CN115109128B - Peanut bacterial wilt effect protein RipXV, encoding gene and application thereof - Google Patents

Abstract

The invention discloses a peanut ralstonia solanacearum effector protein RipXV, and a coding gene and application thereof. The amino acid sequence of the peanut bacterial wilt effect protein RipXV is shown as SEQ ID No.2, and the nucleotide sequence of the coding gene RipXV is shown as SEQ ID No. 1. The RipXV gene is transiently overexpressed in Nicotiana benthamiana, and can induce death of plant cells. The delta ripXV mutant is constructed by a homologous recombination method, and the pathogenicity of the inoculated peanut is enhanced, which shows that RipXV possibly is a nontoxic effector protein and plays an important role in the peanut pathogenicity process. The RipXV target protein AhRFL1 was screened from a peanut bacterial wilt-induced cDNA library by yeast two-hybrid technology. The invention has important significance for resolving the molecular mechanism of interaction of the bacterial wilt and the host and establishing the comprehensive control technical strategy of the plant bacterial wilt.

Description

Peanut bacterial wilt effect protein RipXV, encoding gene and application thereof

Technical Field

The invention relates to the field of plant disease control research, in particular to peanut bacterial wilt effect protein RipXV, a coding gene thereof and potential value and application prospect in plant bacterial wilt.

Background

Bacterial wilt caused by ralstonia solanacearum (Ralstonia solanacearum) is one of the important plant diseases in the world. The pathogenic bacteria have the characteristics of multiple host ranges, abundant physiological species, wide geographical distribution, rapid pathogenic variation, various disease conditions, difficult control and the like, and the plant diseases caused by the pathogenic bacteria cause huge economic losses. At present, the infection and pathogenic mechanism of the bacterial wilt disease are not completely clear, so that the search for an effective acting target is vital to the disease control from the pathogenic mechanism of the bacterial wilt. The type III secretion system is a pathogenicity determinant of bacterial wilt, which activates or inhibits host defensive responses by injecting large amounts of effector proteins into host cells using T3SS in injection form, thereby causing host disease or inducing hypersensitivity responses in non-host plants.

Plant pathogens secrete a class of proteins that interact with the R gene product of a host plant to stimulate the immune system of the plant when they infect the host plant, known as effector proteins. The gene encoding the pathogen effector inducing resistance mediated by the R gene is defined as Avr gene. Effector proteins include toxic proteins, a class of proteins necessary to fully exert toxicity by pathogenic bacteria entering host cells, and non-toxic proteins; nontoxic proteins are a class of proteins that can be recognized directly or indirectly by plants and can trigger allergic reactions. Most effector proteins can change the cellular structure and function of hosts and accelerate the infection of pathogenic bacteria, so that plants evolve R proteins which can specifically recognize different effector proteins. The interaction of plants with pathogenic bacteria is controlled by specific interactions between the avr (avirulence) gene of the pathogen and the corresponding allele of the plant disease resistance (R) gene, i.e. "gene-to-gene" interactions. In this model, specific receptors encoded by plant disease resistance genes recognize metabolites produced by the non-toxic gene products or exciton-mediated catalytic activity of pathogenic bacteria, which in turn trigger the defense response of the plant. This model was used to demonstrate that specific recognition of pathogenic Avr proteins by plant R proteins was demonstrated, with the former being a ligand for the latter. Although these models provide a simple model parallel to the immune system, the relationships they indicate are still lacking, and only a few examples of the current cases are able to demonstrate the direct interaction between the R protein and its cognate Avr protein.

Based on the result of the complete genome sequence analysis of the early-stage peanut ralstonia solanacearum RS-P.362200 in the subject group, the experiment obtains an effector protein RipXV of a III type secretion system through bioinformatics analysis, and the construction of an excessive expression vector can generate strong allergic necrosis reaction through agrobacterium-mediated transient expression of Nicotiana benthamiana. By constructing a delta RipXV mutant strain by using a homologous recombination method, inoculating a host can obviously enhance pathogenicity of the bacterial wilt, which shows that RipXV possibly is a nontoxic gene and plays a key role in interaction of the bacterial wilt and plants. Meanwhile, through yeast double-hybrid screening, ripXV interacting NBS-LRR disease-resistant proteins AhRFL1 in a host are obtained, and AhRFL is presumed to be possibly involved in defense reaction of the host to the bacterial wilt, so that gene resources can be provided for genetic improvement of plant bacterial wilt resistance. At present, less interaction between the bacterial wilt and a host is researched, and the application starts from the bacterial wilt effector protein and the host target protein thereof, researches the interaction between RipXV and AhRFL1, provides a new thought for revealing the pathogenesis and prevention of the bacterial wilt, and has important scientific significance.

Disclosure of Invention

The invention aims to provide a peanut bacterial wilt effect protein RipXV, a coding gene and application thereof, provides a theoretical basis for a bacterial wilt pathogenesis and peanut bacterial wilt resistance, and has important application prospect.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The invention firstly provides a peanut bacterial wilt effect protein RipXV, and the amino acid sequence of the peanut bacterial wilt effect protein RipXV is shown as SEQ ID NO. 2.

The invention further provides a gene for encoding the peanut ralstonia solanacearum effector protein RipXV, wherein the gene is RipXV, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention also provides an overexpression vector containing the peanut ralstonia solanacearum effector protein RipXV, and the construction method of the overexpression vector comprises the following steps: based on Gataway system, constructing an entry vector pDONR207-RipXV through BP reaction, and constructing a plant expression vector pK7WG2.0-RipXV driven by a 35S promoter through LR reaction, namely the over-expression vector containing the peanut bacterial wilt effect protein RipXV.

The invention also provides a knockout carrier containing the peanut bacterial wilt effect protein RipXV, and the construction method of the knockout carrier comprises the following steps: according to the complete gene sequence of the peanut ralstonia solanacearum RS-P.362200, cloning RipXV an upstream 657 bp fragment (U) and a downstream 643 bp fragment (D), and constructing a knockout vector pK18mobSacB-U-D by a digestion connection method.

The invention also provides a reconversion vector containing the peanut ralstonia solanacearum effector protein RipXV, and the construction method of the reconversion vector comprises the following steps: according to the complete gene sequence of the peanut ralstonia solanacearum RS-P.362200, cloning an upstream 657 bp fragment (U), a RipXV fragment (1062 bp) and a downstream 643 bp fragment (D), and constructing a compensation vector pK18mobSacB-U-RipXV-D by an enzyme digestion connection method.

The invention also provides a protein which interacts with the peanut bacterial wilt effect protein RipXV, wherein the protein is NBS-LRR disease-resistant protein AhRFL, the nucleotide sequence of the protein is shown as SEQ ID No.3, and the protein sequence is shown as SEQ ID No. 4. AhRFL1 is based on yeast two hybrid screening: constructing a bait carrier PGBKT-RipXV, and screening to obtain the RipXV interaction protein according to a mixed cDNA library of the root and the leaf induced by the bacterial wilt.

The invention also provides application of the peanut ralstonia solanacearum effector protein RipXV in stimulating plant defense response, anaphylactic response and/or stimulating plant disease resistance gene expression as an effector protein.

The invention also provides application of the peanut ralstonia solanacearum effector protein gene RipXV in culturing transgenic engineering bacteria with enhanced pathogenicity to peanuts.

Furthermore, the method for cultivating the transgenic engineering bacteria with enhanced pathogenicity to the peanuts is to knock out RipXV genes in the original strain and screen the transgenic engineering bacteria with enhanced pathogenicity to the peanuts; the starting strain is peanut ralstonia solanacearum (Ralstonia solanacearum).

The invention has the beneficial effects that:

According to the application, through the sequencing analysis of the whole genome of the peanut ralstonia solanacearum Rs-P.362200, a triple effector protein RipXV is predicted, and the coding gene RipXV of the triple effector protein RipXV is transiently overexpressed on Nicotiana benthamiana and can cause strong allergic necrosis reaction. The pathogenicity of the bacterial wilt is obviously enhanced after RipXV gene is knocked out, which is the report of RipXV affecting the pathogenicity of the bacterial wilt for the first time. The application further screens RipXV host target NBS-LRR disease-resistant protein AhRFL1 for the first time through yeast two-hybrid. The research result of the application has important scientific significance for revealing the pathogenic mechanism of the bacterial wilt on the peanuts and the bacterial wilt resistance mechanism of the peanuts.

Drawings

Fig. 1: construction of pK7WG2.0-RipXV overexpression vector.

Fig. 2: a RipXV knockout and anaplerotic strain schematic diagram is constructed based on a homologous recombination method. Delta RipXV is a knockout mutant of effector protein RipXV and C.DELTA. RipXV is a complementation strain of effector protein Delta RipXV.

Fig. 3: ripXV knockout and anaplerosis strains are used for constructing PCR amplification electrophoresis gel running results. WT represents the result of PCR amplification of wild type bacterial wilt strain, delta RipXV represents the result of PCR amplification of knockout strain, C Delta RipXV represents the result of PCR amplification of anaplerotic strain, and "-" represents negative control.

Fig. 4: the transient overexpression of the bacterial wilt effector RipXV gene in the leaf of Nicotiana benthamiana causes strong cell necrosis. The HR response results show that transient overexpression RipXV causes strong allergic death in the lamina of nicotiana benthamiana; DAB staining results showed RipXV that caused the HR reaction to produce large amounts of H ₂O₂ and trypan blue staining results showed RipXV that caused the HR reaction to produce large amounts of callus.

Fig. 5: ripXV mutations and anaplerotic strains the phenotype after inoculation with peanut.

Fig. 6: ripXV and AhRFL in yeast. The positive control was pGBKT7-53+pGADT7-T, and the negative control was pGBKT7-lam+ PGADT7-T.

Detailed Description

Construction RipXV of an overexpression vector

According to the complete genome sequence of the peanut ralstonia solanacearum Rs-P.362200, the full-length gene CDS sequence of RipXV is queried, and a specific primer (RipXV-attB 1-F:5' -GGGGACAAGTTTGTACAAAAAAGCA) is designed

GGCTTCATGAAAAGATTTATGAGG-3', ripXV-attB2-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCGCTCAATAGCTTTTC-3') carrying out PCR amplification by taking peanut bacterial wilt genome DNA as a template and adopting PRIMESTAR ^® Max of Takara company, wherein a PCR reaction system is as follows: 1. 2X PRIMESTAR ^® Max of the DNA template of [ mu ] L, and 0.5 [ mu ] L of each forward and reverse primer, and supplementing water to 20 [ mu ] L. The reaction conditions are as follows: pre-denaturation at 95 ℃ 5 min;95℃for 30 s,60℃for 30 s,72℃for 1 min,30 cycles. And (3) performing gel cutting, purifying and recycling after agarose gel electrophoresis detection of the PCR product, and performing BP reaction on the target gene fragment and the pDONR207 in a no-load connection manner: ripXV PCR product 1 u L, pDONR207 empty 1 u L, BP enzyme 0.25 u L,25 ℃ connection overnight, the connection product is transformed into E.coli DH5 alpha competent cells, screening positive clones for sequencing, correctly read through cloning extraction plasmid, construction of entry vector pDONR207-RipXV. The entry vector plasmid and the overexpression vector pk7wg2.0 were subjected to LR reaction: pDONR207-RipXV plasmid 1. Mu.L, pEarley gate201 empty 1. Mu.L, LR enzyme 0.25. Mu.L, 25℃overnight ligation, transformation of E.coli, verification of positive clones, construction of the 35S CaMV promoter driven overexpression vector pK7WG2.0-RipXV. The construction process of the over-expression vector is schematically shown in FIG. 1.

Construction of Delta RipXV deletion mutant and C Delta RipXV make-up vector

According to the bacterial wilt Rs-P.362200, a RipXV upstream 657 bp sequence U and a downstream 643 bp sequence D are found, a specific primer RipXV-1-F（5'-CGGAATTCAGCTTCCCATCGAACACTG-3'）,RipXV-1-R（5'-CGGGATCCGATAGACAATCCTTGAATTTTC-3'）,RipXV-2-F（5'-CGGGATCCGTATGATCTGCTCAGAGTCG-3'）,RipXV-2-R（5'-CCAAGCTTTCAACATCAAGGCGATCC-3'）, is designed, PCR amplification is carried out by taking peanut bacterial wilt genome DNA as a template, PCR premix liquid of a company is adopted for amplification, and a PCR reaction system is as follows: 1. genomic DNA template of [ mu ] L, PCR premix of 5 [ mu ] L, forward and reverse primers of 0.5 [ mu ] L respectively, and water of 3 [ mu ] L. The reaction conditions are as follows: 94 ℃ 5 min;94 ℃ for 30 s,60 ℃ for 30 s,72 ℃ for 1 min,28 cycles; storing at 72deg.C for 10min at 25deg.C. And detecting the PCR product by agarose gel electrophoresis, purifying and recovering the PCR product after no impurity band. The desired fragment and empty pK18mobsacb were recovered by restriction enzyme EcoRI, bamHI, hindIII, respectively, and U and D were ligated to pK18mobsacb vector by T4 ligase, respectively, under the following conditions: t4 interest 2 [ mu ] L, T4 buffer 1 [ mu ] L, pK18mobsacb [ mu ] L (100 ng), U/D2 [ mu ] L (100 ng), H ₂ O4 [ mu ] L,16 ℃ connection overnight. The ligation product was transformed into E.coli, positive clones were confirmed by PCR, and the construction of RipXV knockout vector pK18mobsacb-U-D was completed, and the schematic diagram of the vector construction process is shown in the left panel of FIG. 2.

The construction method of the repayment vector comprises the following steps: according to the whole genome sequence of the peanut bacterial wilt Rs-P.362200, a specific primer is designed, PCR amplification is carried out by taking bacterial wilt genome DNA as a template (construction of a PCR reaction system and a knockout vector), meanwhile, cloning comprises up-stream 657 bp (U), ripXV (1062 bp) and down-stream 643 bp (D) total 2362 bp fragments (the specific primer is used for CRipXV-XhoI-F：5'-ATTACTCGAGTCGCCTACGCTACAATCTCCTG-3',CRipXV-BamHI-R：5'-ACCAGGATCCTCATCGCTCAATAGCTTTTCTG-3'）. respectively cutting and recovering a target fragment and empty load pK18mobsacb by using restriction endonucleases XhoI and BamHI, U-RipXV-D is connected to the pK18mobsacb vector by using T4 ligase (connection conditions are the same above). The connection product is transformed into escherichia coli, positive cloning is verified by PCR, and construction of a RipXV-compensating vector pK18mobSacB-U-RipXV-D is completed, and a vector construction process is shown in a right diagram of fig. 2.

Example 3A delta RipXV knockout mutation and a C delta RipXV anaplerotic strain were obtained by the triparental binding method

The construction method of the mutant strain and the anaplerotic strain mainly adopts a three-parent combination method, a monoclonal of host bacterial wilt peanut is selected and cultured in a 4 mL BG liquid culture medium (containing 100 mg/L polymyxin B) in a constant temperature shaking table at 28 ℃, and when the concentration of the bacterial cells OD ₆₀₀ =0.6, all bacterial cells are collected in 2mL EP tubes; picking helper MT616, culturing in 4 mL LB liquid culture medium (containing 34 mg/L clindamycin) at 37deg.C in a constant temperature shaker, and collecting all thallus with 2mL EP tubes when thallus concentration OD600 = 1.0; picking donor bacteria pK18mobSacB-U-RipXV-D or pK18mobsacb-U-D into LB liquid medium (containing 50 mg/L kanamycin) of 4 mL, culturing in a constant temperature shaking table at 37 ℃, and collecting all bacteria by 2mL EP tubes when the concentration of bacteria OD ₆₀₀ =2.0; centrifuging three types of thalli 5000 rpm to min, washing the collected thalli twice with sterile water, and then suspending the thalli again with sterile water; the cell concentration OD ₆₀₀ is adjusted to 1.0 by a spectrophotometer; host bacteria, rhizoctonia solani, helper bacteria MT616, donor bacteria pK18mobSacB-U-RipXV-D or pK18mobsacb-U-D were then used in accordance with 2:1:1, uniformly mixing the components in a volume ratio; and centrifugally collecting thalli, suspending the thalli again by 50 mu L of sterile water, uniformly spotting the thalli on a BG flat plate by a pipetting gun, and culturing the flat plate in a constant temperature incubator at 28 ℃ in an inverted manner after the flat plate bacterial liquid is dried, so as to induce 2-3 d. Scraping mixed bacteria on a plate, suspending the mixed bacteria with 100 mu L of sterile water, uniformly coating the mixed bacteria on a BG plate (containing 100 mg/L polymyxin B and 50 mg/L kanamycin) (marking the edges of the plate with peanut bacterial liquid for negative control), placing the plate in a constant temperature incubator at 28 ℃, and culturing for two days; the bacterial wilt with good growth on the plate is selected, genomic DNA is extracted, and amplified by specific primers RipXV-1-F, ripXV-2-R, for example, the amplified band is consistent with the designed segment (two bands, one at 1.28 kb and one at 2.92 kb), which indicates that the vector is successfully transferred into bacterial wilt.

5-6 Bacterial wilts with good growth vigor and consistent PCR band sizes are selected and streaked on a BG (PB+Kan) plate for 1-2 days (a small amount of peanut bacterial wilt liquid is streaked on the edge of the plate to be used as a negative control), and the bacterial wilt is selected and streaked on the BG plate for 1 d. Bacterial wilt with good growth vigor is picked and streaked on BG and BGS plates respectively (colony containing sac carrier does not grow or grows slowly on sucrose-containing culture medium). After 1 d, picking up bacterial wilt which grows well on the BG plate but does not grow or grows slowly on the BGS plate, shaking the bacterial wilt at 28 ℃ for 5-6 h, and then coating 100 mu L on the BGS plate for culturing 1-2 d. After 2d, well grown bacterial wilt was picked and streaked overnight on BG and BG plates (containing 100 mg/L polymyxin B and 50 mg/L kanamycin), respectively (plates with grids were streaked with antibiotics first and then BG plates). Bacterial wilt not growing on BG plates (containing 100 mg/L polymyxin B and 50 mg/L kanamycin) was picked up, genomic DNA was extracted, amplified with specific primers RipXV-1-F, ripXV-2-R, and bacterial wilt was used as a positive control. And then selecting PCR banded ralstonia solanacearum to carry out genome extraction, and verifying by using specific primers.

The mutation and anaplerotic strain RipXV was obtained by the method described above, and PCR identification of the mutation and anaplerotic strain specific primers is shown in FIG. 3.

Example 4 transient overexpression RipXV coding gene of Benshi tobacco leaves

And (3) transforming the constructed RipXV over-expression plasmid into agrobacterium tumefaciens GV3101, culturing in a 28 ℃ incubator for 2 d, selecting a clone with good growth vigor, adding the clone into a corresponding antibiotic culture medium, placing the clone into a shaking table for shake culture, setting the temperature to be 28 ℃, culturing overnight at the rotating speed of 250 rpm, and verifying whether the transformation is successful or not by PCR. Positive clones that were confirmed to be correct were added to 50mL medium to expand the shaking until an OD ₆₀₀ value of 0.5 was measured. Placing into a centrifuge, centrifuging for 10 min at 4000 rpm, re-suspending with MES buffer, adjusting OD ₆₀₀ to 0.7-0.8, and incubating in an incubator at 28deg.C for 2 h. Selecting healthy Benshi tobacco in 5-6 leaf period, 3 plants as experimental group, 3 plants as control group, selecting two leaves of penultimate plant and third plant, puncturing the leaves by 1 mL syringe needle, injecting agrobacterium liquid at the punctured part by agrobacterium infiltration method, and the injection area is a circle with diameter of 1 cm. The tobacco phenotype was recorded by observation and photographing after 48 h greenhouse cultures at 25 ℃. After transient overexpression 48 h in Nicotiana benthamiana leaves, the infected plants were stained with 3,3' -Diaminobenzidine (DAB) and lactophenol-ethanol-trypan blue. The leaf pieces of Nicotiana benthamiana after injection were incubated overnight at room temperature in 1 mg/mL DAB solution, boiled in a 3:1:1 ethanol/lactic acid/glycerol solution for 5: 5 min, and then decolorized in absolute ethanol. Cell death was detected using trypan blue staining, and leaves after injection were boiled in trypan blue solution (10 mL lactic acid, 10 mL glycerol, 10 g phenol, 30mL absolute ethanol, and 10 mg trypan blue) for 2 minutes, stained overnight at room temperature, and transferred to chloral hydrate solution (2.5 g/mL) for 20 minutes to remove staining. The staining results were observed under an optical microscope. As shown in fig. 4, transient expression RipXV can generate strong allergic necrosis reaction of the nicotiana benthamiana, and DAB staining results show that the transient over-expression RipXV gene can release a large amount of active oxygen, and trypan blue staining results show that the transient over-expression RipXV gene can generate a large amount of calluses.

Influence of RipXV on the pathogenicity of ralstonia solanacearum

The wild-type strain (WT), mutant strain, and anaplerotic strain were cultured with 15 mL BG (100 mg/L PB ⁺) in a constant temperature shaker at 28℃at 250 rpm. When OD ₆₀₀ =0.4 is to be measured, the two-and three-functional peanut leaves are inoculated by a leaf-cutting inoculation method, and the growth condition of peanut plants after inoculation is observed and recorded after 7 days. The phenotype of the bacterial wilt inoculated peanuts is shown in fig. 5, the wild strain (WT) shows disease resistance 7 days after inoculating the bacterial wilt-resistant peanut variety Guangdong oil 92, the mutant Delta RipXV strain shows wilting of leaves 7 days after inoculating the bacterial wilt-resistant peanut variety Guangdong oil 92, the plant shows typical bacterial wilt symptoms, and the anaplerotic strain C Delta RipXV recovers the disease resistance of the leaves 7 days after inoculating the bacterial wilt-resistant peanut variety Guangdong oil 92.

Example 6 screening and validation of the interaction of ralstonia solanacearum effector protein RipXV with target proteins

The PCR product of RipXV was obtained by amplification using RipXV-BD-NcoI-F (5'-AGGACCATGGACCATGAAAAGATTTATGAG-3') and RipXV-BD-BamHI-R (5'-ATTAGGATCCTCATCGCTCAATAGCTTTTC-3') as primers and the pDONR207-RipXV plasmid as a template, and the PCR product and the bait vector PGBKT7 were digested simultaneously with NcoI and BamHI to recover the target bands, after which RipXV was ligated to the PGBKT7 vector using T4 DNA ligase (ligation conditions were the same). The ligation product was transformed into E.coli, positive clones were verified by PCR and sent to sequencing company for verification to obtain the bait vector pGBKT7-RipXV. The self-activation and toxicity detection of the bait carrier are verified through pGBKT7-RipXV transformed yeast Y2H Gold yeast strain, the self-activation phenomenon is avoided, and the subsequent screening library test can be performed. Preparing competent cells of pGBKT7-RipXV yeast positive clone, transforming with peanut bacterial wilt induced root-leaf mixed cDNA library plasmid, coating SD/-His/-Leu/-Trp plate with transformed thallus, picking clone with normal cloning point SD/-Ade/-His/-Leu/-Trp + x-alpha-gal plate growing on SD/-His/-Leu/-Trp plate, and turning blue on four-missing plate after 2-3 d as candidate positive clone. The plasmids of candidate positive clones were extracted, verified by PCR, and the PCR products were sequenced by company, and the coding sequence of the interaction target protein AhRFL was queried by alignment at PGR (http:// peanutgr. Fafu. Edu. Cn /). Cloning AhRFL full-length gene CDS sequences onto PGADT vectors, co-transferring PGADT-AhRFL 1 and PGBKT7-RipXV plasmids onto Y2H yeast competent cells, plating SD/-Leu/-Trp plates, picking clone shaking, diluting the bacterial solution according to a 10 ⁰/10^-1/10^-2/10^-3 gradient, taking 5. Mu.L to SD/-Ade/-His/-Leu/-Trp +X-alpha-gal plates, and changing the colony to blue after 3 days, indicating RipXV and AhRFL interactions (FIG. 6).

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

SEQUENCE LISTING

<110> Fujian university of agriculture and forestry

<120> Peanut bacterial wilt effect protein RipXV, coding gene and application thereof

<130>

<160> 14

<170> PatentIn version 3.3

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gaactgagca ccagagtaac taaaaccata ttcttaatat gtggtttgtt tccaagtggt 900

cgtgatcatg aacatgaagt tctcattgaa gagctgtcac aatatgcaat ccgactaggc 960

tggaggaaag atgaagtgag agcagccatc aatgacctca ttgagtctag tttgctgatg 1020

ctttctgata aaagcaaaga ccatatcata atgagtagtc tggtgcgtga cattgctaga 1080

aagatggttt atgaggatat ccctgccagc aagatggtta atgtttgtga ttacatgttt 1140

gcacaatata atcaggaaga tttccagtgg cgtgtagcat tacatcgact tcttcacttg 1200

ttgtatggca gtgcaaatga gctggcatct tcatcaacga ccaatgcaac atttgctgaa 1260

gagggtcttg aattccaaga tgtgtcgtcc tattcctatc caaaggttgc tacagttcct 1320

aagctatcag ggaaaatggt gaattttaaa aggctgtgtc aggtggacaa atcattcctg 1380

cctctgctca agaaagcgtg tgagaaccac ccccagctga ttcatagcca gaaaaaccac 1440

tctgaaatag taacacaaag cgcctttgat agcttagggc gggtgctgtt tctgttgaaa 1500

aatgttgaga agagggattg ggtgtatctg gaagatgagt tacgtattct ctggaagcag 1560

ttaaacgagt tcaaatttga tttggaatgg ttaagtcctt atgtgaaaga ggtgttctct 1620

accactaaga tcaccaggat tgaagtactt cgggagaaag agaaggattt gaaagacgaa 1680

gctgctaagc tgcgtgcact gcttggagct gcagaggacg aagctgctaa gctgcgtgcc 1740

cggcttgggg ttgttgagga cggagcttct gagctacgcg aacgactcaa ggtgaccgag 1800

aacaaagctg cagatgttag ggctgaagtt gtccgcagag aatctgagat gattgaatct 1860

gcaattgcta ttgttatgga gtaa 1884

<210> 4

<211> 627

<212> PRT

<213> Peanut (Arachis hypogaea)

<400> 4

Met Val His Glu Gly Glu Arg Ile Ile Lys Met Glu Glu Gly Ile Ser

1 5 10 15

Val Thr Asp Asp Tyr Asp Ser Gly Tyr Leu Met Gln Phe Gln Ser Arg

20 25 30

Glu Ser Thr Phe Asp Gln Ile Leu Glu Ala Leu Lys Asp Cys Lys Ile

35 40 45

Ser Gln Val Ala Leu Ser Gly Met Gly Gly Ser Gly Lys Thr Thr Leu

50 55 60

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

65 70 75 80

Val Val Phe Val Pro Val Thr Ser Thr Pro Ser Phe Met Lys Ile Gln

85 90 95

Gly Glu Ile Ala Asp Gln Leu Ser Leu Arg Leu Gly Glu Glu Ala Gly

100 105 110

Leu Ala Ala Arg Gln Arg Lys Lys Ser Met Thr Ser Lys Glu Leu Val

115 120 125

Leu Ile Ile Leu Asp Asp Val Leu Asp Arg Leu Asn Leu Glu Lys Ile

130 135 140

Gly Ile Phe Asn Gly Cys Asn Gly Leu Gln Leu Arg Thr Met Gly Arg

145 150 155 160

Arg Met Ser Cys Lys Val Leu Met Thr Thr Ser Thr Gln Glu Ala Val

165 170 175

Tyr Arg Met Gly Cys Gln Pro Ser Ile Asn Leu Ser Leu Leu Thr Glu

180 185 190

Gly Glu Ala Cys Asp Leu Leu Arg Lys His Ala Asp Ile Ser Asp Gly

195 200 205

Ser Pro Glu Ser Ile Val Glu Val Ala Lys Gln Val Ala Lys His Cys

210 215 220

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

225 230 235 240

Lys Thr Val Asp Gln Trp Lys Glu Val Leu Lys Thr Met Gln Met Leu

245 250 255

Pro Val Lys Ser Leu Gln Cys Glu Ala Asp Thr Ser Lys Phe Ile His

260 265 270

Gln Ile Ile Glu Val Gly Tyr His Glu Leu Ser Thr Arg Val Thr Lys

275 280 285

Thr Ile Phe Leu Ile Cys Gly Leu Phe Pro Ser Gly Arg Asp His Glu

290 295 300

His Glu Val Leu Ile Glu Glu Leu Ser Gln Tyr Ala Ile Arg Leu Gly

305 310 315 320

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

325 330 335

Ser Leu Leu Met Leu Ser Asp Lys Ser Lys Asp His Ile Ile Met Ser

340 345 350

Ser Leu Val Arg Asp Ile Ala Arg Lys Met Val Tyr Glu Asp Ile Pro

355 360 365

Ala Ser Lys Met Val Asn Val Cys Asp Tyr Met Phe Ala Gln Tyr Asn

370 375 380

Gln Glu Asp Phe Gln Trp Arg Val Ala Leu His Arg Leu Leu His Leu

385 390 395 400

Leu Tyr Gly Ser Ala Asn Glu Leu Ala Ser Ser Ser Thr Thr Asn Ala

405 410 415

Thr Phe Ala Glu Glu Gly Leu Glu Phe Gln Asp Val Ser Ser Tyr Ser

420 425 430

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

435 440 445

Phe Lys Arg Leu Cys Gln Val Asp Lys Ser Phe Leu Pro Leu Leu Lys

450 455 460

Lys Ala Cys Glu Asn His Pro Gln Leu Ile His Ser Gln Lys Asn His

465 470 475 480

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

485 490 495

Phe Leu Leu Lys Asn Val Glu Lys Arg Asp Trp Val Tyr Leu Glu Asp

500 505 510

Glu Leu Arg Ile Leu Trp Lys Gln Leu Asn Glu Phe Lys Phe Asp Leu

515 520 525

Glu Trp Leu Ser Pro Tyr Val Lys Glu Val Phe Ser Thr Thr Lys Ile

530 535 540

Thr Arg Ile Glu Val Leu Arg Glu Lys Glu Lys Asp Leu Lys Asp Glu

545 550 555 560

Ala Ala Lys Leu Arg Ala Leu Leu Gly Ala Ala Glu Asp Glu Ala Ala

565 570 575

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

580 585 590

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

595 600 605

Glu Val Val Arg Arg Glu Ser Glu Met Ile Glu Ser Ala Ile Ala Ile

610 615 620

Val Met Glu

625

<210> 5

<211> 49

<212> DNA

<213> Artificial sequence

<400> 5

ggggacaagt ttgtacaaaa aagcaggctt catgaaaaga tttatgagg 49

<210> 6

<211> 47

<212> DNA

<213> Artificial sequence

<400> 6

ggggaccact ttgtacaaga aagctgggtc tcgctcaata gcttttc 47

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<400> 7

cggaattcag cttcccatcg aacactg 27

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence

<400> 8

cgggatccga tagacaatcc ttgaattttc 30

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence

<400> 9

cgggatccgt atgatctgct cagagtcg 28

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence

<400> 10

ccaagctttc aacatcaagg cgatcc 26

<210> 11

<211> 32

<212> DNA

<213> Artificial sequence

<400> 11

attactcgag tcgcctacgc tacaatctcc tg 32

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence

<400> 12

accaggatcc tcatcgctca atagcttttc tg 32

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<400> 13

aggaccatgg accatgaaaa gatttatgag 30

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence

<400> 14

attaggatcc tcatcgctca atagcttttc 30

Claims (2)

1. The use of ralstonia solanacearum effector protein RipXV as an effector protein to stimulate plant allergic necrosis, characterized in that: through an agrobacterium-mediated transient expression system, the peanut bacterial wilt effector protein RipXV is transiently overexpressed in the Nicotiana benthamiana to excite the Nicotiana benthamiana to generate allergic necrosis reaction; the amino acid sequence of the peanut ralstonia solanacearum effector protein RipXV is shown as SEQ ID NO. 2.

2. The application of the peanut ralstonia solanacearum effector protein gene RipXV in culturing transgenic engineering bacteria with enhanced peanut pathogenicity is characterized in that: knocking out RipXV genes in an original bacterial strain, namely peanut bacterial wilt (Ralstonia solanacearum) in a homologous recombination mode to obtain transgenic engineering bacteria with enhanced pathogenicity to peanuts; the nucleotide sequence of RipXV gene is shown in SEQ ID NO. 1.