口虾蛄卵黄蛋白原(Vg)基因的初步研究

为研究口虾蛄Oratosquilla oratoria卵黄蛋白原(vitellogenin,Vg)基因,利用c DNA末端快速扩增(RACE)技术克隆口虾蛄Vg基因c DNA全长序列(Gen Bank登陆号:KR422400),并应用相对实时荧光定量PCR技术检测雌雄口虾蛄不同组织、不同发育时期卵巢Vg mRNA的表达量。结果表明:口虾蛄Vg基因全长7727 bp,其中5'端非编码区为36 bp,开放阅读框(ORF)为7521 bp,3'端非编码区为170 bp,共编码2506个氨基酸;预测的氨基酸序列中存在1个长度为20个氨基酸的信号肽,1个类似于枯草蛋白酶的内切蛋白酶识别位点(RSKR),3个潜在的N-连接糖基化位点,1个在无脊椎和脊椎动物卵黄蛋白原中都比较保守的KALGNVG基序;对其结构域分析表明,口虾蛄Vg蛋白含有4种保守结构域,分别为卵黄蛋白原N端结构域(Vitellogenin_N)、血管性血友病因子(v WFD)和两种未知功能结构域(domain of unknown function)DUF1943与DUF1081;经NCBI BLASTP同源性比较,口虾蛄Vg氨基酸序列与其他甲壳类的相似性为46%~52%;系统发育分析结果显示,口虾蛄Vg单独聚为一支;实时定量PCR结果显示,口虾蛄Vg基因在性腺和肝胰腺中均有表达,在肌肉中均不表达,卵巢中Vg mRNA的表达量显著高于其他组织(P0.05),且口虾蛄Vg mRNA的相对表达量随着卵巢的发育不断增加(P0.05),在成熟期时达到最高值,随后显著下降,Vg mRNA表达量在卵巢中的变化规律可以作为了解口虾蛄性腺发育的有效指标。本研究结果为口虾蛄Vg蛋白的功能研究奠定了基础。

Vitellogenin(Vg) gene in mantis shrimp Oratosquilla oratoria

In the present study, the full length cDNA of vitellogenin( Vg) gene was cloned in mantis shrimp Ora-tosquilla oratoria using rapid amplification of cDNA ends (RACE) (GenBank accession No. KR422400), and ex-pression levels of Vg gene mRNA in various tissues, especially in ovary within different ovarian development stages, were investigated by qPCR. The results showed that the Vg gene was 7727 bp long, with 36 bp of 5' untranslated region( UTR) , 170 bp of 3'UTR and 7521 bp of the coding region encoding a size of 2506 amino acids predicted protein. The predicted protein contained a signal peptide(20 amino acids long) , three N-glycosylation site, a con-sensus cleavage site RSKR and a conserved KALGNVG motif that was conservative in invertebrates and vertebrate. Moreover, there were 4 conserved domains, Vitellogenin N, vWFD, DUF1943 and DUF1081, in the predicted protein. The deduced amino acids sequence showed 46%-52% identity with other known decapods Vg by using BLASTP online analysis. Phylogenetic tree rvealed that the Vg formed a clade separated from the decapod species Vg. The qPCR indicated that the Vg mRNA were found in both gonad and hepatopancreas, and had significantly higher expression level of Vg mRNA in ovary than in other tissues (P<0. 05), without expression in muscle. In the ovary, however, the expression of Vg mRNA was found to be significantly increased from immature stage to mature stage ( P<0 . 05 ) , then dropped immediately ( P<0 . 05 ) . The expression profile in ovary well was responding to the annual cycle of vitellogenesis. The findings will provide basic information for understanding of the whole function of Vg gene in the mantis shrimp.