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引用本文:陈凯锋,董迎辉,姚韩韩,林志华,孙长森.缢蛏(Sinonovacula constricta)氨氮胁迫应答miR-8245a-5p靶基因GOT验证及其表达特征分析.海洋与湖沼,2020,51(2):388-394.
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缢蛏(Sinonovacula constricta)氨氮胁迫应答miR-8245a-5p靶基因GOT验证及其表达特征分析
陈凯锋1, 董迎辉1, 姚韩韩1, 林志华1, 孙长森2
1.浙江万里学院生物与环境学院 浙江省水产种质资源高效利用技术研究重点实验室 宁波 315100;2.浙江万里学院 宁海海洋生物种业研究院 宁波 315604
摘要:
为鉴定缢蛏(Sinonovacula constricta)氨氮胁迫应答相关miRNA靶基因及其表达特征,选择氨氮胁迫下缢蛏肝胰腺组织中表达水平差异显著的miR-8245a-5p进行研究。靶基因验证结果显示,缢蛏GOT基因表达量极显著高于未胁迫组(P<0.01),表明GOT是miR-8245a-5p的靶基因。缢蛏GOT基因的ORF长1227bp,编码408个氨基酸;氨基酸多重序列比对和系统进化树分析发现,缢蛏等贝类同源性较高,与其他物种同源性较低。基因时空特征表达分析结果显示,GOT基因在缢蛏肝胰腺中的表达量明显高于其余6个组织(P<0.01);140mg/L氨氮胁迫下缢蛏GOT基因在肝胰腺中的表达量1h内急剧上调,此后上调逐步减弱,60mg/L氨氮水平下第12h上调才达峰值;不同氨氮水平暴露下缢蛏肝胰腺中GOT活力整体均呈现先升后降趋势,但在较高氨氮浓度水平下同样表现出更快速的应答特征。这些结果为后续深入研究缢蛏miR-8245a-5p及GOT基因调控氨氮解毒机制提供了理论基础。
关键词:  缢蛏(Sinonovacula constricta)  miRNA  天冬氨酸转氨酶(GOT)  氨氮胁迫
DOI:10.11693/hyhz20191200271
分类号:Q789;S968.3
基金项目:国家现代贝类产业技术体系项目,CARS-49号;浙江省农业新品种选育重大科技专项,2016C02055-9号;宁波市"科技创新2025"重大专项项目,2019B10005号;宁波市"环境科学与工程"一流学科项目。
Identification And Characteristic Expression analysis of miR-8245a-5p target gene related to ammonia nitrogen stress in Sinonovacula constricta
CHEN Kai-Feng1, DONG Ying-Hui1, YAO Han-Han1, LIN Zhi-Hua1, SUN Chang-Sen2
1.Key Laboratory of Aquatic Germplasm Resource of Zhejiang Province, Zhejiang Wanli University, Ningbo 315100, China;2.Ninghai Institute of Marine Biology Seed Industry, Zhejiang Wanli University, Ningbo 315604, China
Abstract:
To identify the miRNA target genes and their expression characteristics of Sinonovacula constricta under ammonia nitrogen stress, miR-8245a-5p with significant expression difference in hepatopancreas, was studied in detail. The Result of qRT-PCR (Quantitative Reverse Transcription-Polymerase Chain Reaction) in target gene verification showed that GOT gene expression changed most significantly (P<0.01), indicating that GOT gene was the target gene of mir-8245a-5p. Bioinformatic analysis showed that the length of Sc-GOT gene was 1227bp, encoding 408 amino acids. Multiple sequence alignment and phylogenetic analysis indicated S. constricta had higher homology to scallop and oyster, but lower homology to other species on the GOT gene sequences. The results of qRT-PCR in different tissues presented that expression level of Sc-GOT gene in hepatopancreas was very significantly higher than in other tissues (P<0.01). Under 140mg/L ammonia nitrogen stress, the expression level of GOT gene in hepatopancreas increased abruptly after 1h, and then the upregulated level gradually decreased; and under 60mg/L ammonia nitrogen stress, the upregulated level reached peak value after 12h. Under different ammonia nitrogen levels, the GOT enzyme activity in hepatopancreas presented early increase and later decrease trend, but higher ammonia nitrogen level prompted a faster response. These results provide a theoretical basis for further study on the regulation mechanism of miR-8245a-5p and GOT gene.
Key words:  Sinonovacula constricta  miRNA  glutamic oxaloacetic transaminase (GOT)  ammonia nitrogen stress
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