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Peanut ralstonia solanacearum effect protein RipAU and application thereof in peanut bacterial wilt resistance

A technology of effector protein, R. solanacearum, applied in the field of plant pathology and crop disease control

Active Publication Date: 2022-06-24
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the interaction between R. solanacearum and the host. This application starts from the type III effector protein and its host target gene to study the interaction between RipAU and AhSBT1.7, which is helpful for revealing the pathogenicity of R. solanacearum The mechanism and prevention provide new ideas and have important scientific significance

Method used

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  • Peanut ralstonia solanacearum effect protein RipAU and application thereof in peanut bacterial wilt resistance
  • Peanut ralstonia solanacearum effect protein RipAU and application thereof in peanut bacterial wilt resistance
  • Peanut ralstonia solanacearum effect protein RipAU and application thereof in peanut bacterial wilt resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction RipAU overexpression vector

[0025] according to RipAU The CDS sequence of the full-length gene (the nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2), and specific primers ( RipAU -attb1-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATGCCGAACAAGCGATTGCGTCTGAGC-3', RipAU -attb2-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTCGGCCGGCGCTTTCCGATGC-3') was amplified by PCR using the R. PCR reaction system: 1 µL of genomic DNA as template, 5 µL of PCR master mix, RipAU -attb1-F / RipAU 0.5 µL of -attb2-R forward and reverse primers, 3 µL of water. Reaction conditions: 94°C for 5 min; 28 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min; 72°C for 10 min and storage at 25°C. The PCR product was detected by agarose gel electrophoresis, and the RipAU product was purified and recovered. The target gene fragment was ligated with the pDONR207 empty vector for BP reaction: the purified RipAU product was ...

Embodiment 2

[0026] Example 2 Construction RipAU Knockout and complement vector

[0027] According to R. solanacearum Rs-P.362200, the upstream 653bp sequence U of RipAU (the nucleotide sequence of U is shown in SEQ ID NO.5) and the downstream 518bp sequence D (the nucleotide sequence of D is shown in SEQ ID NO.6) are found. , design specific primers ( RipAU -U-XbaI-F1:5’-CTAGTCTAGACTAGATCAACTGGATCGACGACACTGTATCG-3’, RipAU -U-SalI-R1:5'-GCGTCGACGTCTTGGCCATAGCGGCCGCGGGGCGGCCTGGGACAGATCGGCCAGGGCA-3', RipAU -D-SalI-F2:5'-GCGTCGACGTCTTGGCCATAGCGGCCGCGGCGGGCGCATCGGCACATCGCCTGCC-3', RipAU-D-SphI-R2:5'-ACATGCATGCATGTAGGGCTCCAGATGGTGGAACAAATCGC-3') PCR amplification was carried out with the genomic DNA of R. arachis Rs-P.362200 as the template, and the PCR premix of Kangwei Company was used for amplification. PCR reaction system 2: 1 µL of genomic DNA as template, 5 µL of PCR master mix, 0.5 µL of forward and reverse primers, and 3 µL of water. Reaction conditions: 94°C for 5 min; 28 cyc...

Embodiment 3

[0029] Example 3 Construction of RipAU mutant and complementing strains

[0030] The main methods for constructing mutant and complementing strains are as follows: pick a single clone of R. arachis-P.362200 and place it into 50 mL of BG+PB (polymyxin sulfate B) liquid medium (BG medium formula: peptone 10g / L, yeast extract powder 1g / L, tyrosine 1g / L, agar powder 15g / L, glucose 5g / L, polymyxin sulfate B 50mg / L), cultivated in a constant temperature shaker at 28°C, and tested. Bacterial concentration OD 600 When = 0.6, all cells were collected by centrifugation; the helper bacteria was Escherichia coli MT616, and a single clone was picked in 50 mL LB+Cm (chloramphenicol) medium (LB medium formula: yeast extract powder 3 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agarose 15 g / L; chloramphenicol 30 mg / L), cultured in a constant temperature shaker at 37 °C, and the bacterial concentration OD to be measured 600 When = 1.0, all cells were collected by centrifugation; the donor b...

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Abstract

The invention provides peanut ralstonia solanacearum effect protein RipAU, and belongs to the field of research on plant pathology and crop disease control. The peanut ralstonia solanacearum RS-P.362200 effect protein RipAU has an amino acid sequence as shown in SEQ ID No.2, and the nucleotide sequence of the coding gene is as shown in SEQ ID No.1. The invention also discloses a preparation method of the Ralstonia solanacearum RS-P.362200 effect protein The RipAU gene is transiently expressed in nicotiana benthamiana, and an allergic necrosis reaction can be generated. After the RipAU is knocked out, the pathogenicity of the ralstonia solanacearum to the peanuts can be remarkably reduced, and it is indicated that the RipAU serves as a virulence factor. According to a yeast two-hybrid method, a target protein AhSBT1.7 of RipAU is screened out. The invention has important application value in the aspects of disclosing the pathogenesis of ralstonia solanacearum and preventing and treating peanut bacterial wilt.

Description

technical field [0001] The invention belongs to the technical field of plant pathology and crop disease prevention and control, and specifically discloses the application of R. Background technique [0002] Ralstonia solanacearum, one of the top ten most studied plant pathogens in the world, is pathogenic to many plant species, including not only dicotyledonous herbs, but also dicotyledonous woody plants, as well as monocotyledonous plants. R. solanacearum persists in the soil, invades the root system of the host plant, and eventually forms a high-density population in the xylem vessels, clogging the vascular bundles, reducing the flow of water from the roots to the aerial tissues, and causing the plants to produce blight symptoms. [0003] Peanut is an important oil crop in the world, and is known as the four major oil crops together with rape, soybean and sesame. As one of the important peanut production, consumption and import and export countries in China, peanuts play ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/57C12N15/84A01H5/00A01H6/82C12N15/74A01N63/50A01P1/00
CPCC07K14/195C12N9/52C12Y304/21062C12N15/74C12N15/8281A01N63/50A01N63/20Y02A50/30
Inventor 庄伟建陈坤陈华张冲庄宇慧蔡铁城杨强王珊珊李梦轲
Owner FUJIAN AGRI & FORESTRY UNIV
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