与家蚕细胞凋亡相关的Caspase基因BmIce的克隆和序列分析

Cloning and Sequence Analysis of the Caspase Gene, BmIce, Related to the Apoptosis in the Silkworm

DrIce基因在果蝇的细胞调亡代谢途径中起着重要作用。采用tblastn程序将DrICE在家蚕EST数据库中进行同源性检索,检索到的EST序列进行电子延伸,根据延伸结果设计引物,用RT-PCR检测和克隆测序验证,成功地克隆到家蚕第二种Caspase基因的全长cDNA(GenBank登录号为:AY885228)。克隆的cDNA长1410bp,ORF长825bp,编码275个氨基酸残基,预测分子量为31.8kD,等电点为6.15,基因名定为BmIce。将BmIce的cD-NA与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,外显子/内含子边界处均符合GT-AG规则。BmICE在氨基酸水平上与果蝇DrICE、线虫的CED-3的一致性分别为34%和29%,在第169个氨基酸残基处具有QACRG的五肽活性位点,该活性位点是Caspase家族的典型结构。与果蝇Caspase家族聚类分析中,BmICE与果蝇中具有短的N端结构域的DrICE、DCP-1及DECAY聚为一类,根据其结构分析和聚类分析,推测家蚕BmIce基因可能参与细胞凋亡的执行作用。

The Drlce plays an important role in the progress of apoptosis in Drosophila. The animo acid sequence of Drosophila DrlCE was searched against the silkworm EST database with tBLASTn program. The ESTs with high score were clustered and assembled to a consensus sequence. By using the primers based on the consensus sequence, we cloned the second Caspase gene of Bombyx mori, termed it as Bmlce （ GeneBank accession number, AY885228）. The length of the Bmlce cDNA was 1410 base pairs in length and encoded 275 amino acids, with the predicted molecular weight of 31.8 kD and isoelectric point of 6. 15. An alignment of the cDNA sequence with the silkworm genome sequences revealed that there were 6 exons in Bmlce and all of them conformed the GT-AG rule. The deduced amino acid sequence of the Bmlce shares 34% identity with Drosophila DrlCE and 29% with C. elegans CED-3. There is an active site of pentapeptide QACRG（GIn-Ala-Cys-Arg-Gly） in BmlCE, which was an important site for Caspase involved in catalysis activity, A clustering analysis showed that the BmlCE was classified into a cluster of the short prodomain family with DrlCE, DECAY, DCP-1. These results of the structure and cluster information for Bm/ce indicated that this gene is likely to involve in the process of apoptisis effect function.