Abstract: [Objective] This project aims to construct a bait vector for yeast two-hybrid system of TA gene in Acidovorax citrulli in order to lay the foundation for exploring the gene function involved in the pathogenic mechanism.[Method]The TA gene was amplified by PCR and cloned into the vector pGBKT7.The verified pGBKT7-TA was then transformed into the yeast strain AH109.The activities of report genes ADE2,HIS3 and LacZ were used to detect the self activation of pGBKT7-TA,and the toxicity of pGBKT7-TA was investigated in nutrient deficient medium.[Result]The constructed bait vector pGBKT7-TA had neither self-activation nor toxic effect in the yeast strain AH109.[Conclusion]The constructed bait vector can be used for the follow-up work of the yeast two hybrid system,which has laid a foundation for further screening of proteins interacting with TA from the cDNA library of cucumber cotyledons infected by Acidovorax citrulli.

摘要： [目的]构建西瓜食酸菌TA基因的酵母双杂交诱饵载体,为探究TA基因编码产物参与西瓜食酸菌致病的机制奠定基础.[方法]通过PCR扩增TA基因,并定向克隆到载体pGBKT7,获得可用于酵母双杂交的诱饵载体pGBKT7-TA,经酶切鉴定和测序验证为正确后将pGBKT7-TA转化到酵母菌株AH109中;进一步用ADE2、HIS3、LacZ 报告基因活性检测法检测pGBKT7-TA的自激活作用,用固体及液体营养缺陷型培养基培养法检测pGBKT7-TA的毒性作用.[结果]诱饵载体pGBKT7-TA对酵母菌株AH109没有自激活及毒性作用.[结论]所构建的诱饵载体可用于酵母双杂交系统的后续工作,为进一步从被西瓜食酸菌侵染的黄瓜子叶cDNA文库中筛选与TA互作的蛋白奠定了基础.