| eSRKs酵母双杂交诱饵载体的构建及其自激活作用的检测 |
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| 引用本文： | 杨红,朱利泉,彭一波,罗兵,杨昆,张贺翠,吴志刚,黄丹,高启国.eSRKs酵母双杂交诱饵载体的构建及其自激活作用的检测[J].西南农业大学学报,2011,33(4). |
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| 作者姓名： | 杨红 朱利泉 彭一波 罗兵 杨昆 张贺翠 吴志刚 黄丹 高启国 |
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| 作者单位： | 西南大学农学与生物科技学院;西南大学重庆市蔬菜重点实验室; |
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| 基金项目： | 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) |
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| 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.
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| 关 键 词： | eSRKs 酵母双杂交 诱饵载体构建 自激活作用 |
Re-construction of eSRKs Bait Vector and Detection of Its Self-Activating Effect in a Yeast Two-Hybrid System |
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| YANG Hong,ZHU Li-quan,PENG Yi-bo,LUO Bing,YANG Kun,ZHANG He-cui,WU Zhi-gang,HUANG Dan,GAO Qi-guo.School of Agronomy , Biotechnology,Southwest University,Chongqing ,China,.Chongqing Key Laboratory of Olericulture.Re-construction of eSRKs Bait Vector and Detection of Its Self-Activating Effect in a Yeast Two-Hybrid System[J].Journal of Southwest Agricultural University,2011,33(4). |
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| Authors: | YANG Hong ZHU Li-quan PENG Yi-bo LUO Bing YANG Kun ZHANG He-cui WU Zhi-gang HUANG Dan GAO Qi-guoSchool of Agronomy Biotechnology Southwest University Chongqing China Chongqing Key Laboratory of Olericulture |
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| Institution: | YANG Hong1,ZHU Li-quan1,PENG Yi-bo1,LUO Bing1,YANG Kun1,ZHANG He-cui1,WU Zhi-gang1,HUANG Dan1,GAO Qi-guo21.School of Agronomy and Biotechnology,Southwest University,Chongqing 400716,China,2.Chongqing Key Laboratory of Olericulture |
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| Abstract: | 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 in... |
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| Keywords: | eSRKs yeast two-hybrid vector construction bait protein autonomous reporter activation |
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