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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。
Fusarium oxysporum secretes several specific small cysteine-rich proteins (15.8 ~ 29.9 kD) into the xylem to initiate virulence in the interaction with the host, and it is called SIX (Secreted in xylem) protein . Among them, SIX6 protein is a virulence factor. Fus (Fusarium oxysporum f.sp. niveurn), a watermelon-specific Fusarium oxysporum f.sp. niveurn, is a pathogenic fungus that causes watermelon wilt. In order to understand the interaction between FonSIX6 and its signal transduction pathway, the FonSIX6 gene was cloned and the coding region of FonSIX6 was fused with the DNA binding domain of yeast GAL4 to construct the yeast bait protein expression vector pGBKT7-SIX6 Yeast strain Y2H Gold, tested to be non-toxic and self-activating, can be used in yeast two-hybrid studies. At the same time, a yeast expression library was constructed with internationally recognized Fusarium wilt material PI296341-FR. The yeast two-hybrid method was used to screen 14 interacting target proteins. The analysis and screening of the proteins mainly involved in energy metabolism, photosynthesis and gene expression regulation of the host, speculated that FonSIX6 target site is mainly to destroy the host energy system and affect the photosynthesis, and the host response to it is the regulation of disease resistance genes expression.