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Application of gene PlRACK1 in regulation and control of growth, oxidation resistance and pathogenicity of peronophythora litchii

A technology of downy mildew and pathogenicity of litchi, which is applied in the field of plant genetic engineering, can solve the problems of harm and no disease-resistant litchi planting varieties have been found, and achieve the goal of reducing tolerance, reducing pathogenicity, and slowing down the growth rate. Effect

Pending Publication Date: 2022-07-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Litchi frost blight caused by Lychee downy mildew is the most serious damage to litchi, and no effective disease-resistant litchi cultivars have been found

Method used

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  • Application of gene PlRACK1 in regulation and control of growth, oxidation resistance and pathogenicity of peronophythora litchii
  • Application of gene PlRACK1 in regulation and control of growth, oxidation resistance and pathogenicity of peronophythora litchii
  • Application of gene PlRACK1 in regulation and control of growth, oxidation resistance and pathogenicity of peronophythora litchii

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] experiment material

[0031] Test strains, plants and vectors

[0032] The test strain is Phytophthora litchii wild type strain (Peronophythora litchii, Wild Type, referred to as WT) is a conventional Phytophthora, which can be obtained through commercial channels or natural isolation, Escherichia coli (Eschrichia coli) strain DH5α, can be obtained through commercial channels The inoculated plant material for the test was the young leaves of Litchi (Feizixiao) (collected from the horticultural practice orchard of South China Agricultural University); the oomycete knockout and transformation vectors pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA (already in the literature " Yufeng,Fang,Linkai,et al.Efficient Genome Editing in theOomycetePhytophthora sojaeUsing CRISPR / Cas9[J].Current Protocols in Microbiology,2017”), pBSSK (disclosed in the document “A CRISPR / Cas9-mediated in situcomplementation method for Phytophthora sojae” mutants[J].Molecular PlantPathology, 2021, 22(3)", th...

Embodiment 2

[0095] Preparation of Phytophthora litchi protoplasts and PEG-mediated transformation

[0096] Phytophthora litchi wild-type strain WT was activated on nutrient pea solid plate medium. The mycelium block was taken into a conical flask, and 50 mL of NPB liquid medium was added for cultivation. Three flasks were co-cultured and cultured in the dark at 25°C for 3 d, with shaking every 12 h. Filter with gauze, collect mycelia, squeeze gently with tweezers and add them to the enzymolysis solution (composition of enzymolysis solution: weigh 0.15g of lysing enzyme (Lysing Enzymes, SIGMA) and 0.06g of cellulase (Cellulase, SIGMA) ) in a sterilized beaker, add 10mL of 0.8mol·L to the ultra-clean bench -1 Mannitol, 8mL sterile ddH 2 O, 800μL 0.5mol·L -1 KCl, 800μL 0.5mol·L -1 MES-KOH and 400μL of 0.5mol·L -1 CaCl 2 , after fully dissolving, transfer to 50mL centrifuge tube of 50mL centrifuge tube (for use), mix gently, enzymolysis for 40-45min at 25°C, 40rpm. After the mycelium w...

Embodiment 3

[0099] Validation and Determination

[0100] (1) Validation of fluorescence quantitative analysis of PlRACK1 knockout transformants

[0101] The hyphae of Phytophthora litchi wild-type WT and transformants were extracted with the All-In-One DNA / RNA Mini-Preps Kit from BBI Company, and the genome was performed with the FastKing cDNA first-strand synthesis kit from Tiangen. DNA removal and reverse transcription cDNA synthesis, the quantitative primers PlActin-F and PlActin-R were designed with the PlActin gene as the internal reference gene, and the quantitative primers PlRACK1-qPCR-F and PlRACK1-qPCR-R were designed with the CDS sequence of the PlRACK1 gene as the target gene. SYBR Premix Ex Taq TM kit (TAKARA) was used for real-time quantitative PCR, and the expression of PlRACK1 gene was analyzed to verify whether the PlRACK1 gene was successfully knocked out.

[0102] The wild-type WT and transformants of Phytophthora lychee were extracted by CTAB method. Using the genomic ...

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Abstract

The invention provides application of a gene PlRACK1 in regulation and control of growth, oxidation resistance and pathogenicity of peronophythora litchii. Experiments prove that upstream and downstream sequences of a gene PlRACK1 are amplified respectively, then an NPT II gene sequence is amplified and connected with a PBSSK carrier, and after homologous recombination, knockout is carried out through a polyethylene glycol mediated protoplast transformation technology and a CRISPR / Cas9 knockout strategy. Due to deletion of the gene PlRACK1, compared with a wild type, the obtained mutant has the advantages that the growth rate of peronophythora litchii is obviously slowed down, the pathogenicity of the peronophythora litchii is obviously reduced, and the tolerance of the peronophythora litchii to oxidative stress is obviously reduced. The invention proves that the gene PlRACK1 is necessary for the growth, development and pathogenicity of peronophythora litchii. The research is helpful for deeply illuminating the pathogenic molecular mechanism of peronophythora litchii, and a target gene is provided for developing effective bactericides.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and particularly relates to the application of a gene PlRACK1 in regulating the growth, anti-oxidation and pathogenicity of Phytophthora litchi. Background technique [0002] Peronophythora litchii belongs to Chromista and Oomycota. The lychee frost blight caused by Phytophthora lychee is the most serious damage to lychees, and no effective disease-resistant lychee varieties have been found. Studies have shown that the successful infection of Phytophthora lychee in lychee mainly depends on a series of pathogenic factors. Therefore, fully excavating lychee pathogenic genes and carrying out functional research can effectively control the harm of Phytophthora lychee and breed disease-resistant varieties. important meaning. [0003] Studies have shown that signal transduction and protein translation play an important role in the growth and development of oomycetes, sexual or asexual reprodu...

Claims

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Application Information

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IPC IPC(8): C12N15/80C07K14/37C12N15/31C12N1/15A01N57/16A01P3/00C12R1/645
CPCC12N15/80C07K14/37A01N57/16
Inventor 孔广辉黄伟雄李雯周文喆宋雨马俊妮蔡旭敏习平根李敏慧姜子德
Owner SOUTH CHINA AGRI UNIV
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