三疣梭子蟹Drosha和Exportin 5基因克隆及其在性腺发育过程中的表达分析

摘要

In the present study, full-length complementary DNA (cDNA) of Drosha and Exportin 5 involved in the pathway of microRNA (miRNA) biogenesis of the swimming crab Portunus trituberculatus were cloned by rapid amplification of cDNA ends (RACEs). The length of the Drosha gene is 3443 bp, which is predicted to encode a polypeptide of 1038 amino acids, containing two ribonuclease (RNase) Ⅲ domains (RⅢDa and RⅢDb) and a double-stranded RNA binding domain (dsRBD). The full-length of Exportin 5 is 5000 bp, which is predicted to encode a polypeptide of 1208 amino acids, containing an importin-β N-terminal domain (IBN-N) and an exportin 1-like domain (XPO-1). Homology analysis revealed that Drosha in P. trituberculatus is highly similar to those in some species and shares the highest similarity with that in Marsupenaeus japonicus (94％). Quantitative real-time PCR (qRT-PCR) analysis showed that Drosha and Exportin 5 are expressed in all the tested tissues, and were highly expressed in the ovary and hepatopancreas tissues (P<0.05). At different stages of testis development, the expression of the genes Drosha and Exportin 5 shared the same trend, and both showed the highest expression at stage Ⅱ. During ovarian development, the expression level of the genes Drosha and Exportin 5 increased gradually from phase Ⅱ to phase Ⅴ. The results suggest that the genes Drosha and Exportin 5 cooperatively regulate the gonadal development of P. trituberculatus in an miRNA-dependent manner. Furthermore, the results provide useful information for studies on the regulation of gonadal development in P. trituberculatus.%本研究利用RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)Drosha和Exportin 5基因的cDNA全长序列,Drosha基因长度为3443 bp,编码1038个氨基酸,预测蛋白质包含2个相邻的RNA酶Ⅲ结构域(RⅢDa和RⅢDb)和1个双链RNA结合结构域(dsRBD).Exportin 5基因全长为5000 bp,编码1208个氨基酸,预测蛋白质包含1个Importin-βN-末端结构域(IBN-N)和1个核输出蛋白结构域(XPO-1).同源分析显示,三疣梭子蟹Drosha氨基酸序列与其他物种高度相似,与日本对虾(Marsupenaeus japonicus)相似度最高,达94％.qRT-PCR分析结果显示,Drosha和Exportin 5基因在三疣梭子蟹各组织中均有表达,其在卵巢和肝胰腺中的表达量最高(P<0.05);在精巢不同发育时期,2个基因表达量的变化趋势一致,均在Ⅱ期(精母细胞增殖、分化期)表达量最高;在卵巢发育过程中,2个基因表达量的变化趋势也大致相同,随着卵巢发育(Ⅱ～Ⅴ期)逐渐升高.研究表明,Drosha和Exportin 5基因可能通过调控miRNA的合成来影响三疣梭子蟹性腺发育过程,为深入解析三疣梭子蟹性腺发育调控机制提供了重要参考.