CN106434689A

CN106434689A - Plant disease-resistant essential gene ShORR-1 and application thereof

Info

Publication number: CN106434689A
Application number: CN201610642207.8A
Authority: CN
Inventors: 李成伟; 张怡; 裴冬丽; 徐克东; 刘坤; 王岩; 于德水; 张菊; 胡立宗; 王健; 谭光轩; 马红珍
Original assignee: Zhoukou Normal University
Current assignee: Zhoukou Normal University
Priority date: 2016-08-02
Filing date: 2016-08-02
Publication date: 2017-02-22
Anticipated expiration: 2036-08-02
Also published as: CN106434689B

Abstract

The invention provides a plant disease-resistant essential gene named as ShORR-1 gene, wherein the gene is oriented from tomato solanum genus. The plant disease-resistant essential gene ShORR-1 is characterized in that the nucleotide sequence thereof is shown in 1) or 2): 1) the nucleotide sequence of the gene is shown as SEQ ID NO.1; 2) on the basis of the nucleotide sequence defined in 1), the nucleotide sequence of the gene as the activity of the plant disease resistance through the deletion/substitution/insertion/mutation of a basic group. When the gene disclosed in the invention is transformed into a plant tissue, the resistance of transgenic tomatoes and transgenic tobaccos to powdery mildew, late blight and verticillium wilt is greatly improved. The plant disease-resistant essential gene ShORR-1 disclosed in the invention has an important meaning in improving the disease resistance of tomatoes and tobaccos. Therefore, not only the conventional breeding is effectively directed, but also an excellent gene resource is provided for the transgenic breeding of tomatoes and tobaccos. As a result, the plant disease-resistant essential gene ShORR-1 has a wide application prospect in the tomato and tobacco production.

Description

A kind of plant disease-resistant indispensable gene ShORR-1 and application thereof

Technical field

The present invention relates to a kind of plant disease-resistant indispensable gene and application thereof, belong to biological technical field.

Background technology

Tomato is a kind of well for the Solanaceae model plant of genetic research, has multiple genetic linkages of abundant mark Figure and physical map, its genome sequencing has also completed and has announced (http：//solgenomics.net/).Tomato has Very big economic worth, is one of widest vegetable crop of a kind of cultivation.According to the statistics of FAO, within 2013, world tomato is total Yield has reached 1.639 hundred million tons, accounts for the 14.43% of world vegetables (containing melon) total output.Tomato in China total output reaches 0.506 hundred million tons, account for the 30.8% of world's tomato production total amount.Visible tomato is in China or even world's vegetables production and supply There is very important status.But pest and disease damage has a strong impact on tomato production, do not only result in the underproduction and increase because of the administration of agricultural chemicals yet Add tomato production cost and environmental pollution, it is estimated that, pest and disease damage can cause the tomato underproduction to reach 30%-80%, because spraying agriculture Medicine increases cost and reaches 10%-15%.

Tomato powdery mildew is a kind of very serious worldwide plant disease, particularly particularly tight on hothouse production tomato Weight (Jones et al., 2001), tomato powdery mildew not only affects tomato yield and has an effect on tomato quality.Due to disease-resistant tomato product Planting less, the method for this fungal disease of preventing and treating is mainly by administration bactericide at present, causes vegetable pesticide residue and environment is dirty Dye, is therefore the obstacle that pollution-free tomato produces.The pathogenic bacteria of this disease are tomato powdery mildew (Oidium Neolycopersici), tomato powdery mildew is a kind of live body parasitical fungi, only infects tomato epidermal cell.In recent years, tomato is white Powder disease is also found in China and again and again breaks out in various places, and we have also carried out morphology and ribose to tomato powdery mildew pathogenic bacteria The analysis of body transcriptional units spacer region (ITS) sequence, specify that this pathogenic bacteria are tomato powdery mildew China fungus strain, and in the world Carry out reporting (Li et al., 2008).

Tomato late blight is to be drawn by the parasitic type pathogen-phytophthora infestans (Phytophthora infestans) of half live body The fungal disease rising, always one of the great disease of tomato production in world wide.Not only endanger south in China's late blight Tomato production, produces to north tomato in greenhouse and also result in very big harm, and the general time underproduction is up to 30%, and morbidity is serious Time can reach more than 80% (Xue Minju etc., 2002；Feng Lanxiang etc., 2004；Zhao Tongmin etc., 2006).In recent years, due to kind Eggplant whole year production, Protected production environment are beneficial to late blight generation, chronic administration agricultural chemicals causes the germ resistance to the action of a drug to improve, China is raw The factors such as state environment is various, the late blight that phytophthora infestans causes frequently break out become anniversary disease (Xue Minju etc., 2002；Zhao Tong Quick etc., 2006).

Tomato verticillium wilt is to be passed saprophytic fungus by a kind of soil that verticillium dahliae (Verticillium dahliae) causes Disease, is to affect the important disease that tomato production particularly tomato in greenhouse produces, in recent years this disease in China by secondary Disease is wanted to become Major Diseases (Lei Na etc., 2011).Although 2 tomato resisting verticillium gene Ve1 and Ve2 are cloned (Kawchuk Et al., 2001), the disease resistance response signal pathway of Ve1 gene mediated and the signal pathway with Cf gene mediated thereof overlapping also It is interpreted (Fradin et al., 2009), but, still unclear with the interaction mechanism of tomato to this germ.

Tomato Rcr1 and Rcr2 is that Cf-9 mediates to the indispensable gene (Hammond-carrying Avr9 Fulvia fulva Ciferri Resistant reaction Kosack et al., 1994)；Rcr3 is that Cf-2 mediates to indispensable gene (the Dixon et carrying Avr leaf mycete Resistant reaction Al., 2000；Rooney et al., 2005).Rar1 and Rar2 is found to be barley powdery mildew (Bgh) disease-resistant gene Mla, Mih Indispensable gene with Mlk mediation Resistant reaction；Ror1 and Ror2 is mlo mediation Resistant reaction indispensable gene (Freialdenhoven et al, 1994and 1996) (Fig. 1, quoted from Li, 2005).Although this kind of indispensable gene is initially often It is found specifically planting sick body system, but more and more research shows that these indispensable genes take part in different R gene mediated Resistant reaction, such as RAR, SGT1 and the indispensable gene (Azevedo that HSP90 is the Resistant reaction participating in different R gene mediateds Et al., 2002；Takahashi et al., 2003；Austin et al., 2002).Therefore, studying indispensable gene can be Lasting and resistance of wide spectrum mechanism provides foundation.

Virus induced gene silencing (virus induced gene silencing, VIGS) be a kind of rapidly and efficiently Functional genome's science study method (van Kammen, 1997；Senthil-Kumar and Mysore, 2014), overcome biography The limitation of system functional genomics investigative technique, have quick, do not need Plant Transformation and base can be carried out to gene family Because of reticent advantage.In addition, VIGS can also between species and in species different genetic backgrounds plant between carry out gene The comparison (Burch-Simith et al., 2004) of function.VIGS has been used successfully to understand that various plants is disease-resistant instead Should, for example, the qualification of the tobacco mosaic virus (TMV) Resistant reaction indispensable gene of tobacco NPR1 gene mediated and functional analysis (Liu et Al., 2002a＆b)；Function Identification in the bacterial spot of tomato Resistant reaction that Pto mediates for NPR1 and the TGA transcription factor (Ekengren et al., 2003)；Leaf mold induction the quick expressing gene of tomato functional analysis (Rowland et al., 2005).The VIGS of TRV viral vectors mediation has the advantage (Senthil-such as quick, the easy operation that other viral vectors do not have Kumar and Mysore, 2014), in addition, the VIGS of TRV mediation can work in growing point separate living tissue and can reach To the reticent effect (Ratcliffet al., 2001) of housekeeping gene.

Content of the invention

It is an object of the invention to provide a kind of plant disease-resistant indispensable gene, entitled ShORR-1, derive from tomato genus kind Eggplant (Solanum lycopersicum), it is characterised in that its nucleotide sequence for example following 1) or 2) shown in：

1) its nucleotides sequence is classified as nucleotide sequence shown in SEQ ID NO.1；

2) 1) on the basis of the nucleotide sequence that limits through base disappearance, replacement, insert or sudden change forms and has and plants The nucleotide sequence of thing anti-disease activity.

The protein of described gene code, its amino acid sequence is SEQ ID NO.2.

The recombinant vector of described nucleotide sequence.

Described carrier for for clone pMD18-T carrier, for express pSAT6-GFP-N1, pCAMBIA1300, PCAMBIA2300 carrier and the pYY13 carrier for VIGS.

The transgenic cell line of described nucleotide sequence or recombinant bacterium.

Described nucleotides sequence is listed in the application during plant tissue is expressed.

Described plant tissue is root, stem, leaf, flower or seed.

Described plant is monocotyledon or dicotyledon.

Described monocotyledon is paddy rice, wheat, Chinese sorghum and corn, described dicotyledon be tomato, tobacco, soybean and Potato.

Application in cultivating disease-resistant plants for the albumen described in described gene or claim 2, described disease-resistant for powdery mildew, evening Epidemic disease and verticillium wilt.

The technique effect of the present invention：First by gene constructed for the ShORR1 carrier pYY13 mediating VIGS to TRV, it is thus achieved that Recombinant vector and zero load are proceeded to Agrobacterium and are used for further converting, utilize Agrobacterium to soak by recombinant vector pYY13-ShORR1 Mildew-resistance wild-type tomato (S.habrochiates G1.1560) blade is converted by profit method；Then ShORR1 gene is sunk The blade inoculation powdery mildew of silent and unloaded adjoining tree has carried out susceptibility and microscopic analysis.Susceptibility and microscopic analysis show, After inoculation powdery mildew 7d, comparison disease-resistant plant G1.1560 not carrying out ShORR1 gene silencing shows as disease-resistant, and its blade connects The powdery mildew conidium major part planted does not has rudiment, and the haustorium sprouting sporogenesis then causes it to infect epidermal leaf cells Allergic reaction (meronecrosis), it is impossible to obtain sufficient nutrition from blade cell, and then mycelial growth be suppressed, it is impossible to shape The conidium of Cheng Xin and complete whole vegetative propagation cycle (Fig. 1).The disease-resistant plant G1.1560 blade of ShORR1 gene silencing Show as susceptible, the powdery mildew conidium of inoculation on its blade can normally rudiment, the haustorium of formation does not cause and infects leaf The allergic reaction of piece cell, mycelia normal growth simultaneously defines conidiophore and new conidium, can complete whole nothing In the sexual reproduction cycle (Fig. 1), carry out infecting of a new round.Result shows, ShORR1 gene is the pass of tomato powder mildew resistance reaction Key gene.

The VIGS utilizing TRV to mediate analyzes effect in tomato verticillium wilt Resistant reaction for the ShORR1 and shows, inoculation Huang withers The height of sick pathogenic bacteria verticillium dahliae resists the tomato (S.lycopersicum LA1221) of withered, verticillium wilt after one week, carries The unloaded Agrobacterium-mediated Transformation of pYY13 and clear water inoculation adjoining tree excised leaf do not show susceptible symptom (Fig. 2)；Carry pYY13- The ShORR1 gene silencing plant excised leaf of ShORR1 recombinant vector Agrobacterium-mediated Transformation shows obvious susceptible symptom (Fig. 2). Result shows, ShORR1 gene is the key gene of tomato verticillium wilt Resistant reaction.

ShORR1 gene recombined vector pYY13-ShORR1, unloaded and tomato phytoene dehydrogenase will be carried (PDS) TRV-VIGS carrier (pYL252) of recombinating proceeds to Agrobacterium respectively, will carry pYY13-by agroinfiltration method ShORR1 Agrobacterium and carry pYL252 Agrobacterium and jointly infiltrate eight these uncured tobaccos of week old (Nicotiana benthamiana) leaf In piece, simultaneously using clear water with carry pYL252 agroinfiltration this uncured tobacco blade as comparison.Table is observed after inoculation 10～15d Type changes, and after tobacco leaf albefaction, is processing and is inoculating late disease bacteria pathogenic bacteria (note on the blade of adjoining tree respectively：This Research discovery, this life cigarette extremely strong disease resistance to phytophthora infestans performance).Through DAB and Trypan Blue, under the microscope to inoculation Blade is observed, and experiment shows, ShORR1 gene silencing this uncured tobacco blade shows as susceptible (Fig. 3 A-to phytophthora infestans B, arrow indication is phytophthora infestans mycelia), create zoospore and mycelia, and adjoining tree blade table reveals allergic reaction (meronecrosis), produces without zoospore and mycelia and mycelia.Result shows, ShORR1 gene is that this life cigarette P. infestans resistant is anti- The key gene answered.

Brief description

Fig. 1 represents the powder mildew resistance tomato S.lycopersicum G1.1560 inoculation of the reticent ShORR-1 gene of VIGS The phenotype of powdery mildew and microexamination；A shows as susceptible after showing ShORR-1 gene silencing blade inoculation powdery mildew, red arrow Show powdery mildew bacterium colony；The newborn conidium stalk that B is formed after showing ShORR-1 gene silencing rear blade inoculation powdery mildew；C shows comparison Plant inoculation powdery mildew shows as disease-resistant；Cause the allergic reaction being infected blade (thin after D adjoining tree blade inoculation powdery mildew Born of the same parents are downright bad).

Fig. 2 represents resistance to verticillium wilt tomato (S.lycopersicum LA1221) inoculation of the reticent ShORR-1 gene of VIGS The Phenotypic Observation of verticillium dahliae spore suspension；A shows that ShORR-1 gene silencing plant shows in verticillium dahliae spore suspension For susceptible, adjoining tree shows as disease-resistant, and B shows that ShORR-1 gene silencing plant and adjoining tree all show as resisting in clear water Disease, red arrow indication is reticent plant leaf, and black arrow indication is adjoining tree blade.

Fig. 3 represents phenotype and the microexamination of this uncured tobacco inoculation phytophthora infestans of the reticent ShORR-1 gene of VIGS；A After showing ShORR-1 gene silencing plant leaf inoculation phytophthora infestans, showing as susceptible phenotype, red arrow shows newborn mycelia；B After showing ShORR-1 gene silencing plant leaf inoculation phytophthora infestans, the newborn mycelia passing in Stoma of Leaves；C shows that comparison is planted Strain blade inoculation phytophthora infestans shows as disease-resistant phenotype；After D shows adjoining tree blade inoculation phytophthora infestans, show allergy Reaction (non-viable non-apoptotic cell).

Fig. 4 represents the Semiquatitative RT-PCR assay result of tomato ShORR-1 and PDS gene silencing；Left：Comparison tomato plant；Right： Reticent tomato plant；20th, the 25th, the 30th, 35 different PCR cycle number is represented；Actin transcript is internal reference comparison.

Fig. 5 represents expression in tomato different tissues for the ShORR-1.

Fig. 6 represents that ShORR-1 infects the expression change of tomato different times at tomato powdery mildew.

Fig. 7 represents that ShORR-1 infects the expression change of tomato different times at phytophthora infestans.

Fig. 8 represents that ShORR-1 infects the expression change of tomato different times in verticillium dahliae.

Detailed description of the invention

Below by example, the present invention is further elaborated, its object is to be best understood from present invention and unrestricted Protection scope of the present invention.

The present invention intends being analyzed the gene of ShORR-1 interaction of genes and the associated signal paths of participation thereof further, And analyze whether ShORR-1 gene has broader spectrum of resistance effect.By analyzing this gene at Different Kinds of Pathogens microorganism induction Tomato effect protein activate immune response (ETI) and pathogen-associated molecular pattern activate immune response (PTI) in work With, theoretical with pathogenic microorganism interaction to abundant plant, and provide foundation and gene for tomato broad-spectrum disease resistance breed of variety Source.

Embodiment

Embodiment one VIGS tests

1 materials and methods

1.1 vegetable materials, pathogen and cultural method thereof

Tomato variety for examination includes tomato cultivation kind (S.lycopersicum)：Moneymaker(MM)、Microtom (MT)、LA1221；Collections of Wild Tomato Species Lycopersicon：S.habrochaites G1.1560；This uncured tobacco：N.benthamiana.Powdery mildew comes The susceptible tomato plant MM blade preserving from Zhoukou Normal University of Henan Province, is stored on the MM plant of climate box, temperature：20± 2 DEG C, relative humidity (RH)：75%, light dark cycles：16h/8h.Phytophthora infestans T124 biological strain is by the Chinese Academy of Agricultural Sciences Biotechnology research institute Li Junming researcher presents, and expands numerous growth, temperature on rye culture medium：20±2℃.Verticillium dahliae Given by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute plant protection research department Zhu Heqin researcher, Czapek's medium expands numerous life Long, temperature：25±2℃.All of plant strain growth in condition good growing weather room, temperature：20 ± 2 DEG C, light dark cycles： 16h/8h, luminous intensity：150μmol/m²·s.

1.2 used carrier

VIGS experiment silent carrier used is that the double base of RNA1 and RNA2cDNA of Tobacco rattle virus (TRV) expresses load Body, respectively TRV1 (pYL192) and TRV2 (pYY13).TRV1 carrier includes the RNA polymerase, motion egg that coding RNA relies on The gene of white and 16K albumen, is the helper viral vector of VIGS system.TRV2-LIC carrier include capsid protein (Cp) gene, MCSs (MCS) etc., for the structure of genes of interest.PYY13 is derived from carrier pYL170 (Burch-Smith et Al., 2006).In order to clone purpose fragment with high throughput in TRV2-VIGS carrier, have employed the company of being independent of of an improvement Clone (ligation independent cloning, the LIC) method connecing, is added to two LIC joints on pYL170, is formed Carrier pYY12, joint comprises the sequence of 15bp and middle Pst I restriction enzyme site.Then pass through Pst I to be digested, Insert a ccdB gene between two LIC sites, form carrier pYY13 (Fig. 1).CcdB is a lethal gene, can be fast Speed, filter out recombinant vector exactly.

The extraction of 1.3 RNA, cDNA synthesis and RT-PCR amplifying target genes

The extracting method of tomato total serum IgE carries out (TaKaRa) according to total RNA extraction reagent box operational manual.With 1 μ g's RNA does template, synthesizes the first chain cDNA according to cDNA synthetic agent box (TaKaRa) operating instruction.Phytoene dehydrogenase Gene (Phytoene desaturase, PDS) is the whether effective reporter gene of inspection VIGS carrier system.According to tomato PDS Gene order (Gene Bank accession number M88683), choose be positioned at sequence in the middle part of PDS gene for target fragment design upstream and Downstream primer.Use the method for electronic cloning, carry out cDNA step to tomato powdery mildew disease-resistant gene candidate sequence and move, move according to step After sequence, design is with the primer of TRV2-LIC vector junctions.Upstream primer：5’-CGACGACAAGACCCT-gene specific Sequence-3 ', downstream primer：5’-GAGGAGAAGAGCCCT-gene specific sequence-3 ', underscore is LIC joint sequence, gene Specific primer sequence is shown in Table 1-1.RT-PCR reaction system (20 μ L)：10 × PCR reaction buffer 2 μ L, dNTP mixture is (each 2.5mmol/L) 1.6 μ L, Mg²⁺(25mmol/L) 1.6 μ L, each 1 μ L of primer (10pm/ μ L), Taq polymerase (5U/ μ L) 0.2 μ L, Template cDNA 2 μ L, ddH₂O10.6μL.PCR reaction condition：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.

The primer sequence of table 1 RT-PCR amplifying target genes

Table1 The sequences of primers used in RT-PCR of objective genes

The structure of 1.4 TRV2-LIC recombinant vectors

The PCR primer of genes of interest, according to precious biological (Takara) Agarose Gel DNA Purification Kit Kit operation is purified.After restriction enzyme Pst I single endonuclease digestion TRV2-LIC plasmid, then use T₄Archaeal dna polymerase is digested Genes of interest and TRV-LIC carrier.T₄Archaeal dna polymerase is digested genes of interest reaction system：Genes of interest 6 μ L, T₄DNA is polymerized Enzyme (5U/ μ L) 0.5 μ L, dATP (2.5mmol/L) 0.5 μ L, BSA (0.1%) 1 μ L, 10 × buffer 1 μ L, ddH₂O 1μL.T₄ Archaeal dna polymerase is digested TRV2-LIC carrier reaction system：Plasmid single endonuclease digestion product 6 μ L, T₄Archaeal dna polymerase (5U/ μ L) 0.5 μ L, DTTP (2.5mmol/L) 0.5 μ L, BSA (0.1%) 1 μ L, 10 × buffer 1 μ L, ddH₂O 1μL.Matter after being finally digested Grain and genes of interest carry out LIC connection, reaction system：Plasmid 1 μ L, genes of interest 6 μ L；Reaction condition：65 DEG C, 2min, then 22 DEG C, 10min.Connecting product transformed competence colibacillus E.coli DH5 α, coating LB flat board (50mg/L Kana) is cultivated, and 37 DEG C overnight It is inverted to cultivate and form single bacterium colony.

The qualification of 1.5 TRV2-LIC recombinant vectors

TRV2-LIC empty plasmid carries a ccdB gene, a kind of albumen disturbing e. coli dna gyrase of codified Escherichia coli are had lethal effect by matter, if carrier occurs from connecting, the e. coli host cell converting these carriers will be dead Dying, only after exogenous dna fragment is connected with carrier, the expression of ccdB gene is blocked, and recombinant plasmid could be at large intestine bar Bacterium survives, is thus advantageous to efficiently obtain positive colony.Formed after picking TRV2-LIC recombinant plasmid transformed E.coli DH5 α Monoclonal, extract plasmid enter performing PCR identify.Positive plasmid conversion, to competence Agrobacterium GV3101, is coated with LB flat board (50mg/L Kana, 50mg/L Rif) cultivates, and is inverted for 28 DEG C and cultivates 2d, selects positive colony conversion Agrobacterium GV3101.

1.6 Agrobacteriums are infected and plant strain growth

With four leaf phase tomato G1.1560, LA1221 and six leaf phase N.benthamiana as experiment material.TRV1、TRV2- LIC (unloaded), TRV2-LIC (recombinant plasmid) convert Agrobacterium GV3101 by freeze-thaw method.Respectively picking Agrobacterium (TRV1 and TRV2-LIC) monoclonal is in 28 DEG C of overnight incubation, respectively takes 10mL nutrient solution, 3000rpm, 20min, centrifuges and collect bacterial sediment, It is resuspended in buffer solution (10 μm of ol/L MES, 10 μm of ol/L MgCl respectively₂, 200 μm of ol/L acetosyringones) in, OD₆₀₀= 1.0, the Agrobacterium GV3101 containing TRV1 is mixed with containing TRV2-LIC (recombinant plasmid) Agrobacterium GV3101 1: 1 ratio, Room temperature 100rpm low-speed oscillation 2h.Draw the bacterium solution of activation with the disposable 2mL syringe removing syringe needle, use compression method injection It is inoculated in the tomato true leaf back side (Mandar et al., 2008).With TRV2-LIC (unloaded) as negative control.Infect tomato in Overnight incubation under 20 DEG C of dark conditions, is 20 ± 2 DEG C in temperature afterwards, light dark cycles be 16h/8h climatic chamber in cultivate.Agriculture After bacillus bacterium solution injection G1.1560 10d, the conidium of fresh powdery mildew is rinsed from the blade of severe infections, uses Water is diluted to 2 × 10⁴It is used for inoculating after the concentration of spore/mL.The change of the disease-resistant phenotype of tomato G1.1560 is observed after one week. Every kind of silent carrier inoculation 10 strain tomato seedling, experiment is repeated 3 times.

After agroinfection N.benthamiana 10d, good with aseptic bamboo let picking growth conditions in superclean bench Good phytophthora infestans mycelia is coated in rib, blade tip and limb edge etc. respectively, and picking hyphae length keeps consistent, a Zhou Houguan Examine the change of the disease-resistant phenotype of N.benthamiana.Every kind of silent carrier inoculation 10 strain N.benthamiana seedling, experiment weight Multiple 3 times.

After agroinfection tomato LA1221 10d, new for the LA1221 newly growing branch is invaded respectively profit 10⁵Spore/mL In the spore suspension of verticillium dahliae and clear water, after one week, observe the change of the disease-resistant phenotype of tomato G1.1560.Every kind of reticent load Body inoculates 10 strain tomato LA1221 seedling, and experiment is repeated 3 times.

1.7 tissue Microscopic analysis

Leaf tissue H₂O₂Detection use benzidine (3,3 '-diaminobenzidine tetrahydrochloride, DAB) dyeing detection method, powdery mildew uses platform to expect blue decoration method (Huang et al., 1998).Comprise the following steps that：

(1) DAB dyeing：Take the fresh blade with handle, its petiole is cut into inclined-plane, insert the DAB (pH 3.8) of 1mg/mL In solution, the inclined-plane of the dipped petiole of solution.It is placed under illumination standing 8h, be visually observed henna DAB solution suitable Vein and reach the top of blade.Reaction time can adjust according to the size of blade and actual conditions.

(2) fix：It after DAB dyeing rear blade is sheared along middle main lobe arteries and veins, then is cut into the little of 1.5cm × 1.5cm Block, is directly immersed in fixer (absolute ethyl alcohol: glacial acetic acid=3: 1), to sloughing color, generally overnight processes.

(3) platform expects blue dyeing：Leaf tissue proceeds to expect containing the platform of 0.3% in blue solution, outwards winding test tube lid gently, It is placed in the water-bath of heating (70 DEG C-80 DEG C), make dye liquor boiling 1min.Leaf tissue in dye liquor overnight.

(4) decolour：Rinsing the leaf tissue being colored 3 times with the chloral hydrate of 2.5g/mL, sample can be in hydration Trichloroacetaldehyde is placed the several months.

1.8 semi-quantitative RT-PCR analysis

Extract adjoining tree and the total serum IgE of reticent plant.Do template with the RNA of 1 μ g, according to cDNA synthetic agent box (TaKaRa) operating instruction synthesis the first chain cDNA.Use gene transcripts in the reticent plant of semiquantitive RT-PCR detection Silencing efficiency.Tomato Actin gene (GenBank accession number U60480) is reference gene, and Semiquatitative RT-PCR assay primer sequence is shown in Table 4-2.PCR reaction system (20 μ L)：10 × PCR reaction buffer 2 μ L, dNTP mixture (each 2.5mmol/L) 1.6 μ L, Mg²⁺ (25mmol/L) 1.6 μ L, each 1 μ L of primer (10pm/ μ L), Taq polymerase (5U/ μ L) 0.2 μ L, template cDNA 2 μ L, ddH₂O 10.6μL.PCR reaction condition：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, and 30 are followed Ring；72 DEG C extend 10min.In order to avoid PCR primer amount reaches the impact on experimental result for the plateau, carry out the 20th, the 25th, 30 and The amplification of 35 different periods, 1% agarose gel electrophoresis detection pcr amplification product.

The primer sequence of table 2 Semiquatitative RT-PCR assay

Table2 The sequences of primers used in semi-quantitative RT-PCR

1.9 full length gene amplifications

Carrying out gene walking according to tomato whole genome sequence, design comprises the primer of complete reading frame, and RT-PCR expands The full length gene of the ShORR-1 sequence of G1.1560 simultaneously checks order.The corresponding upstream region of gene primer of amplification ShORR-1 5 '- ATTACTCTCTTCATAAACTCATTTCCA-3 ', downstream primer 5 '-TCTGCTGCTATTTCTGCCACT-3 '.PCR reactant System (20 μ L)：10 × PCR reaction buffer 2 μ L, dNTP mixture (each 2.5mmol/L) 1.6 μ L, Mg²⁺(25mmol/L)1.6μ L, each 1 μ L of primer (10pm/ μ L), pfu high-fidelity enzyme (5U/ μ L) 0.2 μ L, template cDNA 2 μ L, ddH₂O 10.6μL.PCR is anti- Answer condition：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, 30 circulations；72 DEG C of extensions 10min.Pcr amplification product is cloned on pMD18-T carrier after purification, in conversion to E.coli DH5 α competent cell, and plasmid PCR identifies that positive bacteria falls behind, and transfers to Nanjing Genscript Biotechnology Co., Ltd. to check order.

1.10 ShORR-1 gene space are expressed and inoculation Different Kinds of Pathogens thing expression analysis

In order to analyze the change of ShORR-1 gene expression after expression and pathogen infection in different tissues, take MT The root of tomato, stem, leaf, flower, green fruit and erythrocarpus, extract RNA, the expression of reverse transcription post analysis ShORR-1.Simultaneously Take about 4w MT in age tomato plant, be respectively adopted rendezvous method inoculating tomato powdery mildew and phytophthora infestans in tomato leaf, root-pouring method Inoculation verticillium dahliae, in the root of MT tomato, analyzes the expression change of ShORR-1 gene at different point in time sampling.Real When quantitative fluorescent PCR reaction system：10 μ L 2 × SYBR Premix Ex Taq, 0.4 μ L PCR Forward Primer, 0.4 μ L PCR Reverse Primer, 2.0 μ L DNA profilings, 7.2 μ L ddH₂O；Reaction condition is：Denaturation 95 DEG C, 10sec； 95 DEG C, 5sec；58 DEG C, 30sec, totally 40 circulations of this reaction system, at BIO-RAD CFX96^TMIn Real-time System Analyze the expression of gene, with relative quantification 2^-ΔΔCT method carries out relative quantitative assay to expression, with tomato Actin as internal reference Gene.

The primer sequence of table 3 real-time quantitative qRT-PCR

Table3 The sequences of primers used in real-time RT-PCR

Embodiment two ShORR-1 gene VIGS Phenotypic Observation

The clone of 2.1 ShORR-1 genes

Clone the tomato powdery mildew resisting candidate ShORR-1 of 510bp with the cDNA of wild disease-resistant tomato G1.1560 for template Gene order, the sequence owing to deriving from the DE-TDF of order-checking is shorter, uses the method for bioinformatics, according to tomato full genome After group sequence carries out gene walking, obtain the sequence with its homology, Primer5 Software for Design primer.

The structure of 2.2 TRV2-LIC recombinant vectors and qualification

The tomato powder mildew resistance reaction candidate gene that RT-PCR amplification obtains uses this chapter method 1.4 to carry with TRV2-LIC Body connects, and Transformed E .coli DH5 α obtains positive colony, and positive plasmid converts to Agrobacterium GV3101, by plasmid PCR, survey Sequence successfully filters out the agrobacterium strains containing TRV2-LIC recombinant vector.

2.3 ShORR-1 gene VIGS Phenotypic Observations

2.3.1 powdery mildew resistance analysis

In order to analyze ShORR-1 gene silencing is how to affect disease-resistant to O.neolycopersici of G1.1560 plant Property, carrying out tissue microscopic analysis to ShORR-1 gene silencing plant and adjoining tree, germ dyeing uses platform to expect blue decoration method. Susceptibility and microscopic analysis show, after inoculation powdery mildew 7d, do not carry out the comparison disease-resistant plant of ShORR-1 gene silencing G1.1560 shows as disease-resistant, and on its blade, the powdery mildew conidium major part of inoculation does not has rudiment, and sprouts sporogenesis Haustorium then causes it to infect the allergic reaction of epidermal leaf cells (meronecrosis), it is impossible to obtain sufficient battalion from blade cell Support, and then mycelial growth is suppressed, it is impossible to form new conidium and complete the whole vegetative propagation cycle (Fig. 1 C, D). The disease-resistant plant G1.1560 blade of ShORR1 gene silencing shows as susceptible, the powdery mildew conidium energy of inoculation on its blade Enough normal rudiments, the haustorium of formation does not cause the allergic reaction infecting blade cell, and mycelia normal growth simultaneously defines mitogenetic Sporophore and new conidium, can complete whole vegetative propagation cycle (Figure 1A, B), carry out infecting of a new round.Result table Bright, ShORR1 gene is the key gene of tomato powder mildew resistance reaction.

2.3.2 verticillium dahliae Analysis of Resistance

2.3.3 phytophthora infestans Analysis of Resistance

Carrying out susceptibility and microexamination analysis to ShORR-1 gene silencing plant and adjoining tree, experiment shows, This uncured tobacco blade of ShORR1 gene silencing shows as to phytophthora infestans that susceptible (Fig. 3 A-B, arrow indication is phytophthora infestans Mycelia), create zoospore and mycelia, and adjoining tree blade table reveals allergic reaction (meronecrosis), without zoospore Produce with mycelia and mycelia.Result shows, ShORR1 gene is the key gene of this life cigarette P. infestans resistant reaction.2.4 VIGS RT-PCR detection after Chen Mo

The method using Semiquatitative RT-PCR assay have detected TRV2-LIC-PDS, TRV2-LIC-ShORR1 at molecular level The 20th, transcript degree after Chen Mo, carried out to genes of interest fragment and Actin reference gene with the cDNA of synthesis respectively for template 25th, 30 and 35 cyclic amplifications.Electrophoresis detection finds, in PDS, ShORR1 gene silencing plant leaf, expression is significantly lower than Comparison, is less than about 60% (Fig. 4) of comparison.

2.4 ShORR-1 sequence pair answer the total length of gene

Carrier T PCR sequencing PCR records ShORR-1 sequence pair and answers the full length sequence of gene is 974bp, wherein comprises complete reading code Frame sequence length is 807bp, sequence following SEQ ID NO.1；

NCBI ORF-finder is used to predict that its coded protein is 268 amino acid, sequence following SEQ ID NO.2

The space expression analysis of 2.5 ShORR-1 genes and inoculation Different Kinds of Pathogens thing are analyzed

Take root, stem, leaf, flower, green fruit and the erythrocarpus of MT tomato, the expression analysis discovery to ShORR-1, its Expression in leaf is apparently higher than other tissues (Fig. 5), thus it is speculated that express in the main leaf of ShORR-1.Inoculate three kinds of inhomogeneities The fungal pathogens of type, in different point in time sampling analyses, result shows ShORR-1 expression after powdery mildew infects 12h Reach a peak, after 72h, reach top (Fig. 6)；Similar with powdery mildew expression pattern, ShORR-1 is at phytophthora infestans First peak expression occur after infecting 3d, after infecting 14d, expression reaches the highest (Fig. 7)；And ShORR-1 takes turns branch beautiful greatly After bacterium inoculation 60h, expression reaches the highest, declines (Fig. 8) afterwards.Thus speculating, ShORR-1 is various pathogenic bacteria and tomato Interaction in all play an important role.

According to sequence analysis, ShORR-1 is the new gene of a Unknown Function, has not still had any function with its homology The gene known is found.

Above example is only in order to illustrating, and unrestricted technical scheme, although with reference to above-described embodiment to this Invention has been described in detail, and it will be understood by those within the art that：Still the present invention can be modified or Equivalent, any modification or partial replacement without departing from the spirit and scope of the present invention, it all should be covered the present invention's In the middle of right.

Claims

1. a plant disease-resistant indispensable gene, it is characterised in that its nucleotide sequence such as 1) or 2) shown in：

2) 1) on the basis of the nucleotide sequence that limits through base disappearance, replacement, insert or sudden change forms and has Genes For Plant Tolerance The nucleotide sequence of sick activity.

2. the protein of gene code described in claim 1, its amino acid sequence is SEQ ID NO.2.

3. contain the recombinant vector of nucleotide sequence described in claim 1.

4. recombinant vector according to claim 2, it is characterised in that described carrier for for clone pMD18-T carrier, PSAT6-GFP-N1, pCAMBIA1300, pCAMBIA2300 carrier for expression and the pYY13 carrier for VIGS.

5. contain transgenic cell line or the recombinant bacterium of nucleotide sequence described in claim 1.

6. nucleotides sequence described in claim 1 is listed in plant tissue the application expressed.

7. application according to claim 6, it is characterised in that described plant tissue is root, stem, leaf, flower or seed.

8. application according to claim 6, it is characterised in that described plant is monocotyledon or dicotyledon.

9. application according to claim 8, it is characterised in that described monocotyledon is paddy rice, wheat, Chinese sorghum and corn, Described dicotyledon is tomato, tobacco, soybean and potato.

10. application in cultivating plant disease-resistant kind for the albumen described in gene described in claim 1 or claim 2；Described anti- Sick for powdery mildew, late blight and verticillium wilt.