巴西橡胶树胶胞炭疽病菌CgE6基因RNAi突变体的构建

Construction and Expression Analysis of CgE6 RNAi Mutant in Colletotrichum gloeosporioides from Hevea brasiliensis

为了研究橡胶树胶孢炭疽病菌效应蛋白基因CgE6的功能,笔者利用RNAi技术原理,构建了可用于转化丝状真菌的RNAi双元重组载体p Silent-CgE6-FR,通过PEG介导法遗传转化橡胶树胶孢炭疽病菌原生质体,在抗性培养基上获得了CgE6的RNAi转化子并任意选取抗性转化子进行分子鉴定。结果表明,所检测的转化子基因组中都有抗性标记的整合;与野生型菌株相比,所检测的转化子中目标基因CgE6的表达均出现不同程度的降低,其中1,2,7号转化子的表达水平最低;本实验成功构建了有效的CgE6效应蛋白基因的RNAi突变体,为进一步研究该效应蛋白的功能奠定了基础。

Abstrat：RNA interference （RNAi） technology is one of the important methods for studying gene function,but it is not widely used in filamentous fungi.The RNAi binary vector pSilent-CgE6-FR was constructed based on the principle of RNAi technology to investigate the function of effector gene CgE6 in Colletotrichum gloeosporioides from Hevea brasiliensis.Transformation was done by using the PEG mediated method,and CgE6 RNAi transformants were screened on culture medium with hygromycin.Seven transformants randomly selected were identified resistant at a molecular level.The results of PCR amplification for hygromycin gene were positive,and the semi-quantitative RT-PCR analysis demonstrated that the transformants had a lower expression of CgE6 but at different levels than those of the wild type strain,of which the transformants 2 and 7 showed a minimum expression of CgE6.All these data showed that CgE6 RNAi mutants were successfully constructed,which would help to further study the functions of CgE6.