摘要
为了研究香蕉束顶病毒与香蕉寄主致病的互作分子机制，本文报道利用Make Your Own＂Mate＆Plate＂ Library System试剂盒成功构建感染BBTV香蕉叶片的cDNA文库。通过改良CTAB法提取感染BBTV香蕉叶片的总RNA，采用SMART法反转录合成双链cDNA，经靳，酶切并去除短片段之后，与经同样酶切的pGADT7-SfiI载体连接，利用电转法将重组载体转化到大肠杆菌宿主细胞中，获得初级cDNA文库，最后以初级文库100万克隆为基数扩增，得到扩增文库并提取质粒。结果得到库容量大于2．0X10。的初级文库，检测表明文库cDNA插入片段长度主要分布在700—2000bp，文库重组率为87．5％。结果表明，该文库质量较好，可用于后续酵母双杂交互作蛋白筛选试验，本研究为开展病毒与寄主互作的研究奠定基础。
BBTV is a major limiting factor for banana production. In this report, we presented a BBTV infected banana （family Musaceae） leaf cDNA library constructed by using a kit named Make Your Own ＂Mate＆PlateTM＇＇ Library System. Total RNA was extracted from BBTV-infected banana leaves by using modified CTAB method. The first stand eDNA was synthesized by reverse transcription of mRNA with SMART technique. To generate eDNA library, the eDNA was digested with Sill and then sub-cloned into Sill side of pGADT7 vector. The recombinant plasmids were transferred into E. coli via electroporation. The result of the plasmids from amplified cDNA library showed that the library contained 2.0 x 106 independent clones and the size of most inserts were 700-2 000 bp in the library. The recombination rate of library was 87.5%. These results indicated that the library was suitable for screening proteins interacting with BBTV coded protein. It will facilitate the investigation of interaction between BBTV and the host.
出处
《植物研究》
CAS
CSCD
北大核心
2013年第6期713-717,共5页
Bulletin of Botanical Research
基金
国家自然科学基金项目(31070131)
国家科技支撑计划课题(2007BAD48B01)
中央级公益性科研院所基本科研业务专项(ITBB110303)
