摘要
参考大豆疫霉的CRISPR/Cas9基因组编辑技术体系,针对荔枝霜疫霉RXLR效应蛋白编码基因Pl Avh133设计了20 bp的sgRNA靶向序列,结合同源替换的方式对该基因进行敲除。利用聚乙二醇(PEG)介导的原生质体转化,共获得了58个具有G418抗性的转化子,通过PCR和测序分析证明其中5个转化子的Pl Avh133基因被敲除,敲除效率约为8.6%。荧光定量PCR分析证实Pl Avh133敲除突变体中该基因不表达。本研究结果为荔枝霜疫霉的基因功能研究提供了重要的技术基础。
The CRISPR/Cas9 genome editing system,which was already proven to be feasible in Phytophthora sojae,was applied in P.litchii in this study.In order to establish this system,a20 bp sgRNA target sequence of the RXLR effector encoding gene PlAvh133 was designed and homologous recombination strategy was utilized.Through PEG-mediated protoplast transformation,a total of 58 transformants with G418 resistance were obtained.PCR assay and sequencing analysis showed that PlAvh133 was knocked out in five of the transformants and the rate of gene knockout was about 8.6%.The q PCR analysis confirmed that PlAvh133 was not expressed in the knockout mutants.The results of this study will provide an important technical basis to study of the function of P.litchii gene.
作者
司徒俊键
江立群
邵毅
孔广辉
习平根
姜子德
SITU Junjian;JIANG Liqun;SHAO Yi;KONG Guanghui;XI Pinggen;JIANG Zide(College of Plant Protection/Guangdong Province Key Laboratory of Microbial Signals and Disease Control,South China Agricultural University,Guangzhou 510642,China;Rice Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510642,China;Maoming Branch,Guangdong Laboratory for Lingnan Modern Agriculture,Maoming 525000,China)
出处
《菌物研究》
CAS
2020年第3期181-188,共8页
Journal of Fungal Research
基金
国家荔枝龙眼产业技术体系建设项目(CARS-32)
广东省乡村振兴战略专项(403-2018-XMZC-0002-90)。
