| 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 |
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| 引用本文： | 罗 兵,朱利泉,薛丽琰,张贺翠,彭一波,杨 红,杨 昆,高启国,李成琼,王小佳.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[J].西南农业大学学报,2012,34(8):079-085. |
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| 作者姓名： | 罗 兵 朱利泉 薛丽琰 张贺翠 彭一波 杨 红 杨 昆 高启国 李成琼 王小佳 |
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| 作者单位： | 西南大学植物生理生物化学实验室;常熟理工学院生物与食品工程学院;西南大学重庆市蔬菜学重点实验室 |
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| 基金项目： | 国家自然科学基金资助项目(30971849) |
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| 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.
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| 关 键 词： | 甘蓝 SCR蛋白 同源重组 酵母双杂交 诱饵载体 自激活 |
RapidConstructionofBaitVectorContainingSCR
fromBrassicaoleracea L.byHomologousRecombination
andEvaluationofItsSelf-ActivationintheYeastTwo-hybridSystem |
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| LUO Bing,ZHU Li-quan,XUE Li-yan,ZHANG He-cui,PENG Yi-bo,YANG Hong,YANG Kun,GAO Qi-guo,LI Cheng-qiong,WANG Xiao-jia.RapidConstructionofBaitVectorContainingSCR
fromBrassicaoleracea L.byHomologousRecombination
andEvaluationofItsSelf-ActivationintheYeastTwo-hybridSystem[J].Journal of Southwest Agricultural University,2012,34(8):079-085. |
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| Authors: | LUO Bing ZHU Li-quan XUE Li-yan ZHANG He-cui PENG Yi-bo YANG Hong YANG Kun GAO Qi-guo LI Cheng-qiong WANG Xiao-jia |
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| Institution: | 1.Plant Physiology and Biochemistry Laboratory of Southwest University,Chongqing 400716,China;2.School of Biology and Food Engineering,Changshu Institute of Technology,Changshu Jiangsu 215500,China;3.Key Laboratory in Olericulture of Chongqing,Southwest University,Chongqing 400716,China |
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| Abstract: | 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.. |
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| Keywords: | Brassica oleracea L SCR protein homologous recombination yeast two-hybrid system bait plasmid self-activation |
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