橡胶树14-3-3蛋白基因的酵母双杂交诱饵载体的构建及自激活和毒性检测

Construction of Bait Vector Containing 14-3-3 Protein Gene from Hevea brasiliensis in Yeast Two-hybrid System and Identification of Its Toxicity and Autonomous Activation Effects

以克隆到的3个编码橡胶树14-3-3蛋白的基因HbGF14a、HbGF14b、HbGF14c为基础,构建酵母双杂交诱饵载体pGBKT7-HbGF14a、pGBKT7-HbGF14b、pGBKT7-HbGF14c,并对其进行自激活和毒性实验鉴定。自激活实验结果表明,3个诱饵载体没有自激活性;毒性实验结果表明,pGBKT7-HbGF14a、pGBKT7-HbGF14b、pGBKT7-HbGF14c表达的蛋白对酵母菌无毒性,酵母生长良好,结果表明,构建好的诱饵载体均可用于下一步的酵母双杂交实验。这为进一步的诱饵载体与橡胶树胶乳cDNA酵母表达文库的杂交做好了前期准备。

Three genes encoding 14-3-3 protein,designated HbGF14a、HbGF14b、HbGF14c were amplified from Hevea brasiliensis by the PCR and then fused with pGBKT7 vector.After confirmed by using sequence analysis,the plasmids were transformed into the cell of yeast strain Y2H Gold,and tested in terms of toxicity and transcriptional activation by nutrition disfigurement assay.As a result,three genes were successfully amplified and cloned into the pGBKT7 vector.The yeast strain Y2H Gold cells harboring bait plasmids grew well on SD/-Trp plate,without toxicity and autonomous activation effect,which lays the foundation for using yeast two-hybrid system to screen the protein interacting with 14-3-3 protein.