葱蝇非滞育蛹的全长cDNA文库构建  

Construction of Full-length cDNA Library of Non-diapause Pupae of the Onion Maggot,Delia antiqua

作　　者：黎万顺[1] 陈斌[1] 冯国忠[1] 李廷景[1] 

机构地区：[1]重庆师范大学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：《重庆师范大学学报（自然科学版）》2010年第1期21-25,共5页Journal of Chongqing Normal University:Natural Science

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

摘　　要：葱蝇具有兼性滞育的特性且与果蝇近缘，是昆虫滞育分子机理和冬滞育和夏滞育专化基因比较研究的理想模式种，本研究目的在于构建葱蝇非滞育蛹的全长cDNA文库，为进一步的滞育专化基因筛选、克隆和表达分析奠定基础。利用RNAiso试剂提取葱蝇非滞育蛹的总RNA，采用SMART技术合成全长cDNA，将限制性内切酶Sfi I消化后的cDNA克隆到质粒载体pDNR－LIB，转化入大肠杆菌（DH10B），获得原始文库。经过涂平板测定表明，原始文库的库容量为2．3×10^7个单克隆；随机挑取15个单克隆，通过PCR快速鉴定，插入片段大小在0．4～1．2kb之间，平均大小在0．9kb，重组率达100％。这些结果表明本研究所构建文库的代表性和重组片段的序列完整性达到了用于目的基因的分离筛抗和克隆表达的建库要求。The onion maggot, Delia antiqua, has the characteristics of summer and winter-diapause,and is close to Drosophila Melanogaster in phelogenetics. It is an ideal model species for the studies of the molecular mechanism of insect diapause and the comparison with winter-and summer-diapause-specific genes. The study aims at constructing full-length cDNA library of summer-diapause pupae of the onion maggot,Delia antiqua, in order to provide a base for further screening, cloning and expression analysis of diapause-specific genes. In this study, total RNA is extracted from non-diapause pupae of onion maggot, D. antiqua using RNAiso. Double-stranded cDNAs are synthesized with SMART technique and digested by Sfi I ,and then the cDNAs are ligated with the vector pDNR-LIB. The ligation mixture is transformed into E. coli DH10B by eletroporation. According to the evaluation of quality, the capacity of primary library is 2.3 x 107 efu/mL. The results from random picking 15 clones show that the inserted fragments ranging from 0. 4 to 1.2 kb by PCR amplification, with an average size of 0.9 kb, and the recombination rate is 100 %. These results show that a full-length eDNA library with high quality on Delia antiqua non-diapause pupae is well constructed. This indicates that the library is of high quality for cloning target genes and expressing target proteins.

关 键 词：葱蝇 非滞育蛹 全长CDNA文库 SMART技术 

分 类 号：Q966[生物学—昆虫学]