平菇疏水蛋白Po.hyd基因RNAi载体构建及转化

Construction of RNAi Vector and Transformation of Hydrophobin Gene Po.hyd in Pleurotus ostreatus

为研究平菇疏水蛋白基因Po.hyd的功能,首次构建了Po.hyd的RNAi载体,并通过根癌农杆菌介导获得了平菇转化子。通过聚合酶链式反应(polymerase chain reaction,PCR)扩增Po.hyd序列,利用Golden Gate克隆法构建了干扰载体p1302-GG-ihyd,将其导入根癌农杆菌GV3101中,并在乙酰丁香酮的诱导下完成根癌农杆菌对平菇幼嫩菌丝的介导转化。转化子经PCR检测、荧光显微镜观察、实时定量荧光PCR技术(real-time PCR)以及Southern杂交等技术进行验证。结果显示,本研究最终获得1株可稳定遗传的平菇转化子,该转化子具有潮霉素抗性,且在荧光显微镜下可检测到绿色荧光信号,Po.hyd基因的表达量为野生型的43%,干扰载体T-DNA片段以单拷贝的形式整合在转化子基因组内。

RNAi vector for Pleurotus ostreatus hydrophobin gene, Po.hyd, was constructed and transformants were obtained by Agrobacterium tumefaciens mediated transformation. Po.hyd was cloned by PCR method using the Golden Gate technology to build the interference vector p1302-GG-ihyd, which had a neck ring structure. Finally, this vector was transferred into A. tumefaciens GV3101 under the induction of acetosyringone(AS), the young mycelia were mediated by A. tumefaciens, and the transformants were detected and identified by PCR amplification, fluorescence microscopy, real-time PCR and Southern blot technique. One genetically stable P. ostreatus transformant was obtained, which was hygromycin resistant. The green fluorescence signal could be detected under a fluorescence microscope, and the expression of Po.hyd was 43% compared with the wild type strain. The exogenous DNA was integrated into the genome in a single copy. This result will lay the preliminary foundation for further studies on the function of Po.hyd.