氨氮胁迫下中国明对虾(Fenneropenaeus chinensis)谷氨酸脱氢酶基因的表达分析

cDNA Cloning and Expression Analysis of Glutamate Dehydrogenase in Chinese Shrimp (Fenneropenaeus chinensis) Exposed to Ambient Ammonia

中文关键词: 中国明对虾  谷氨酸脱氢酶  基因克隆  基因表达  氨氮胁迫

英文关键词: Fenneropenaeus chinensis  Glutamate dehydrogenase  Gene cloning  Gene expression  Ammonia stress

中文摘要:

      采用RACE技术克隆获得中国明对虾(Fenneropenaeus chinensis)谷氨酸脱氢酶GDH基因(FcGDH)。FcGDH基因全长1779 bp，包括1个1659 bp的开放阅读框(ORF)，编码552个氨基酸，预测分子量大小为61.3 kDa，理论等电点为6.54。同源性分析显示，FcGDH氨基酸序列与其他动物高度保守，其中，与凡纳滨对虾最为相似，高达98%，其次为中华绒螯蟹，为89%。系统进化树分析显示，FcGDH氨基酸序列与凡纳滨对虾GDH聚为一支，之后依次为：中华绒螯蟹、黑腹果蝇、埃及按蚊。组织表达分析发现，FcGDH基因在肌肉、鳃、肝胰腺、胃、肠、淋巴和血淋巴中均有表达，其中，肌肉中表达量最高。氨氮胁迫后，FcGDH基因在肌肉和肝胰腺组织中变化显著，在胁迫后期，FcGDH基因表达量均上调，且与对照组相比差异显著(P<0.05)，说明FcGDH基因在氨氮解毒代谢过程中发挥了重要作用。

英文摘要:

      Chinese shrimp (Fenneropenaeus chinensis) is an ecologically and economically important shrimp species. During the culture, F. chinensis were exposed to a series of stressors that adversely affect biological activities including growth rate. Ammonia, a product of protein degradation and bacterial activity, is a strong stressor in shrimp aquaculture. Glutamate dehydrogenase (GDH) is an abundant and ubiquitous mitochondrial enzyme that catalyzes reversible amination of glutamate. cDNA of GDH from F. chinensis (FcGDH) was cloned by rapid amplification of cDNA ends (RACE). The FcGDH cDNA was 1779 bp in size, and it included a 1659-bp open reading frame (ORF) that encoded a 522 amino-acid polypeptide of which the isoelectric point (pI) was 6.54 and the molecular mass was 61.3 kDa. Homology analysis revealed that the amino acid sequence of FcGDH was highly conserved with its homologs in other arthropod. The similarities between FcGDH and GDHs of Litopenaeus vannamei and Eriocheir sinensis were 98% and 89% respectively. Phylogenetic analysis showed that FcGDH was in the same branch with that of L. vannamei and then in the same branches with those of E. sinensis, Drosophila melanogaster, and Aedes aegypti in order. The tissue expression analysis showed that FcGDH was detected in all tested tissues including muscle, gill, hepatopancreas, stomach, intestine, lymph, and hemocytes. The highest expression of FcGDH was in the muscle that was an amino acid pool and the major tissue for protein deposition. After exposure to ambient ammonia, the expression of FcGDH gene was up-regulated significantly in muscles compared to the control group (P<0.01). The expression level of FcGDH in hepatopancreas was down-regulated significantly at 3 h (P<0.05), and was then stabilized up to 24 h. The expression of FcGDH was increased significantly after 48 h and reached the maximum at 72 h compared to the control group (P<0.01). These results implied that FcGDH might play an important role in the process of ammonia detoxification.