葱蝇夏滞育蛹的全长cDNA文库构建  

Construction of a Full-Length cDNA Library of Summer-Diapause Pupae of the Onion Maggot,Delia antiqua

作　　者：冯国忠[1] 黎万顺[1] 陈斌[1] 李莹[1] 

机构地区：[1]重庆师范大学生命科学院,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：《西南大学学报（自然科学版）》2010年第6期89-94,共6页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30870340);重庆市自然科学基金资助项目(CSTC2008BB1365);重庆市自然科学基金重点资助项目(CSTC2008BA5030)

摘　　要：葱蝇具有夏滞育的特性且与果蝇近缘.为构建葱蝇夏滞育蛹的全长cDNA文库,并为进一步的夏滞育专化基因筛选、克隆和表达分析奠定基础,利用RNAiso试剂盒提取葱蝇夏滞育蛹的总RNA,采用SMART技术合成全长cDNA,限制性酶切消化后将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.随机挑取克隆,经PCR快速鉴定,插入片段大小在0.35～2.2kb之间,平均大小约为0.9kb,重组率达100%,获得了高质量的葱蝇夏滞育蛹cDNA文库.The onion maggot （Delia antiqua） has the characteristic of summer-diapause and is close to Drosophila rnelanogaster in phelogenetics. In order to construct a full-length cDNA library of summer-diapause pupae of D. antiqua so as to lay a foundation for further screening, cloning and expression analysis of summer-diapause-specific genes, total RNA was extracted from summer-diapause pupae of D. antiqua using RNAiso kit. Double-stranded cDNAs were synthesized with SMART technique and digested by the enzyme Sfi I , and then the cDNAs were ligated into the vector pDNR-LIB. The ligation mixture was transformed into E. coli DHIOB by eletroporation. According to the evaluation on quality, the titer of the primary library was 2.4 × 10^7 cfu/mL. Random clones showed that the size of the inserted fragments ranged from 350 bp to 2.2 kb by PCR amplification, with an average of 0.9 kb, and the recombination rate was 99~. These results showed that a full-length of cDNA library with high quality on D. antiqua summer-diapause pupae was successfully constructed.

关 键 词：葱蝇 夏滞育蛹 全长CDNA文库 SMART技术 

分 类 号：Q969.454.1[生物学—昆虫学]