家蚕一个DDRGK超家族成员的克隆及分析

Cloning and Characterization of a Novel Member of the DDRGK Gene Family from the Silkworm,Bombyx mori

DDRGK类蛋白是在动物及植物中发现的约有300个氨基酸残基的蛋白家族,含有一个高度保守的DDRGK基序,但功能还未知。在对家蚕幼虫cDNA文库的测序中发现了含有该保守结构域的EST序列,经过电子克隆、RT-PCR,获得该基因完整的cDNA序列,将其命名BmDDRGK(GenBank登录号FJ645927)。然后构建了重组质粒转化到大肠杆菌BL21和Rosetta中诱导表达,但经SDS-PAGE检测未检测到重组蛋白的表达。利用Real-timePCR技术对BmDDRGK在家蚕五龄幼虫期和蛹期不同组织的mRNA表达水平进行了鉴定。绝对定量结果显示BmDDRGK在家蚕五龄幼虫期和蛹期的各个组织中均有较为明显的表达,在五龄幼虫期,以中肠、马氏管、睾丸表达量较高,在蛹期,以翅原基、睾丸的表达量较高。

DDRGK is a family of proteins of approximately 300 residues found in plants and animals which contain a highly conserved DDRGK motif.However,the function of this family is still unknown,up to now.The ESTs of DDRGK are found through searching cDNA library of silkworm puper.A full length cDNA is found by EST cloning and RT-PCR methords,named this gene BmDDRGK（GenBank Accession No.FJ645927）.The recombinant plasmid is transformed into E.coli BL21 and Rosetta.But recombinant protein is undetectable by SDS-PAGE analysis with the IPTG induction.The mRNA expression lever of BmDDRGK in the tissues of 5th larva and pupae is analyzed by real-time PCR.And result shows that BmDDRGK is expressed obviously in every tissue in the two stages by absolute quantitation analysis.Furthermore,it is expressed highly in midgut,malpighian tubule,testis of 5th larva,highly in wing disk,testis of pupae.