家蚕钙网蛋白的基因结构与初步表达谱研究

钙网蛋白(calreticulin,CRT)是内质网/肌浆网主要的Ca2+结合蛋白,在细胞功能的调节方面发挥重要作用。利用双向电泳技术及质谱技术研究家蚕脂肪体蛋白组的变化,通过分析检索蛋白质组数据,获得了钙结合蛋白的氨基酸序列。通过电子克隆、cDNA3′末端快速扩增(3′rapid amplification of cDNA ends,3′RACE)方法克隆了家蚕钙网蛋白基因cDNA全长序列,命名为BmCRT(GenBank登录号:FJ360528)。BmCRT基因cDNA全长1 705 bp,开放阅读框序列(ORF)长1 197 bp,编码398个氨基酸。氨基酸序列分析显示,BmCRT与其它物种的CRT都具有保守的N-结构域、脯氨酸富集结构域和C-端结构域。BmCRT基因组结构分析表明:该基因由7个外显子和6个内含子组成。分析BmCRTmRNA在不同发育时期的表达变化显示,该基因在家蚕幼虫眠期的mRNA表达量比食桑期高,而在4龄和5龄食桑期mRNA表达量没有随着生长变化而变化。

Expression Profile and Gene Structure Analysis of Calreticulin From the Silkworm, Bombyx mori

Calreticulin(CRT) is an essential Ca2+binding protein of endoplasmic reticulum or sarcoplasmic reticulum,which plays important roles in regulation of many cell functions.Using methods of two-dimensional gel electrophoresis and mass spectrometry,we investigated differential expression proteins in fat body of the silkworm,Bombyx mori.The protein sequence of calreticulin was obtained via the analysis and retrieving protein database.We cloned the cDNA of calreticulin from silkworm through electronic cloning and 3′RACE(3′ rapid amplification of cDNA ends).Then we designated it as BmCRT(GenBank accession number:FJ360528).The full-length of BmCRT cDNA was 1 705 bp and harbored an ORF of 1 197 bp,which encoded a deduced polypeptide of 398 amino acids.The deduced amino acid had a high similarity to that of the CRT reported in other species.It also had several conserved domains,such as the conserved N-domain,P-domain and C-domain.Genomic structure analysis results suggested that BmCRT contained 7 exons and 6 introns.The expression profile of BmCRT at different development stages indicated that the expression level of BmCRT was higher at the molting stage than that of the feeding stage.In addition,the expression level of BmCRT was steady during the whole feeding stage of 4th and 5th instar larvae.