TcLr19PR1、TcLr19PR2和TaLr19TLP1的酵母双杂交诱饵载体的构建及鉴定

Construction and identification of yeast two-hybrid bait vectors of TcLr19PR1,TcLr19PR2 and TaLr19TLP1

在前期研究的基础上,将成功克隆得到3个病程相关蛋白PR1、PR2和PR5的序列,分别命名为TcLr19PR1、TcLr19PR2和TaLr19TLP1。将这3个基因分别连接到酵母双杂交诱饵载体pGBKT-7上,并转化到感受态菌株Y2HGold中,检测其毒性和自激活性。结果显示,成功构建了包含目的基因的诱饵重组载体pGBKT-7-TcLr19PR1、pGBKT-7-TcLr19PR2和pGBKT-7-TaLr19TLP1;将转化产物涂布于SD/-Trp/X平板上,生长良好,并出现阳性克隆;毒性检测中,将3个诱饵重组载体与空载体在SD/-Trp液体培养基中的生长情况进行对比,发现诱饵无毒性;自激活检测试验中,重组载体无法在二缺、三缺和四缺培养基中正常生长,证明诱饵无自激活性。因此,本研究成功构建的3个诱饵重组载体可用于3个PR蛋白互作蛋白的筛选,为进一步研究其在小麦与叶锈菌互作中的分子机理奠定基础。

Based on the previous studies,three pathogenesis-related proteins of PR1 ,PR2,and PR5 named TcLr19PR1 , TcLr19PR2 and TaLr19TLP1 have been successfully cloned. In this study,three target genes were attached to yeast two-hybrid bait vector PGBKT-7, and then they were transformed into yeast Y2HGold competent cells to test its self-activated activity and toxicity. The results showed that these recombinant bait vectors grew well on SD/-Trp/X agar medium. In SD/-Trp culture, bait vectors grew faster than pGBKT-7, which indicated that three bait plasmids were not toxic to yeast cells. In the self-activated activity experiments, the yeast strains containing bait plasmids could not grow on SD/-Trp/-leu DO supplement,SD/- His/-leu/-Trp DO supplement, and SD/-Ade/-His/-leu/-Trp DO supplement, which proved that they didn＇t have self-activated activity. Three baits were successfully constructed and could be used to screen targeted protein interacting with 3 PR proteins. The experiment lay the foundation for understanding the molecular mechanism of the interaction between wheat and leaf rust pathogen in the future.