辣椒疫霉菌效应因子RxLR23克隆与基因敲除体系建立

Cloning of RxLR23 and Establishment of Gene Knockout System of Phytophthora Capsici

在辣椒疫霉侵染寄主的过程中,病原菌能够分泌大量的Rx LR效应因子,协助病原菌干扰寄主植物的抗病反应。长期以来,由于缺乏有效的定向诱变和基因置换的技术严重阻碍了关于疫霉菌的致病性的遗传研究。为了深入开展辣椒疫霉Rx LR效应因子的功能研究,本项研究以辣椒疫霉菌株SD33总DNA为模板,设计引物,克隆出基因Rx LR23的序列。以Rx LR23基因序列为模版设计sg RNA构建p YF2.3G-ribo-sg RNA重组载体以及设计构建同源修复供体载体p Bluescript II KS+重组载体。利用CRISPR/cas9系统敲除基因Rx LR23,经G418抗性筛选和PCR扩增验证,成功获得了基因Rx LR23的4个敲除突变体。

In the process of infection of Phytophthora capsici,pathogenic bacteria can secrete a large number of RxLR effectfactors to help pathogens interfering with the disease resistance of host plants.For a long time,the lack of efficient techniquesfor directed mutagenesis and gene replacement has seriously hampered genetic study on pathogenicity of Phytophthorapathogenicity.In order to understand the function of RxLR effectors in Phytophthora capsici,using the Phytophthora capsicibacterial strain SD33DNA as template,we designed primers to clone the sequence of gene RxLR23,and it was used as thetemplate to design sgRNA to construct pYF2.3G-ribo-sgRNA recombinant vector and to design the homologous recombinantdonor vector pBluescript II KS+recombinant vector.The CRISPR/Cas9system was used to knockout gene RxLR23,afterG418resistance screening and PCR amplification verification,4knockout mutants of gene RxLR23were obtainedsuccessfully.