陆地棉酵母双杂交cDNA文库及VdSCP7诱饵载体的构建

由大丽轮枝菌(Verticillium dahliae)引起的棉花黄萎病是制约我国棉花生产的首要病害,严重影响棉花的产量与品质。为研究棉花与黄萎病菌互作的分子机制,本研究以国审棉百棉1号为材料,提取被大丽轮枝菌侵染的棉花根部总RNA,检测质量并用DNase Ⅰ处理后,用RNA 5'末端的模板转换方法,即SMART技术合成双链cDNA,再经酶切、过柱纯化去除短片段后,连接至pGADT7载体上构建棉花cDNA文库。VdSCP7是定位于寄主细胞核的大丽轮枝菌效应因子,能激发棉花的免疫反应,增强棉花抗病性。以大丽轮枝菌cDNA为模板扩增VdSCP7,产物纯化后重组到载体pGBKT7上构建诱饵载体pGBKT7-SCP7。诱饵载体经酶切及测序鉴定后,转化到酵母AH109中,鉴定诱饵蛋白毒性并检测诱饵载体是否存在自激活活性。结果表明,获得了库容量为2×10⁶ CFU的棉花cDNA文库,文库片段多样性良好,文库重组率约94%,质粒文库滴度约2.0×10⁹ CFU·mL^-1,满足酵母双杂交cDNA文库构建要求。酶切及测序结果表明,诱饵载体pGBKT7-SCP7构建成功且经过鉴定后无毒性、无自激活活性。本研究结果为棉花抗黄萎病基因的筛选及VdSCP7和棉花互作机制的解析奠定了基础。

Construction of a Yeast Two-Hybrid cDNA Library of Cotton(Gossypium hirsutum L.)and VdSCP7 Bait Vector

Verticillium wilt caused by Verticillium dahliae is one of the major diseases that restrict cotton production in China, which seriously affects cotton yield and quality. To study the molecular mechanism of interaction between cotton and Verticillium dahliae, the construction of yeast two-hybrid (Y2H) cDNA library was performed to explore interacting protein of SCP7 in cotton. In this study, the total RNA of cotton roots infected by Verticillium dahliae was extracted from the national certification cotton Baimian1. The quality of the total RNA was detected, and then the RNA was treated with DNase Ⅰ. The double-stranded cDNA was synthesized by SMART technology and purified after digestion, then ligated to pGADT7 to construct a cotton cDNA library. VdSCP7 is a Verticillium dahliae effector located in the host cellular nucleus, which can stimulate the immune response and enhance the disease resistance of cotton. VdSCP7 gene was amplified by using Verticillium dahliae cDNA as template. The product was purified and then recombined into pGBKT7 to construct bait vector pGBKT7-SCP7. After digestion and sequencing, the bait vector pGBKT7-SCP7 was transformed into yeast AH109 to identify the toxicity of bait protein and detect the self-activating activity of bait vector. The results showed that a cotton cDNA library with a library capacity of 2×10⁶ CFU was obtained, and the diversity of library fragment was good and the recombination rate was about 94%. The titer of the plasmid library was about 2.0×10⁹ CFU mL^-1, which reached the requirements for constructing yeast two-hybrid cDNA library. The enzyme digestion and sequencing results showed that the bait vector pGBKT7-SCP7 was successfully constructed and identified as no toxicity and self-activation. The results laid a the foundation for the screening of Verticillium wilt resistance genes in cotton and the analysis of the interaction mechanism between VdSCP7 and cotton.