三疣梭子蟹蜕皮激素受体EcR基因的cDNA克隆及表达分析

Cloning and expression analysis of EcR gene in Portunus trituberculatus

中文关键词: 三疣梭子蟹  蜕皮激素受体  基因克隆  急性盐度胁迫  RT-PCR

英文关键词: Portunus trituberculatus  EcR  Gene cloning  Salinity stress  RT-PCR

中文摘要:

      通过简并引物扩增及SmartTM Race技术，首次克隆了三疣梭子蟹Portunus trituberculatus蜕皮激素受体EcR基因cDNA全长序列。该基因全长2996bp，编码一个由503个氨基酸组成的多肽，预测理论等电点pI为7.33，分子量大小为55.69kDa。同源性及系统进化分析表明，三疣梭子蟹EcR基因与青蟹、拟穴青蟹、大西洋招潮蟹、陆地蟹、美洲螯虾、褐虾、日本对虾的同源性分别为94%、90%、88%、84%、82%、73%、66%。荧光定量RT-PCR结果表明，EcR在肝胰腺、卵巢、肌肉、眼柄、鳃和心脏中均有分布，在肝胰腺中表达量最高，在心脏中最低。高盐（45）胁迫下，EcR基因的表达量在24h后显著低于对照组，总体呈下降趋势；低盐（11）胁迫条件下，EcR转录组的表达量呈现先降低后上升的趋势，在12h达到最低，之后逐渐上升，在48h之后显著高于对照组（P<0.05）。

英文摘要:

      The complete cDNA sequence of EcR gene in Portunus trituberculatus was first cloned through RT-PCR and SmartTM Race technology. The length of the EcR gene is 2,996bp and encodes the protein of 503 amino acids. Bioinformatics analysis deduced the EcR gene protein molecular weight of 55.69 kDa with a theoretical pI 7.33. Blast analysis revealed that the similarities of EcR with C. maenas, Scylla paramamosain, U. pugilator, Gecarcinus lateralis, Homarus americanus, Crangon crangon, and Marsupenaeus japonicas were 94%, 90%, 88%, 84%, 82%, 73%, 66%, respectively. Real-time fluorescent quantitative PCR was used to assess the mRNA expression of EcR in different tissues and its expression level of EcR after the salinity stress. The results showed that EcR expression existed in all tested tissues of P. trituberculatus, including hepatopancreas, muscle, eyes, gills, and heart. The highest expression level was observed in hepatopancreas, while the lowest in heart. The expression level of EcR was significantly lower than those of the control group at salinity of 45 after 24 h and declined generally. At salinity 11, the EcR expression presented the trend of rising after the first decreases and reaches the lowest level at 12h. After that the expression level rose gradually and significantly higher than those in the control group after 48h.