葱蝇冬滞育蛹的全长cDNA文库的构建  

Construction of full-length cDNA library of winter-diapause pupae of the onion maggot,Delia antique

作　　者：黎万顺 冯国忠 陈斌 何正波 

机构地区：[1]昆虫与分子生物学研究所重庆高校生物活性物质工程研究中心重庆高校动物生物学重点实验室,重庆400047

出　　处：《昆虫知识》2010年第1期53-58,共6页Entomological Knowledge

基　　金：国家自然科学基金(No.30870340);重庆市自然科学基金(No.CSTC2008BB1365)资助;重庆市自然科学基金重点项目(No.CSTC2008BA5030)

摘　　要：葱蝇Delia antiqua(Meigen)具有冬滞育的特性且与黑腹果蝇Drosophila melanogaster近缘。该研究目的在于构建葱蝇冬滞育蛹的全长cDNA文库,为进一步的冬滞育专化基因筛选、克隆、和表达分析奠定基础。利用RNAiso试剂盒提取葱蝇冬滞育蛹的总RNA,采用SMART技术合成全长cDNA,经SfiⅠ酶切消化后,将cDNA克隆到质粒载体pDNR-LIB。经鉴定,原始文库的滴度为2.4×107cfu/mL。随机挑取15个克隆,经PCR快速鉴定,插入片段大小在0.5～3kb之间,平均大小在1kb左右,重组率达100%。结果表明该研究获得高质量的葱蝇冬滞育蛹cDNA文库。The onion maggot,Delia antiqua（Meigen）,is characterized by winter-diapause and is close to Drosophila melanogaster in phelogenetics. This study aims to construct full-length cDNA library of winter-diapause pupae of D. antique,in order to provide a base for further screening,cloning and expression analysis of winter-diapause-specific genes. In this study,total RNA was extracted from winter-diapause pupae of D. antiqua using RNAiso kit. Double-stranded cDNAs were synthesized with SMART technique and digested by the enzyme SfiI,and then the cDNAs were ligated into the vector pDNR-LIB. The ligation mixture was transformed into E. coli DH10B by eletroporation. According to the evaluation on quality,the titer of primary library was 2.4×107 cfu/mL. The results from random sampling of 15 clones showed that the inserted fragments ranged from 0.5 kb to 3 kb by PCR amplification,with an average size of 1 kb,and the recombination rate was 100 %. These results showed that a full-length cDNA library with high quality on D. antiqua winter-diapause pupae was well constructed.

关 键 词：葱蝇 冬滞育蛹 全长CDNA文库 SMART技术 

分 类 号：S433[农业科学—农业昆虫与害虫防治] Q96[农业科学—植物保护]