CN106434689B - A kind of plant disease-resistant indispensable gene ShORR-1 and its application - Google Patents
A kind of plant disease-resistant indispensable gene ShORR-1 and its application Download PDFInfo
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Abstract
1) or 2) the present invention provides a kind of plant disease-resistant indispensable gene, entitled ShORR-1 gene derives from Solanum tomato, which is characterized in that its nucleotide sequence is as shown in: 1) its nucleotides sequence is classified as nucleotide sequence shown in SEQ ID NO.1；2) missing on the basis of nucleotide sequence 1) limited through base, substitution, insertion or mutation form and have the active nucleotide sequence of plant disease-resistant.After gene-transformed plant tissue disclosed by the invention, transgene tomato and tobacco leaf can be significantly improved to the resistance of powdery mildew, late blight and verticillium wilt.Plant disease-resistant indispensable gene ShORR-1 disclosed by the invention is of great significance to the disease-resistant performance for improving tomato and tobacco, conventional breeding can not only effectively be instructed, but also favorable genes resource can be provided for the transgenic breeding of tomato and tobacco, it has broad application prospects in tomato and tobacco leaf production.
Description
Technical field
The present invention relates to a kind of plant disease-resistant indispensable gene and its applications, belong to field of biotechnology.
Background technique
Tomato is a kind of Solanaceae model plant for being used for genetic research well, has multiple genetic linkages of abundant label
Figure and physical map, genome sequencing have also been completed and have been announced (http://solgenomics.net/).Tomato has
Very big economic value is a kind of one of widest vegetable crop of cultivation.According to the statistics of FAO, world tomato is total within 2013
Yield accounts for about the 14.43% of world vegetables (containing melon) total output up to 1.639 hundred million tons.Tomato in China total output reaches
0.506 hundred million tons, account for about the 30.8% of world's tomato production total amount.It can be seen that tomato is in China or even world's vegetables production and supply
With very important status.But pest and disease damage seriously affects tomato production, does not only result in the underproduction and also increases because of the application of pesticide
Add tomato production cost and environmental pollution, it is estimated that, pest and disease damage can cause the tomato underproduction up to 30%-80%, because spraying agriculture
Medicine increases cost and reaches 10%-15%.
Tomato powdery mildew is a kind of worldwide plant disease very serious, especially tight especially on hothouse production tomato
Weight (Jones et al., 2001), tomato powdery mildew not only influences tomato yield and has an effect on tomato quality.Due to disease-resistant tomato product
Kind is less, prevents and treats the method for this fungal disease at present mainly by application fungicide, causes vegetable pesticide residue and environment is dirty
Dye, therefore be an obstacle of pollution-free tomato production.The pathogenic bacteria of the disease are tomato powdery mildew (Oidium
Neolycopersici), tomato powdery mildew is a kind of living body parasitical fungi, only infects tomato epidermal cell.In recent years, tomato is white
Powder disease is also found in China and breaks out again and again in various regions, we have also carried out morphology and ribose to tomato powdery mildew pathogenic bacteria
The analysis of body transcriptional units spacer region (ITS) sequence specifies that the pathogenic bacteria are tomato powdery mildew China fungus strain, and in the world
It is reported (Li et al., 2008).
Tomato late blight is drawn by half living body parasitism type pathogen-phytophthora infestans (Phytophthora infestans)
The fungal disease risen, is always one of great disease of tomato production in world wide.In China, late blight not only endangers south
Tomato production also results in very big harm to the production of northern tomato in greenhouse, and up to 30%, morbidity is serious for the general time underproduction
Time can achieve 80% or more (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 conducive to late blight generation, chronic administration pesticide causes germ drug resistance to improve, China is raw
The factors such as state environment multiplicity, late blight caused by phytophthora infestans frequently break out become anniversary disease (Xue Minju etc., 2002；Zhao Tong
It is quick etc., 2006).
Tomato verticillium wilt is a kind of soil biography saprophytic fungus as caused by verticillium dahliae (Verticillium dahliae)
Disease is the important disease for influencing tomato production especially tomato in greenhouse production, and the disease is in China via secondary in recent years
Disease is wanted to become Major Diseases (Lei Na etc., 2011).Although 2 tomato resisting verticillium genes Ve1 and Ve2 are cloned (Kawchuk
Et al., 2001), the disease resistance response signal pathway of Ve1 gene mediated and its overlapping with the signal pathway of Cf gene mediated
It is interpreted (Fradin et al., 2009), it is still, still unclear to the interaction mechanism of the germ and tomato.
Tomato Rcr1 and Rcr2 are that Cf-9 is mediated to the indispensable gene (Hammond- for carrying Avr9 Fulvia fulva Ciferri Resistant reaction
Kosack et al., 1994)；Rcr3 is that Cf-2 is mediated to indispensable gene (the Dixon et for 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
The indispensable gene of Resistant reaction is mediated with Mlk；Ror1 and Ror2 is that mlo mediates 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 it is more and more research shows that these indispensable genes take part in different R gene mediateds
Resistant reaction, such as RAR, SGT1 and HSP90 are the indispensable gene (Azevedo for participating in the Resistant reaction of different R gene mediateds
Et al., 2002；Takahashi et al., 2003；Austin et al., 2002).Therefore, research indispensable gene can be
Persistently and resistance of wide spectrum mechanism provides foundation.
Virus induced gene silencing (virus induced gene silencing, VIGS) be it is a kind of rapidly and efficiently
Functional genome's science study method (van Kammen, 1997；Senthil-Kumar and Mysore, 2014), overcome biography
Unite functional genomics investigative technique limitation, have quickly, do not need Plant Transformation and can to gene family carry out base
The advantages of because of silencing.In addition to this, VIGS can also carry out gene between the plant of different genetic backgrounds between species and in species
The comparison (Burch-Simith et al., 2004) of function.VIGS has been used successfully to understand that various plants are disease-resistant anti-
It answers, for example, identification and functional analysis (the Liu et of the tobacco mosaic virus (TMV) Resistant reaction indispensable gene of tobacco NPR1 gene mediated
Al., 2002a&b)；Function Identification of the NPR1 and TGA transcription factor in the bacterial spot of tomato Resistant reaction that Pto is mediated
(Ekengren et al., 2003)；Leaf mold induce the quick expressing gene of tomato functional analysis (Rowland et al.,
2005).The VIGS that TRV viral vectors mediates has many advantages, such as (the Senthil- quick, easy to operate that other viral vectors do not have
Kumar and Mysore, 2014), in addition, the VIGS that TRV is mediated can work in growing point separate living tissue and can reach
To the effect (Ratcliffet al., 2001) of housekeeping gene silencing.
Summary of the invention
The purpose of the present invention is to provide a kind of plant disease-resistant indispensable gene, entitled ShORR-1 derives from tomato genus kind
Eggplant (Solanum lycopersicum), it is characterised in that its nucleotide sequence is for example following 1) or 2) shown:
1) its nucleotides sequence is classified as nucleotide sequence shown in SEQ ID NO.1；
2) missing on the basis of nucleotide sequence 1) limited through base, substitution, insertion or mutation form and have and plant
The nucleotide sequence of object anti-disease activity.
The protein of the gene coding, amino acid sequence are SEQ ID NO.2.
The recombinant vector of the nucleotide sequence.
The carrier be the pMD18-T carrier for clone, pSAT6-GFP-N1, pCAMBIA1300 for expression,
PCAMBIA2300 carrier and pYY13 carrier for VIGS.
The transgenic cell line or recombinant bacterium of the nucleotide sequence.
The nucleotides sequence is listed in the application in plant tissue expression.
The plant tissue is root, stem, leaf, flower or seed.
The plant is monocotyledon or dicotyledon.
The monocotyledon be rice, wheat, sorghum and corn, the dicotyledon be tomato, tobacco, soybean and
Potato.
Albumen described in the gene or claim 2 is cultivating the application in disease-resistant plants, described disease-resistant for powdery mildew, evening
Epidemic disease and verticillium wilt.
Technical effect of the invention: it first by the gene constructed carrier pYY13 for mediating VIGS to TRV of ShORR1, obtains
Recombinant vector and zero load are transferred to Agrobacterium and are used to further convert, soaked using Agrobacterium by recombinant vector pYY13-ShORR1
Profit method converts mildew-resistance wild-type tomato (S.habrochiates G1.1560) blade；Then heavy to ShORR1 gene
The blade inoculation powdery mildew of silent and unloaded adjoining tree has carried out susceptibility and microscopic analysis.Susceptibility and microscopic analysis show,
After being inoculated with powdery mildew 7d, the control disease-resistant plant G1.1560 for not carrying out ShORR1 gene silencing shows as disease-resistant, connects on blade
The powdery mildew conidium of kind does not have rudiment largely, and the haustorium for sprouting sporogenesis then causes it to infect epidermal leaf cells
Allergic reaction (meronecrosis), sufficient nutrition can not be obtained from blade cell, and then mycelia growth is suppressed, can not shape
The conidium of Cheng Xin and the entire vegetative propagation period (Fig. 1) of completion.The disease-resistant plant G1.1560 blade of ShORR1 gene silencing
Show as it is susceptible, the powdery mildew conidium being inoculated on blade can normal rudiment, the haustorium of formation do not cause to infect leaf
The allergic reaction of piece cell, mycelia normal growth simultaneously form conidiophore and new conidium, can complete entire nothing
In the sexual reproduction period (Fig. 1), carry out infecting for a new round.The result shows that ShORR1 gene is the pass of tomato powder mildew resistance reaction
Key gene.
Effect of the VIGS analysis ShORR1 mediated using TRV in tomato verticillium wilt Resistant reaction shows that inoculation Huang withers
The highly resistance of sick pathogenic bacteria verticillium dahliae is withered, verticillium wilt tomato (S.lycopersicum LA1221) after a week, carry
PYY13 zero load Agrobacterium-mediated Transformation 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).
The result shows that ShORR1 gene is the key gene of tomato verticillium wilt Resistant reaction.
ShORR1 gene recombined vector pYY13-ShORR1, zero load and tomato phytoene dehydrogenase will be carried
(PDS) recombination TRV-VIGS carrier (pYL252) is transferred to Agrobacterium respectively, will carry pYY13- by agroinfiltration method
ShORR1 Agrobacterium and carrying pYL252 Agrobacterium infiltrate eight week old this uncured tobacco (Nicotiana benthamiana) leaves jointly
In piece, while using clear water and this uncured tobacco of pYL252 agroinfiltration blade is carried as control.Table is observed after 10~15d of inoculation
Type variation is inoculated with late disease bacteria pathogenic bacteria (note: originally after tobacco leaf albefaction on the blade of processing and adjoining tree respectively
The study found that this life cigarette shows extremely strong disease resistance to phytophthora infestans).Through DAB and Trypan Blue, under the microscope to inoculation
Blade is observed, and experiment shows that ShORR1 this uncured tobacco of gene silencing blade shows as susceptible (Fig. 3 A- to phytophthora infestans
B, arrow meaning are phytophthora infestans mycelia), zoospore and mycelia are produced, and adjoining tree blade shows allergic reaction
(meronecrosis), no zoospore and mycelia and mycelia generate.The result shows that ShORR1 gene is that this life cigarette P. infestans resistant is anti-
The key gene answered.
Detailed description of the invention
Fig. 1 shows the powder mildew resistance tomato S.lycopersicum G1.1560 of VIGS silencing ShORR-1 gene inoculations
The phenotype and microexamination of powdery mildew；A shows as susceptible, red arrow after showing ShORR-1 gene silencing blade inoculation powdery mildew
Show powdery mildew bacterium colony；B shows the newborn conidium stalk formed after ShORR-1 gene silencing rear blade inoculation powdery mildew；C shows control
Plant inoculation powdery mildew shows as disease-resistant；Cause the allergic reaction for being infected blade (thin after D adjoining tree blade inoculation powdery mildew
Born of the same parents' necrosis).
Fig. 2 indicates resistance to verticillium wilt tomato (S.lycopersicum LA1221) inoculation of VIGS silencing ShORR-1 gene
The Phenotypic Observation of verticillium dahliae spore suspension；A shows that ShORR-1 gene silencing plant shows in verticillium dahliae spore suspension
To be susceptible, adjoining tree shows as disease-resistant, and B shows that ShORR-1 gene silencing plant and adjoining tree show as resisting in clear water
Disease, red arrow meaning are silencing plant leaf, and black arrow meaning is adjoining tree blade.
Fig. 3 indicates the phenotype and microexamination of this uncured tobacco inoculation phytophthora infestans of VIGS silencing ShORR-1 gene；A
After showing ShORR-1 gene silencing plant leaf inoculation phytophthora infestans, susceptible phenotype is shown as, red arrow shows newborn mycelia；B
After showing ShORR-1 gene silencing plant leaf inoculation phytophthora infestans, the newborn mycelia that is pierced by Stoma of Leaves；C shows that control is planted
Strain blade inoculation phytophthora infestans show as disease-resistant phenotype；After D shows adjoining tree blade inoculation phytophthora infestans, allergy is shown
It reacts (non-viable non-apoptotic cell).
The Semiquatitative RT-PCR assay result of Fig. 4 expression tomato ShORR-1 and PDS gene silencing；It is left: control tomato plant；It is right:
Silencing tomato plant；20,25,30,35 different PCR cycle numbers is indicated；Actin transcript is internal reference control.
Fig. 5 indicates expression quantity of the ShORR-1 in tomato different tissues.
Fig. 6 indicates that ShORR-1 infects the expression quantity variation of tomato different times in tomato powdery mildew.
Fig. 7 indicates that ShORR-1 infects the expression quantity variation of tomato different times in phytophthora infestans.
Fig. 8 shows ShORR-1 to change in the expression quantity that verticillium dahliae infects tomato different times.
Specific embodiment
Below by example, the present invention is further elaborated, limits its object is to be best understood from the content of present invention
Protection scope of the present invention.
The present invention quasi- further gene to ShORR-1 interaction of genes and its associated signal paths of participation are analyzed,
And analyze whether ShORR-1 gene has broader spectrum of resistance effect.By analyzing the gene in Different Kinds of Pathogens microorganism induction
Tomato effect protein activation immune response (ETI) and pathogen-associated molecular pattern activation immune response (PTI) in work
With, it is theoretical to abundant plant and pathogenic microorganism interaction, and foundation and gene are provided for tomato broad-spectrum disease resistance breed of variety
Source.
Embodiment
One VIGS of embodiment experiment
1 materials and methods
1.1 vegetable materials, pathogen and its cultural method
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 saved from Zhoukou Normal University, 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 present, expands numerous growth, temperature: 20 ± 2 DEG C on rye culture medium.Verticillium dahliae
It is given by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute plant protection research department Zhu Heqin researcher, expands numerous life on Czapek's medium
It is long, temperature: 25 ± 2 DEG C.All plant strain growths are in condition good growing weather room, temperature: 20 ± 2 DEG C, light dark cycles:
16h/8h, luminous intensity: 150 μm of ol/m2·s。
1.2 used carrier
The double base that VIGS tests RNA1 and RNA2cDNA that silent carrier used is Tobacco rattle virus (TRV), which is expressed, to be carried
Body, respectively TRV1 (pYL192) and TRV2 (pYY13).TRV1 carrier includes RNA polymerase, the movement egg that coding RNA relies on
White and 16K albumen gene, is the helper viral vector of VIGS system.TRV2-LIC carrier include capsid protein (Cp) gene,
Multiple cloning sites (MCS) etc., the building for target gene.PYY13 is derived from carrier pYL170 (Burch-Smith et
Al., 2006).In order to clone target fragment with high throughput in TRV2-VIGS carrier, using the company of not depending on of an improvement
Clone (ligation independent cloning, the LIC) method connect, two LIC connectors are added on pYL170, are formed
Carrier pYY12, connector include the sequence of 15bp and the Pst I restriction enzyme site of centre.Then pass through Pst I digestion, In
A ccdB gene is inserted between two sites LIC, is formed carrier pYY13 (Fig. 1).CcdB is a lethal gene, can be fast
Speed accurately filters out recombinant vector.
Extraction, cDNA synthesis and the RT-PCR amplifying target genes of 1.3 RNA
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 to examine the whether effective reporter gene of VIGS carrier system.According to tomato PDS
Gene order (Gene Bank accession number M88683), choose be located at PDS gene in the middle part of sequence be target fragment design upstream with
Downstream primer.Using the method for electronic cloning, cDNA step is carried out to tomato powdery mildew disease-resistant gene candidate sequence and is moved, is moved according to step
Sequence afterwards, design have the primer of TRV2-LIC vector junctions.Upstream primer: 5 '-CGACGACAAGACCCTGene specific
Sequence -3 ', downstream primer: 5 ' -GAGGAGAAGAGCCCTGene specific sequence -3 ', underscore are 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, primer (10pm/ μ L) each 1 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 initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s,
72 DEG C of extension 1min, 30 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
The primer sequence of 1 RT-PCR amplifying target genes of table
Table1 The sequences of primers used in RT-PCR of objective genes
The building of 1.4 TRV2-LIC recombinant vectors
The PCR product of target gene, according to treasured biology (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 digestion
Target gene and TRV-LIC carrier.T4Archaeal dna polymerase digestion target gene reaction system: target gene 6 μ L, T4DNA polymerization
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 digestion 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.Finally by the matter after digestion
Grain carries out LIC with target gene and connects, reaction system: 1 μ L of plasmid, 6 μ L of target gene；Reaction condition: 65 DEG C, 2min, then
22 DEG C, 10min.Connection product transformed competence colibacillus E.coli DH5 α, coating LB plate (50mg/L Kana) culture, 37 DEG C overnight
It is inverted culture and forms single colonie.
The identification of 1.5 TRV2-LIC recombinant vectors
TRV2-LIC empty plasmid carries a ccdB gene, a kind of albumen for interfering e. coli dna gyrase of codified
Matter has lethal effect to Escherichia coli, if carrier occurs from connecting, the e. coli host cell for converting these carriers will be dead
It dies, only after exogenous dna fragment is connect with carrier, the expression of ccdB gene is blocked, and recombinant plasmid could be in large intestine bar
It survives in bacterium, therefore is conducive to efficiently obtain positive colony.It is formed after picking TRV2-LIC recombinant plasmid transformed E.coli DH5 α
Monoclonal, extract plasmid carry out PCR identification.Positive plasmid is converted to competence Agrobacterium GV3101, is coated with LB plate
2d is cultivated in (50mg/L Kana, 50mg/L Rif) culture, 28 DEG C of inversions, selects positive colony conversion Agrobacterium GV3101.
1.6 Agrobacteriums are infected and plant strain growth
Using four leaf stage tomato G1.1560, LA1221 and six leaf phase N.benthamiana as experimental material.TRV1,TRV2-
LIC (zero load), TRV2-LIC (recombinant plasmid) convert Agrobacterium GV3101 by freeze-thaw method.Pick them separately Agrobacterium (TRV1 and
TRV2-LIC) monoclonal respectively takes 10mL culture solution in 28 DEG C of overnight incubations, and 3000rpm, 20min, thalline were collected by centrifugation precipitates,
It is resuspended in buffer (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 1: 1 ratio of TRV2-LIC (recombinant plasmid) Agrobacterium GV3101,
Room temperature 100rpm low-speed oscillation 2h.The bacterium solution that activation is drawn with the disposable 2mL syringe for removing syringe needle, is injected using compression method
It is inoculated in the tomato true leaf back side (Mandar et al., 2008).With TRV2-LIC (zero load) for negative control.Infect tomato in
Overnight incubation under 20 DEG C of dark conditions is later 20 ± 2 DEG C in temperature, cultivates in the climatic chamber that light dark cycles are 16h/8h.Agriculture
After bacillus bacterium solution injects G1.1560 10d, the conidium of fresh powdery mildew is rinsed from the blade of severe infections, uses
Water is diluted to 2 × 104For being inoculated with after spore/mL concentration.The variation of the disease-resistant phenotype of tomato G1.1560 is observed after a week.
Every kind of silent carrier is inoculated with 10 plants of tomato seedlings, and experiment is repeated 3 times.
It is good with sterile bamboo stick picking growth conditions in superclean bench after Agrobacterium infects N.benthamiana 10d
Good phytophthora infestans mycelia is coated in leaf vein, blade tip and limb edge etc. respectively, and picking hyphae length is consistent, and is seen after a week
Examine the variation of the disease-resistant phenotype of N.benthamiana.Every kind of silent carrier is inoculated with 10 plants of N.benthamiana seedling, experiment weight
It is 3 times multiple.
After Agrobacterium infects tomato LA1221 10d, the new branch of the LA1221 newly grown is invaded to profit respectively 105Spore/mL
In the spore suspension and clear water of verticillium dahliae, the variation of the disease-resistant phenotype of tomato G1.1560 is observed after a week.Every kind of silencing carries
Body is inoculated with 10 plants of tomato LA1221 seedling, and experiment is repeated 3 times.
1.7 tissue Microscopic analysis
Leaf tissue H2O2Detection using benzidine (3,3 '-diaminobenzidine tetrahydrochloride,
DAB detection method) is dyed, powdery mildew expects blue decoration method (Huang et al., 1998) using platform.Specific step is as follows:
(1) DAB is dyed: taking the fresh blade with handle, its petiole is cut into inclined-plane, be inserted into the DAB (pH 3.8) of 1mg/mL
In solution, the inclined-plane of the dipped petiole of solution.It is placed under illumination and stands 8h, the DAB solution for being visually observed brownish red is suitable
Vein reach blade top.Reaction time can adjust according to the size of blade and actual conditions.
(2) fixed: after DAB dyeing rear blade is sheared along intermediate main lobe arteries and veins, then to be cut into the small of 1.5cm × 1.5cm
Block, be directly immersed in fixer (dehydrated alcohol: glacial acetic acid=3: 1), and until sloughing color, usually processing overnight.
(3) platform expects blue dyeing: leaf tissue is transferred to expects in blue solution containing 0.3% platform, and test tube lid is gently unscrewed,
(70 DEG C -80 DEG C) are placed in the water-bath of heating, dye liquor boiling 1min is made.Leaf tissue is stayed overnight in dye liquor.
(4) it decolourizes: rinsing leaf tissue 3 times be colored with the chloral hydrate of 2.5g/mL, sample can be in hydration
The several months is placed in trichloroacetaldehyde.
1.8 semi-quantitative RT-PCR analysis
Extract the total serum IgE of adjoining tree and silencing plant.Template is done with the RNA of 1 μ g, according to cDNA synthetic agent box
(TaKaRa) operating instruction synthesizes the first chain cDNA.Using gene transcripts in semiquantitive RT-PCR detection silencing plant
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, primer (10pm/ μ L) each 1 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 initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 30 are followed
Ring；72 DEG C of extension 10min.In order to avoid PCR product amount reaches influence of the plateau to experimental result, 20,25,30 and have been carried out
The amplification of 35 different recurring numbers, 1% agarose gel electrophoresis detect pcr amplification product.
The primer sequence of 2 Semiquatitative RT-PCR assay of table
Table2 The sequences of primers used in semi-quantitative RT-PCR
The amplification of 1.9 full length genes
Gene walking is carried out according to tomato whole genome sequence, design includes the primer of complete reading frame, RT-PCR amplification
The full length gene of the ShORR-1 sequence of G1.1560 and sequencing.Amplification ShORR-1 corresponds to upstream region of gene primer 5 '-
ATTACTCTCTTCATAAACTCATTTCCA-3 ', downstream primer 5 '-TCTGCTGCTATTTCTGCCACT-3 '.PCR reactant
It is (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, primer (10pm/ μ L) each 1 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 initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extensions
10min.Pcr amplification product is cloned into after purification on pMD18-T carrier, converts the plasmid into E.coli DH5 α competent cell
PCR identifies that positive bacteria falls behind, and Nanjing Genscript Biotechnology Co., Ltd. is transferred to be sequenced.
The expression of 1.10 ShORR-1 gene spaces and inoculation Different Kinds of Pathogens object expression analysis
In order to analyze the variation of ShORR-1 gene expression quantity after expression quantity and pathogen infection in different tissues, MT is taken
Root, stem, leaf, flower, green fruit and the erythrocarpus of tomato extract RNA, the expression quantity of reverse transcription post analysis ShORR-1.Simultaneously
About 4w age MT tomato plant is taken, rendezvous method inoculating tomato powdery mildew and phytophthora infestans are respectively adopted in tomato leaf, root-pouring method
Verticillium dahliae is inoculated in the root of MT tomato, puts the expression quantity variation of sampling analysis ShORR-1 gene in different times.It is 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 are as follows: 95 DEG C of initial denaturation, 10sec；
95 DEG C, 5sec；58 DEG C, 30sec, which recycles for 40 totally, in BIO-RAD CFX96TMIn Real-time System
The expression quantity for analyzing gene, with relative quantification 2-ΔΔCT method carries out relative quantitative assay to expression quantity, using tomato Actin as internal reference
Gene.
The primer sequence of 3 real-time quantitative qRT-PCR of table
Table3 The sequences of primers used in real-time RT-PCR
Two ShORR-1 gene VIGS Phenotypic Observation of embodiment
The clone of 2.1 ShORR-1 genes
The tomato powdery mildew resisting candidate ShORR-1 of 510bp has been cloned using the cDNA of wild disease-resistant tomato G1.1560 as template
Gene order, since the sequence of the DE-TDF from sequencing is shorter, using the method for bioinformatics, according to tomato full genome
After group sequence carries out gene walking, the sequence homologous with it, Primer5 software Design primers are obtained.
The building and identification of 2.2 TRV2-LIC recombinant vectors
The tomato powder mildew resistance reaction candidate gene that RT-PCR is expanded is carried using this chapter method 1.4 and TRV2-LIC
Body connection, Transformed E .coli DH5 α obtain positive colony, and positive plasmid is converted 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 is analyzed
It is how to influence G1.1560 plant pair O.neolycopersici disease-resistant to analyze ShORR-1 gene silencing
Property, tissue microscopic analysis is carried out to ShORR-1 gene silencing plant and adjoining tree, germ dyeing expects blue decoration method using platform.
Susceptibility and microscopic analysis are shown, after being inoculated with powdery mildew 7d, do not carry out the control disease-resistant plant of ShORR-1 gene silencing
G1.1560 shows as disease-resistant, and the powdery mildew conidium being inoculated on blade does not have rudiment largely, and sprouts sporogenesis
Haustorium then causes it to infect the allergic reaction of epidermal leaf cells (meronecrosis), and sufficient battalion can not be obtained from blade cell
It supports, and then mycelia growth is suppressed, new conidium can not be formed and completes entire vegetative propagation period (Fig. 1 C, D).
The disease-resistant plant G1.1560 blade of ShORR1 gene silencing shows as susceptible, to be inoculated on blade powdery mildew conidium energy
Enough normal rudiments, the haustorium of formation do not cause the allergic reaction for infecting blade cell, and mycelia normal growth simultaneously forms mitogenetic
Sporophore and new conidium can complete entire vegetative propagation period (Figure 1A, B), carry out infecting for a new round.As a result table
Bright, ShORR1 gene is the key gene of tomato powder mildew resistance reaction.
2.3.2 verticillium dahliae Analysis of Resistance
Effect of the VIGS analysis ShORR1 mediated using TRV in tomato verticillium wilt Resistant reaction shows that inoculation Huang withers
The highly resistance of sick pathogenic bacteria verticillium dahliae is withered, verticillium wilt tomato (S.lycopersicum LA1221) after a week, carry
PYY13 zero load Agrobacterium-mediated Transformation 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).
The result shows that ShORR1 gene is the key gene of tomato verticillium wilt Resistant reaction.
2.3.3 phytophthora infestans Analysis of Resistance
Susceptibility and microexamination analysis are carried out to ShORR-1 gene silencing plant and adjoining tree, experiment shows
This uncured tobacco blade of ShORR1 gene silencing to phytophthora infestans show as it is susceptible (Fig. 3 A-B, arrow meaning be phytophthora infestans
Mycelia), zoospore and mycelia are produced, and adjoining tree blade shows allergic reaction (meronecrosis), no zoospore
It is generated with mycelia and mycelia.The result shows that ShORR1 gene is the key gene of this life cigarette P. infestans resistant reaction.2.4 VIGS
RT-PCR is detected after silencing
TRV2-LIC-PDS, TRV2-LIC-ShORR1 are had detected in molecular level using the method for Semiquatitative RT-PCR assay
Transcript degree after silencing, 20 is carried out respectively to target gene fragment and Actin reference gene using the cDNA of synthesis as template,
25,30 and 35 cyclic amplifications.Electrophoresis detection finds that expression is significantly lower than in PDS, ShORR1 gene silencing plant leaf
Control, is less than about 60% (Fig. 4) of control.
2.4 ShORR-1 sequences correspond to the overall length of gene
It is 974bp that carrier T PCR sequencing PCR, which measures ShORR-1 sequence and corresponds to the full length sequence of gene, wherein including complete reading code
Frame sequence length is 807bp, the following SEQ ID NO.1 of sequence；
Use NCBI ORF-finder predict its coding protein for 268 amino acid, the following SEQ ID NO.2 of sequence
The space expression of 2.5 ShORR-1 genes is analyzed and inoculation Different Kinds of Pathogens object analysis
Root, stem, leaf, flower, green fruit and the erythrocarpus for taking MT tomato, the expression analysis discovery to ShORR-1,
Expression quantity in leaf is apparently higher than other tissues (Fig. 5), thus it is speculated that expresses in the main leaf of ShORR-1.It is inoculated with three kinds of inhomogeneities
The fungal pathogens of type put sampling analysis in different times, the results showed that ShORR-1 expression quantity is after powdery mildew infects 12h
Reach a peak, top (Fig. 6) is reached after 72h；Similar with powdery mildew expression pattern, ShORR-1 is in phytophthora infestans
There is first peak expression after infecting 3d, expression quantity reaches highest (Fig. 7) after infecting 14d；And ShORR-1 is in big beautiful wheel branch
Expression quantity reaches highest after bacterium inoculation 60h, declines (Fig. 8) later.Thus speculate, ShORR-1 is in various pathogenic bacteria and tomato
Interaction in play an important role.
It is analyzed according to sequence, ShORR-1 is the new gene of a Unknown Function, and still any function homologous not with it be
The gene known is found.
Above embodiments are only to illustrate, the technical solution being not intended to limit the present invention, although referring to above-described embodiment to this
Invention is described in detail, those skilled in the art should understand that: can still modify to the present invention or
Equivalent replacement should all cover of the invention without departing from the spirit or scope of the invention, or any substitutions
In scope of the claims.
Claims (10)
1. a kind of plant disease-resistant indispensable gene, it is characterised in that its nucleotides sequence is classified as nucleotide sequence shown in SEQ ID NO.1.
2. the protein of the coding of gene described in claim 1, amino acid sequence is SEQ ID NO.2.
3. the recombinant vector containing nucleotide sequence described in claim 1.
4. recombinant vector according to claim 3, it is characterised in that the carrier be for clone pMD18-T carrier,
For pSAT6-GFP-N1, pCAMBIA1300, pCAMBIA2300 carrier of expression and for Gene Silencing
PYY13 carrier.
5. transgenic cell line or recombinant bacterium containing nucleotide sequence described in claim 1.
6. nucleotides sequence described in claim 1 is listed in the application expressed in plant tissue.
7. application according to claim 6, it is characterised in that the plant tissue is root, stem, leaf, flower or seed.
8. application according to claim 6, it is characterised in that the plant is monocotyledon or dicotyledon.
9. application according to claim 8, it is characterised in that the monocotyledon be rice, wheat, sorghum and corn,
The dicotyledon is tomato, tobacco, soybean and potato.
10. albumen described in gene or claim 2 described in claim 1 is cultivating the application in plant disease-resistant kind, described anti-
Disease is powdery mildew, late blight and verticillium wilt.
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