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

摘要

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 Y2 HGold 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.%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 Y2 HGold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。