanamycin (or other appropriate antibiotic) as the selectivity of the agent. Only transformed kosmidou DNA seedlings could detoxify selectivity of the agent and to survive. Sprouts, surviving in this selection, transferred to soil and tested in relation to phenotype nim or their progeny tested in relation to phenotype nim. Transgenic plants that no longer had a phenotype nim, are consistent with cosmides(s)that contain (s) functionally active gene NIM1.

2. The complementation in the transient expression system

The ability to clone DNA repair (complementarity) mutation nim1 tested in two time systems expression.

In the first system of the plant Arabidopsis with mutation of the nim1 containing the transgene PR1-luciferase (PR1-lux)was used as the recipient of the material in the bombing. These plants was obtained by transformation of plants of ecotype Columbia using design PR1-lux by vacuum infiltration with subsequent selection of the collected seeds for resistance to kanamycin as described above. Was carried out by self-pollination of the transformed plants that expressed luciferase activity after induction INC., and received homozygot the e plants. They were crossed with nim1 plants. When the Express analysis of the progeny plants resulting from the crossing, which was homozygous for the gene nim1 and PR1-lux, was used for identification of DNA clones that can restore the phenotype nim1. In this case, plants are first treated INC., as described above in example 1.1. After 2 days, these plants were collected, sterilized their surface and were planted on agar medium GM. Then leaf tissue was bombarded by kosmidou, clones P1 or YOU (or poklonny) of the NIM1-area and after one day was measured by luciferase activity in the leaves. Clones that induce luciferase activity, contain NIM1 gene.

In the second system of a plant with a mutation nim1 handled INC. (as described above in example 1.1) and after 2 days was bombarded cloned DNA (kosmidou, P1 clones, YOU and/or YAC or subclones) of the locus NIM1-region and the reporter plasmid. Reporter plasmid contained the luciferase gene under the control of the promoter of Arabidopsis PR1 (PR1-lux). In nim1 plants INC does not activate the PR1 promoter (as described above in example 1.2) and, thus, could not induce luciferase activity of reporter plasmids. However, when a co-transformed clone DNA contained the recovered gene NIM1, INC. is not induced PR1 promoter, which can be seen on indukti the luciferase activity. After one day after co-bombardment was measured by luciferase activity in the whole plant. The DNA clones (Comedy, clones P1 or YOU or subclones), which induced luciferase activity significantly above baseline levels, contain NIM1 gene.

3. Changes of transcripts in line with the phenotype nim1

Since plants with a phenotype nim1 have mutations in the gene NIM1, it seems likely that in some lines the gene is modified in such a way that it is not transcribed mRNA is produced or deviating from the norm (size) of the mRNA. To assess this fact in lines nim1 conducted blot analysis of RNA.

RNA was isolated from plant ecotypes Ws and Ler of these lines (after treatment with water, INC. or BTK) and used for Northern blots. These blots hybridized with DNA fragments isolated from the clones sets of DNA sequences locus NIM1. DNA fragments that are characteristic lines nim1 with aberrant expression of RNA (abnormal in size or concentration)probably identify gene NIM1 (or part of it). The DNA fragment and okrujayushaya region DNA sequenced and used for isolation of cDNA by screening the library, or by PCR with reverse transcription), which also sequenced. The clone, which was isolated fragment, or a dedicated cDNA was used for supporting the surveillance of complementaly phenotype nim1 in stable and transient expression systems.

Example 3: determination of the DNA sequence of a gene NIM1

1. Genomic sequencing

Genomic clones, which may contain NIM1 gene, sequenced using methods known in this field. They include YOU-04, P1-18 and Comedy of NIM1-field.

For example, Comedy were digested using enzymes restricts and the fragments obtained from the insert cloned in the vector General purpose, such as pUC18 or Bluescript. The largest clones P1 and YOU randomly tsalala and fragments cloned in the vector General purpose. Fragments in these vectors sequenced by standard methods (for example, using "primerno walk" or by creating deletions, insertions). The obtained sequences were combined into sets of sequences.

The sequence of the insert complementarios clone contains a gene NIM1. Approximate beginning and end of the NIM1 gene deduced deduction based on generalized data on DNA sequences, sequence motifs such as TATA boxes, open reading frames present in the sequence, normal codons, data kosmidou complementaly, about the relative position PDAF markers and additional available data (see example 4, below).

2. Sequencing of cDNA

Kosmidou(s) or larger clones that contain the gene NIM1 (as described in the above example 2), was used for cDNA selection. Used clones or DNA fragments as probes for screening cDNA library of Arabidopsis wild-type. Selected cDNA sequenced as described for sequencing of cosmid, and used in tests of complementaly. This full-size cDNA cloned in an acceptable plant expression vector under the control of a constitutive promoter. These constructs were used for proximate analyses, as described previously. Alternatively, cDNA cloned in the binary expression vector designed for expression in plant tissues and for Agrobacterium mediated transformation of plants, as described above in example 2. Sequenced cDNA containing the gene NIM1 (as defined in complementaly, selection using a closely related PDF-marker selection using kosmidou fragment or other deductive analysis).

Genes from plants Ws-O and nim1 were isolated and sequenced. Genes were obtained from cosmid cDNA library using fragment selected NIM1 gene as a probe. Alternatively, genes or cDNA was isolated by PCR, using as matrix-specific gene NIM1 primers and genomic DNA or cDNA. Similarly, were isolated and sequenced nim1 alleles from other lines nim1 (see example 1.1 above).

Example 4: OPI is the W gene NIM1 and derived by deduction sequence protein

The DNA sequence of the gene NIM1 cDNA or determined as described above in example 3 method. This sequence was analyzed using DNA analysis, for example, the programs included in the software package Genetics Computer Group (GCG)software package Sequencer or Staden or any other software package for DNA testing.

In particular, the beginning and the end of the gene was determined on the basis of the analysis of open reading frames, the presence of a stop codon and a potential start codon, the presence of potential motives promoter (such as the TATA-box), the presence of polyadenylation signals, etc. Predicted amino acid sequence also taken out by way of deduction based on open reading frame. The sequence of both DNA and protein was used to explore the database of homologous sequences, such as transcription factors, enzymes, or the motives of these genes or proteins.

Example 5: Isolation of homologues of the gene NIM1

The gene of Arabidopsis NIM1, can be used as a probe for screening by hybridization under low stringency library of genomic DNA or cDNA to identify homologues NIM1 from other plant species. In an alternative embodiment, was used for this PCR amplification using primers based on the sequence of the gene of Arabidopsis NIM1, and use the cation of genomic DNA or cDNA as template. Gene NIM1 can be selected from maize, wheat, rice, barley, rapeseed, sugar beet, potato, tomato, beans, cucumbers, grapes, tobacco, and other interest of cultures, the sequence of which is known. Having a set of sequences of homologues NIM1 gene, using PCR amplification can be created by the new primers from conserved regions of the gene to identify homologues of NIM1 more remote from the point of view of the relationship of the species.

Example 6: the Complementation of the gene nim1-1 using genomic Fragments

1. Design kosmidou set of sequences Kemeny set of sequences NIM1-region were designed using CsCl-purified DNA from WAS, WAS and P1-18. Dnstring clones were mixed in equimolar amounts and was partially digested with restrictase Sau3A. Fragments of length 20-25 TPN allocated in the sucrose gradient were pooled and filled with dATP and DSTF. Plasmid pCLD04541 used as a T-DNA kosmidou vector. This plasmid contains the replicon with a wide range of hosts, derived from pRK290, the gene of resistance to tetracycline for breeding bacteria and nptII gene for plant breeding. The vector was digested with XhoI and filled dCTP and dTTP. Then the resulting fragments were leading with vector. Legirovannoi the mixture was Packed and transducible PCs in the mm E. coil XL1-blue MR (firm Stratagene). Was carried out by screening the resulting transformants by hybridization with clone WAS, WAS and P1-18, and provided positive clones. Kosmidou DNA was isolated of these clones and obtained DNA template using KF EcoRI/MseI and HindIII/MseI. The scheme received PDAF-fingerprints were analyzed to determine the order komenich clones. Selected set of 15 polupereprevshy of cosmid covering nim-region (Fig). Cosignee DNA was also digested with EcoRI, PST, BssHII and SgrAI. This allowed us to estimate the size komenich inserts and confirm the presence of overlapping areas between different kominami that is installed with PDF-fingerprints.

2. Identification of a clone containing the gene NIM

Comedy obtained from clones covering the NIM1-region, was built in Agrobacterium tumefaciens AGL-1 by transferring conjugate method technovillage mating with strain-helper NV (pRK2013). Then Comedy used to transform susceptible to kanamycin line nim1-1 Arabidopsis by vacuum infiltration (Mindrinos and others, 1994, Cell 78, 1089-1099). Seeds of infected plants were collected and germinated on GM agar-plates containing 50 mg/ml kanamycin as the selectivity of the agent. Only seedlings transformed kosmidou DNA could carry out detoxification selectivity agent and survive. Arr is siteline two weeks after sowing the seeds, survivors with this selection, transferred to soil and tested in relation to phenotype nim according to the above-described method. Transgenic plants that no longer had a phenotype nim, are consistent with cosmides(s)that contain (s) functionally active gene NIM1.

3. Analysis of the phenotype of nim1 transformants

Transferred to the soil the plants were grown in the phytotron for approximately one week after transplantation. 300 μm INC. made in a light mist that completely cover the plants using the atomized romanobritish type "chromister". After 2 days, the leaves were collected for extraction of RNA and analysis of gene expression of PR-1. Then the plants were sprayed Peronospora. parasitica (isolate EmWa) and were grown in conditions of high humidity in a climatic chamber at a daily temperature of 19°/night temperature of 17° and when the light cycle 8 h light/16 h of dark. After 8-10 days after infection by the fungus plants investigated and based on the growth of the fungus was evaluated as positive or negative. Plant lines Ws, and nim1 treated in the same way, served as controls in each experiment.

Total RNA was extracted from collected tissue using the buffer for the extraction of LiCl/phenol (Verwoerd and others, NAR 17: 2362). RNA samples were dispersed on a formaldehyde/agarose gel and were bottiroli on the membrane type GeneScreen Plus (DuPont company). The blots guy who was ridesafely probe which was a marked using the32R cDNA PR-1. The resulting blots were exposed to film to determine what transformants possessed the ability to Express PR-1 after processing INC. The results are summarized in table 16.

Table 16 shows the complementation phenotype nim1 kominami clones.

Table 16
Name cloneThe number of transformantsThe number of plants in which the INC was induced PR-1/total number of investigated plants (%)
A830/3 (0%)
A1184/18 (22%)
C2101/10 (10%)
C7331/32 (3%)
D2814/49 (8%)
D565/6 (83%)
E11010/10 (100%)
D712936/36 (100%)
E890/9 (0%)
F1260/6 (0%)
E610/1 (0%)
E7340/4 (0%)
WS-control (wild type)NP28/28 (100%)
phenotype im1-1 (control) NP0/34 (0%)
NP stands for "not applicable".

Example 7: Sequencing, the field of gene NIM1 length 9.9 TPN

DNA WAS (25 μg, obtained from the firm's Key Gene) was used as source DNA for sequence analysis. It was shown that this clone of YOU is a clone, which completely covers the region complementary to the nim1 mutants. DNA was randomly split using the method developed in the laboratory Cold Spring Harbor. In General this method is that DNA clone YOU were digested in the gun-to-average molecular weight of approximately 2 TPN the ends of the split-off fragments were repairable using a two-stage Protocol using dNTP, polymerase of phage T4 and fragment maple (firm Boehringer). DNA preparirovanie ends were dispersed in 1%agarose gel with a low melting point and are cut out of the gel region from 1.3 TPN to 2.0 TPN DNA was isolated from a slice of the gel by the method of freezing and thawing. Then DNA was mixed with the cleaved with EcoRV pBRKanF4 and ligated overnight at 4°C. Plasmid pBRKanF4 is derived pBRKanF1 received from Kolavi br Vanderbilt University. hat for K.S., Gene 134(1), 83-87 (1993)). The strain E. coli DH5a transformed legirovannoi mixture and transformed the mixture was sown on plates, operasie kanamycin and 5-bromo-4-chloroindole-β -D-galactosidase (X-gal). For plasmid isolation were collected 1600 white or light blue colonies KanR. Individual colonies were made in 96-well tablets with deep holes (firm Polyfiltronics, No. U508), which contained 1.5 ml TV+Kan (kanamycin) (50 μg/ml). The tablets were covered and placed for 16 h in a shaker rotating platform at 37°C. Plasmid DNA was isolated using the Wizard Plus 9600 Miniprep system (firm Promega, No. A) according to the manufacturer's recommendations.

Plasmids sequenced by the method of Dye Terminator chemistry (company Applied BioSystems, set PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, P/N 402078) and primers designed for sequencing obeh circuits plasmids. Data were obtained using the DNA sequencers type ABI 377. Approximately 75% of these reactions gave useful information about the sequence. Sequence obrabatyvali and combined into sets of sequences of fragments using a sequencing machine Sequencher 3.0, Gene Codes Corporation), Staden gap4 (Roger Staden, e-mail: rs@mrc-lmb.cam.ac.uk and PHRED (Phil Green, e-mail: phg@u-washington.edu). The largest set of sequences (approximately 76 TPN) blocked complementarily area to an average depth of 7 independent signals/base.

By analyzing complementaly revealed characterized by the overlapping of cosmid D7 E1 and the area approximately 9,9 TPN, which contains nim1-areas of the ü. Created the primers flanking the insertion site of the vector and is specific to Caracas Comedy, using the software Oligo 5.0 Primer Analysis Software (National firm Biosciences, Inc.). DNA was isolated from cosmid D7 and E1 using a modified method using ammonium acetate (P.L. Traynor, 1990 BioTechniques 9(6): 676.) This DNA directly sequenced using the above method Dye Terminator chemistry. The resulting sequence has allowed to define the ends complementary area.

Also designed a truncated version of the BamHI-EcoRV fragment, getting a design that does not contain any gene region 3 "Gene 3" (Fig). The following approach was necessary because of the presence of the HindIII sites in Bam-Spe-region DNA. Design BamHI-EcoRV was completely digested with Spel and then to conduct a double cleavage was divided into two different reaction mixtures. One aliquot was digested with BamHI, a another using HindIII. BamHI-SpeI fragment length 2816 base pairs and HindIII-SpeI fragment length 1588 base pairs was isolated from agarose gels (set for extraction type QiaQuick Gel) and was in the lead with split using BamHI-Hindlll the plasmid pSGCG01. DH5a transformed legirovannoi mixture. The formed colonies were subjected to screening for the correct insert by cleavage with HindIII followed by obtaining DNA using Wizard Magic MiniPreps, Promega. The clone containing the correct design, built using electroporation into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.

Example 8: Identification region of the gene NIM1 alleles by sequencing

1. Genetic analysis

To determine the dominance of different mutants exhibiting the phenotype nim1, pollen of wild-type plants were transferred to the stigma of nim1 mutants-1, -2, -3, -4, -5, -6. If the mutation is dominant, the phenotype nim1 should appear in the resulting plants of the F1 generation. If the mutation is recessive, then the resulting F1 plants should show the phenotype of the wild type.

The data presented in table 17, indicate that when nim1-1, -2, -3, -4 and -6 were crossed with wild type, the resulting F1 plants showed wild type phenotype. Thus, these mutations are recessive. In contrast, all of the F1 progeny obtained by crossing nim1-5 × wild type, showed a phenotype nim1, which indicates the dominance of this mutation. After processing, INC. no sporulation .parasitica not detected in wild-type plants, while on the F1 plants were growing and some sporulation .parasitica. However, the phenotype nim1 these F1 plants was less strict compared to those that were observed when the mutation nim1-5 g was monigotes.

To determine allelism pollen from resistant to kanamycin nim1-1-mutant plants were transferred to the stigma nim1-2, -3, -4, -5, -6. To confirm their hybrid origin of the seed; the seeds obtained from the crosses were planted in tablets with the environment of Murashige and Skoog B5 containing kanamycin at a concentration of 25 µg/ml Resistant to kanamycin (F1) plants transferred to soil and explored in relation to phenotype nim1. Because the F1 progeny obtained by crossing mutant nim1-5 wild type Ws, showed a phenotype nim1, was also analyzed F2, obtained from crossing nim1-5 × nim1-1.

As can be seen from table 17, all of the resulting F1 plants showed a phenotype nim1-1. Thus, mutations nim1-2, -3, -4, -5, -6 not fully recovered with the help of nim1-1; all of these plants were included in one group of complementaly and, therefore, were allelic. Analysis of the F2 progeny obtained from crossing nim1-5 × nim1-1 showed that it also manifests the phenotype nim1, confirming that the nim1-5 is a nim1 alleles.

2. The sequence analysis and subclavian NIM1-area

Area length of 9.9 TPN containing NIM1-region, were analyzed for the presence of open reading frames in all six frames using Sequencher 3.0 software package GCG. As candidate genes we identified 4 areas that contain large ORFS gene region 1-4). These 4 region amplified by PCR from the DNA of the parent wild-type and six different nim1-allelic variants. The primers for the amplification were selected using Oligo 5.0 (firm National Biosciences, Inc.) and synthesized firm Integrated DNA Technologies, Inc. PCR products were separated on a 1.0-percentage agarose gels and purified using the kit for the extraction of the gel type QIAquick. Purified genomic PCR products directly sequenced using the primers used for the initial amplification, with additional primers designed for sequencing any areas that are not covered by the primers in the initial amplification. Average coverage of these gene regions was approximately 3.5 readings/base.

Sequences were processed and merged using Sequencher 3.0. Replacement bases specific to different nim1 alleles were detected only in the area designated as gene region 2.

Provisions are shown in table 18, correspond pig and correspond to the upper chain region of length 9,9 TPN depicted on Fig. Sequenced the open reading frame of the gene of the areas indicated on Fig as 1, 2, 3 and 4, with replacement in various nim1 alleles are shown in table. In accordance with Fig described replacement are at the top of the chain in the direction 5’→3’.

It is obvious that the gene NIM1 would the cloned and that it lies within the gene region 2, since the detected amino acid substitutions or changes to the sequence within the open reading frame of the gene region 2 in all six nim1 alleles. At the same time for at least one of the nim1 alleles revealed no changes in open reading frames in the gene regions 1, 3 and 4. Therefore, only one gene within a region of length 9,9 TPN, which can be a NIM1, is a gene region 2, NIM1 gene.

A section dedicated in table 18 ecotype Ws indicates the substitutions in the Arabidopsis ecotype Ws relative of Arabidopsis ecotype Columbia. Fig, 14, 15 and others, on which a given sequence, refer to the Arabidopsis ecotype Columbia, which in the experiments contained the gene of the wild type. See replacing represent amino acid substitutions in the gene region 2 or in NIM1-region, and they are shown as replace in pairs of bases in other areas.

On Fig given 4 different panels describing the cloning of the gene NIM1 and the entire area length of 9.9 TPN On Fig shows the sequence of the full field length of 9.9 TPN in the same orientation as on Fig. On Fig given region sequence-specific gene NIM1, which corresponds to the gene region 2 on Fig; sequence on Fig contains a gene NIM1. On Fig in single-letter code provides serial amino acid is inost and shown full size; cDNA and RACE-product, which are indicated by capital letters in the DNA sequence.

Some of allelic mutations that were detected is shown above the DNA sequence and partially marked nim1-allele, which had the replacement.

Sequence analysis of the region and sequencing of the various nim1 alleles (see below) allowed us to identify kosmidou the region that contains the gene nim1. This area was linearizable getting BamH1-EcoRV-restriction fragment length ~5,3 TPN Kosmidou DNA from D7 and plazamedia DNA from pBlueScriptI(pBSII) were digested with BamHI and EcoRV (NEB). Fragment length 5.3 TPN of D7 was isolated from agarose gels and purified using the kit for the extraction of the gel type QIAquick (No. 28796, the company Qiagen). The fragment ligated overnight with cleaved with Bam-EcoRV a plasmid pBSII and legirovannoi mixture was used to transform the E. coli strain DH5a. Selected colonies containing the insert were isolated DNA and its structure was confirmed by cleavage with HindIII. Then for the transformation of Arabidopsis designed Bam-EcoRV fragment into the binary vector (pSGCG01).

3. The analysis Northern blot of four gene regions

Identical Northern blots were generated from the RNA samples isolated from the treated water, SC, BTK and INC lines Ws and nim1, as described previously (Delaney and others, 1995, PNAS 92, 6602-6606). These blots hybridized with PCR products derived from che ireh gene regions, identified in the field of gene NIM1 length of 9.9 TPN for Only one gene region containing the NIM1 gene (gene 2)identified by hybridization with RNA samples, indicating that only NIM1-area contains the transcribed gene that can be detected (Fig and table 18).

Table 18 shows the variability of the sequence nim1-allele.

Table 18
 Region gene
Allele/ecotype1 (base 590-. 1090)2 (NIM1) (base 1380-4100)3 (base 5870-6840)4 (base 8140-9210)
nim1-1no changet built-in 2981: replacement AA and termination of protein before pubertyno changeno change
nim1-2no changeg and 2799: His on Tightno changeno change
nim1-3no changedividing t 3261: replacement AA and termination of protein before pubertyno changeno change
nim1-4no changeC on t in 2402: Arg to Lysno changeno change
nim1-5no changeC on t in 2402: Arg to Lysno changeno change
nim1-6g and in 734: Asp Lysg and 2670: Gin stop codonno changeno change
WS (no comparison with Columbia)no changeand g in 1607: Il on Leu

and with at 2344: intron

t on g 2480: Gln Pro

g with at 2894: Ser on TGR

ggc-deletion in 3449: Troubleshooting l

C on t in 3490: Ala to Thr

C on t in 3498: Ser to Asn

and t 3873: non-coding

g and 3992: non-coding

g and 4026: non-coding

g and 4061: non-coding
t and 5746 and t 5751 t and 5754 s on t 6728 and t 6815 t with the C 6816t on g 8705 g on t 8729 g on t 8739 g on t 8784 with on and in 8789 s on t 8812 and g in 8829 t in g in 8856 and with 9004 and t 9011 and g 8461
detected RNAnoYesnono

Provisions listed in the table correspond Fig, which includes a sequence length of 9.9 TPN All alleles with nim1-1 and nim1-6 sootvetstvuut strain WS. Columbia-0 denotes wild type

By performing additional experiments complementaly also been shown that gene region 2 (Fig) contains functionally active gene NIM1. BamHI/HindIII-fra is ment genomic DNA, containing gene region 2, was isolated from Comedy D7 and cloned in the binary vector pSGCG01 containing the gene that determines resistance to kanamycin (Fig; Steve Goff, personal communication). This plasmid was used to transform Agrobacterium strain GV3101 and positive colonies were selected on medium containing kanamycin. To confirm that the selected colony contained the plasmids used PCR. Sensitive to kanamycin plants nim1-1 were infected with these bacteria, as described previously. The resulting seeds were collected and sown on GM-arap containing 50 μg/ml kanamycin. Selected surviving plants transferred to soil and tested against complementaly. Transgenic plants and control Ws - and nim1 plants were sprayed with 300 μm INC. After 2 days, the leaves were collected for extraction of RNA and analysis of gene expression of PR-1. Then the plants were sprayed Peronospora. parasitica (isolate EmWa) and were grown as described previously. 10 days after infection by the fungus plants investigated and evaluated depending on the fungus growth as positive or negative. All 15 of the transformed plants, as well as the control and Ws plants refused negative in the growth of the fungus after processing INC., while the control nim1 plants were positive for growth of the fungus. RNA was extracted from these transformants and control plants is analyzed, as described previously. The control Ws-plants and all 15 of transformants after processing INC. discovered the induction of the gene PR-1, and the control nim1-plants after treatment INC is not revealed induction of PR-1.

4. Selection NIM1 cDNA

A cDNA library of Arabidopsis created in the expression vector 1YES (Elledge and others, 1991, PNAS 88, 1731-1735), were sown and the resulting plaques were transferred to filters. The filters are hybridized with labeled using32R-PCR product derived from a gene region containing nim-1. By screening approximately 150,000 plaques there were 14 positive clones. Each plaque purified and isolated plasmid DNA. cDNA inserts were tsalala from the vector using EcoRI, purified on agarose gel and sequenced. The sequence obtained from the longest cDNA is shown in Fig. For evidence that was obtained 5’-end of cDNA used set Gibco BRL 5’ RACE, following the manufacturer's instructions. The resulting RACE products sequenced and found that they include additional grounds given for Fig. The transcribed region is present in both clones cDNA and detected in the RACE-the product, and it is shown in capital letters on Fig. Replacement alleles are indicated above the DNA chain. Uppercase letters indicate the sequence present in the cDNA clone or detected after PCR with RACE.

Example 9: the Characteristics of erotica gene NIM1

Designed multiple number sequences using the software Clustal V (Higgins Desmond G. and Paul M Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5: 151-153) as part of the DNA* (1228 South Park Street, Madison, Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994).

It was found that the amino acid sequence of certain areas of the NIM1 protein homologous amino acid sequences of four different protein products of the cDNA of Fig. Homologues were identified using sequences NIM1 found in GenBank BLAST. Comparison with homology regions for products NIM1 and rice cDNA is shown in Fig. Fragments of the NIM1 protein having from 36 to 48% amino acid sequence identical to the amino acid sequences of the four products of rice.

Example 10: Phenotypic characteristics of different alleles nim1

1. Analysis of chemical reactivity in the nim1 alleles

Analyzed the differences between the various nim1 alleles from the point of view of chemical induction of PR gene expression and resistance to Peronospora. parasitica (see Fig and 18).

Mutant plants were treated with chemical inducers and then analyzed in terms of PR gene expression and resistance to disease.

2. Plant growth and chemical processing

Seeds of wild-type and the seeds obtained from the plants with which each of Alela nim1 (nim1-1, -2, -3, -4, -5, -6), were planted in a growing medium type MetroMix 300, covered with a transparent plastic dome and kept in the dark at a temperature of 4°C for 3 days. After keeping at 4°C for 3 days the plants were transferred to 2 weeks in the phytotron. Approximately 2 weeks after sowing germinated sprouts gave 4 true leaves. After this plant was treated with N2Oh, 5 mm SK, 300 μm BTK or 300 μm INC. Chemical compounds were made in a light mist to full coverage leaves with the help of romanoarchives type "chromister". The treated water control plants were returned to growth in the climate chamber and processed chemical compounds plants were kept in a separate, but identical climatic chambers. After 3 days the plants were divided into 2 groups. From one group were extracted and analyzed RNA. The second group was infected P. parasitica.

3. Inoculation Peronospora. parasitica and analysis

The isolate P. parasitica "EmWa" represents the isolate RR compatible with ecotype Ws. Compatible isolates are isolates capable of causing disease in a particular host. The isolate P. parasitica "NoCo" incompatible with the ecotype Ws, but is compatible with the ecotype Columbia. Incompatible pathogens are recognized by potential host, causing the host response, which prevents the development of disease. On the 3rd day after treatment the water or processing of chemical compounds plants infected compatible isolate "EmWa". Isolate "NoCo" was inoculable only treated water plants. After inoculation the plants were closed with a transparent plastic dome to maintain high humidity required for successful infection with P. parasitica, and were placed in a growth chamber with a day temperature of 19°/ / night temperature of 17°and a light cycle of 8 h light/16 h darkness.

Through various time intervals after inoculation the plants were analyzed under a microscope to assess the development of symptoms. Under increased sporulation of fungi can be detected at very early stages of the disease. Each pot was determined by the percentage of plants that showed sporulation 5, 6, 7, 11 and 14 days after inoculation, and also assessed the density of sporulation.

On Fig the assessment of disease development in plants with different nim1 alleles after inoculation .parasitica. The most characteristic points were the 5th and 6th day after inoculation. On the 5th day after inoculation the plants nim1-4 after all treatments inducing chemicals found ~80%infection, clearly indicating that this allele/genotype is the most susceptible to the disease. On the 6th day after inoculation the plants nim1, -2, -3, -4 and -6 after all treatments inducing chemicals found significant incidence. However, in plants the deposits nim1-5 on the 6th day after all the treatments found less contamination, than the wild-type Ws. Therefore, nim1-5 represents the allele, the most disease resistant in comparison with various nim1 alleles. Probably nim1-2 is intermediate in sensitivity to diseases after treatment of BTK, but not after treatments other inducing agents.

Expression of PR-1 indicates that the nim1-4 is the least sensitive to all of the studied inducing chemicals (Fig), and nim1-5 shows elevated levels of expression of PR-1 in the absence of inducers. These results on the gene expression of PR-1 are consistent with the assessment of the incidence, which was performed using .parasitica (Fig), and suggests that nim1 alleles can lead to resistance or sensitivity.

The above samples were used for analysis of gene NIM1 gene (Fig). The wild type mRNA NIM1 was present in the untreated control samples. After processing IC, INC., BTK or intrusion compatible pathogen mRNA NIM1 accumulated to high levels. In plants with nim1 alleles compared to wild type were observed difference in the number of transcripts NIM1 (mRNA). The number of copies of mRNA NIM1 untreated mutant plants was lower than that detected in the wild type, except nim1-2 and -5, where the number of copies was close. Plants Catalani nim1-1, -3 and -4 had a smaller number of transcripts NIM1, and in plants with mutations nim1-6 found a very small accumulation of mRNA NIM1. Increase the number of copies of mRNA NIM1 after processing IC, INC. or BTK was observed in plants nim1-1, -2, -3, but not observed in plants nim1-5 or-6. However, this increase was smaller than that detected in wild-type plants. After pathogen infection extra band hybridization with a cDNA probe NIM1 detected in wild-type plants and mutant plants, and the number of copies of mRNA NIM1 was increased compared to untreated controls, with the exception of nim1-6.

On Fig the data evaluation for disease resistance, obtained on the basis of detection of contamination of plants with different nim1 alleles, and NahG plants through various time intervals after inoculation with Peronospora. parasitica. WsWT denotes the parent line Ws wild type infected nim1 alleles. Various nim1 alleles are indicated in the table, which also included data on NahG-plant. Data NahG-plant previously published (Delaney and others, Science 266, pages 1247-1250 (1994)). NahG-type Arabidopsis is also described in WO 95/19443.

Gene NahG represents a gene from Pseudomonas putida, which converts salicylic acid to catechol, thereby preventing the accumulation of salicylic acid, the required signal component transduction SAR the plants. So, Arabidopsis plants with the gene NahG not have normal SAR. In addition, they have found a significantly higher sensitivity to pathogens. Therefore NahG plants are a type of universal sensitive control. In addition, NahG plants still retain the ability to respond to chemical inducers INC. and BTK, as shown in the two bottom panels on Fig.

As follows from Fig, alleles nim1-4 and nim1-6 are probably the most simple that can be easily found in earlier periods of time, as described above under "Results" in this description, as also follows from the results obtained using panel EmWa-BTK (the bottom panel in the drawing). In addition, allele nim1-5 gives the most pronounced response and INC and BTK and, therefore, represents the weakest allele nim1.

In NahG plants found a very good response to both the INC and BTK, and it is very similar to the reaction of the allele nim1-5. However, in later periods of time, day 11 in the drawing, disease resistance induced in NahG plants, begins to disappear, indicating a marked difference between INC and BTK in the sense that the induced INC. resistance disappears much faster and more noticeable in comparison with the resistance induced in NahG plants with BTK. From these experiments what s seen that INC and BTK induce very good resistance in plants wsk EmWa, a nim1-1, nim1-2 and other alleles~*P18 62Xnim1 not actually respond to SC or INC in relation to disease resistance.

On Fig specified percentage of plants that showed sporulation after infection by a strain of P. parasitica EmWa, and each histogram represents the number of days after infection, during which determined the resistance to diseases.

Northern analysis of the expression of one of the SAR genes PR1 also conducted on these samples, as shown Fig. On Fig shown that wild-type plants give very good response to salicylate, INC., BTK, as well as infection by a pathogen, which is clearly evidenced by the increased gene expression of PR1. On the other hand, plants with allele nim1-1 show only a limited response to all chemical inducers, including the pathogen.

Induction of pathogen at least several times lower for allele nim1-1 than wild-type. For alleles nim1-2, nim1-3 and nim1-6 found the response to various treatments, similar reactions allele nim1-1. However, for allele nim1-4 found no expression in response to any of the inductors used. Essentially all detected expression was a background level. For allele nim1-5 detected a very high background level is compared with the treated water contact is the ol, this background level was maintained in all treatments; however, there was a limited induction or no induction under the influence of chemical inducers.

NahG plants are quite intuitive control due to the fact that they are not able to induce PR-1 in the presence of SC; with the other hand and INC and BTK induce a very high level of expression of PR-1. Infection with a pathogen similar to the effect IC; processed EmWa NahG plants was not observed in the expression of PR-1.

The same RNA samples that were obtained in the study of induction, also probed gene NIM1 using a full-size cDNA clone as a probe. On Fig you can see that INC induces NIM1 gene in the plant Ws with the wild-type allele. However, the mutant allele nim1-1 shows the lower primary level of expression of the gene NIM1 and not able to appear INC. This is similar to what was obtained for allele nim1-3 and allele nim1-6. Allele nim1-2 shows approximately normal levels in the untreated sample and shows the induction, a similar induction pattern of the wild type, as well as allele nim1-4. Allele nim1-5, apparently, has higher base level of expression of the gene NIM1 and more pronounced expression in the induction of chemical inducers.

Induction NIM1 chemical inducers of resistance and other inductors agree on the I with its role in protection against pathogens, and is an additional proof of the fact that according to the invention detected the desired gene in a region of length 9,9 TPN

1. The selected DNA molecule, providing resistance of plants to diseases, with a DNA molecule comprising NIM1 gene, which encodes the amino acid sequence represented in SEQ ID NO: 3.

2. The selected DNA molecule according to claim 1, comprising the nucleotide sequence represented in SEQ ID NO: 2.

3. The selected DNA molecule according to claim 1, length approximately 9,9 TPN, which encodes the NIM1 gene product.

4. The selected DNA molecule according to claim 1, comprising the nucleotide sequence represented in SEQ ID NO: 1.

5. The selected DNA molecule of claim 1 encoding the amino acid sequence of the NIM1 gene product, performance is undertaken in SEQ ID NO: 2.

6. Recombinant vector comprising active in the plant promoter functionally linked to a DNA molecule according to claims 1 to 5, and provides the expression of the protein, which activates systemic acquired resistance in plants.

7. Recombinant vector according to claim 6, where the specified vector capable of stably transforming a cell of the host.

8. Recombinant vector according to claim 6, where the specified vector capable of transforming a plant, the seeds of a plant, plant tissue or plant cell.

9. The method of imparting disease resistance to plant cells, plants and their seed, wherein said plant cells, plants or their progeny are transforming DNA molecule according to any one of claims 1 to 5.



 

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