CN115109128A - Peanut ralstonia solanacearum effect protein RipXV and coding gene and application thereof - Google Patents

Abstract

The invention discloses a ralstonia solanacearum effect protein RipXV as well as a coding gene and application thereof. The ralstonia solanacearum effect protein RipXVThe amino acid sequence of (A) is shown as SEQ ID No.2, and the coding gene thereofRipXVThe nucleotide sequence of (A) is shown in SEQ ID No. 1. Transient overexpression in Nicotiana benthamianaRipXVGene, which can induce plant cell death. Construction by homologous recombination△ripXVThe mutant strain, inoculated peanut, shows enhanced pathogenicity, and shows that RipXV is probably a nontoxic effector protein and plays an important role in the pathogenic process of peanut. The target protein AhRFL1 of RipXV is screened from the cDNA library of peanuts induced by ralstonia solanacearum by a yeast two-hybrid technology. The invention has important significance for analyzing the molecular mechanism of the interaction between the ralstonia solanacearum and the host and establishing a comprehensive control technical strategy of the plant bacterial wilt.

Description

Peanut ralstonia solanacearum effect protein RipXV and coding gene and application thereof

Technical Field

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

Background

Ralstonia solanacearum (L.), (L.)Ralstonia solanacearum) The caused bacterial wilt is one of important plant diseases in the world. The pathogenic bacteria have the characteristics of wide host range, rich physiological race, wide geographical distribution, quick pathogenic variation, various disease conditions, difficult control and the like, and the caused plant diseases cause huge economic loss. At present, the infection and pathogenic mechanism of the ralstonia solanacearum are not completely clear, so that starting from the pathogenic mechanism of ralstonia solanacearum, the search of an effective action target is of great importance for the prevention and treatment of diseases. The III type secretion system is a pathogenicity determining factor of ralstonia solanacearum, and the ralstonia solanacearum utilizes a large amount of effector proteins injected into host cells to activate or inhibit the defense reaction of the host, thereby causing host diseasesOr inducing the non-host plant to generate a hypersensitivity reaction.

Plant pathogens, when infecting host plants, secrete a class of proteins, called effector proteins, that interact with the host plant R gene product to stimulate the plant's immune system. The gene encoding the pathogen effector inducing R gene mediated resistance is defined as the Avr gene. The effector proteins include toxic proteins and non-toxic proteins, and the toxic proteins are necessary proteins for completely exerting toxicity of pathogenic bacteria entering host cells; non-toxic proteins are proteins that can be recognized directly or indirectly by plants and trigger allergic reactions. Most effector proteins can change the cell structure and function of a host and accelerate the infection of pathogenic bacteria, so that R proteins capable of specifically recognizing different effector proteins are evolved by plants. The interaction of plants with pathogenic bacteria is controlled by specific interactions between the avr (avirulence) gene of the pathogen and the allele of the corresponding disease resistance (R) gene of the plant, i.e. "gene-gene" interactions. In the model, a specific receptor coded by a plant disease-resistant gene recognizes a nontoxic gene product of pathogenic bacteria or a metabolite generated by exciton-mediated catalytic activity, and further triggers a defense reaction of the plant. This model serves to illustrate the specific recognition of the pathogenic Avr protein by the plant R protein, where the former is the ligand for the latter. Although these models provide a simple model parallel to the immune system, the relationships they suggest remain poorly demonstrated, and only a few examples are currently available to demonstrate direct interactions between the R protein and its cognate Avr protein.

Based on the result of the whole genome sequence analysis of the early-stage ralstonia solanacearum RS-P.362200 of the subject group, the experiment obtains an effect protein RipXV of a III type secretion system through bioinformatics analysis, and constructs an over-expression vector to generate strong anaphylactic necrosis reaction through agrobacterium-mediated transient expression of Nicotiana benthamiana. Construction by homologous recombination△RipXVThe mutant strain and inoculated host can obviously enhance the pathogenicity of ralstonia solanacearum, and the RipXV is possibly an avirulence gene and plays a key role in the interaction process of the ralstonia solanacearum and plants. Simultaneously, NBS-LRR disease-resistant protein AhRFL of RipXV interaction in host is screened by yeast double-hybrid1, the AhRFL1 is presumed to be possibly involved in the defense reaction of a host to ralstonia solanacearum and can provide gene resources for the genetic improvement of bacterial wilt resistance of plants. The method is less in interaction research between ralstonia solanacearum and a host at present, and the method starts from ralstonia solanacearum effect protein and host target protein thereof, researches the interaction between RipXV and AhRFL1, provides a new thought for disclosing the pathogenic mechanism and preventing and treating ralstonia solanacearum, and has important scientific significance.

Disclosure of Invention

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

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

the invention firstly provides a ralstonia solanacearum effect protein RipXV, and the amino acid sequence of the ralstonia solanacearum effect protein RipXV is shown in SEQ ID NO. 2.

The invention further provides a gene for coding the ralstonia solanacearum effect protein RipXV, and the gene isRipXVThe nucleotide sequence is shown in SEQ ID NO. 1.

The invention also provides an overexpression vector containing the ralstonia solanacearum effector protein RipXV, and the construction method of the overexpression vector comprises the following steps: gate entry vector pDONR 207-based construction by BP reaction based on Gataway systemRipXVConstruction of 35S promoter-driven plant expression vector pK7WG2.0-RipXVThe expression vector is the overexpression vector containing the ralstonia solanacearum effector protein RipXV.

The invention also provides a knockout vector containing the ralstonia solanacearum effector protein RipXV, and the construction method of the knockout vector comprises the following steps: cloning based on the whole gene sequence of ralstonia solanacearum RS-P.362200RipXVAn upstream 657 bp fragment (U) and a downstream 643 bp fragment (D) are used for constructing a knockout vector pK18mobSacB-U-D by an enzyme digestion connection method.

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

The invention also provides a protein interacting with the peanut ralstonia solanacearum effector protein RipXV, wherein the protein is NBS-LRR disease-resistant protein AhRFL1, 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 was obtained based on a yeast two-hybrid screen: construction of the bait vector PGBKT7-RipXVAnd further screening to obtain the interactive protein of RipXV according to the mixed cDNA library of the rhizopus inducive roots and leaves of the ralstonia solanacearum.

The invention also provides application of the ralstonia solanacearum effector protein RipXV in stimulating plant defense reaction and anaphylactic reaction and/or stimulating plant disease-resistant gene expression as an effector protein.

The invention also provides the ralstonia solanacearum effect protein geneRipXVThe application in culturing transgenic engineering bacteria with enhanced pathogenicity to flowers.

Furthermore, the method for cultivating the transgenic engineering bacteria with enhanced pathogenicity to the peanuts is to use the original strainsRipXVKnocking out genes, and screening to obtain transgenic engineering bacteria with enhanced pathogenicity to the peanut; the starting strain is ralstonia solanacearum (A)Ralstonia solanacearum）。

The invention has the beneficial effects that:

through the whole genome sequencing analysis of ralstonia solanacearum Rs-P.362200, the application predicts a triple-type effector protein RipXV and instantaneously over-expresses the coding gene of the triple-type effector protein RipXV on Nicotiana benthamianaRipXVA strong allergic necrosis reaction is induced. Knock-outRipXVThe pathogenicity of the ralstonia solanacearum can be obviously enhanced after the gene is obtained, and the first report that RipXV influences the pathogenicity of the ralstonia solanacearum is reported. The invention further screens host target NBS-LRR disease-resistant protein AhRFL1 of RipXV for the first time through yeast two-hybrid. The research result of the invention has the effects of disclosing the pathogenic mechanism of ralstonia solanacearum on the peanuts and the bacterial wilt resistance mechanism of the peanutsImportant scientific significance.

Drawings

FIG. 1: pK7WG2.0-RipXVSchematic diagram of overexpression vector construction.

FIG. 2: a schematic diagram of RipXV knockout and anaplerosis strains is constructed based on a homologous recombination method.△RipXVIs a knockout mutant of the effector protein RipXV,C△RipXVis an effector protein△RipXVThe anaplerotic strain of (1).

FIG. 3: RipXV knockout and complementation strain construction PCR amplification electrophoresis gel running results. WT shows the result of PCR amplification of a wild type Ralstonia solanacearum strain,△RipXVshows the PCR amplification result of the knockout strain,C△RipXVthe results of PCR amplification of the complemented strain are shown, and "-" shows a negative control.

FIG. 4 is a schematic view of: transient overexpression of ralstonia effect protein in Nicotiana benthamiana leavesRipXVThe gene causes intense cell necrosis. HR reaction results show transient overexpressionRipXVCauses intense allergic death in nicotiana benthamiana leaves; DAB staining result displayRipXVCause HR reaction to generate a large amount of H ₂ O ₂ Trypan blue staining results showRipXVCausing the HR reaction to produce large amounts of calluses.

FIG. 5:RipXVthe mutant and complementing strains of (a) inoculate a postnatal phenotype.

FIG. 6: the interaction between RipXV and AhRFL1 in yeast is verified. The positive control was pGBKT7-53+ pGADT7-T and the negative control was pGBKT7-Lam + PGADT 7-T.

Detailed Description

[ example 1 ] constructionRipXVOverexpression vectors

Inquiring according to the whole genome sequence of ralstonia solanacearum Rs-P.362200RipXVCDS sequence of full-length gene, design of specific primer: (RipXV-attB1-F：5’-GGGGACAAGTTTGTACAAAAAAGCA

GGCTTCATGAAAAGATTTATGAGG-3’，RipXV-attB 2-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCGCTCAATAGCTTTTC-3') performing PCR amplification using Ralstonia arachidicola genomic DNA as a template, using PrimeSTAR from Takara ^® Max is used for amplification, and the PCR reaction system is as follows: 1 μ L of DNA template, 10 μ L of 2 XPrimeSTAR ^® Max, 0.5 muL of each forward primer and reverse primer, and water is supplemented to 20 muL. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 1 min. Carrying out agarose gel electrophoresis detection on a PCR product, then cutting, purifying and recovering gel, and carrying out no-load connection on a target gene fragment and pDONR207 to carry out BP reaction:RipXVthe PCR product of 1 muL, pDONR207 no-load 1 muL, BP enzyme 0.25 muL, 25 ℃ connection overnight, the connection product is transformed into Escherichia coli DH5 alpha competent cells, positive clones are screened for sequencing, correctly read-through clone extraction plasmids are constructed, and an entry vector pDONR207-RipXV. The entry vector plasmid and the over-expression vector pK7WG2.0 were subjected to an LR reaction: pDONR207-RipXVPlasmid 1 uL, pEarley Gate201 no-load 1 uL, LR enzyme 0.25 uL, 25 ℃ connection overnight, transformation of Escherichia coli, verification of positive clone, construction of 35S CaMV promoter driven overexpression vector pK7WG2.0-RipXV. The construction process of the overexpression vector is schematically shown in FIG. 1.

[ example 2 ]△RipXVDeletion mutants andC△RipXVconstruction of a complementation vector

Finding out an upstream 657 bp sequence U and a downstream 643 bp sequence D of RipXV according to Ralstonia solanacearum Rs-P.362200, designing specific primers RipXV-1-F (5'-CGGAATTCAGCTTCCCATCGAACACTG-3'), RipXV-1-R (5'-CGGGATCCGATAGACAATCCTTGAATTTTC-3'), RipXV-2-F (5'-CGGGATCCGTATGATCTGCTCAGAGTCG-3') and RipXV-2-R (5'-CCAAGCTTTCAACATCAAGGCGATCC-3'), carrying out PCR amplification by taking Ralstonia arachidicola genome DNA as a template, and carrying out amplification by adopting PCR premix of Kangji company, wherein the PCR reaction system is as follows: 1 muL of genome DNA template, 5 muL of PCR premix, 0.5 muL of forward and reverse primers and 3 muL of water. The reaction conditions are as follows: 5 min at 94 ℃; 30 s at 94 ℃, 30 s at 60 ℃,1 min at 72 ℃ and 28 cycles; preserving at 72 deg.C for 10 min and 25 deg.C. And (3) detecting the PCR product by agarose gel electrophoresis, purifying and recovering the PCR product after no impurity band. The target fragment and the empty pK18mobsacb are respectively cut and recovered by restriction enzymes EcoRI, BamHI and HindIII, and U and D are respectively connected to the pK18mobsacb vector by T4 ligase under the following connection conditions: t4 ligase 2 μ L, T4 buffer 1 μ L, pK18mobsacb 1 μ L (100 ng), U/D2 μ L (100 ng), H ₂ O4. mu.L, 16 ℃ ligation overnight.The ligation product is transformed into escherichia coli, positive cloning is verified by PCR,RipXVthe construction of the knockout vector pK18mobsacb-U-D is completed, and the schematic diagram of the vector construction process is shown in the left figure of FIG. 2.

The construction method of the anaplerotic vector comprises the following steps: according to the whole genome sequence of ralstonia solanacearum Rs-P.362200, specific primers are designed, ralstonia solanacearum genome DNA is used as a template for PCR amplification (construction of a PCR reaction system and a knockout vector), and meanwhile, 2362 bp fragments (CRipXV-XhoI-F: 5'-ATTACTCGAGTCGCCTACGCTACAATCTCCTG-3', CRipXV-BamHI-R: 5'-ACCAGGATCCTCATCGCTCAATAGCTTTTCTG-3') which are totally included in upstream 657 bp, (U), RipXV (1062 bp) and downstream 643 bp (D) are cloned. The target fragment and the pK18mobsacb were recovered by digestion with restriction enzymes XhoI and BamHI, respectively, and U-RipXV-D was ligated to the pK18mobsacb vector using T4 ligase (ligation conditions were as above). The ligation product is transformed into Escherichia coli, positive cloning is verified by PCR, the RipXV complementation vector pK18mobSacB-U-RipXV-D is constructed, and the schematic diagram of the vector construction process is shown in the right graph in figure 2.

[ example 3 ] Triparental conjugation method△RipXVKnock-out mutations andC△RipXVanaplerotic strain

The construction method of the mutant strain and the anaplerotic strain is mainly carried out by adopting a three-parent combination method, selecting the host bacterium, namely the pseudomonas solanacearum, and monoclonally culturing the host bacterium pseudomonas solanacearum in a 4 mL BG liquid culture medium (containing 100 mg/L polymyxin B), and culturing the host bacterium in a constant temperature shaking table at 28 ℃ until the thallus concentration OD is up to ₆₀₀ When =0.6, all the cells were collected in a 2 mL EP tube; selecting helper MT616, culturing the single clone in an LB liquid culture medium (containing 34 mg/L clindamycin) of 4 mL in a constant temperature shaking table at 37 ℃, and collecting all thalli by using an EP (enhanced EP) tube of 2 mL when the thalli concentration OD600= 1.0; the donor bacteria pK18mobSacB-U-RipXV-D or pK18mobSacB-U-D were selected and monoclonal to 4 mL LB liquid medium (containing 50 mg/L kanamycin), cultured in a shaker at 37 ℃ until OD of the bacterial concentration ₆₀₀ When =2.0, all the cells were collected in a 2 mL EP tube; centrifuging the three thalli at 5000 rpm for 5 min, washing the collected thalli twice with sterile water, and then resuspending the thalli with the sterile water; the cell concentration OD was measured by a spectrophotometer ₆₀₀ Adjusting the temperature to 1.0; then the host bacterium is culturedRalstonia arachidicola, helper MT616, donor pK18mobSacB-U-RipXV-D or pK18mobSacB-U-D were as follows 2: 1:1, uniformly mixing; and centrifugally collecting the thallus, resuspending the thallus in 50 muL of sterile water, uniformly dropping the thallus on a BG flat plate by using a pipette gun, and inversely culturing the flat plate in a constant-temperature incubator at 28 ℃ after the flat plate bacterial liquid is dried, and inducing for 2-3 d. The mixed bacteria on the plate is scraped and suspended by 100 muL sterile water, a BG plate (containing 100 mg/L polymyxin B and 50 mg/L kanamycin) is uniformly coated (a small amount of ralstonia solanacearum bacterial liquid is streaked at the edge of the plate to be used as negative control), and the plate is placed in a constant temperature incubator at the temperature of 28 ℃ for two days; picking the ralstonia solanacearum with good growth condition on the plate, extracting genome DNA, and amplifying the ralstonia solanacearum by using specific primers RipXV-1-F and RipXV-2-R, wherein if the amplified bands are consistent with the sizes of the designed fragments (two bands, one is at 1.28 kb and the other is at 2.92 kb), the carrier is successfully transferred into the ralstonia solanacearum.

Selecting 5-6 ralstonia solanacearum with good growth vigor and consistent PCR strip size to perform streak culture on a BG (PB + Kan) plate for 1-2 days (a little of ralstonia solanacearum bacterial liquid is streaked at the edge of the plate to be used as negative control), and selecting the ralstonia solanacearum to perform streak culture on the BG plate for 1 d. Well-growing ralstonia solanacearum is picked and streaked on BG and BGS plates respectively (colonies containing sac vector do not grow or grow slowly on the sucrose-containing medium). After 1 d, selecting the ralstonia solanacearum which grows well on the BG plate but does not grow or grows slowly on the BGS plate, shaking the ralstonia solanacearum for 5-6 h at 28 ℃, and then coating 100 mu L of the ralstonia solanacearum on a BGS flat plate for culturing for 1-2 d. After 2 days, well-growing ralstonia solanacearum was streaked on BG and BG plates (containing 100 mg/L polymyxin B and 50 mg/L kanamycin) for overnight culture (with plates with grids, first with antibiotic, and then with BG plates). Bacterial wilt cells which do not grow on BG plates (containing 100 mg/L polymyxin B and 50 mg/L kanamycin) were picked, genomic DNA was extracted, and amplified with specific primers RipXV-1-F and RipXV-2-R, and bacterial wilt cells were used as a positive control. And selecting pseudomonas solanacearum without bands in PCR (polymerase chain reaction) for genome extraction, and verifying by using a specific primer.

Mutant and complementation strains of RipXV were obtained by the above method, and PCR identification of the primers specific for the mutant and complementation strains is shown in FIG. 3.

Example 4 transient overexpression of RipXV coding Gene in Nicotiana benthamiana leaves

And (3) transforming the constructed RipXV overexpression plasmid into Agrobacterium tumefaciens GV3101, culturing for 2 d in an incubator at 28 ℃, selecting well-growing clones, adding the clones into a corresponding antibiotic culture medium, placing the corresponding antibiotic culture medium into a shaking table for shake culture, setting the temperature at 28 ℃, the rotating speed at 250 rpm, performing overnight culture, and verifying whether the transformation is successful by PCR. Positive clones verified to be correct were added to 50 mL of medium and shaken until OD was measured ₆₀₀ The value was 0.5. Placing into a centrifuge, centrifuging at 4000 rpm for 10 min, re-suspending with MES buffer solution, and separating OD ₆₀₀ Adjusting to 0.7-0.8, and incubating in an incubator at 28 ℃ for 2 h. Healthy Nicotiana benthamiana in the 5-6 leaf stage is selected, 3 strains are used as an experimental group, 3 strains are used as a control group, the penultimate leaf and the third leaf of the plant are selected, a 1 mL syringe needle is used for puncturing the leaves, agrobacterium liquid is injected at the punctured position through an agrobacterium infiltration method, and the injection area is a circle with the diameter of 1 cm. After incubation in the greenhouse at 25 ℃ for 48 h, the tobacco phenotype was recorded by observing the photographs. After 48 h of transient overexpression in Nicotiana benthamiana leaves, infected plants were stained with 3, 3' -Diaminobenzidine (DAB) and lactol-ethanol-trypan blue. The injected Nicotiana benthamiana leaves are placed in 1 mg/mL DAB solution for incubation at room temperature overnight, boiled in 3:1:1 ethanol/lactic acid/glycerol solution for 5 min, and then placed in absolute ethanol for decolorization. Cell death was detected using trypan blue staining, the injected leaves were boiled in trypan blue solution for 2 minutes, trypan blue solution (10 mL lactic acid, 10 mL glycerol, 10 g phenol, 30mL absolute ethanol and 10 mg trypan blue), stained overnight at room temperature, transferred to chloral hydrate solution (2.5 g/mL) and boiled for 20 minutes to remove the stain. The staining results were observed under an optical microscope. Transient expression, as shown in FIG. 4RipXVCan make the Nicotiana benthamiana generate strong allergic necrosis reaction, and DAB staining results show transient overexpressionRipXVThe gene can release a large amount of active oxygen, and trypan blue staining results show that the transient overexpression is realizedRipXVGenes can produce large amounts of calluses.

Example 5 Effect of RipXV on the virulence of Ralstonia solanacearum

Wild type strain (WT), mutant strain, and anaplerotic strain were treated with 15 mL BG (100 mg/L PB) in a constant temperature shaker at 28 ℃ at 250 rpm ⁺ ) And (5) culturing. OD to be measured ₆₀₀ And when the number is not less than 0.4, inoculating inverted two and inverted three functional leaves of the peanut by a leaf cutting inoculation method, and observing and recording the growth condition of the peanut plant after inoculation 7 days later. The phenotype of the bacterial wilt after inoculation and flowering is shown in FIG. 5, the wild type strain (WT) shows disease resistance 7 days after inoculation of peanut Guangdong oil for resisting bacterial wilt 92, and the mutant△RipXVThe leaf blades of the peanut variety Guangdong oil resistant to the bacterial wilt appear withered 7 days after the bacterial strain is inoculated with 92 days of the Guangdong oil, the plant shows typical symptoms of the bacterial wilt, and the leaf blades of the peanut variety Guangdong oil resistant to the bacterial wilt recover the disease resistance 7 days after the replanting bacterial strain C delta RipXV is inoculated with 92 days of the Guangdong oil resistant to the bacterial wilt.

Example 6 screening and verification of Ralstonia solanacearum Effector protein RipXV interaction target protein

RipXV-BD-NcoI-F (5'-AGGACCATGGACCATGAAAAGATTTATGAG-3') and RipXV-BD-BamHI-R (5'-ATTAGGATCCTCATCGCTCAATAGCTTTTC-3') were used as primers and pDONR207-RipXV plasmid was used as a template to amplify a PCR product of RipXV, the PCR product and a bait vector PGBKT7 were digested simultaneously with NcoI and BamHI, and after the target band was recovered, RipXV was ligated to a PGBKT7 vector using T4 DNA ligase (ligation conditions were the same as above). The ligation product was transformed into E.coli, positive clones were verified by PCR and sent to the sequencer to verify, obtaining the bait vector pGBKT 7-RipXV. The self-activation and toxicity detection of the bait vector are verified by pGBKT7-RipXV transformation yeast Y2H Gold yeast strain, the self-activation phenomenon is avoided, and the subsequent screening library test can be carried out. Preparing competent cells of pGBKT7-RipXV yeast positive clones, transforming root and leaf mixed cDNA library plasmids induced by ralstonia solanacearum with peanuts, coating SD/-His/-Leu/-Trp plates with transformed thalli, selecting the clone points SD/-Ade/-His/-Leu/-Trp plates which grow normally on the SD/-His/-Leu/-Trp plates, and taking the clones which turn blue on the four-deficiency plates after 2-3 d as candidate positive clones. Plasmids of candidate positive clones were extracted, verified by PCR, and the PCR products were sequenced by the company and aligned to query the coding sequence of the interacting target protein AhRFL1 in PGR (http:// peanutgr. Cloning the CDS sequence of AhRFL1 full-length gene into PGADT7 vectorCo-transforming PGADT7-AhRFL1 and PGBKT7-RipXV plasmid into Y2H yeast competent cell, coating SD/-Leu/-Trp plate, selecting clone, shaking, and performing cell culture according to the method of 10 ⁰ /10 ^-1 /10 ^-2 /10 ^-3 The bacterial suspension was diluted in gradient, 5. mu.L was applied to SD/-Ade/-His/-Leu/-Trp + X-. alpha. -gal plates, and the colonies became blue after 3 days, indicating that the interaction between RipXV and AhRFL1 existed (FIG. 6).

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> peanut ralstonia solanacearum effect protein RipXV and coding gene and application thereof

<130>

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 1062

<212> DNA

<213> Ralstonia solanacearum)

<400> 1

atgaaaagat ttatgagggc gattggcgta gggtcgagcc gaagcacccg gaccaactac 60

gtagagccgc aagctgatga cgcacctgac tcaaacgcca gttccaattc atcaccggaa 120

aggcctacct tgaggcgctc cccgaaccct gcttttgcga gcctgcctcc gcgcgccaaa 180

gataaggcgg tcgcattgga caattcgttg cgaagaaatt tgaattatat tccatccgat 240

ttggaaaact atgctcgcgc tgccctgaat agggtggagt atactgcgac atcaggcgag 300

atgactgatc ttgatattga aaatattcac catctcgttg gggcatataa tgacagattt 360

tctgggctgc atttaaaaaa tcacgaatcc cctcgatcat ttttcgagga attcatggat 420

tctggggagt ctgtttggcg ttccgtcgtt aggctaagca tgaatgatcg tcatcatgtg 480

gcaatcgatg ttcgagttga tgacgacaag cgaaccatga taataataga gtcggcgctg 540

gcgcataatc ctcgtcagcc aggcgtattt cttcaggggt atgagcagct tagtggtaaa 600

ttggcaagct acgccggaga ggatggcggt atggctgtag tggagttggg ggtgcaaaaa 660

tcgaattacg attgcattat ttactcattg aataactccc ttgctgcata tcaaaagaat 720

gaggtttttg atgaaatgca tgcgagcctg agggcgatag ggaggtgttt cggtaggtac 780

gatgggaagt caactataca aagcggtatc gagcttatcg atggaagcaa agttctgcca 840

gcaatcttct tcaagcatgc acactccaga gcaacaattg atgatgttct tgagaatcaa 900

ccggatcttg agggaagaaa tgtaagtacg ggcagcgaaa atcctcatca aacgttgtcg 960

cagcgagtgc gagatttcag aattgagcga gacgatagag ggtatagcat gtccattgaa 1020

gcatccaggt tgcgaaaaat cagaaaagct attgagcgat ga 1062

<210> 2

<211> 353

<212> PRT

<213> Ralstonia solanacearum)

<400> 2

Met Lys Arg Phe Met Arg Ala Ile Gly Val Gly Ser Ser Arg Ser Thr

1 5 10 15

Arg Thr Asn Tyr Val Glu Pro Gln Ala Asp Asp Ala Pro Asp Ser Asn

20 25 30

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

35 40 45

Asn Pro Ala Phe Ala Ser Leu Pro Pro Arg Ala Lys Asp Lys Ala Val

50 55 60

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

65 70 75 80

Leu Glu Asn Tyr Ala Arg Ala Ala Leu Asn Arg Val Glu Tyr Thr Ala

85 90 95

Thr Ser Gly Glu Met Thr Asp Leu Asp Ile Glu Asn Ile His His Leu

100 105 110

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

115 120 125

Glu Ser Pro Arg Ser Phe Phe Glu Glu Phe Met Asp Ser Gly Glu Ser

130 135 140

Val Trp Arg Ser Val Val Arg Leu Ser Met Asn Asp Arg His His Val

145 150 155 160

Ala Ile Asp Val Arg Val Asp Asp Asp Lys Arg Thr Met Ile Ile Ile

165 170 175

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

180 185 190

Gly Tyr Glu Gln Leu Ser Gly Lys Leu Ala Ser Tyr Ala Gly Glu Asp

195 200 205

Gly Gly Met Ala Val Val Glu Leu Gly Val Gln Lys Ser Asn Tyr Asp

210 215 220

Cys Ile Ile Tyr Ser Leu Asn Asn Ser Leu Ala Ala Tyr Gln Lys Asn

225 230 235 240

Glu Val Phe Asp Glu Met His Ala Ser Leu Arg Ala Ile Gly Arg Cys

245 250 255

Phe Gly Arg Tyr Asp Gly Lys Ser Thr Ile Gln Ser Gly Ile Glu Leu

260 265 270

Ile Asp Gly Ser Lys Val Leu Pro Ala Ile Phe Phe Lys His Ala His

275 280 285

Ser Arg Ala Thr Ile Asp Asp Val Leu Glu Asn Gln Pro Asp Leu Glu

290 295 300

Gly Arg Asn Val Ser Thr Gly Ser Glu Asn Pro His Gln Thr Leu Ser

305 310 315 320

Gln Arg Val Arg Asp Phe Arg Ile Glu Arg Asp Asp Arg Gly Tyr Ser

325 330 335

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

340 345 350

Arg

<210> 3

<211> 1884

<212> DNA

<213> peanut (Arachis hypogaea)

<400> 3

atggttcatg aaggagagcg aattatcaag atggaagagg gtatttctgt cacagatgat 60

tatgattctg gatatttgat gcagttccag tccagagaaa gcactttcga tcaaattttg 120

gaagcactaa aagattgtaa gatctctcag gttgcattgt ctggcatggg tggatcagga 180

aagacaacct tagcaaaaca agtgggtgaa aaggccaagc tgttgaacat cttcaacctt 240

gttgtattcg ttcctgtgac atctactcca agtttcatga agatacaagg tgagatagct 300

gaccaattga gtctcagatt gggagaagaa gctggtctag ctgcaagaca gagaaaaaaa 360

tcaatgacaa gtaaagaact ggttcttata attttggatg atgtgttgga caggctcaac 420

ctggaaaaga tagggatttt taatggttgt aatggattgc agcttagaac catgggtagg 480

cgtatgagct gtaaagtcct catgactaca agtactcaag aagcagtata taggatggga 540

tgccaaccaa gcattaacct gtctttatta actgaaggag aagcttgcga tcttttgagg 600

aagcatgcag acattagtga cggctcccct gaatcaattg ttgaagtagc aaaacaagtt 660

gcgaaacatt gtcatggttt accccttgca agttcagtga ttggatccac tttaagagga 720

aaaactgttg accagtggaa ggaagtatta aaaacaatgc agatgctacc agtgaagagt 780

ttgcaatgtg aagctgacac atctaaattt attcaccaga ttatagaagt aggctatcat 840

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 (9)

1. The ralstonia solanacearum effect protein RipXV is characterized in that the amino acid sequence of the ralstonia solanacearum effect protein RipXV is shown in SEQ ID No. 2.

2. Peanut ralstonia solanacearum effect protein geneRipXVCharacterized in thatRipXVA gene encoding the Ralstonia arachidicola effector protein RipXV of claim 1, whereinRipXVThe nucleotide sequence of the gene is shown in SEQ ID No. 1.

3. A gene comprising the ralstonia solanacearum effector protein of claim 2RipXVThe overexpression vector of (3).

4. A gene comprising the ralstonia solanacearum effector protein of claim 2RipXVThe knock-out vector of (1).

5. A transgenic plant comprising the ralstonia solanacearum effector protein gene of claim 2RipXVThe anaplerotic vector.

6. The protein interacting with the ralstonia solanacearum effector protein RipXV of claim 1, wherein the protein is NBS-LRR type disease-resistant protein AhRFL1, the nucleotide sequence of which is shown in SEQ ID No.3, and the protein sequence of which is shown in SEQ ID No. 4.

7. The use of the ralstonia solanacearum effector protein RipXV of claim 1 as an effector protein for eliciting a defense response, an allergic response in plants and/or for eliciting the expression of a disease resistance gene in plants.

8. The ralstonia solanacearum effector protein gene of claim 2RipXVThe application in culturing transgenic engineering bacteria with enhanced pathogenicity to flowers.

9. The use of claim 8, wherein the method for cultivating genetically engineered bacteria with enhanced pathogenicity to flowers is to use the genetically engineered bacteria in the original strainRipXVKnocking out genes, and screening to obtain transgenic engineering bacteria with enhanced pathogenicity to the peanut; the starting strain is peanut ralstonia solanacearum (A) (B)Ralstonia solanacearum）。

Images