中华大蟾蜍精巢组织全长cDNA文库的构建及泛素延伸蛋白基因Bg-ubi52的获得  

Construction of Testis cDNA Library and Sequence Analysis of Ubiquitin/L40e Extension Gene in Bufo bufo gargarizans

作　　者：陈文[1] 郑萍萍[1] 聂刘旺[1] 

机构地区：[1]安徽师范大学生命科学学院,芜湖241000

出　　处：《动物学杂志》2007年第1期20-28,共9页Chinese Journal of Zoology

基　　金：国家自然科学基金(No.30640048);生物环境与生态安全安徽省高校重点实验室专项基金;安徽省学术与技术带头人专项基金

摘　　要：运用SMART技术构建了中华大蟾蜍（Bufo bufo gargarizans）精巢全长cDNA文库。提取中华大蟾蜍精巢总RNA，用Clontech公司SMART^TM cDNA文库构建试剂盒反转录合成第一链cDNA，LD-PCR扩增获得全长cDNA双链；经SfiⅠ酶切、层析柱分离后，500bp以上的片段与λTriplEx2载体连接并包装，建成原始文库。经鉴定，原始文库滴度为2．21×10^6pfu／ml，重组率为91％；文库扩增后的滴度为2．94×10^9pfu／ml，重组率为93．7％。插入片段大小分布于0．4～2．0kb之间，平均长度约为1．0kb，说明已构建文库质量较高，为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因，全长561bp，包含完整的5′和3′非编码区，编码128个氨基酸，即泛素的76个氨基酸后融合了52个氮基酸的核糖体L40蛋白。A full-length cDNA hbrary from the testis of Chinese Large Toad （Bufo bufo gargarizans） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reversely transcripted into full-length cDNA using Powerscript^TM reverse transcriptase. First-strand cDNA was amplified by long-distance PCR. After Sfi Ⅰ digestion and Chroma spin-400 fractionation, cDNAs （〉 500 bp） were hgated to λ TriplEx2 vector and were packaged with Gigapack Ⅲ Gold Packaging Extract. The optimal primary library contained 2.21×10^6 pfu/ml clones and the amplified library had a titer of 2.94×10^9 pfu/ml, in which over 90% clones were recombinant and the average size of inserted cDNAs was about 1.0 kb. These results show that the testis cDNA library can be used for screening genes. A full-length gene with 5＇ and 3＇ untranslated regions was isolated from the cDNA library. Sequence analysis showed that this 561 bp cDNA encoded a 128-amino acid ubiquitin/L40e extension protein.

关 键 词：中华大蟾蜍 精巢 CDNA文库 SMART技术 泛素延伸蛋白基因 

分 类 号：Q78[生物学—分子生物学] Q953