红麻酮脂酰辅酶A合成酶(KCS)酵母双杂交诱饵载体构建及自激活检测  

The Study on Self Activation of Ketoacyl-Coenzyme A Synthase in Yeast Two Hybrid Systerm of Hibiscus Cannabinus

作　　者：张高阳[1,2] 邓接楼[1] 黄思齐[2] 李德芳[2] ZHANG Gaoyang;DENG Jielou;HUANG Siqi;LI Defang(College of Life Sciences, ShangRao Normal University, Shangrao, Jiangxi 334001,China;Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205,China)

机构地区：[1]江西上饶师范学院生命科学学院,江西上饶334001 [2]中国农业科学院麻类研究所,长沙410205

出　　处：《中国麻业科学》2018年第2期75-78,共4页Plant Fiber Sciences in China

基　　金：江西省教育厅科学技术研究项目(GJJ161047);农业部麻类生物学与加工重点实验室开放课题(201602);上饶师范学院2014年博士科研启动基金(001055)

摘　　要：酮脂酰辅酶A合成酶(KCS)是超长链脂肪酸合成过程中的限速酶,参与超长链脂肪酸和蜡质的合成,在植物抗旱和应对生物胁迫反应中发挥重要调控作用。研究采用PCR方法从红麻中(茎皮)分离KCS基因片段,将该基因按正确方式插入酵母双杂交表达质粒pGBKT7中,酶切鉴定正确后,用PEG/Li Ac方法转化酵母MaV203,经抗性筛选后,用X-Gal对转化酵母进行自激活检测。结果表明,KCS基因的DNA结合结构域与酵母GAL4转录因子的上游激活位点(UAS)结合,可激活报告基因半乳糖苷酶的表达。经删除KCS基因DNA binding区域后重新转化酵母,结果表明,不携带KCS基因DNA binding区域的截短蛋白可以作为诱饵蛋白来研究与之相互作用的蛋白。Ketoacyl coenzyme A synthetase （KCS） is the limiting enzyme in synthesis process of long chain fatty acid and is involved in the synthesis of long chain fatty acids and wax, which plays an important regulatory role in response to a biological and plant drought stress. In this study we uses the PCR method to isolate KCS gene fragments from kenaf and inserts them into yeast express plasmid pG- BKT7 in the correct direction. After the identification of enzyme digestion, PEG/LiAc method has been used to transform MaV203 yeast, and X - Gal is used to detect report gene p - galactosidaseuse activity. The results show that the DNA binding domain of KCS can combine with （UAS） sites of GAL4 transcription factor, which could activate p - galactosidaseuse gene express. When deleting the DNA binding do-main of KCS gene, self activation disappeared which, showed that KCS protein can be used as bait to study the interaction of proteins.

关 键 词：酮脂酰辅酶A合成酶 酵母双杂交 自激活 红麻 

分 类 号：S563.5[农业科学—作物学]