马铃薯晚疫病菌RxLR效应因子RD24基因克隆及其PVX表达载体构建与鉴定

Cloning of Potato Phytophthora infestans RxLR Effector Gene RD24 and Constructing and Identifying of Its PVX Expression Vector

马铃薯晚疫病病原菌致病疫霉菌(Phytophthora infestans)在侵入寄主细胞的同时分泌效应因子(Effector),该类蛋白在晚疫病致病过程中发挥着关键作用,是抑制植物免疫的重要因素。PITG_RD24是马铃薯晚疫病菌位于细胞核核的一个重要效应因子。为了快速鉴定效应因子RD24在马铃薯中瞬时表达的状况及抗性情况,构建效应子RD24的PVX表达载体是十分必要的。本研究利用PCR、酶切、分子克隆等生物学技术,将RD24片段连接插入至PVX (pGR106)载体中,然后用热击法将连接产物转化至大肠杆菌DH10B感受态细胞中。通过PCR筛选、质粒提取、测序比对,结果表明成功获得含有目的表达载体pGR106·RD24的质粒,可为下一步筛选马铃薯广谱抗病基因研究提供必备载体。

The pathogen Phytophthora infestans,which causes potato late blight,delivers effectors inside the living host cells,the effector proteins play an important role for the infection of the pathogen,and are the critical reasons for the pathogen to suppress plant immunity.PITG_RD24 is one of the important effectors of P.infestans which is located in the nucleus of plant cell.To identify the transient expression and resistance of RD24 effector in potato,it is necessary to construct the expression vector of PVX·RD24(pGR106·RD24).In this study,the fragment of RD24 is inserted to empty vector of pGR106 by the methods of PCR,restriction enzyme digestion and molecular cloning.Then the connected product and plasmid is transformed into DH10B Agrobacterium tumefaciens strains by heat shock method.The expression vector of pGR106·RD24 is successfully obtained after PCR screening,plasmid extraction,contrasting sequence,and the study can provide the necessary vector for screening potato resistant genes.