西瓜cDNA表达文库构建及镰刀菌致病因子FonSIX6互作蛋白的筛选和鉴定

Construction of cDNA Expression Library of Watermelon and Screening and Identification of Interaction Partner of FonSIX6,a Virulence Factor from Fusarium oxysporum f. sp. niveurn

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白(15.8～29.9kD)进入木质部中启动致病力,被称为SIX(Secreted in xylem)蛋白。其中,SIX6蛋白是一个致病因子。西瓜专化型尖孢镰刀菌Fon(Fusarium oxysporum f.sp. niveurn)是引起西瓜枯萎病的病原真菌。为了解与FonSIX6存在相互作用的西瓜蛋白及其信号传导途径,克隆了FonSIX6基因,将FonSIX6的编码区与酵母GAL4的DNA结合功能区融合,构建成酵母诱饵蛋白表达载体pGBKT7-SIX6,进而转化到酵母菌株Y2H Gold中,经检测证实不具有毒性和自激活功能,可以用于酵母双杂交研究。同时,以国际上公认的抗枯萎病材料PI296341-FR构建酵母表达文库。采用酵母双杂交的方法,筛选到14个相互作用靶蛋白。分析筛选到的蛋白主要参与寄主的能量代谢、光合作用和基因表达调控,推测FonSIX6作用的靶位点主要是破坏寄主能量系统并影响光合作用,而寄主对其响应的方式是调控抗病基因的表达。

The fungal pathogen Fusarium oxysporum f. sp. niveum（Fon）that causes Fusarium wilt in watermelon is a soilborne fungus. During interactions between F. oxysporum and host plant,the fungus secretes several effector proteins into the plant xylem to promote virulence. These effectors are small cysteine-rich proteins and are called SIX（Secreted in Xylem）proteins. Among them,the SIX6 was proved to be a virulence factor. To understand function and signaling pathway of FonSIX6 in watermelon, FonSIX6 was cloned from Fon. To construct the bait vector pGBKT7-SIX6 for yeast two-hybrid,the encoding region of FonSIX6 was fused to the GAL4 DNA-binding domain in yeast expression vector pGBKT7. Then bait vector was transformed into Y2H gold strain. Transcriptional activation and toxicity test showed that the bait could be used in yeast two hybrid screens. PI296341-FR,a watermelon line that was resistant to all Fon races was used to construct the cDNA expression library in yeast cells. By yeast two hybrid screens,14 interacting proteins were identified that interact with FonSIX6. These proteins involved in various pathways such as energy metabolism,photosynthesis and regulation of gene expression. According to our results,FonSIX6 may target the host energy and photosynthesis system to promote virulence,while host regulated resistant gene expression response to FonSIX6.