辣椒疫霉效应分子RxLR19781与其互作蛋白的酵母互作一对一验证

One-to-one Verification of the Interaction between the Phytophthora capsici Effector Molecule RxLR19781 and Its Interacting Protein

辣椒疫霉菌(Phytophthora capsici)引起的辣椒疫病是毁灭性土传病害,短期内暴发成灾,该病害严重发生常给辣椒生产造成严重损失。植物病原卵菌分泌大量的胞内效应分子,即RxLR效应分子,能干扰寄主植物细胞的正常生理代谢和功能,在病原卵菌侵染寄主及与寄主植物互作过程发挥着重要的作用。酵母双杂交(Yeast two hybrid,Y2H)系统是研究蛋白质相互作用最常用的方法,它既可以在cDNA文库中筛选互作的未知靶标蛋白,同时还可以验证两个目的蛋白之间的互作关系。本研究分别以辣椒疫霉c DNA基因组,辣椒cDNA基因组为模板,克隆了辣椒疫霉效应因子Rx LR19781以及其疑似互作蛋白泛素连接酶E2 10和辣椒脂质转移蛋白EARLI 1,并将它们分别成功构建到PGBKT7和PGADT7载体上。运用酵母共转化技术,将带有AD载体的互作蛋白和带有BD载体的效应因子共同转化到酵母感受态Y2HGold中,对它们是否存在互作关系进行了深入研究。结果表明,两个疑似互作蛋白都与效应因子RxLR19781互作。但由于酵母双杂交存在一定的假阳性,因此该效应分子与互作蛋白的互作有待进一步深入的研究。

Capsicum blight caused by Phytophthora capsici is a devastating soil-borne disease,which is a disaster in a short period of time.The serious occurrence of this disease often causes serious losses to pepper production.Plant pathogenic oocysts secrete a large number of intracellular effector molecules,namely RxLR effector molecules,which can interfere with the normal physiological metabolism and function of host plant cells,and play an important role in the process of infecting host and host plants with pathogenic oomycetes.The yeast two-hybrid(Y2H)system is the most commonly used method for studying protein interactions.It can not only screen for unknown target proteins in cDNA libraries;but also verify the interaction between two proteins of interest.The Phytophthora cDNA genome and the capsicum cDNA genome were used as templates to clone the P.capsici effect factor RxLR19781 and its suspected interacting protein ubiquitin ligase E210 and capsicum lipid transfer protein EARLI 1.They were successfully constructed on the PGBKT7 and PGADT7 vectors,respectively.Using yeast co-transformation technology,the interaction protein with AD vector and the effector factor with BD vector were transformed into yeast competent Y2HGold,and their interaction relationship was studied.The results showed that both suspected interaction proteins interacted with the effector RxLR19781.However,due to the existence of certain false positives in yeast two-hybrid,the interaction between the effector molecule and the interacting protein is required to further study out.