西瓜食酸菌TA基因酵母双杂交诱饵载体的构建及其自激活作用的检测

Construction and Self-activation Detection of Bait Vector for Yeast Two-hybrid System of TA gene in Acidovorax citrulli

【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p GBKT7,获得可用于酵母双杂交的诱饵载体p GBKT7-TA,经酶切鉴定和测序验证为正确后将p GBKT7-TA转化到酵母菌株AH109中;进一步用ADE2、HIS3、Lac Z报告基因活性检测法检测p GBKT7-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p GBKT7-TA的毒性作用。【结果】诱饵载体p GBKT7-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c DNA文库中筛选与TA互作的蛋白奠定了基础。

[ 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 expXoring 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.