摘要
为研究甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)与S位点受体激酶(SRK)胞外域高变区(eSRKs)的相互作用,构建了eSRKs基因的酵母双杂交诱饵载体,检测其自激活作用,并验证是否适用于后续的相互作用研究.以甘蓝B3为材料,通过RT-PCR技术获得eSRKs目的基因片段,将其克隆到pGBKT7载体中,构建酵母双杂交诱饵载体pGBKT7-eSRKs;测序正确后,将重组质粒转入Y2HGold,检测其表达产物对酵母细胞有无毒性以及对报告基因有无自激活作用,结果获得了正确的eSRKs基因片段,并成功克隆到pGBKT7诱饵载体中,且转化在有诱饵载体pGBKT7-eSRKs的Y2HGold在SD/-Trp营养缺陷平板上生长良好,表明表达产物对酵母细胞无毒性;显色反应结果表明对报告基因也无自激活作用,为下一步利用酵母双杂交系统检测SCR蛋白与SRK蛋白胞外域高变区的相互作用奠定基础.
Self-incompatibility of Brassica oleracea var.capitata L.is determined by the female S-locus receptor kinase（SRK） and the male S-locus cysteine-rich protein/S-locus protein 11（SCR /SP11）.In this study a yeast two-hybrid system was adopted to investigate the interaction between SCR/SP11 and the extracellular hypervariable region of SRK（eSRKs）,a bait expression vector of eSRKs was construct and its self-activating effect was detected.A fragment of eSRKs was amplified from B.oleracea L.＇B3＇ by RT-PCR,cloned into the bait expression vector pGBKT7 and transformed into Y2HGold yeast cells after being verified by sequencing.Toxicity and self-activation of the bait protein were detected by culture in SD/-Trp.This research proved that eSRKs was successfully amplified and cloned into pGBKT7.The vectors pGBKT7-eSRKs were transformed into Y2HGold as well and these cells exhibited neither toxicity nor self-activation.The bait expression vector of eSRKs was constructed successfully,which laid the foundation for detecting SCR interaction with the bait protein eSRKs using the yeast two-hybrid system.
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第4期58-63,共6页
Journal of Southwest University(Natural Science Edition)
基金
国家自然科学基金资助项目(30971849)
高校基本科研业务专项基金资助项目(XDJKC109)
关键词
eSRKs
酵母双杂交
诱饵载体构建
自激活作用
eSRKs
yeast two-hybrid
vector construction
bait protein
autonomous reporter activation
