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本文以羽化3天内的大草蛉Chrysopa pallens(Rambur)雌雄触角为材料提取总RNA,以此为模板,通过SMARTscribeTM Reverse Transcriptase反转录酶反转录合成第一链cDNA,引物扩增获得双链cDNA,经CHROMA SPIN+TE-1000 Column分级分离后,收集0.4-2.0 kb之间的片段重组于质粒载体pSMART2IFD并电转化至大肠杆菌Escherichia coli DH5ɑ,最终构建获得大草蛉雌雄触角全长cDNA文库。结果显示构建的雌雄触角cDNA初级文库滴度分别2.1×106pfu/mL和1.8×106pfu/m L,重组率均达到93.0%以上,插入cDNA的片段大小范围为0.4-2.0 kb,主要集中在0.5-1.0 kb,平均长度为549.0 bp,表明构建获得的文库质量较高,可保证大规模全长c NA的获得。该文库的构建丰富了大草蛉基因序列信息,为大量克隆大草蛉嗅觉相关基因,深入揭示大草蛉嗅觉识别机制奠定基础,也为合理利用大草蛉进行生物防治提供理论依据。
In this paper, the total RNA was extracted from the female and male antennae of Chrysopa pallens (Rambur) within 3 days of eclosion. The first strand cDNA was reverse transcribed by SMARTscribeTM Reverse Transcriptase reverse transcriptase and the double-stranded cDNA After separation by CHROMA SPIN + TE-1000 Column, fragments between 0.4-2.0 kb were collected and recombined into the plasmid vector pSMART2IFD and electroporated into Escherichia coli DH5ɑ. Finally, the full-length cDNA library of M. grandis male and female was obtained. The results showed that the titers of cDNA primary libraries of male and female antennae were 2.1 × 106pfu / mL and 1.8 × 106pfu / mL, respectively, and the recombination rates reached above 93.0%. The inserted cDNA fragments ranged from 0.4-2.0 kb in size, mainly in the 0.5- 1.0 kb with an average length of 549.0 bp, indicating that the quality of the library obtained by the construction is high enough to ensure the availability of large-scale full-length cNAs. The construction of this library enriches the information of the gene sequence of S. grandis, and provides a theoretical basis for cloning a large number of genes related to olfactory related genes of S. grandis and revealing the mechanism of olfactory recognition in greater depth. It also provides a theoretical basis for the biological control of S. grandis.