›› 2008, Vol. 51 ›› Issue (8): 785-791.

• 研究论文 •    下一篇



  • 出版日期:2010-07-27 发布日期:2008-08-20
  • 通讯作者: 苗雪霞

Cloning and expression of a G protein alpha subunit gene BmGα73B from Bombyx mori.

ZHANG Yu-Ping   

  • Online:2010-07-27 Published:2008-08-20

摘要: 异三元G蛋白是真核细胞感知外界信号后将信号传递到胞内的重要分子,在生物中参与了广泛的信号转导途径,如光、神经递质和激素等。为了研究G蛋白在家蚕Bombyx mori中的生理功能及其作用机理,我们运用生物信息学方法在已有的家蚕基因组数据库中找到了一段与G蛋白alpha亚基()同源性很高的序列。通过设计特异性引物,运用PCRRACE技术,成功地克隆了一个家蚕基因的全长cDNA序列。该基因全长1 509 bp (GenBank登录号:EU914850),开放阅读框(ORF)为1 158 bp,编码385个氨基酸。BlastDNAstar等软件分析发现该基因编编码的蛋白质与其他物种已知的具有一定的保守性,将它命名为BmGα73BRT-PCR扩增检测该基因在家蚕不同组织器官和不同发育时期的转录表达活性,结果表明它在不同发育时期的家蚕各组织器官中都有表达。从组织水平上看,BmGα73B在中肠中表达量最高,在马氏管、头部和神经索等组织中也有适量表达。在家蚕的不同发育时期中,转录水平峰值出现在幼虫期,在蛹早期也有适量的表达,而在预蛹期、蛹后期和成虫期几乎没有表达。结果说明BmGα73B可能参与了家蚕生长前期的中肠发育过程,为进一步研究G蛋白在家蚕发育过程的作用奠定了一定的基础。

关键词: G蛋白, α-亚基, 家蚕, 克隆, 表达, RT-PCR

Abstract: The heterotrimeric GTP-binding proteins are vital molecules in eukaryotic cell signal transduction;they mediate extracellular signals to pass down to the downstream effectors. In order to investigate the physiological function and action mechanism of the silkworm G protein which takes part in multiple signal transduction pathways, we found a known G protein alpha subunit homolog in silkworm genome database by means of bioinformatics. A novel G protein alpha subunit from Bombyx mori was cloned by PCR and RACE using specific primers and named BmGα73B. The full length BmGα73B gene (GenBank accession no. EU914850) was 1 509 bp with a 1 158 bp ORF coding for 385 amino acid residues. Sequence analysis aided by Blast and DNAstar revealed that the BmGα73B protein contained all the well conserved domains and motifs that were critical sites for interaction with receptors and other binding effectors compared with the already known Gα sequences in other species. RT-PCR was conducted to investigate the BmGα73B expression in different tissues and at different developmental stages. The results indicated that BmGα73B was widely expressed in B. mori tissues, especially rich in the midgut; it was also obviously present in head, Malpighian tubule and other tissues. The stage specific expression patterns indicated that BmGα73Bwas mainly expressed in larvae, also existed in early pupae, but less or not expressed in later pupae and adults. The results suggest that BmGα73B may play crucial roles at the early developmental stage of the B. mori midgut, which could be a clue to further investigate the function of G proteins in silkworm development.

Key words: G protein, alpha subunit, Bombyx mori, cloning, expression, RT-PCR