论文部分内容阅读
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 the full-length cDNA library of beet armyworm antennae. Methods: The total RNA was extracted from the beet armyworm by TRIzol reagent. The first strand cDNA was reverse transcribed by SMAR-TScribeTM reverse transcriptase. The double-stranded cDNA was amplified by primer and digested with Proteinase K, After fractionation and CHROMA SPIN-400Column fractionation, fragments between 400-2000 bp were collected and recombined into the transformed pUC19 vector and transformed into E. coli Escherichia coli DH5α to construct a full-length cDNA library of beet armyworm. Results: The titer of the library and recombination rate analysis showed that the constructed cDNA primary library had a titer of 1.6 × 107pfu / ml, a recombination rate of 94%, an insert size of 0.5-3.0 kb and an average length of more than 1 kb , Indicating that the library obtained by the construction is a high-quality library. Conclusion: The construction of this library lays the foundation for cloning olfactory related genes of beet armyworm in the future.