【作者】 李玉萍； 夏润玺； 王欢； 李喜升； 刘彦群； 魏兆军； 姜德富； 鲁成； 向仲怀；

【Author】 Yu-ping Li1,Run-xi Xia1,Huan Wang1,Xi-sheng Li2,Yan-qun Liu1,3,Zhao-jun Wei 4,De-Fu Jiang2,Cheng Lu 3,Zhong-huai Xiang 3 (1. College of Bioscience and Biotechnology,Shenyang Agricultural University,Shenyang 110161,China 2. Sericultural Institute of Liaoning Province,Fengcheng 118100,China 3. The Key Sericultural Laboratory of Agricultural Ministry,Southwest University,Chongqing 400716,China 4. School of Biotechnology and Food Engineering,Hefei University of Technology,Hefei 230009,China)

【机构】 沈阳农业大学生物科学技术学院； 辽宁省蚕业科学研究所； 农业部蚕桑学重点开放实验室西南大学； 合肥工业大学食品与生物工程学院；

【摘要】 我们成功构建了柞蚕Antheraea pernyi蛹的全长cDNA文库。总RNA提取自处于滞育后期的单个新鲜雌蛹。所构建文库的滴度是5×105cfu/ml,重组率达到95%。表达序列标签（EST）测序得到了250个有效序列,它们可以聚成175个单基因簇,其中包括24个重叠群和151的单拷贝。生物信息学分析结果表明,这175个单基因簇中有97个（55.4%）在数据库中有同源序列（其中只有5个是柞蚕已知基因）,37个（21.2%）在数据库中有EST同源序列但缺乏功能注释,41个（23.4%）为新的EST。通过EST测序,我们从该文库中鉴定了柞蚕KK-42结合蛋白基因（ApKK42-BP;GenBank登录号FJ744151）,该基因与天蚕Antheraea yamamai的KK-42结合蛋白基因在氨基酸序列上的一致性高达95%,而KK-42结合蛋白基因与天蚕的幼虫滞育解除有关。蛋白质序列分析表明,ApKK42-BP是一个胞外蛋白而不是膜蛋白,其信号肽位于第1-18。ApKK42-BP包括2个推测的保守功能域abhydro_lipase和abhydrolase₁,这一结果表明该蛋白可能是一种脂肪酶。基于EST数量的表达发现,ApKK42-BP在滞育的后期是一个高表达基因,表明该基因与柞蚕的蛹滞育解除也有关系。更多还原

【Abstract】 In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm,Antheraea pernyi,the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the post-diapause stage. The titer of the library was 5 × 105 cfu/ml and recombination rate reached 95%. Expressed sequence tag（EST） analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes,97（55.4%） were known genes but only five from A. pernyi,37（21.2%） were known ESTs without function annotation,and 41（23.4%） were novel ESTs. By EST sequencing,we identified a gene coding KK-42-binding protein in A. pernyi（named as ApKK42-BP;GenBank accession no. FJ744151） that shared 95% identity with the homologue in Antheraea yamamai that involved in larva-diapause termination. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18,and contained two putative conserved domains,abhydro_lipase and abhydrolase₁,suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of post-diapause stage,suggesting it may also be involved in pupa-diapause termination.更多还原