大麦条纹花叶病毒诱导大麦cDNA文库的构建

Construction of the cDNA Library of Barley Infected by the Barley Stripe Mosaic Virus

构建病毒诱导的寄主植物cDNA文库是利用酵母双杂交方法筛选与病毒相互作用寄主因子的前提条件.该文以大麦条纹花叶病毒接种大麦,在ELISA跟踪检测的病毒复制和运动初期,采集大麦叶片,用TRIZOL法提取总RNA,利用SMART原理进行反转录PCR获得dscDNA,SfiⅠ/XhoⅠ共切后1.1%琼脂糖凝胶检测并分段回收dscDNA,分别与SfiⅠ/XhoⅠ共切的载体连接后,共同转化X-blue感受态细胞构建文库.文库的容量和质量检测结果表明:文库的容量为1.27×106,插入片段大于500 bp,集中在1.2 kb左右,符合酵母双杂交筛选文库的要求.

It is necessary to construct the cDNA library of plant infected by virus before screening the host-virus-interaction gene of the host. In an experiment reported in this paper, barley was inoculated with Barley Stripe Mosaic Virus. At the initial stage of virus replication and movement, leaves infected by Barley Stripe Mosaic Virus were collected and total RNA was extracted from them. DscDNA was synthe- sized by RT-PCR with SMART method. The dscDNA was digested by Sfi I/Xho I and separated into different parts based on their size by 1.1 % agarose gel. Each part of the dscDNA was ligated separately to the vectors digested by Sfi I/Xho I. All the recombinant vectors were electroporated into E. coil X-blue to construct the cDNA library. The detection result showed that the cDNA library contained 1.27 × 10^6 recombinant clones and that all the inserted cDNA fragments were more than 500 bp in size and most were a-round 1.2 kb.