CAJ | 학술논문

利用 RACE 技术克隆获得中国明对虾(Fenneropenaeus chinensis)天门冬氨酸转氨酶 GOT 基因(FcGOT)。FcGOT 基因cDNA全长为1910 bp,其中,开放阅读框1284 bp,编码427个氨基酸。同源性分析表明,中国明对虾天门冬氨酸转氨酶 GOT氨基酸序列与其他节肢动物高度保守,与克氏原螯虾(Procambarus clarkii)和桔粉蚧壳虫(Planococcus citri)的同源性分别为78%和73%。系统进化分析表明, FcGOT基因氨基酸序列与克氏原螯虾GOT聚为一支。组织表达分析发现FcGOT基因在肝胰腺、鳃、血细胞、肌肉、心脏、淋巴中均有表达,其中肝胰腺中表达量最高。氨氮胁迫后,荧光定量PCR分析结果表明, FcGOT基因在肝胰腺和鳃组织中的表达与对照组相比具有显著差异(P<0.05),表明 FcGOT 基因在氨氮代谢方面具有重要的作用,参与了中国明对虾机体的急性氨氮胁迫应答反应。
이용 RACE 기술극륭획득중국명대하(Fenneropenaeus chinensis)천문동안산전안매 GOT 기인(FcGOT)。FcGOT 기인cDNA전장위1910 bp,기중,개방열독광1284 bp,편마427개안기산。동원성분석표명,중국명대하천문동안산전안매 GOT안기산서렬여기타절지동물고도보수,여극씨원오하(Procambarus clarkii)화길분개각충(Planococcus citri)적동원성분별위78%화73%。계통진화분석표명, FcGOT기인안기산서렬여극씨원오하GOT취위일지。조직표체분석발현FcGOT기인재간이선、새、혈세포、기육、심장、림파중균유표체,기중간이선중표체량최고。안담협박후,형광정량PCR분석결과표명, FcGOT기인재간이선화새조직중적표체여대조조상비구유현저차이(P<0.05),표명 FcGOT 기인재안담대사방면구유중요적작용,삼여료중국명대하궤체적급성안담협박응답반응。
Fenneropenaeus chinensis is an important mariculture species in China. In aquaculture environments ammo-nia is a common toxic substance. In recent years, higher frequencies of ammonia nitrogen toxicity in shrimps have been reported. Therefore, it is necessary to investigate ammonia metabolism by F. chinensis. As an important member of the AAT-like family, the enzyme aspartate aminotransferase (GOT) is involved in many aspects of ammonia metabolism including participating in inosine monophosphate transdeamination, and the urea and citric acid cycles. Therefore, de-tailed understanding of the regulation of GOT is of great significance. In this study, we successfully cloned the aspartate aminotransferase cDNA of F. chinensis (FcGOT). The FcGOT cDNA, which was 1 910 bp in length, contained a 5′-untranslated region(UTR) of 83 bp, a 3′UTR of 543 bp, and an open reading frame (ORF) of 1 284 bp, encoded a 427 amino-acid polypeptide. FcGOT protein exhibited typical AAT-like family features, including a Lys catalytic residue and 10 pyridoxal-5′-phosphate binding sites suggesting that it belongs to the AAT-I superfamily. Homology analysis revealed that the amino acid sequence of FcGOT was highly conserved with its homologs of other arthropods. The si-milarities of FcGOT with the GOT of Procambarus clarkia and Planococcuscitri were 78% and 73%, respectively. Phylogenetic analysis showed that FcGOT was in the same branch as P. clarkia. Tissue expression analysis showed that FcGOT transcripts were detected in all tested tissues, including hepatopancreas, gill, muscle, hemocytes, heart and lymph. The highest expression level of FcGOT was in the hepatopancreas. To study the biological functions of FcGOT in F. chinensis, the hepatopancreas and gill were sampled 1, 6, 12, 24, 48 and 72 h after exposure to different concen-trations of ammonia nitrogen. The expression of the FcGOT gene was significantly different compared with the control group (P<0.05), and the expression conditions differed between hepatopancreas and gill. In hepatopancreas, the gene expression was significantly down-regulated immediately after the shrimps were exposed to 16 mg/L ammonia-N, and down-regulated after treatment with 4 mg/L ammonia-N for 48 h. In gill tissue, gene expressions were up-regulated promptly after ambient ammonia stress and reached a peak at 12 h. The reason for the discrepant expression between the two tissues may be that the gene expressions were affected by organizational distance. The results imply that FcGOT might play an important role in shrimp ammonia metabolism and be involved in responses to acute ammonia stress.