家蚕细胞凋亡相关基因BmICAD的克隆、序列和功能分析及其在精巢中特异表达的初步研究

Molecular Cloning,Sequence and Function Analyses and Specific Expression of Gene BmICAD Related to Apoptosis in Silkworm,Bombyx mori

DREP-1基因在果蝇的细胞凋亡过程中对DNA的降解有重要调控作用。本研究将该基因序列在家蚕EST数据库中进行同源性检索,把检索到的EST序列进行电子延伸和克隆重叠群拼接,根据拼接结果设计引物进行PCR扩增并克隆测序验证,首次成功克隆了家蚕第一个ICAD基因BmICAD的cDNA:该cDNA全长844 bp,ORF长522 bp,编码含有174个氨基酸的蛋白;预测分子量为19.6 kDa,等电点为4.23,所编码蛋白与果蝇DREP-1的一致性(identity)为36%;虽然Northern印记杂交未检测到信号,但是RT-PCR结果显示,该基因在精巢中特异表达。

The DREP-1 gene plays an important role in the process of apoptosis in Drosophila. The amino acid sequence of Drosophilo ICAD was searched against the silkworm EST database. The resulted ESTs were clustered and assembled into a consensus sequence. Primers based on the consensus sequence were designed and the full length cDNA of ICAD gene, designated BmICAD, was cloned firstly from Bombyx mori. This BmICAD cDNA was 844 bp in length, containing a 522 bp open reading frame （ORF）, which encoded a protein of 174 amino acids; the molecular weight of this protein was estimated to be 19.6 kD and the isoelectric point was 4.23. The BmICAD protein shares 36% identity with Drosophila DREP-1. No signal has inspected by Northern blot, but RT-PCR analysis showed that BmICAD was testis-specific expressed at the mRNA level. Structure and clustering analysis indicated that this gene might be involved in the process of apoptosis that regulated the degradation of DNA.