同源重组快速构建甘蓝SCR酵母双杂交诱饵载体及其自激活作用的检测  

Rapid Construction of Bait Vector Containing SCR from Brassica oleracea L.by Homologous Recombination and Evaluation of Its Self-Activation in the Yeast Two-hybrid System

作　　者：罗兵[1,2] 朱利泉[1] 薛丽琰[1] 张贺翠[1] 彭一波[1] 杨红[1] 杨昆[1] 高启国[3] 李成琼[3] 王小佳[3] 

机构地区：[1]西南大学植物生理生物化学实验室,重庆400716 [2]常熟理工学院生物与食品工程学院,江苏常熟215500 [3]西南大学重庆市蔬菜学重点实验室,重庆400716

出　　处：《西南大学学报（自然科学版）》2012年第8期79-85,共7页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30971849)

摘　　要：采用巢式PCR获得甘蓝SCR的成熟肽编码区,利用同源重组技术首次将其克隆到pGBKT7载体中,构建酵母双杂交系统的诱饵载体pGBKT7-SCR,结果表明:通过巢式PCR获得了正确的甘蓝SCR成熟肽编码区,并成功构建到pGBKT7诱饵载体中,且转化有诱饵载体的Y2HGold在SD/-Trp营养缺陷平板上生长良好,而在SD/-His-Trp和SD/-Trp/X-a-Gal/AbA营养缺陷平板上皆不能生长,说明对报告基因无自激活作用;且毒性实验也表明,SCR蛋白对酵母没有毒害作用.The mature coding sequence of S-locus cysteine-rich gene（SCR） in Brassica oleracea L.was amplified by nested-PCR and cloned into the yeast plasmid pGBKT7 by homologous recombination.The recombinant bait plasmid pGBKT7-SCR was verified by sequencing before transformation into competent yeast cells.Sequence analysis showed that the SCR sequence was right and the yeast vector of pGBKT7-SCR was constructed correctly.The transformed Y2HGold Yeast（pGBKT7-SCR） could grow on the SD/-trp plates,while no colony could survive on the SD/-his-trp and SD/-Trp/X-a-Gal/AbA pate,indicating that the bait vector of pGBKT7-SCR would not self-activate the reporter gene.Furthermore,the recombinant bait plasmid showed no toxic effect on yeast Y2HGold cell.These results suggested that the recombinant bait plasmid pGBKT7-SCR can be used to study the interactions between SCR and SRK by the yeast two-hybrid system and lay a foundation for obtaining insight into the mechanism of self-incompatibility in B.oleracea L..

关 键 词：甘蓝 SCR蛋白 同源重组 酵母双杂交 诱饵载体 自激活 

分 类 号：Q78[生物学—分子生物学]