摘要
目的:构建甜菜夜蛾触角全长cDNA文库。方法:利用TRIzol试剂提取甜菜夜蛾触角总RNA,以此为模板,通过SMAR-TScribeTM反转录酶反转录合成第一链cDNA,引物扩增获得双链cDNA,经Proteinase K消化、SfiⅠ酶切和CHROMA SPIN-400Column分级分离后,收集400～2 000 bp之间的片段重组于改造的pUC19载体并转化至大肠杆菌Escherichia coli DH5α,最终构建获得甜菜夜蛾触角全长cDNA文库。结果:对文库进行滴度测定和重组率分析,结果表明构建的cDNA初级文库滴度为1.6×107pfu/ml,重组率为94%,插入片段大小为0.5～3.0 kb,平均长度在1 kb以上,表明构建获得的文库是一个高质量的文库。结论:该文库的构建为今后克隆甜菜夜蛾嗅觉相关基因奠定了基础。
Objective:To construct full length cDNA library from the antenna of Spodoptera exigua.Method:Total RNA was isolated from the antenna of S.exigua using TRIzol reagent.The SMARTScribeTM reverse transcriptase was used to synthesize and anchor the first-strand cDNA adaptor primers.And the primers were used to amplify the double-strand cDNA.The PCR products were digested by proteinase K and SfiⅠ.Then they were size fracted using CHROMA SPIN-400 column.The cDNAs ranged from 400 to 2 000 bp were recombined to the modified pUC19 vector.The ligation mixture was transformed into Escherichia coli DH5α.Finally,the library was constructed.Result:The recombinant titer and the recombinant rate were determined for the constructed library.The titer and recombinant rate of primary constructed cDNA library were 1.6×107 pfu/ml and 4%.The size of the inserted fragments ranged from 0.5 to 3.0 kb with average size was above 1kb.These indicated that the constructed library was with high quality.Conclusion:This library is beneficial for further cloning the olfactory genes from S.exigua.
出处
《生物技术》
CAS
CSCD
北大核心
2012年第4期11-14,共4页
Biotechnology
基金
国家自然科学基金项目(31160354)
云南省应用基础研究面上项目(2008CD140)资助
