诸氏鲻虾虎鱼卵黄蛋白原基因全长cDNA的克隆及表达

Full-length cDNA cloning and expression analysis of vitellogenin gene in Mugilogobius chulae

为了探讨诸氏鲻虾虎鱼(Mugilogobius chulae)卵黄蛋白原组织分布及17β-雌二醇(E2)暴露对雄性诸氏鲻虾虎鱼Vg的影响,作者采用RT-PCR、RACE方法克隆并分析了诸氏鲻虾虎鱼卵黄蛋白原(Vg)基因的全长cDNA序列,并对Vg在诸氏鲻虾虎鱼体内的组织表达分布及E2诱导后不同时间表达规律进行了研究。结果表明:获得的Vg cDNA序列全长5 067 bp,开放阅读框(ORF)含4 992 bp,编码1 663个氨基酸,含有信号肽、多丝氨酸区域,推测其编码氨基酸分子量为186.2 ku,等电点为9.31。荧光定量PCR结果显示,Vg在诸氏鲻虾虎鱼肝脏中表达量最高。E2诱导后,诸氏鲻虾虎鱼肝脏中Vg m RNA的表达量第1天达到小高峰,第3天达到高峰,第7天开始明显下降,第11天仍能维持较高水平。本研究成功克隆了诸氏鲻虾虎鱼Vg基因全长cDNA序列,诸氏鲻虾虎鱼肝脏是Vg主要合成场所,E2诱导雄性诸氏鲻虾虎鱼Vg的表达作为生物标志物用于近海环境雌激素类物质的检测具有良好的应用前景。

To explore the tissue distribution of the vitellogenin（Vg） gene in Mugilogobius chulae and the effect of the pollutant 17β-estradiol on male M. chulae, the full length of vitellogenin（Vg） cDNA in M. chulae was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE） techniques using total RNA extracted from the liver of female fish. Meanwhile, the expression of Vg m RNA of M. chulae was determined by real-time fluorescent quantitative PCR. The full length of M. chulae＇s Vg cDNA sequence contains 5 067 bp nucleotides, with a complete open reading frame（ORF） of 4 992 bp, which encodes 1 664 amino acid proteins with a deduced protein molecular weight of 186.2 ku. The ORF contains signal peptides and a polyserine domain. The real-time RT-PCR result showed that M. chulae＇s Vg m RNA was primarily detected in the liver. Furthermore, Vg m RNA expression levels at different time points of E2 treatment of male fish were determined. M. chulae＇s Vg m RNA expression levels reached the peak on the third day and then the expression levels declined on the seventh day. These results indicated that the full length of Vg cDNA in M. chulae was cloned successfully. This study suggests that Vg in male M. chulae can be considered as a valid biomarker for offshore environmental monitoring of estrogenic substances.