葱蝇冬滞育蛹的全长cDNA文库的构建
Construction of fulllength cDNA library of winterdiapause pupae of the onion maggot, Delia antique.
黎万顺，冯国忠，陈斌**，何正波
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作者单位：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
中文关键词：葱蝇，冬滞育蛹，全长cDNA文库，SMART技术
英文关键词：Delia antiqua, winterdiapause pupae, fulllength cDNA library, SMART technique
中文摘要： 葱蝇Delia antique-LIB。经鉴定，原始文库的滴度为2.4×107 cfu/mL。随机挑取15个克隆，经PCR快速鉴定，插入片段大小在0.5～3 kb之间，平均大小在1 kb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。(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
英文摘要： 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 winterdiapause pupae of D. antique, in order to provide a base for further screening, cloning and expression analysis of winter-diapausespecific genes. In this study, total RNA was extracted from winterdiapause 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.