棉花(Gossypium hirsutum)ROP1基因酵母双杂交体系的诱饵载体构建及鉴定

Construction and Identification of the Bait Vector Containing Gossypium hirsutum ROP1 Gene in Yeast Two-hybrid System

ROP是高等植物里一类分布广泛的信号小G蛋白,在植物生长、发育、抗逆、抗病和激素信号转导过程中起重要调控作用。本研究用PCR体外扩增技术获得棉花GhROP1基因片段,将目的基因与诱饵质粒载体pGBKT7通过双酶切定向重组,构建诱饵质粒pGBKT7-GhROP1,并成功的转入到Y187酵母菌通过缺陷性培养基培养进行毒性和自激活检测。结果表明,成功克隆棉花GhROP1基因,该基因开放阅读框共有591 bp。双杂交诱饵表达载体pGBKT7-GhROP1构建成功,并成功转化酵母菌Y187;结果显示含表达载体的酵母菌Y187在SD/-Trp平板上长势良好,说明表达蛋白对酵母菌无毒害作用。在SD/-Leu,SD/-Trp-Leu,SD/-Trp-Leu-His-Ade平板上则没有生长,说明转化pGBKT7-GhROP1质粒后的Y187酵母菌无自激活发生。本研究结果为下一步利用酵母杂交系统筛选GhROP1蛋白相互作用的蛋白提供理论参考。

ROP is a class of widely distributed signal small G protein in higher plants,which plays an important role in plant growth,development,stress resistance,disease resistance and hormone signal transduction.In this study,the cotton GhROP1 gene fragment was obtained by PCR amplification.The bait plasmid pGBKT7-GhROP1 was constructed through double enzyme digestion and directional recombination of target gene GhROP1 and bait plasmid vector p GBKT7.The p GBKT7-GhROP1 was transferred to yeast strain Y187.The self-activation detection was conducted by the culture on defective medium.The results showed that the GhROP1 gene was successfully cloned,and the open reading frame of the gene was 591 bp.The yeast two-hybrid bait expression vector of GhROP1 gene was constructed and successfully transferred to Y187 yeast.The yeast strain Y187 containing the expression vector had a good growth on the SD/-Trp plate,but no growth on the SD/-Leu,SD/-Trp-Leu-His-Ade plate;The results indicated the recombined bait plasmid pGBKT7-GhROP1 had no toxicity on yeast and no self-activation effect on the reporter gene.The research laid a foundation for screening out the interact protein of bait protein GhROP1.