外源ABA处理大麦胚的均一化cDNA文库的构建

Construction of a Normalized cDNA Library of Barley Embryo Treated with Exogenous ABA

构建外源ABA处理大麦胚的均一化cDNA文库是利用酵母双杂交方法筛选与靶蛋白相互作用蛋白的前提条件。为给大麦抗逆基因的研究及抗逆转基因大麦材料的创制奠定基础,利用5μmol·L-1ABA和蒸馏水分别处理大麦种子0h、6h、12h、24h,剥取大麦的胚,用热酚法提取总RNA,利用SMART原理进行反转录PCR并进行均一化处理获得ds-cDNA。经SfiⅠ酶切的ds-cDNA与pGADT7-Rec载体连接后,转化大肠杆菌感受态细胞DH10B构建文库。结果表明,文库的容量为1.1×106,插入片段大于1 000bp,集中在1 000~3 000bp之间,符合酵母双杂交文库的要求。

It is necessary to construct the cDNA library of barley embryo before screening the proteins interacted with target proteins.In the experiment reported in this paper,barley seeds were treated with5μmol·L-1 ABA and distilled water for 0h,6h,12hand 24h,respectively.Barley embryos were stripped and total RNA was extracted.Ds-cDNA was synthesized by RT-PCR with SMART method.The ds-cDNA which digested by Sfi I was ligated to the vectors pGADT7-Rec.All the recombinant vectors were electroporated into DH10Bto construct the cDNA library.The detection results showed that the cDNA library contained 1.1×106 recombinant clones and that all the inserted cDNA fragments were more than 1kb in size,between 1kb and 3kb and most were around 1.2kb,which meets the requirement of the yeast two-hybrid library.