Gateway技术构建酵母双杂分析用辣椒cDNA文库  

Construction of a cDNA Library of Pepper for Yeast Two-hybrid Analysis Using Gateway Technology

作　　者：邱爱连[1] 刘林林[1] 蔡汉阳[1] 朱万里[1] 黄木坤[1] 陈雄[1] 何水林[1] 张健[1] 

机构地区：[1]福建农林大学生命科学学院,福建福州350002

出　　处：《江西农业大学学报》2010年第4期791-796,共6页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然基金项目(30571128);福建省自然科学基金重点项目(2008J003;2008J0049);福建省创新人才项目(2007F3013)

摘　　要：构建cDNA文库是利用酵母双杂交技术开展特定cDNA分离的重要基础工作。利用Gateway技术,在成功提取辣椒总RNA并分离mRNA的基础上,合成了3种读码框的双链cDNA,通过BP反应构建其相应的入门文库,再通过LR反应将cDNA迅速且定向地重组到酵母双杂分析用目的载体pDESTTM22上,构建成高质量目的文库。经检测入门文库和目的文库滴度均达到(4.0～5.0)×106cfu/mL,文库总容量为(2.4～3.0)×107cfu,平均插入片段大小为1250bp,其质量完全满足于进一步利用酵母双杂体系分析目的基因互作蛋白cDNA。It’s an important prerequisite work to construct a cDNA library for isolating a specific cDNA using yeast two-hybrid technology.In this study,after successfully extracting total RNA and isolating mRNA of pepper,the authors synthesized three-frame cDNAs,performed BP reactions to generate corresponding entry libraries and then performed LR reactions to transfer cDNA into destination vector of pDESTTM22 directionally and quickly to form a high-quality destination library using Gateway technology.It has been detected that both entry library and destination library in this report have a high titer of （4.0 ～ 5.0） × 106 cfu/mL and contain a total clones of （2.4 ～ 3.0） × 107 cfu,with an average insert size of about 1 250 bp,it can be suitable for analyzing the interaction protein cDNA of the interested destination gene of pepper by two-hybrid system.

关 键 词：辣椒 入门文库 目的文库 GATEWAY 酵母双杂 

分 类 号：Q785[生物学—分子生物学]