家蚕Caspase家族基因 BmCaspase-X 的克隆与功能分析

【目的】本研究旨在克隆获得家蚕 Bombyx mori Caspase家族基因，并通过RNAi技术初步分析其在家蚕细胞水平细胞凋亡中的功能。【方法】 用cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆家蚕Caspase家族一个新基因，用RNAi和流式细胞术初步分析该基因在家蚕细胞凋亡中的功能。【结果】将克隆获得的家蚕Caspase家族基因命名为 BmCaspase-X，其全长为2 105 bp，开放阅读框长为1 494 bp，编码497 aa，分子量为57.8 kDa，等电点为5.29。BmCaspase-X含有Caspase特有的结构位点。系统进化分析表明，BmCaspase-X与家蚕ICE同其他昆虫Caspase-4同源物先聚在一起，再与具有长前体结构域的昆虫其他因子聚为一类。RNAi初步结果显示该基因干扰后没有出现明显的细胞凋亡。【结论】BmCaspase-X具有典型Caspase特征，属于Caspase家族成员。目前RNAi实验结果表明 BmCaspase-X 未能引起家蚕细胞凋亡的改变。本文为进一步研究该基因的功能奠定基础。

Cloning and functional analysis of a Caspase gene BmCaspase-X in Bombyx mori

【Aim】 The present study aims to clone Caspase family gene in Bombyx mori and to investigate its function preliminarily using RNAi. 【Methods】 A new gene of Caspase family was cloned by rapid amplification of cDNA ends (RACE), and then its functions in apoptosis were analyzed by RNAi and flow cytometry. 【Results】 The cloned Caspase family gene was denominated BmCaspase-X in B.mori, the full length is 2 105 bp, and the open reading frame is 1 494 bp, coding a protein of 497 aa with an estimated molecular mass of 57.8 kD and pI of 5.29. BmCaspase-X contains the typical characteristics of Caspase family. Phylogenetic analysis showed that BmCaspase-X and ICE of the silkworm cluster with Caspase-4 homologs from other insects firstly, and then cluster with Caspase members with long precursor structure domain from other insects. RNAi of BmCaspase-X in BmE-SWU1 cells did not cause significant apoptosis effect. 【Conclusion】 The new BmCaspase-X, with the typical characteristics of Caspase family, should be classified into the Caspase family. The preliminary RNAi data show that BmCaspase-X seems not to cause apoptosis in silkworm cells. This study lays a foundation for further functional investigation on BmCaspase-X.