铜绿丽金龟雌虫触角全长cDNA文库的构建及质量分析

为了研究铜绿丽金龟(Anomala corpulentaMotschulsky)成虫嗅觉相关蛋白的功能,本文以铜绿丽金龟雌成虫触角为原始材料提取总RNA,利用SMART技术构建了铜绿丽金龟雌虫触角全长cDNA文库。经测定,原始文库滴度为6.74×106pfu/mL,扩增后的文库滴度为1.08×1010pfu/mL,文库重组率为98.98%。随机挑取24个单克隆,通过菌液PCR检测得知文库插入片段的大小在0.5~2.0kb之间,片段平均长度为1.0kb左右。经过菌液PCR并测序筛选cDNA文库,本试验获得了两条编码气味结合蛋白的全长基因序列AcorOBP1和AcorOBP2。以上数据表明,已获得高质量的铜绿丽金龟触角全长cDNA文库,并筛选、克隆获得了气味结合蛋白基因序列。这为进一步研究触角内气味结合蛋白的功能奠定了基础。

Construction and quality analysis of a full-length cDNA library from antennae of Anomala corpulenta Motschulsky

In order to study the function of the olfactory associated proteins in Anomala corpulentaMotschulsky, a full-length cDNA library was constructed based on SMART technique. Total RNA was extracted from the female antennae of A.corpulenta. The titer of primary library was 6.74×106pfu/mL, and the recombinant rate was 98.98%.The amplified library showed a titer of 1.08×1010pfu/mL. Twenty-four monoclonal colonies had been picked randomly and detected by PCR. The results showed that the sizes of amplified fragments were 0.5-2.0 kb, with an average of 1.0 kb. By PCR and sequencing, this study obtained two full-length gene sequencesAcorOBP1 and AcorOBP2 that coding odorant binding proteins. These experiment data showed that a high-quality full-length cDNA library of the female antennae of A.corpulentahad been constructed successfully, and also have screened and cloned the odorant binding protein gene sequences, which laying solid foundation for further research on the function of odorant binding proteins.