中国对虾(Fenneropenaeus chinensis)p38 MAPK基因克隆及表达分析

应用RACE克隆技术获得中国对虾(Fenneropenaeus chinensis) p38 MAPK基因全长cDNA序列,并对该序列进行分析.结果显示,中国对虾p38 MAPK基因全长为1563 bp,开放阅读框长1098 bp,5′非编码区长122 bp,3′非编码区长343 bp,将该基因命名为Fcp38.氨基酸序列分析推测,该基因编码365个氨基酸,分子量为41.77 kDa,理论等电点为5.68.同源性分析表明,Fcp38基因与凡纳滨对虾和日本囊对虾的p38相似性最高,为98％.通过比对发现,该基因除含p38家族特有的标志性Thr-Gly-Tyr双磷酸化位点和底物结合位点Ala-Thr-Arg-Trp,还具有p38家族关键功能位点ED.系统进化分析显示,Fcp38与凡纳滨对虾和日本囊对虾的p38聚为一支.荧光定量PCR结果显示,Fcp38基因在肠、鳃、胃、心脏、淋巴、肝胰腺、肌肉、血细胞中均有表达,以在肌肉中表达量最高.氨氮胁迫后,该基因在中国对虾肌肉、血细胞、鳃、心脏、肠和胃中的相对表达量均显著增加,且有不同的时空表达趋势,表明Fcp38基因可能在中国对虾应对环境胁迫过程中起着重要作用.

The cDNA Cloning and Expression Analysis of p38 MAPK Gene of Fenneropenaeus chinensis

In this study we employed the RACE method to sequence the full-length cDNA of p38 MAPK gene of Fenneropenaeus chinensis for the first time and named it as Fcp38. The full-length cDNA sequence contained 1563 bp, including a 122-bp 5''-UTR, a 343-bp 3''-UTR, and a 1098-bp open reading frame (ORF) that encoded 365 amino acid residues. The isoelectric point (pI) of this peptide was 5.68, and the molecular mass was 41.77 kDa. Homology analysis revealed that the amino acid sequence of Fcp38 was highly similar to the p38 MAPK sequences in other species. The sequence similarity reached 98% between F. chinensis and Litopenaeus vannamei and Marsupenaeus japonicus. Fcp38 had a conserved Thr-Gly-Tyr (TGY) motif, a substrate-binding site Ala-Thr-Arg-Trp (ATRW), and an ED (ERK docking) motif. This structure played a critical role in the interaction between p38 MAPK and other molecules. The phylogenetic analysis showed that p38 of F. chinensis was in the same branch with L. vannamei and M. japonicus. The expression of Fcp38 gene in different tissues was also analyzed with quantitative real-time PCR. The results showed that Fcp38 existed in all the tested tissues including the intestine, gill, stomach, heart, hepatopancreas, muscles and hemocytes, and the expression was the highest in muscles. Real-time PCR analysis showed that ammonia-N stress significantly up-regulated the expression of Fcp38 in the muscles, hemocytes, gill, heat, intestine and stomach, and that there was a spatiotemporal pattern for the expression of Fcp38. These results implied that Fcp38 might play an important role in the response to the environmental stresses