CN1952143A - Cloning of gene against meloidogyne of capsicum and application thereof - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering technologies. The invention specifically involves the separation and cloning of a DNA fragment. The said DNA fragment gives the plants the tolerant capacity against diseases caused by edlworm. Inserting the DNA fragment into proper vector to obtain plant expressing vector (conservation number: CCTCC-M205110), introducing the plant expressing vector into plant through Agrobacterium-mediated method and the transgenic plants can generate resistance against edlworm. As one of the implementation of cases, nematode resistant transgenic tomatoes have been obtained.

Description

The clone of gene against meloidogyne of capsicum and application thereof

Technical field

This patent belongs to plant genetic engineering field.Be specifically related to from resistance capsicum genome to separate, the clone obtains genjie eelworm resistance gene, this gene imported to not have in the vegetable material of root knot nematode resistance then, as the tomato to the root knot nematode sensitivity, obtains transgenosis tomato of preventing root-knot Nematode disease plant.

Background technology

Plant root-knot nematode (Meloidogyne spp.) disease is a kind of world disease, the production of serious threat crop.The host range of root knot nematode extensively (reaching 2000 various plants), route of transmission is various, is a kind of soil-borne disease source of extremely difficult control.The root knot nematode that has been found that in the world has (the Karssen et al. of kind more than 80,1999), China has identified 25 kinds, and what wherein distribution was the widest, harm is the most serious is Meloidogyne incognita (M.incognita), javanese root knot nematode (M.javanica) and peanut root-knot nematode (M.arebaria).According to statistics, in the four big cause of diseases that cause plant infectious diseases, the harm of plant nematode surpasses bacterium and virus, is only second to fungal disease, the about 1,000 hundred million dollars of (Sasser of loss that cause its every year world agriculture to produce; Freekman, 1987), and wherein root knot nematode loss that important cash crop are caused is annual just up to tens billion of dollars (Barker et al., 1986).The loss that China every year causes the harm of various vegetables because of nematode is up to more than 30 hundred million yuans.

Up to the present, root knot nematode is still lacked direct effectively preventing method, mainly adopt some indirect means, comprise crop rotation control, physical control, chemical prevention and biological control etc.Wherein crop rotation control is widely used, but can't reach the requirement of efficent rotation to some special economic crops or particular locality, has bigger limitation, nor is applicable to the control of the root knot nematode of host range broad.Physical control such as soil high-temperature sterilization, time-consuming, and only effective with the soil of the interior degree of depth to 30cm, and root knot nematode has past deep layer active ability (Cartia ﹠amp; Greco, 1978).Country such as chemical prevention such as America and Europe mostly adopts monobromethane to carry out soil-fumigating, because the cost height, and monobromethane may destroy earth's ozone layer, and some countries such as the U.S. have made laws and banned use of.Biological control mainly is the nemic natural enemy biology that utilizes nature all, and its mechanism of action to nematode is divided into three classes such as predation, parasitism, antagonism or poisoning generally.What research was maximum at present is Nematophagous fungi, though certain progress is arranged, also has very big distance from practical application.In fact, the trend that root knot nematode is rapid expansion and spreads on producing, as seen these methods effect in the generation of containment root knot nematode is very limited, and contaminate environment, reduces product quality, is detrimental to health the Sustainable development of influence agricultural.

This shows control root knot nematode, seed selection and to utilize disease-resistant crop varieties be direct, effective, the most economic approach.Traditional sexual hybridization method is cultivated the sick kind of nematicide, have that anti-source material lacks, selection cycle is long, the expense height, defectives such as the hereditary burden of bad proterties, have a strong impact on the realization of breeding objective, so utilize engineered method to obtain genjie eelworm resistance gene and it is transformed in the specified plant, the plant that obtains to have anti-root knot nematode characteristic is a new breeding approach.

Reported in the world at present that the clone has obtained the gene of four nematicide.1) is the anti-cyst roundworm gene Hsl that the clone obtains in beet ^Pro-1(1. Cai D, Kleine M, Kifle S, et al. (1997) .Positional cloning of a gene for nematode resistance insugar beet.Science 275:832-834.); 2) be the anti-tomato root-knot eelworm gene M i that obtained by the method for map based cloning in 1998 in the tomato (2. Stephen B M, Jhon B, Jafar Y, et al. (1998) .The Root Knot Nematode Resistance Gene Mifrom Tomato Is a Member of the Leucine Zipper, Nucleotide Binding, Leucine-Rich Repeat Family ofPlant Genes.The Plant Cell, Vol 10,1307-1319.); 3) the anti-potato cyst roundworm gene Gpa2 that obtains for clone in the potato (3. Van der V., E.A., van der V., et al. (2000) .Homologues of a single resistance-gene cluster in potatoconfer resistance to distinct pathogens:a virus and a nematode.Plant J.23:567-576.), 4) be the anti-potato cyst roundworm gene Hero that the clone obtains in tomato (4. Ernst K, Kumar A., Kriseleit D., et al. (2002) .Thebroad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a largegene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region.The Plant Cell, Vol31 (2): 127-136.).

The anti-root knot nematode breeding research of peppery (sweet) green pepper starts from 20 middle of century.Martin in reported first in 1948 resistance of capsicum variety to root knot nematode.Existing result of study shows, has a plurality of nematicide ospc genes in the Capsicum, the source of these genes, anti-spectral limit, the stability and the disease-resistant mechanism of temperature is had nothing in common with each other, and localized nematicide ospc gene is as shown in table 1.(Hare WW such as Hare, Inheritance of resistance to root-knot nematodes in pepper.Phytopathology, 1957,47:455-459.) first nematicide unnamed gene that he is found is N, this gene is a single dominant gene, anti-Meloidogyne incognita, javanese root knot nematode and peanut root-knot nematode can lost partial resistance more than 28 ℃.(Hendy H such as Hendy, Pochard E, Dalmasso A.Transmissionhereditaire de Ia resistance aux nematodes Melooidigyne Chitwood (Tylenchida) portee par 2 lignees deCapsicum annuum L.:etude de descendances homozygotes issues d ' androgenese.Agronomie, 1985,5 (2): 93-100) by double haploid colony, 5 disease-resistant dominant genes (Me1～5) have been found altogether at PM217 and PM687, Mel wherein, the anti-spectrum of Me3 is wider, and utility value is bigger.(Di Vito M such as Di Vito, Zaccheo G, Catalano F, etal.Effect of temperature on stability of resistance to root-knot nematode (Meloidogyne spp.) IXthEUCARPIA Meeting on Genetics and Breeding of Capsicum ﹠amp; Eggplant, 1995-Budapest, Hungary, 1995,230-232) in several wild capsicum materials, also found several nematicide ospc genes, but not name.

Table 1 has been located as yet not clone's Capsicum (Capsicum spp.) nematicide gene

Gene title source	Feature, characteristic	Hereditary feature	Document
				N Santaka XS Me1 PM 217 Me2 Me3 PM687 Me4 Me5 Yolo Wonder - PI159256 - 46-530/7 - 46-530/7 - 56-547/7 - 56-547/7 - Tabasco - 28-201 - PA135 - PA-426	Anti-Mi, Mj and Ma, to the anti-Mi of temperature sensitive, Mj and Ma, to anti-Mj of temperature sensitive and the anti-Mi of M.sp.Seville, Mj and Ma, to the anti-Mj of the anti-Ma of temperature-stable, the more weak anti-Mi of resistance, Mj and Ma, to the anti-Mi of temperature-stable, Ma, to the anti-Mi of temperature-stable, to the anti-Mi of temperature-stable, Mj, to the anti-Ma of temperature-stable, to the anti-Mi of temperature-stable, Mj and Ma, to the anti-Mi of temperature-stable, Mj and Ma are to the anti-Mi of the anti-Mi of temperature-stable	Dominance single-gene dominance, with the not chain dominance of Me2, with the not chain dominance of Me1, with the chain dominance of Me4, with Me3 chain-two dominant genes of single recessive gene single dominant gene, recessive epistasis is made two dominant genes of single dominant gene mutually, dominant epistasis is made dominant gene of single dominant gene single dominant gene and a recessive gene mutually, dominant gene and N equipotential single dominant gene are with the N equipotential	3 4 5 6 1 2

Annotate: Mi:Meloidigyne incognita (Meloidogyne incognita), Mj:M.javanica (javanese root knot nematode), Ma:M.arenaria (peanut root-knot nematode)

Listed reference is seen shown in the specification sheets end in the table 1.

Though forefathers have located these nematicide genes in capsicum, but have not yet to see the report of gene clone and sequence thereof, do not see that more using above-mentioned related gene cultivates transgenosis nematicide plant, wherein particularly cultivates the bibliographical information of transgenosis tomato of preventing root-knot Nematode disease.The just patent application " new root knot nematode (Meloidogyne) resistant gene and uses thereof " of the Japanese University of tsukuba of discovery in new patent searching (Chinese invention patent application number: mention the application of gene against meloidogyne in plant of Solanaceae (embodiment is Nicotiana gossei and potato transgenic plant) 02829029.1), but the present invention has following difference with it:

The first, gene and source thereof.Gene against meloidogyne in the patent of Japan University of tsukuba application derives from potato; And genjie eelworm resistance gene of the present invention derives from the capsicum genome, is a complete gene, and does not have great the contact with gene against meloidogyne in the patent formerly.

The second, range of application and method.Though mentioning in the patent of preceding application is the Solanaceae plant, does not mention tomato and this gene application method and the effect in tomato in specification sheets and embodiment; The present invention is then for being cloned into gene against meloidogyne from the capsicum genome, then it is used for the cultivation of transgenic plant of the anti-root knot nematode of tomato, and detailed preparation method and effect explanation are arranged.

Summary of the invention

The objective of the invention is to overcome the prior art defective, the clone obtains a genjie eelworm resistance gene from capsicum (Capsicum annuum L.) plant genome, obtain the transgenic Fructus Lycopersici esculenti plant of anti-root knot nematode by genetic transformation, to overcome the not characteristic of anti-root knot nematode of existing tomato plants, implement green cultivation, avoid helping reducing agriculture production cost simultaneously for a large amount of applying pesticides pollution on the environments of control root knot nematode.

The present invention is achieved in that

The present invention mainly is that the clone obtains a kind of genjie eelworm resistance gene from resistance capsicum genome, and its nucleotide sequence is shown in sequence table SEQ ID NO:1; Then this gene is imported to and do not have the plant of root knot nematode resistance, obtain the tomato of preventing root-knot Nematode disease transfer-gen plant as in the tomato variety to the root knot nematode sensitivity.

Techniqueflow of the present invention as shown in Figure 1.

1. the separating clone of gene of the present invention, called after Me

1.1 homologous sequence clone

Cloned genes sequence of the present invention is from the capsicum genome, according to having cloned at present four nematicide genes obtaining in the plant (referring to above-mentioned reference Hsl ^Pro-11., Mi 2., Gpa2 3., Hero is 4.) nucleotide binding site (Nucleotide binding site, NBS) and rich leucine repeat region (Leucine-rich repeat region, LRR) conserved sequence design pair of degenerate primers, with resistance capsicum genomic dna is template, pcr amplification, product reclaims, be cloned among the carrier pMD18T (available from Japanese TaKaRa company), sequence alignment analysis (http://www.ncbi.nlm.nih.gov/BLAST/) is carried out in order-checking (Bioisystech Co., Ltd finishes by Beijing AudioCodes) among the NCBI.

1.2Me the clone of gene

According to above order-checking comparison result, select tomato gene against meloidogyne sequence, carry out aminoacid sequence relatively, the design special primer, and adding the attB site core sequence of 12bp thereafter, through the two-wheeled pcr amplification, obtain to have the Me gene PCR product of attB site 25bp core sequence, the about 5.4kb of its size.

2.Me the application of gene

2.1 plant expression vector construction

After above-mentioned Me gene PCR reaction product reclaims, under the effect of B/P recombinase (available from American I nvitrogen company), carry out B/P reaction (carrying out) with carrier pDONR201 (available from American I nvitrogen company) with reference to Invitrogen company product description, obtain carrier pDAMe, order-checking (Bioisystech Co., Ltd finishes by Beijing AudioCodes), and carry out sequential analysis.Carrier pDAMe selects for use suitable restriction endonuclease (ApaI and NruI) enzyme to cut, target gene fragment is cloned among the plant binary conversion carrier pBI121 of excision gus gene, (this carrier has been preserved in Chinese typical culture collection center (CCTCC), preserving number: CCTCC-M205110) to obtain expression vector pBAMe.This carrier pBAMe structure is seen accompanying drawing 6, and building process as shown in Figure 2.

2.2 plant genetic transforms

Utilize the genetic transformation of Agrobacterium (bacterial strain number and detailed method of operation see embodiment) mediation, carrier pBAMe is changed in the plant such as tomato to the root knot nematode sensitivity, obtain transfer-gen plant with root knot nematode resistance.

2.3 Molecular Identification and inoculation detect

According to carrier T-DNA plot structure, at marker gene (as the NPTII gene) or goal gene (Me gene) design special primer, carry out pcr amplification respectively or/and Southern hybridization is identified, confirm to obtain the verity of anti-root knot nematode transgenic Fructus Lycopersici esculenti plant.When 25-30 days seedling ages, inoculate simultaneously two age root knot nematode larva such as Meloidogyne incognita (M.incognita), confirm the characteristic of the anti-root knot nematode of transfer-gen plant after 40-50 days.

Positively effect compared with prior art of the present invention is:

1, homologous sequence method clone gene is easier, rapid than additive method;

2, it is more economical, direct, effective to utilize transgenic method to obtain the more traditional breeding method of resistant plant, has solved the deficient problem of germ plasm resource, has shortened the breeding time limit greatly;

3, compare with the tradition method of preventing and treating, transgenosis tomato of preventing root-knot Nematode disease plant of the present invention does not need applying pesticides control root knot nematode, has both saved production cost, has protected environment again, is a kind of green agriculture technology.

Description of drawings

Fig. 1 is a techniqueflow chart of the present invention;

Fig. 2 is the vector construction flow process of one of them embodiment of the present invention;

Fig. 3 is degenerated primer pcr amplification result of the present invention;

Fig. 4 is gene against meloidogyne Me pcr amplification result of the present invention;

Fig. 5 is that the enzyme of the intermediate carrier pDAMe that makes up of the present invention is cut detected result

Fig. 6 is the plant expression vector pBAMe figure of the Me gene that makes up of the present invention;

Fig. 7 is that the enzyme of the expression vector pBAMe that makes up of the present invention is cut detected result;

Fig. 8 is that part T0 is for anti-root knot nematode transgenic Fructus Lycopersici esculenti plant PCR detected result among one of them embodiment of the present invention, and 1 is Marker, 2 positive contrasts, and 3 negative contrasts, 4-10 is a transfer-gen plant;

Fig. 9 is anti-root knot nematode transgenic Fructus Lycopersici esculenti plant Southern hybridization of a part T0 generation detected result among one of them embodiment of the present invention, and 1-22 is a transfer-gen plant, 23 negative contrasts;

Figure 10 is the root outward appearance with anti-root knot nematode characteristic of the transgenic Fructus Lycopersici esculenti plant of an embodiment among the present invention;

Figure 11 is the root outward appearance of the non-transgenic tomato sense root knot nematode disease of an embodiment among the present invention.

Embodiment

Below in conjunction with specific embodiment the present invention is made more detailed description.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment 1: the separating clone of Me gene of the present invention

Cloned genes of the present invention is from the capsicum genome, according to having cloned at present four nematicide genes obtaining in the plant (referring to above-mentioned reference Hsl ^Pro-11., Mi 2., Gpa2 3., Hero is 4.) nucleotide binding site (Nucleotide binding site, NBS) and rich leucine repeat region (Leucine-rich repeat region, LRR) conserved sequence design pair of degenerate primers (as described below), with resistance capsicum genomic dna is template, pcr amplification, and reaction system is 25 μ L, include 1 * PCR buffer, 1.5mmol/LMgCl ₂, 0.2mmol/L dNTPs, 0.2mmol/L forward and reverse primer, 1U Taq archaeal dna polymerase, 100ng template.The reaction cycle parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 50 ℃ of 1min, 72 ℃ of 90s, 5 circulations, 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 90s, 25 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.The PCR product reclaims, be connected in the pMD18T carrier, the thermal shock method is (referring to J. Sa nurse Brooker etc., " molecular cloning experiment guide ", the third edition, Jin Dongyan etc. translate, Science Press, 2002, Beijing) transformed into escherichia coli DH5 α, screening positive clone in the LB solid plate that contains 50mg/L ammonia benzyl mycin, 5 cloning and sequencings of picking (Bioisystech Co., Ltd finishes by Beijing AudioCodes), sequencing result shows that one of them clones long 493bp, and this fragment reaches 99% with the homology of the anti-root knot nematode Mi of the tomato of having reported gene.

The designed degenerated primer sequence of the present invention is as follows:

NBS-FW1：5’-TG(G/C)(G/C)(G/A)GG(T/A/C)(T/A)(T/C)(G/A)GG(T/C/G)AAAACTAC-3’；

NBS-RV1：5’-(T/A/C)(G/A)C(T/A)A(A/G)AGG(A/G/C)A(A/G)CCCT(T/C)(T/G/C)ACA-3’。

According to above order-checking comparison result, the sequence at the anti-root knot nematode Mi of the tomato two ends that selection has been reported, the design special primer, and adding the attB site core sequence of 12bp thereafter, in conjunction with the attB primer, with resistance capsicum genomic dna is template, through the two-wheeled pcr amplification, obtains to have the Me gene PCR product of attB site 25bp.

The positive anti-primer of amplification Me gene is respectively:

Me-FW：5′- AAAAAGCAGGCTTCCAATAGCTTCAACATTATT-3′，

Me-RV：5′- AGAAAGCTGGGTGTACTTATAAAAATTATATTTTTTG-3′，

The positive anti-primer of amplification attB is respectively:

attB FW：5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′，

attB RV：5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′。

The PCR reaction system is: 25 μ L volumes include 1 * PCR buffer, 2.0mM/LMgSO ₄, 0.2mmol/L dNTPs, 0.2pmol/L forward and reverse primer, 0.25U pfu enzyme, 0.5U Taq archaeal dna polymerase, 100ng template.The amplified reaction loop parameter is: 94 ℃ of pre-sex change 5min, and 94 ℃ of 30s then, 49 ℃ of 1min, 72 ℃ of 7min, 10 circulations, last 72 ℃ are extended 10min.4 ℃ of preservations.Get the above reaction product of 5 μ L, utilize the attB primer to carry out second and take turns amplification.Reaction system: in 50 μ L reaction systems, add 1 times of PCR buffer respectively, 2.0mmol/LMgSO ₄, 0.2mmol/L dNTPs, 0.2mmol/L forward and reverse primer, the 0.5Upfu enzyme, 1.0U Taq archaeal dna polymerase, template is 5 μ L first round PCR products.The reaction cycle parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 45 ℃ of 1min, 72 ℃ of 6min30s react 5 circulations, 94 ℃ of 30s then, 55 ℃ of 1min, 72 ℃ of 6min30s react 15 circulations, and last 72 ℃ are extended 10min, 4 ℃ of preservations.The PCR product reclaims.Under the effect of B/P recombinase, reclaim product and carry out B/P reaction (carrying out) according to American I nvitrogen company product description with justice expression site recombinant vectors pDONR201, obtain carrier pDAMe, order-checking (Bioisystech Co., Ltd finishes by Beijing AudioCodes).The result shows, the capsicum genjie eelworm resistance gene Me total length 5447bp that the present invention clone obtains contains two introns and two exons, the long 3950bp of mRNA, the open reading frame that comprises 3656bp in the sequence, the 3 ' non-translational region of 86bp 5 ' non-translational region and 104bp.Anti-root knot nematode Mi compares with the tomato of having reported, and two introns of Me gene vary in size, and the Mi gene intron is respectively 1306bp and 75bp, and the Me gene intron then is respectively 1182bp and 315bp; In addition, the amino acid of two genes encodings varies in size, 1257 amino acid of Mi genes encoding, Me gene 1251 amino acid of then encoding; Also have, with respect to the Mi gene, the Me gene has one section insertion sudden change from the common 171bp of 1937 bases of the 1767th base to the, and the deletion mutantion of 189bp altogether of 2989 base places of the 2801st base to the.

Embodiment 2, plant conversion carrier make up

Plasmid pDAMe is cut through ApaI and NruI enzyme, and it is flat to utilize T4 DNA Polymerase to mend then, reclaims big fragment; Simultaneously, plasmid pBI121 cuts through BamHI and SacI enzyme, remove the gus gene fragment, it is flat to utilize T4 DNA Polymerase to mend, use acid phosphatase lipase CIAP dephosphorylation again, reclaim big fragment after the gel electrophoresis, be connected under the effect of T4 dna ligase with preceding recovery product, connect product transformed into escherichia coli bacterial strain DH5 α, screening positive clone in containing the LB solid plate of 50mg/L kantlex, the extracting plasmid carries out that enzyme is cut and PCR identifies, obtains to contain to insert the segmental recombinant clone of purpose, called after pBAMe.Use electrization (referring to J. Sa nurse Brooker etc., " molecular cloning experiment guide ", the third edition, Jin Dongyan etc. translate, Science Press, 2002, Beijing) pBAMe is imported to Agrobacterium LBA4404 (reference: Sakae S.And Masaru N. (2002) Agrobacterium-Mediated Transformationin Liliaceous Ornamental Plants JARQ 36 (3), 119-127) in.The bacillus coli DH 5 alpha that contains this plasmid pBAMe is deposited in Chinese typical culture collection center (CCTCC), deposit number: CCTCC-M205110.Plant expression vector pBAMe of the present invention makes up flow process to be seen shown in the accompanying drawing 2.

Embodiment 3, plant genetic transform

Utilize electrization that plasmid pBAMe is imported among the Agrobacterium LBA4404, transformation traits is stable and to the tomato variety " middle vegetables No. five " (buying from commercial variety) of root knot nematode sensitivity, by Molecular Identification and pest-resistant screening and selfing purifying, obtain the transfer-gen plant or the strain system of isozygotying.

Agrobacterium tumefaciens mediated tomato genetic transformation step is as follows:

1, aseptic seedling and tobacco cell are cultivated

Aseptic seedling is cultivated: with tomato variety " middle vegetables No. five " seed with 70% alcohol disinfecting 1min, 15min then sterilizes in 20% clorox (containing available chlorine 6%), sterile purified water flushing 3 times, seed is inoculated into 1/2 MS minimum medium (referring to Murashige T.andF.Skoog.Physiol.Plant, 1962,15:473-497) in, in 25 ± 2 ℃ of dark places until seed germination, then in intensity of illumination 1800 lx, cultivate under the dark photoperiod condition of 16h light/8h.

Tobacco cell is cultivated: tobacco cell NT1 is suspension culture in KCMS-L substratum (seeing Table 2), weekly 2: 48 by volume dilution subcultures.

2, explant is prepared and the Agrobacterium cultivation

Prepare tobacco nurse substratum KCMS-S (seeing Table 2) the day before yesterday at the separation explant, the tobacco suspension cell of having grown 7 days is drawn 1.5ml transfer on the KCMS-S substratum, cover the sterilization filter paper of a diameter 7cm, incubated overnight is standby in 25 ℃ of dark.

Explant is prepared: behind " middle vegetables No. five " seed germination 6 days, and the cotyledon of aseptic seedling to be cut or downcut with blade, cotyledon has a bit of petiole, is inoculated in pre-the cultivation 1 day in the KCMS-S substratum.

Agrobacterium is cultivated: contain in the LB substratum of 50mg/L kantlex in 28 ℃ of 150 rev/mins of incubated overnight to mid-log phase O.D 1.0 in 10ml containing the single colony inoculation of Agrobacterium LBA4404 that picking on the LB flat board of 50mg/L kantlex contains pBAMe.

3, transformation tissue culture

" middle vegetables No. five " cotyledon explant of 3～4 wares is transferred in the 50ml sterilization triangular flask, infect 5min through the pBAMe Agrobacterium LBA4404 liquid inoculation that contains that is diluted to O.D=0.2～0.3 with MS0.2 (seeing Table 2), and shake triangular flask gently, remove agrobacterium liquid with the suction pipe suction, explant is transferred on the sterilization filter paper, blot residual bacterium liquid, tieback is to the KCMS-S substratum, in 22 ℃ of common cultivations of carrying out 1～2 day; After the cultivation explant is carefully changed on the MRS1 substratum (seeing Table 2).Through cultivating in 1～2 week, here most of cotyledon colour-darkenings, blade wither and bleach gradually; Some cotyledon base portion also can form the callus of a small amount of white loose, but finally can die in succeeding transfer culture; Have only the base portion of minority cotyledon can grow the callus of a small amount of densification gradually and slowly transfer green to, just can be differentiated to form the budlet point after about 3～4 weeks, this explant that has budlet point is changed in the MRS2 substratum (seeing Table 2) of improvement.This moment, budlet point differentiated spire gradually, formed budlet, but also had budlet point only can differentiate one, two leaf, did not have normal vegetative point, can not normal growth.Generally can only form a budlet on the callus, many persons also can differentiate 3～4 buds." middle vegetables No. 5 " this genotype is carried out the transformation frequency statistics to be about about 10%.Unconverted contrast cotyledon explant (Wt) can not break up and grow on selective medium, and cotyledon very rewind down is green, and is last dead.And the cotyledon explant on the non-selective bud division culture medium that does not promptly have a kantlex screening can sprout in the high frequency differentiation, and differentiation frequency can reach 98%.

Select in time to dispose the material that Agrobacterium pollutes in the culturing process.

4, regeneration bud rooting and transplant

When treating that regeneration bud grows to the 1cm left and right sides, RS substratum (seeing Table 2) is put in the bud cutting-out taken root, 2 all backs are good to taking root, and the conversion seedling that grows to about 5cm carries out acclimatization and transplants to disposal plastic cup, survive the extremely big Tanaka of back transplanting.

The table 2 used culture medium prescription of tomato genetic transformation that is used for of the present invention

The substratum code name	Purposes	Prescription
			1/2MS MRS1 MRS2 RS MS0.2 KCMS-L KCMS-S	The selective regeneration of the selective regeneration of the Aseptic seedling culture resuspended tobacco suspension cell of Agrobacterium of selectively taking root is cultivated nurse and is cultivated	The inorganic salts of 1/2MS minimal medium and vitamin+15g/L sucrose+7.5g/L agar; Add water to 1 liter; Lower inorganic salts and the inorganic salts of the inorganic salts of the inorganic salts of the inorganic salts of vitamin+30g/L sucrose+2.0 mg/L zeatin+100mg/L indole-3-acetic acid+100mg/L kanamycins+200mg/L cephalosporin+7.5g/L agar MS minimal medium and vitamin+30g/L sucrose+0.2mg/L zeatin+1.0mg/L indole-3-acetic acid+100mg/L kanamycins+200mg/L cephalosporin+7.5g/L agar 1/2 MS minimal medium+1.0mg/L indolebutyric acid+3% sucrose+7.5g/L agar MS minimal medium+2% sucrose (fluid nutrient medium) MS minimal medium+20mg/L inositol 1+0.65 mg/L aneurin+0.2mg/L 2 with the MS minimal medium, 4-dichlorphenoxyacetic acid+100mg/L potassium dihydrogen phosphate+0.1mg/L kinetin (KT)+3% sucrose KCMS-L+7.5g/L agar

Annotate: it is outside 5.5 that above substratum is removed name from the rolls the pH that is called the KCMS substratum, and the pH of all the other substratum is 5.8.

The Molecular Identification of embodiment 4, transformed plant

Utilize the CTAB method (to see Fulton, T.M etc., Microprep protocol for extraction of DNA from tomato and otherherbaceous plants., Plant Moleculor Biology Reporter, 1995, (13): 207-209) extract transgenic Fructus Lycopersici esculenti plant leaf DNA to be identified.Get the 0.5-1g young leaflet tablet in 1.5ml Eppendorf pipe, mash rapidly, add 700 μ l and extract damping fluid (100mmol/l Tris-HCl, pH=8.0,1.4mol/l NaCl, 20mmol/lEDTA, 2%CTAB, 0.1-2% mercaptoethanol) 65 ℃ of water-bath 30min, the centrifugal 5min of 12000r/m, get supernatant liquor,, add ice-cold equal-volume Virahol with chloroform isoamyl alcohol (24: 1 by volume) extracting 1 time, shake up, leave standstill, the centrifugal 6min of 12000r/m, precipitation is handled the back and is preserved standby with the washing of 75% alcohol, drying, TE dissolving, RNase.With the special primer of goal gene Me, marker gene NPTII, identify respectively by pcr amplification.Reaction system: in 25 μ L reaction systems, add 1 times of PCR buffer respectively, 1.5mmol/L MgCl ₂, 0.2mmol/L dNTPs, 0.2mmol/L forward and reverse primer, 1U Taq archaeal dna polymerase, the 100ng template adds ddH ₂O to 25 μ L.The reaction cycle parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min30s react 35 circulations, and last 72 ℃ are extended 10min, 4 ℃ of preservations.1% detected through gel electrophoresis.Or carry out Southern Blot (referring to J. Sa nurse Brooker etc., " molecular cloning experiment guide ", the third edition, Jin Dongyan etc. translate, Science Press, 2002, Beijing) Molecular Identification with the specific probe of Me, NPTII gene fragment.Goal gene on the pcr amplification pBAMe carrier and (or) the NPTII fragment is template, adopt random priming (referring to J. Sa nurse Brooker etc., " molecular cloning experiment guide ", the third edition, Jin Dongyan etc. translate, Science Press, 2002, Beijing) (α- ³²P) dCTP label probe.Getting 20 μ g plants DNA of plants Bam HI enzyme and cuts and spend the night 1% agarose gel electrophoresis 12h.Transfer to nylon membrane, prehybridization, hybridization is in-70 ℃ of radioautograph 7 days, X-ray sheet developing, photographic recording result.Qualification result shows that the Me gene is inserted in the genome of tomato with different copy numbers, has obtained transfer-gen plant.PCR and Southern qualification result are shown in Fig. 8,9.

The evaluation of resistance of embodiment 5, transfer-gen plant

Inoculation method: the tomato transfer-gen plant is copied 6 parts by vegetative method the present age, and a copy of it is used to reserve seed for planting, all the other the 5 parts evaluation of resistance experiments that are used for transfer-gen plant.Non-transgenic plant to the root knot nematode sensitivity " the vegetables No. five " field planting that will be used for the tomato transfer-gen plant of anti-root knot nematode experiment and be used for negative control is at diameter 25cm, in the flowerpot of high 30cm, transplant a strain tomato in each flowerpot, disinfectant matrix (peat soil: vermiculite: perlite=2: 2: 1) is housed in the flowerpot.After plant survived, every basin was inoculated about 3000 two Meloidogyne incognitas in age (M.incognita) larva.Afterwards, carry out the normal rich water quality management and the ground prevention and control of plant diseases, pest control.Temperature is controlled at 20～30 ℃ in the greenhouse.After 2 months plant is uprooped, the small-mouthed jar of use 0.05% is red after the washing to the greatest extent from the beginning dyes to the root knot nematode pieces of an egg, and this moment, pieces of an egg can be dyed blue look, made evaluation of resistance whether there to be the quantity of pieces of an egg and pieces of an egg on the root.The resistance grade scale that adopts is: 0 grade, do not have pieces of an egg on the root system; 1 grade, 1～5 pieces of an egg is arranged; 2 grades, 6～20 pieces of an egg are arranged; 3 grades, 21～50 pieces of an egg are arranged; 4 grades, 51～100 pieces of an egg are arranged; 5 grades, the pieces of an egg number is above 100.Wherein, 0～2 grade is disease-resistant level (R), and 3～5 grades is to root knot nematode sensitivity (S).Qualification result shows that the present invention has obtained Meloidogyne incognita is had the transgenic Fructus Lycopersici esculenti plant (seeing Table 3) of good resistance, and this transgenic Fructus Lycopersici esculenti plant heritability is stable, physically well develops, and can be used as the germ plasm resource of the anti-root knot nematode new variety of seed selection.The root of transgenosis tomato of preventing root-knot Nematode disease of the present invention and contrast (sense root knot nematode) are that the mode of appearance of root of non-transgenic tomato variety " middle vegetables No. five " is shown in Figure 10,11.Transfer-gen plant among Figure 10 shows as on the root no pieces of an egg or pieces of an egg seldom, is typical case's performance of anti-root knot nematode, the non-transgenic tomato variety among Figure 11 then severe infections Meloidogyne incognita, all found many root knots at whole root.

Table 3 commentaries on classics of the present invention Me gene tomato of preventing root-knot Nematode disease plant resistance is analyzed

The transformed plant numbering	The root knot number	Root knot progression	Resistance	Southern hybridization determines to introduce the number of gene
					ZS1 ZS3 ZS6 ZS12 ZS15 ZS19 ZS18 ZS33 adds 1 and adds 4 and add 10 and add 11 ZSCK and add CK	6±6 4±8 51±12 2±2 4±5 1±2 0±0 58±57 1±1 55±17 2±4 27±7 ＞100 ＞100	2 1 4 1 1 1 0 4 1 4 1 3 5 5	R R S R R R R S R S R S S S	6 1 5 1 2 1 1 7 1 4 1 3

Annotate: the pieces of an egg number is the mean value ± standard error of pieces of an egg number on 5 vegetative propagation plant roots.The grade scale that adopts: 0 grade, do not have pieces of an egg on the root system; 1 grade, 1～5 pieces of an egg is arranged; 2 grades, 6～20 pieces of an egg are arranged; 3 grades, 21～50 pieces of an egg are arranged; 4 grades, 51～100 pieces of an egg are arranged; 5 grades, the pieces of an egg number is above 100.Wherein, 0～2 grade is disease-resistant level (R), and 3～5 grades is susceptible level (S)

The application benefit evaluation of embodiment 6, tomato of preventing root-knot Nematode disease transgenic strain

Domestic and international at present control root knot nematode method the most widely is to use methyl bromide fumigation soil.Monobromethane is a kind of deadly poisonous compound, not only environment is polluted, and has also increased production cost.And cultivation tomato of preventing root-knot Nematode disease kind not only can not pollute environment, meets the requirement that green agriculture is produced, but also can save production cost, and improves Tomato Quality, makes peasant's increasing both production and income.The present invention utilizes the non-transgenic tomato variety " middle vegetables No. five " to the root knot nematode sensitivity to be contrast, new-create transgenosis tomato of preventing root-knot Nematode disease strain ZS12 has been carried out the evaluation studies of economic benefit.Concrete test design is: the sub-district area is 8.0m * 1.2m, the duplicate rows plantation, and every row 15 strains, seeding row spacing is 50cm and 55cm.Four processing: 1. without the soil cultivation transgenosis tomato of preventing root-knot Nematode disease strain ZS12 of methyl bromide fumigation; 2. through the soil cultivation transgenosis tomato of preventing root-knot Nematode disease strain ZS12 of methyl bromide fumigation; 3. without the anti-non-transgenic tomato variety " middle vegetables No. five " of the soil cultivation of methyl bromide fumigation to the root knot nematode sensitivity; 4. through the anti-non-transgenic tomato variety " middle vegetables No. five " of the soil cultivation of methyl bromide fumigation (monobromethane is built new chemical industry company limited available from Linhai City, and concentration is 98%, liquid, 20 yuan/kilogram, consumption is 75 gram/square metre soil) to the root knot nematode sensitivity.Planting respectively in gardening forestry institute of Hua Zhong Agriculture University vegetables research and teaching practice base spring in 2004 and 2005 has in the heliogreenhouse of root knot nematode other cultivation condition unanimities.Add up tomato yield in whole production in cycle, and calculate its output value according to the average price of tomato then.Be used in combination the expense (1.5 yuan/square metre) of monobromethane simultaneously, estimate the using value of cultivation transgenosis tomato of preventing root-knot Nematode disease, as shown in table 4.

The application benefit evaluation of table 4 tomato of preventing root-knot Nematode disease transfer-gen plant of the present invention

Vegetables No. five (S) in the contrast of tomato variety ZS12 of the present invention (R) mean value	Time 2004 2,005 2,004 2005	Tomato yield (Kilograms Per Square Meter)	The output value (Renminbi, unit/square metre)
				Methyl bromide fumigation does not have methyl bromide fumigation 9.1 ± 0.8 9.0 ± 1.0 8.9 ± 0.9 8.8 ± 0.9 9.0 ± 0.9 8.9 ± 1.1 8.5 ± 1.0 5.3 ± 1.1 8.1 ± 1.0 4.9 ± 1.2	Methyl bromide fumigation does not have methyl bromide fumigation 12.02 11.88 14.96 14.78 13.50 13.34 13.22 7.00 13.60 8.24
		Mean value				8.3±1.3 5.1±1.2	13.40 7.62 **

(R) represent anti-root knot nematode, (S) expression is to the root knot nematode sensitivity.The data of output are the mean value ± standard error of 30 strain tomato yields in each sub-district in the table.The expense of the expense of monobromethane is 1.50 yuan/square metre.The average price of tomato in 2004 and 2005 is respectively 1.32,1.68 yuan/kilogram of Renminbi. ^*The newly multiple differential detection P of Deng Kenshi≤0.01.

As known from Table 4, cultivation tomato of preventing root-knot Nematode disease kind has or not methyl bromide fumigation, and is not remarkable to the influence of its output, but utilizes the production cost of every square metre of increase of methyl bromide fumigation to be 1.5 yuan of Renminbi.And susceptible variety " middle vegetables No. five " is not having under the situation of methyl bromide fumigation, and output descends significantly, reaches utmost point conspicuous level, has seriously influenced the productivity effect of tomato.

The reference of table 1

1.Fery R L， Thies J A. Genetic analysis of Resistance to the southern root-knot nematode in Capsicumchinense Jacq.J.Amer.Soc.Hort.Sci.，1998.123(6)：1008-1011.

1.Fery R L.The inheritance of resistance to the southern root-knot nematode in‘Carolina Hot’cayennepepper.J.Amer.Soc.Hort.Sci.，1996，121(6)：1024-1027.

2.Hare W W. Inheritance of resistance to root-knot nematodes in pepper.Phytopathology，1957，47：455-459.

3.Hendy H，Pochard E，Dalmasso A.Transmission hereditaire de Ia resistance aux nematodesMelooidigyne Chitwood (Tylenchida) portee par 2 lignees de Capsicum annuum L.：etude dedescendances homozygotes issues d’and rogenese.Agronomie，1985，5(2)：93-100

4. Vito M D，Saccardo F，Errico A，et al. Genetics of resistance to root-knot nematodes (Meloidogyne spp.)in Capsicum chacoens.e，C.chinense and C.frutescens.J.Genet. & Breed，1993，47：23-26

5. Vito M D，Saccardo F.Resistance of pepper to root-knot nematode (Meloidogyne spp.)：present andfuture. D N Maynard ed，Proceeding of The National Pepper Conference，December 8-11，1996：55-56.

Sequence table

SEQUENCE LISTING

＜110〉Hua Zhong Agriculture University

＜120〉clone of gene against meloidogyne of capsicum and application thereof

<130>

<141>2005-10-14

<160>2

<170>PatentIn version 3.1

<210>1

<211>5447

<212>DNA

＜213〉capsicum (Capsicum annuum)

<220>

<221>Intron

<222>(64)..(1245)

<223>

<220>

<221>primer_bind

<222>(5426)..(5447)

<223>

<220>

<221>gene

<222>(1)..(5447)

<223>

<220>

<221>mutation

<222>(1767)..(1937)

<223>

<220>

<221>mutation

<222>(2801)..(2989)

<223>

<220>

<221>primer_bind

<222>(1)..(21)

<223>

<220>

<221>CDS

<222>(1269)..(1312)

<223>

<220>

<221>CDS

<222>(1628)..(5336)

<223>

<220>

<221>Intron

<222>(1313)..(1627)

<223>

<400>1

tccaatagct tcaacattat ttctcaaaca aagggttctc tagctaaact tcagcctgtg 60

taaaggtaac atcttcttta ttcacagcat aataacaatg aatttggtcg atgtttgaag 120

taagcttgaa attttctctt tctaagtttg tttgatccat ttagattctt ttaaatactt 180

ttggtattta aaggacttgt gaagtcaatg aattgtattt tagtaatctt gcaattctag 240

atctagctat ttgttgttct cctttcaacc aaactacttc ttcaatttgt ctaacaaaaa 300

tatgtcaaaa aggtgacatt ttactctagt tttttatcaa aataagggaa taatatcctg 360

ttatttaact accttttaag cattatgggt ggaaagtaga aagaagaaac ataacagaac 420

agacagtaag ttatgcttta atgagtagat ctgtatagga ttacatattt gtttgacttt 480

tcggtgtttc gattagaaaa cttacaagtt tttaatacac gtatcatttg ttgatttgtc 540

cgtttggcac gtcatctgtg gttacaagtc acatatgaag tatgtccacg agacacaccg 600

aatgtcaagt atagatttct acttgatcat acacaacttt atctgaggtt gatgccaaat 660

ttaaatgact acctaaagct gatattttaa acattaatct tgtacacgaa aacattattc 720

ctattactgt tttctttacc tttaccttat agactttttt ggcagaaaaa agttagacag 780

agacatttga tgatgtttac cattctcatt ctctctttat tttattttct ttacattcac 840

acgcacaata attttcttgt aggttcctta tatgccatat gcacatagac gaatctagga 900

tttgagattt acaagtttct atgtcgacgt catattaata tcaataataa ttagattgac 960

aatcacatgt ttataatatt aagtcgataa ctttcttctt tgtataggtt ggaaaagtaa 1020

tggtaaacga gcaggactcc tttttctttt ttttgtaaat aattaacagt cgtgagattt 1080

tatgtttgtg acttcatgtc ataaacattt tgatgtgtga ttaagattga catttccaat 1140

tgtgcgagtc taaaattact atatgtgaaa atagtgatat tattgattat tcgtattttt 1200

tcatcttctt tctcctgtta aagttttatc tactttttat tcatcaggtc ttgagaaaaa 1260

gtagaatc atg gaa aaa cga aaa gat att gaa gaa gca aac aac tca ttg 1310

Met Glu Lys Arg Lys Asp Ile Glu Glu Ala Asn Asn Ser Leu

1 5 10

gt atgttattta gtggcaacaa acgaggattt tcagacgcta tggatggatt 1362

Val

ctcagagggg aagtttctgt cgaattccgg tgtgaaagca ggtgatacaa aggagacctc 1422

acgtgtgcaa ccacctaaaa tgaaagatgc taatactcag agtacagctc cagagaggcc 1482

ttctgctgtg aatgatgcct caaaccgtgc gggcagtggt gcccctgcta caaaggcaca 1542

ggttgttggt tggccacttt atagagtaaa ctgtaaagta ttgaattata gatatgtggc 1602

tttaaaatgt attattttgg caggt g tta ttt tct gct ctt agc aag gac att 1655

Leu Phe Ser Ala Leu Ser Lys Asp Ile

20

gcc aat gtt cta att ttc cta gag aat gag gaa aat caa aaa gct ctt 1703

Ala Ash Val Leu Ile Phe Leu Glu Ash Glu Glu Asn Gln Lys Ala Leu

25 30 35 40

gac aaa gat caa gtt gaa aag cta aaa ttg aaa atg gca ttt att tgt 1751

Asp Lys Asp Gln Val Glu Lys Leu Lys Leu Lys Met Ala Phe Ile Cys

45 50 55

aca tat gtt cag ctt gcc aag gag tgt cgc gat gca ata ggt act ata 1799

Thr Tyr Val Gln Leu Ala Lys Glu Cys Arg Asp Ala Ile Gly Thr Ile

60 65 70

aac ctt gtg aag ggc cag cat tta gac aga agg acc act aat caa ttg 1847

Asn Leu Val Lys Gly Gln His Leu Asp Arg Arg Thr Thr Ash Gln Leu

75 80 85

gag gat gct ata aag cac cta aca cat gtt gct gta ttt ctc aca aat 1895

Glu Asp Ala Ile Lys His Leu Thr His Val Ala Val Phe Leu Thr Asn

90 95 100

ctg gag aag cgt cac cct gct aat gga ata tct ata cat ctt tct tat 1943

Leu Glu Lys Arg His Pro Ala Asn Gly Ile Ser Ile His Leu Ser Tyr

105 110 115 120

tcc gat ttt gag cag ttt gaa gat ata atg act aga aat aga caa gag 1991

Ser Asp Phe Glu Gln Phe Glu Asp Ile Met Thr Arg Asn Arg Gln Glu

125 130 135

gtt gag aat ctg ctt caa tca ctt ttg gat gat gat gtc ctt act agc 2039

Val Glu Asn Leu Leu Gln Ser Leu Leu Asp Asp Asp Val Leu Thr Ser

140 145 150

ctc acc agt aat atg gat gac tgt atc agc ttg tat cat cgt tct tat 2087

Leu Thr Ser Asn Met Asp Asp Cys Ile Ser Leu Tyr His Arg Ser Tyr

155 160 165

aaa tca gat gcc atc atg atg gat gag caa ttg gac ttc ctc ctc ttg 2135

Lys Ser Asp Ala Ile Met Met Asp Glu Gln Leu Asp Phe Leu Leu Leu

170 175 180

aat ctg tat cat cta tcc aag cat cac gct gaa aag ata ttt cct gga 2183

Asn Leu Tyr His Leu Ser Lys His His Ala Glu Lys Ile Phe Pro Gly

185 190 195 200

gtg act caa tat gaa gtt ctt cag aat gta tgt ggc aac ata aga gat 2231

Val Thr Gln Tyr Glu Val Leu Gln Asn Val Cys Gly Asn Ile Arg Asp

205 210 215

ttc cat ggg ttg ata ctg aat ggt tgc att aag cat gag atg gtt gag 2279

Phe His Gly Leu Ile Leu Asn Gly Cys Ile Lys His Glu Met Val Glu

220 225 230

aat gtc tta cct ctg ttt caa ctc atg gct gaa aga gta gga cac ttc 2327

Asn Val Leu Pro Leu Phe Gln Leu Met Ala Glu Arg Val Gly His Phe

235 240 245

ctt tgg gag gat cag act gat gaa gac tct cgg ctc tcc gag cta gat 2375

Leu Trp Glu Asp Gln Thr Asp Glu Asp Ser Arg Leu Ser Glu Leu Asp

250 255 260

gag gat gaa cac aat gat aga gac tct cga ctc ttc cag cta aca cat 2423

Glu Asp Glu His Asn Asp Arg Asp Ser Arg Leu Phe Gln Leu Thr His

265 270 275 280

cta ctc ttg aag att gtt cca act gaa ctg gag gtt atg cac ata tgt 2471

Leu Leu Leu Lys Ile Val Pro Thr Glu Leu Glu Val Met His Ile Cys

285 290 295

tat aca aat ttg aaa gct tca act tca gca gaa gtt gga cgc ttc att 2519

Tyr Thr Asn Leu Lys Ala Ser Thr Ser Ala Glu Val Gly Arg Phe Ile

300 305 310

aag aag ctc ctg gaa acc tca ccg gat att ctc aga gaa tat atc att 2567

Lys Lys Leu Leu Glu Thr Ser Pro Asp Ile Leu Arg Glu Tyr Ile Ile

315 320 325

caa cta caa gag cat atg tta act gtt att ccc cct agc act tta ggg 2615

Gln Leu Gln Glu His Met Leu Thr Val Ile Pro Pro Ser Thr Leu Gly

330 335 340

gct cga aac att cat gtc atg atg gaa ttc cta tta ctt att ctt tct 2663

Ala Arg Asn Ile His Val Met Met Glu Phe Leu Leu Leu Ile Leu Ser

345 350 355 360

gat atg ccc aag gac ttt att cat cat gac aaa ctt ttt gat ctc ttg 2711

Asp Met Pro Lys Asp Phe Ile His His Asp Lys Leu Phe Asp Leu Leu

365 370 375

gct cat gtt gga aca ctt acc agg gag gta tcg act ctt gta cgt gac 2759

Ala His Val Gly Thr Leu Thr Arg Glu Val Ser Thr Leu Val Arg Asp

380 385 390

ttg gaa gag aaa tta agg aat aaa gag ggt aat aac caa aca aat tgt 2807

Leu Glu Glu Lys Leu Arg Asn Lys Glu Gly Asn Asn Gln Thr Asn Cys

395 400 405

gca acc cta gac ttg ctg gaa aat att gaa ctc ctc aag aaa gat ctc 2855

Ala Thr Leu Asp Leu Leu Glu Asn Ile Glu Leu Leu Lys Lys Asp Leu

410 415 420

aaa cat gtt tat ctg aaa gcc cca aat tca tct caa tgt tgc ttc ccc 2903

Lys His Val Tyr Leu Lys Ala Pro Asn Ser Ser Gln Cys Cys Phe Pro

425 430 435 440

atg agt ggt gga cca ctc ttc atg cat ctt cta cac atg cac tta aat 2951

Met Ser Gly Gly Pro Leu Phe Met His Leu Leu His Met His Leu Asn

445 450 455

gat ttg cta gat tct aat gct ctt att ttc tca ctt ccc att acc ata 2999

Asp Leu Leu Asp Ser Asn Ala Leu Ile Phe Ser Leu Pro Ile Thr Ile

460 465 470

aag aag atc aaa ctt atc aaa gaa gag atc tct gct tta gat gag aac 3047

Lys Lys Ile Lys Leu Ile Lys Glu Glu Ile Ser Ala Leu Asp Glu Asn

475 480 485

att ccc aag gac aga ggt cta atc gtt gtg aac tct ccc aag aaa cca 3095

Ile Pro Lys Asp Arg Gly Leu Ile Val Val Asn Ser Pro Lys Lys Pro

490 495 500

gtt gag aga aag tca ttg gca act gat aaa ata att gta ggt ttt gag 3143

Val Glu Arg Lys Ser Leu Ala Thr Asp Lys Ile Ile Val Gly Phe Glu

505 510 515 520

gag gag aca aac ttg ata ctt aga aag ctc acc agt gga ccc gca gat 3191

Glu Glu Thr Asn Leu Ile Leu Arg Lys Leu Thr Ser Gly Pro Ala Asp

525 530 535

tta gat gtc att tcg atc acc ggt atg ccg ggt tca ggt aaa act act 3239

Leu Asp Val Ile Ser Ile Thr Gly Met Pro Gly Ser Gly Lys Thr Thr

540 545 550

ttg gca tac aaa gta tac aat gat aag tca gtt tct aga cat ttt gac 3287

Leu Ala Tyr Lys Val Tyr Asn Asp Lys Ser Val Ser Arg His Phe Asp

555 560 565

ctt cgt gca tgg tgc acg gtc gat caa gga tat gac gac aag aag ttg 3335

Leu Arg Ala Trp Cys Thr Val Asp Gln Gly Tyr Asp Asp Lys Lys Leu

570 575 580

ttg gat aca att ttc agt caa gtt agt ggc tca gat tca aat ttg agt 3383

Leu Asp Thr Ile Phe Ser Gln Val Ser Gly Ser Asp Ser Asn Leu Ser

585 590 595 600

gag aat att gat gtt gct gat aaa ttg cgg aaa caa ctg ttt gga aag 3431

Glu Asn Ile Asp Val Ala Asp Lys Leu Arg Lys Gln Leu Phe Gly Lys

605 610 615

agg tat ctt att gtc tta gat gat gtg tgg gat act act aca ttg gat 3479

Arg Tyr Leu Ile Val Leu Asp Asp Val Trp Asp Thr Thr Thr Leu Asp

620 625 630

gag ttg aca aga cct ttt ccc gaa gct aag aaa gga agt agg att att 3527

Glu Leu Thr Arg Pro Phe Pro Glu Ala Lys Lys Gly Ser Arg Ile Ile

635 640 645

ttg aca act cga gaa aag gaa gtg gct ttg cat gga aag ctg aac act 3575

Leu Thr Thr Arg Glu Lys Glu Val Ala Leu His Gly Lys Leu Asn Thr

650 655 660

gat cct ctt gac ctt cga ttg cta aga cca gat gaa agt tgg gaa ctt 3623

Asp Pro Leu Asp Leu Arg Leu Leu Arg Pro Asp Glu Ser Trp Glu Leu

665 670 675 680

tta gag aaa agg aca ttt ggt aat gag agt tgc cct gat gaa cta tta 3671

Leu Glu Lys Arg Thr Phe Gly Asn Glu Ser Cys Pro Asp Glu Leu Leu

685 690 695

gat gtc ggt aaa gaa ata gcc gaa aat tgt aaa ggg ctt cct ttg gtg 3719

Asp Val Gly Lys Glu Ile Ala Glu Asn Cys Lys Gly Leu Pro Leu Val

700 705 710

gct gat ctg att gct gga gtc att gct ggg agg gaa aag aaa agg agt 3767

Ala Asp Leu Ile Ala Gly Val Ile Ala Gly Arg Glu Lys Lys Arg Ser

715 720 725

gtg tgg ctt gaa gtt caa agt agt ttg agt tct ttt att ttg aac agt 3815

Val Trp Leu Glu Val Gln Ser Ser Leu Ser Ser Phe Ile Leu Asn Ser

730 735 740

gaa gtg gaa gtg atg aga gtt ata gaa tta agt tat gac cat tta cca 3863

Glu Val Glu Val Met Arg Val Ile Glu Leu Ser Tyr Asp His Leu Pro

745 750 755 760

cat cac ctc aag cca tgc ttg ctt cac ttt gca agt tgg ccg aag gac 3911

His His Leu Lys Pro Cys Leu Leu His Phe Ala Ser Trp Pro Lys Asp

765 770 775

act cct ttg aca atc tat ttg ttg act gtt tat ttg ggt gct gaa gga 3959

Thr Pro Leu Thr Ile Tyr Leu Leu Thr Val Tyr Leu Gly Ala Glu Gly

780 785 790

ttt gtg gaa aag acg gag atg aag ggt ata gaa gaa gtg gtg aag att 4007

Phe Val Glu Lys Thr Glu Met Lys Gly Ile Glu Glu Val Val Lys Ile

795 800 805

tat atg gat gat tta att tcc agt agc ttg gta att tgt ttc aat gag 4055

Tyr Met Asp Asp Leu Ile Ser Ser Ser Leu Val Ile Cys Phe Asn Glu

810 815 820

ata ggt gat ata ctg aat ttc caa att cat gat ctt gtg cat gac ttt 4103

Ile Gly Asp Ile Leu Asn Phe Gln Ile His Asp Leu Val His Asp Phe

825 830 835 840

tgt ttg ata aaa gca aga aag gaa aat ttg ttt gat cgg ata 8ga tca 4151

Cys Leu Ile Lys Ala Arg Lys Glu Asn Leu Phe Asp Arg Ile Arg Ser

845 850 855

agt gct cca tca gat ttg ttg cct cgt caa att acc att gat tat gat 4199

Ser Ala Pro Ser Asp Leu Leu Pro Arg Gln Ile Thr Ile Asp Tyr Asp

860 865 870

gag gag gag gag cac ttt ggg ctt aat ttt gtc atg ttc gat tca aat 4247

Glu Glu Glu Glu His Phe Gly Leu Asn Phe Val Met Phe Asp Ser Asn

875 880 885

aag aaa agg cat tct ggt aaa cac ctc tat tct ttg ggg ata aat gga 4295

Lys Lys Arg His Ser Gly Lys His Leu Tyr Ser Leu Gly Ile Asn Gly

890 895 900

gac cag ctg gat gac agt gtt tct gat gca ttt cac cta aga cac ttg 4343

Asp Gln Leu Asp Asp Ser Val Ser Asp Ala Phe His Leu Arg His Leu

905 910 915 920

agg ctt att aga gtg ttg gac ctg gaa ccc cct tta atc atg gtg aat 4391

Arg Leu Ile Arg Val Leu Asp Leu Glu Pro Pro Leu Ile Met Val Asn

925 930 935

gat tct ttg ctg aat gaa ata tgc atg ttg aat cat ttg agg tac tta 4439

Asp Ser Leu Leu Asn Glu Ile Cys Met Leu Asn His Leu Arg Tyr Leu

940 945 950

aga att cgg aca caa gtt aaa tat ctg cct ttc tct ttc tca aac ctc 4487

Arg Ile Arg Thr Gln Val Lys Tyr Leu Pro Phe Ser Phe Ser Asn Leu

955 960 965

tgg aat cta gaa agt ctg ttt gtg tct aac aaa gga tca atc ttg gta 4535

Trp Asn Leu Glu Ser Leu Phe Val Ser Asn Lys Gly Ser Ile Leu Val

970 975 980

cta tta ccg aga att ttg gat ctt gta aag ttg cga gtg ctg tcc gtg 4583

Leu Leu Pro Arg Ile Leu Asp Leu Val Lys Leu Arg Val Leu Ser Val

985 990 995 1000

ggt gct tgt tct ttc ttt gat atg gat gca gat gaa tca ata ttg 4628

Gly Ala Cys Ser Phe Phe Asp Met Asp Ala Asp Glu Ser Ile Leu

1005 1010 1015

ata gca aag gac aca aag tta gag aac ttg aga ata tta ggg gaa 4673

Ile Ala Lys Asp Thr Lys Leu Glu Asn Leu Arg Ile Leu Gly Glu

1020 1025 1030

ctg ttg att tcc tat tcg aaa gat aca atg aat att ttc aaa agg 4718

Leu Leu Ile Ser Tyr Ser Lys Asp Thr Met Asn Ile Phe Lys Arg

1035 1040 1045

ttt ccc aat ctt cag gtg ctt cag ttt gaa ctc aag gag tca tgg 4763

Phe Pro Asn Leu Gln Val Leu Gln Phe Glu Leu Lys Glu Ser Trp

1050 1055 1060

gat tat tca aca gag caa cat tgg ttc ccg aaa ttg gat tgc cta 4808

Asp Tyr Ser Thr Glu Gln His Trp Phe Pro Lys Leu Asp Cys Leu

1065 1070 1075

act gaa cta gaa aca ctc tgt gta ggt ttt aaa agt tca aac aca 4853

Thr Glu Leu Glu Thr Leu Cys Val Gly Phe Lys Ser Ser Asn Thr

1080 1085 1090

aac cac tgt ggg tcc tct gtt gcg aca aat cgg ccg tgg gat ttt 4898

Asn His Cys Gly Ser Ser Val Ala Thr Asn Arg Pro Trp Asp Phe

1095 1100 1105

cac ttc cct tca aat ttg aaa gaa ctg ttg ttg tat gac ttt cct 4943

His Phe Pro Ser Asn Leu Lys Glu Leu Leu Leu Tyr Asp Phe Pro

1110 1115 1120

ctg aca tcc gat tca cta tca acg ata gcg aga ctg ccc aac ctt 4988

Leu Thr Ser Asp Ser Leu Ser Thr Ile Ala Arg Leu Pro Asn Leu

1125 1130 1135

gaa aat ttg tcc ctt tat gat aca atc atc cag gga gaa gaa tgg 5033

Glu Asn Leu Ser Leu Tyr Asp Thr Ile Ile Gln Gly Glu Glu Trp

1140 1145 1150

aac atg ggg gag gaa gac act ttt gag aat ctc aaa ttt ttg aac 5078

Asn Met Gly Glu Glu Asp Thr Phe Glu Asn Leu Lys Phe Leu Asn

1155 1160 1165

ttg cgt cta ctg act ctt tcc aag tgg gag gtt gga gag gaa tcc 5123

Leu Arg Leu Leu Thr Leu Ser Lys Trp Glu Val Gly Glu Glu Ser

1170 1175 1180

ttc ccc aat ctt gag aaa tta aaa ctg cag gaa tgc ggt aag ctt 5168

Phe Pro Asn Leu Glu Lys Leu Lys Leu Gln Glu Cys Gly Lys Leu

1185 1190 1195

gag gag att cca cct agt ttt gga gat att tat tca ttg aaa ttt 5213

Glu Glu Ile Pro Pro Ser Phe Gly Asp Ile Tyr Ser Leu Lys Phe

1200 1205 1210

atc aaa att gta aag agt cct caa ctt gaa gat tct gct ctc aag 5258

Ile Lys Ile Val Lys Ser Pro Gln Leu Glu Asp Ser Ala Leu Lys

1215 1220 1225

att aag aaa tac gct gaa gat atg aga gga ggg aac gag ctt cag 5303

Ile Lys Lys Tyr Ala Glu Asp Met Arg Gly Gly Asn Glu Leu Gln

1230 1235 1240

atc ctt ggc cag gag gat atc ccc tta ttt aag tagcattttg 5346

Ile Leu Gly Gln Glu Asp Ile Pro Leu Phe Lys

1245 1250

gtcgaacttt gcttgggtgg tgatattgta tatgattaaa atatcctgtg atgagattcc 5406

tcttagtttc ttttaacaaa aaatataatt tttataagta c 5447

<210>2

<211>1251

<212>PRT

＜213〉capsicum (Capsicum annuum)

<400>2

Met Glu Lys Arg Lys Asp Ile Glu Glu Ala Asn Asn Ser Leu Val Leu

1 5 10 15

Phe Ser Ala Leu Ser Lys Asp Ile Ala Asn Val Leu Ile Phe Leu Glu

20 25 30

Asn Glu Glu Asn Gln Lys Ala Leu Asp Lys Asp Gln Val Glu Lys Leu

35 40 45

Lys Leu Lys Met Ala Phe Ile Cys Thr Tyr Val Gln Leu Ala Lys Glu

50 55 60

Cys Arg Asp Ala Ile Gly Thr Ile Asn Leu Val Lys Gly Gln His Leu

65 70 75 80

Asp Arg Arg Thr Thr Asn Gln Leu Glu Asp Ala Ile Lys His Leu Thr

85 90 95

His Val Ala Val Phe Leu Thr Asn Leu Glu Lys Arg His Pro Ala Asn

100 105 110

Gly Ile Ser Ile His Leu Ser Tyr Ser Asp Phe Glu Gln Phe Glu Asp

115 120 125

Ile Met Thr Arg Asn Arg Gln Glu Val Glu Asn Leu Leu Gln Ser Leu

130 135 140

Leu Asp Asp Asp Val Leu Thr Ser Leu Thr Ser Asn Met Asp Asp Cys

145 150 155 160

Ile Ser Leu Tyr His Arg Ser Tyr Lys Ser Asp Ala Ile Met Met Asp

165 170 175

Glu Gln Leu Asp Phe Leu Leu Leu Asn Leu Tyr His Leu Ser Lys His

180 185 190

His Ala Glu Lys Ile Phe Pro Gly Val Thr Gln Tyr Glu Val Leu Gln

195 200 205

Asn Val Cys Gly Asn Ile Arg Asp Phe His Gly Leu Ile Leu Asn Gly

210 215 220

Cys Ile Lys His Glu Met Val Glu Asn Val Leu Pro Leu Phe Gln Leu

225 230 235 240

Met Ala Glu Arg Val Gly His Phe Leu Trp Glu Asp Gln Thr Asp Glu

245 250 255

Asp Ser Arg Leu Ser Glu Leu Asp Glu Asp Glu His Asn Asp Arg Asp

260 265 270

Ser Arg Leu Phe Gln Leu Thr His Leu Leu Leu Lys Ile Val Pro Thr

275 280 285

Glu Leu Glu Val Met His Ile Cys Tyr Thr Asn Leu Lys Ala Ser Thr

290 295 300

Ser Ala Glu Val Gly Arg Phe Ile Lys Lys Leu Leu Glu Thr Ser Pro

305 310 315 320

Asp Ile Leu Arg Glu Tyr Ile Ile Gln Leu Gln Glu His Met Leu Thr

325 330 335

Val Ile Pro Pro Ser Thr Leu Gly Ala Arg Asn Ile His Val Met Met

340 345 350

Glu Phe Leu Leu Leu Ile Leu Ser Asp Met Pro Lys Asp Phe Ile His

355 360 365

His Asp Lys Leu Phe Asp Leu Leu Ala His Val Gly Thr Leu Thr Arg

370 375 380

Glu Val Ser Thr Leu Val Arg Asp Leu Glu Glu Lys Leu Arg Asn Lys

385 390 395 400

Glu Gly Asn Asn Gln Thr Asn Cys Ala Thr Leu Asp Leu Leu Glu Asn

405 410 415

Ile Glu Leu Leu Lys Lys Asp Leu Lys His Val Tyr Leu Lys Ala Pro

420 425 430

Asn Ser Ser Gln Cys Cys Phe Pro Met Ser Gly Gly Pro Leu Phe Met

435 440 445

His Leu Leu His Met His Leu Asn Asp Leu Leu Asp Ser Asn Ala Leu

450 455 460

Ile Phe Ser Leu Pro Ile Thr Ile Lys Lys Ile Lys Leu Ile Lys Glu

465 470 475 480

Glu Ile Ser Ala Leu Asp Glu Asn Ile Pro Lys Asp Arg Gly Leu Ile

485 490 495

Val Val Asn Ser Pro Lys Lys Pro Val Glu Arg Lys Ser Leu Ala Thr

500 505 510

Asp Lys Ile Ile Val Gly Phe Glu Glu Glu Thr Asn Leu Ile Leu Arg

515 520 525

Lys Leu Thr Ser Gly Pro Ala Asp Leu Asp Val Ile Ser Ile Thr Gly

530 535 540

Met Pro Gly Ser Gly Lys Thr Thr Leu Ala Tyr Lys Val Tyr Asn Asp

545 550 555 560

Lys Ser Val Ser Arg His Phe Asp Leu Arg Ala Trp Cys Thr Val Asp

565 570 575

Gln Gly Tyr Asp Asp Lys Lys Leu Leu Asp Thr Ile Phe Ser Gln Val

580 585 590

Ser Gly Ser Asp Ser Asn Leu Ser Glu Asn Ile Asp Val Ala Asp Lys

595 600 605

Leu Arg Lys Gln Leu Phe Gly Lys Arg Tyr Leu Ile Val Leu Asp Asp

610 615 620

Val Trp Asp Thr Thr Thr Leu Asp Glu Leu Thr Arg Pro Phe Pro Glu

625 630 635 640

Ala Lys Lys Gly Ser Arg Ile Ile Leu Thr Thr Arg Glu Lys Glu Val

645 650 655

Ala Leu His Gly Lys Leu Asn Thr Asp Pro Leu Asp Leu Arg Leu Leu

660 665 670

Arg Pro Asp Glu Ser Trp Glu Leu Leu Glu Lys Arg Thr Phe Gly Asn

675 680 685

Glu Ser Cys Pro Asp Glu Leu Leu Asp Val Gly Lys Glu Ile Ala Glu

690 695 700

Asn Cys Lys Gly Leu Pro Leu Val Ala Asp Leu Ile Ala Gly Val Ile

705 710 715 720

Ala Gly Arg Glu Lys Lys Arg Ser Val Trp Leu Glu Val Gln Ser Ser

725 730 735

Leu Ser Ser Phe Ile Leu Asn Ser Glu Val Glu Val Met Arg Val Ile

740 745 750

Glu Leu Ser Tyr Asp His Leu Pro His His Leu Lys Pro Cys Leu Leu

755 760 765

His Phe Ala Ser Trp Pro Lys Asp Thr Pro Leu Thr Ile Tyr Leu Leu

770 775 780

Thr Val Tyr Leu Gly Ala Glu Gly Phe Val Glu Lys Thr Glu Met Lys

785 790 795 800

Gly Ile Glu Glu Val Val Lys Ile Tyr Met Asp Asp Leu Ile Ser Ser

805 810 815

Ser Leu Val Ile Cys Phe Asn Glu Ile Gly Asp Ile Leu Asn Phe Gln

820 825 830

Ile His Asp Leu Val His Asp Phe Cys Leu Ile Lys Ala Arg Lys Glu

835 840 845

Asn Leu Phe Asp Arg Ile Arg Ser Ser Ala Pro Ser Asp Leu Leu Pro

850 855 860

Arg Gln Ile Thr Ile Asp Tyr Asp Glu Glu Glu Glu His Phe Gly Leu

865 870 875 880

Asn Phe Val Met Phe Asp Ser Asn Lys Lys Arg His Ser Gly Lys His

885 890 895

Leu Tyr Ser Leu Gly Ile Asn Gly Asp Gln Leu Asp Asp Ser Val Ser

900 905 910

Asp Ala Phe His Leu Arg His Leu Arg Leu Ile Arg Val Leu Asp Leu

915 920 925

Glu Pro Pro Leu Ile Met Val Asn Asp Ser Leu Leu Asn Glu Ile Cys

930 935 940

Met Leu Asn His Leu Arg Tyr Leu Arg Ile Arg Thr Gln Val Lys Tyr

945 950 955 960

Leu Pro Phe Ser Phe Ser Asn Leu Trp Asn Leu Glu Ser Leu Phe Val

965 970 975

Ser Asn Lys Gly Ser Ile Leu Val Leu Leu Pro Arg Ile Leu Asp Leu

980 985 990

Val Lys Leu Arg Val Leu Ser Val Gly Ala Cys Ser Phe Phe Asp Met

995 1000 1005

Asp Ala Asp Glu Ser Ile Leu Ile Ala Lys Asp Thr Lys Leu Glu

1010 1015 1020

Asn Leu Arg Ile Leu Gly Glu Leu Leu Ile Ser Tyr Ser Lys Asp

1025 1030 1035

Thr Met Asn Ile Phe Lys Arg Phe Pro Asn Leu Gln Val Leu Gln

1040 1045 1050

Phe Glu Leu Lys Glu Ser Trp Asp Tyr Ser Thr Glu Gln His Trp

1055 1060 1065

Phe Pro Lys Leu Asp Cys Leu Thr Glu Leu Glu Thr Leu Cys Val

1070 1075 1080

Gly Phe Lys Ser Ser Asn Thr Asn His Cys Gly Ser Ser Val Ala

1085 1090 l095

Thr Asn Arg Pro Trp Asp Phe His Phe Pro Ser Asn Leu Lys Glu

1100 1105 1110

Leu Leu Leu Tyr Asp Phe Pro Leu Thr Ser Asp Ser Leu Ser Thr

1115 1120 1125

Ile Ala Arg Leu Pro Asn Leu Glu Asn Leu Ser Leu Tyr Asp Thr

1130 1135 1140

Ile Ile Gln Gly Glu Glu Trp Asn Met Gly Glu Glu Asp Thr Phe

1145 1150 1155

Glu Asn Leu Lys Phe Leu Asn Leu Arg Leu Leu Thr Leu Ser Lys

1160 1165 1170

Trp Glu Val Gly Glu Glu Ser Phe Pro Asn Leu Glu Lys Leu Lys

1175 1180 1185

Leu Gln Glu Cys Gly Lys Leu Glu Glu Ile Pro Pro Ser Phe Gly

1190 1195 1200

Asp Ile Tyr Ser Leu Lys Phe Ile Lys Ile Val Lys Ser Pro Gln

1205 1210 1215

Leu Glu Asp Ser Ala Leu Lys Ile Lys Lys Tyr Ala Glu Asp Met

1220 1225 1230

Arg Gly Gly Asn Glu Leu Gln Ile Leu Gly Gln Glu Asp Ile Pro

1235 1240 1245

Leu Phe Lys

1250

Claims (10)

1, separating clone derive from the genomic dna fragmentation of capsicum with gene M e of anti-root knot nematode function, its nucleotide sequence is shown in sequence table SEQ ID NO:1.

2, the described gene of claim 1, its cDNA sequence is shown in sequence table SEQ ID NO:1.

3, a kind of plant expression vector pBAMe is deposited in CCTCC, deposit number: CCTCC-M205110.

4, according to the dna sequence dna of the described gene of claim 1, it is characterized in that: one section insertion sudden change from the common 171bp of 1937 bases of the 1767th base to the is arranged among the sequence table SEQ ID NO:1, and one section deletion mutantion from the common 189bp of 2989 bases of the 2801st base to the.

5, cDNA sequence according to claim 2, it is characterized in that: the cDNA sequence length is 3950bp, comprises the open reading frame of 3656bp, the 3 ' non-translational region of 86bp 5 ' non-translational region and 108bp, 1251 amino acid of encoding.

6, the degenerated primer of claim 1, its dna sequence dna is as follows:

NBS-F1：5’- TG(G/C)(G/C)(G/A)GG(T/A/C)(T/A)(T/C)(G/A)GG(T/C/G)AAAACTAC-3’；

NBS R2：5’- (T/A/C)(G/A)C(T/A)A(A/G)AGG(A/G/C)A(A/G)CCCT(T/C)(T/G/C)ACA 3’。

7, the primer of claim 1, its dna sequence dna is shown in sequence table SEQ ID NO:1.

8, the method for the anti-root knot nematode transgenic Fructus Lycopersici esculenti of production of plant expression vector that comprises gene, the claim 3 of claim 1 and 2.

9, method according to claim 8, it comprises the following steps:

1) utilize PCR to obtain gene against meloidogyne from the capsicum genome, its nucleotide sequence is shown in sequence table SEQ ID NO:1;

2) used high-fidelity pfu enzyme and the general T aq enzyme of PCR is 1: 2 by portion rate in the step 1);

3) make up plant expression vector pBAMe;

4) utilize agrobacterium mediation method to change the expression vector of step 3) over to tomato plants, obtain transformed plant the root knot nematode sensitivity;

5) identify by Molecular Identification and inoculation, obtain the transfer-gen plant of anti-root knot nematode.

10, the application of each described gene of claim 1-5 in cultivating anti-root knot nematode transgenic Fructus Lycopersici esculenti plant.

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