基于CRISPR/Cas9技术靶向突变番茄WRKY3基因

WRKY转录因子在植物生长发育、次生代谢产物合成以及对生物和非生物胁迫的响应等方面发挥重要作用。为探究WRKY3在植株逆境应答中的功能及作用机制，文章通过生物信息法对不同物种WRKY3的结构域及进化树进行分析，发现WRKY3具有较高的保守性；TomExpress数据库显示WRKY3在各种组织中均有表达，且在红果期果实中表达量最高；并构建WRKY3基因的CRISPR敲除载体，利用农杆菌介导法转化番茄获得WRKY3基因的转基因番茄材料，经聚合酶链式反应(polymerase chain reaction, PCR)及测序鉴定发现，已成功获得敲除WRKY3基因的转基因番茄植株。该研究获得了番茄WRKY3基因敲除突变体稳定遗传株系，为探究WRKY3在逆境胁迫下植物的生长发育及果实成熟中所发挥的功能奠定了基础。

WRKY transcription factors play important roles in plant growth and development, secondary metabolite synthesis, and response to biotic and abiotic stresses. In order to investigate the function and mechanism of action of WRKY3 in plant adversity response, this study analyzed the structural domains and evolutionary trees of WRKY3 in different species by bioinformatic method and found that WRKY3 was highly conserved. TomExpress database was used to analyze WRKY3 expression level in different tissues of tomato. The result showed that WRKY3 was expressed in various tissues, and the highest in the fruit at the red-fruit stage. The CRISPR knockdown vector of WRKY3 gene was constructed. WRKY3 transgenic tomato plants were obtained by using Agrobacterium-mediated transformation and identified by polymerase chain reaction (PCR) and sequencing. In this study, the stable genetic knockout plants of tomato WRKY3 was obtained, which lays the foundation for the investigation of the function of WRKY3 in plant growth and development and fruit ripening under adversity stress.