| 斑节对虾谷氨酸脱氢酶基因的克隆及氨氮胁迫对其时空表达的影响 |
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| 引用本文： | 周发林,陈劲松,黄建华,杨其彬,邱丽华,马振华,江世贵.斑节对虾谷氨酸脱氢酶基因的克隆及氨氮胁迫对其时空表达的影响[J].中国水产科学,2016,23(6):1236-1246. |
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| 作者姓名： | 周发林 陈劲松 黄建华 杨其彬 邱丽华 马振华 江世贵 |
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| 作者单位： | 1. 中国水产科学研究院南海水产研究所，农业部南海渔业资源开发利用重点实验室，广东广州 510300;2. 中国水产科学研究院南海水产研究所，农业部南海渔业资源开发利用重点实验室，广东广州 510300; 上海海洋大学水产与生命学院上海 201306 |
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| 基金项目： | 虾产业技术体系项目(CARS-47)，广东省省级科技计划项目(2013B020201001;2014B020202003)，广东省海洋与渔业科技推广专项(A201501A06)，海南省自然科学基金项目(313117)，深圳市生物产业发展专项资金现代农业生物产业推广扶持计划项目(NYSW201400331010053) |
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| 摘 要： | 采用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)获得了斑节对虾(Penaeus monodon)谷氨酸脱氢酶基因(PmGDH)的cDNA序列。该序列全长2386 bp,开放阅读框(ORF)为1677 bp,3?非编码区(UTR)为688 bp,包括含有27个碱基的poly(A)尾,5?非编码区(UTR)为21 bp。ORF可编码558个氨基酸,预测分子量为61.837 kD,理论等电点为6.57。序列含有ELFV dehydrog N与NAD bind 1 Glu DH两个保守结构域,37个磷酸化位点,3个糖基化位点。通过同源性、相似性以及系统进化树分析,斑节对虾的GDH基因与凡纳滨对虾(Litopenaeus vannamei)GDH基因的同源性和相似性最高,并与其聚为一支。采用荧光定量的方法研究了PmGDH基因在斑节对虾不同组织、氨氮胁迫过程中差异表达情况。结果表明, PmGDH的mRNA在各组织中都有表达,其中,在肌肉组织中表达量最高,其次为眼柄神经,在血淋巴与肠组织中表达量最低。96 h氨氮胁迫后,荧光定量PCR分析结果表明PmGDH在肝胰腺和鳃组织中的表达与对照组相比都具有显著差异(P<0.05),都具有不同程度的表达上调。表明PmGDH在氨氮代谢方面具有重要的作用,参与了斑节对虾机体的急性氨氮胁迫应答反应。
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| 关 键 词： | 斑节对虾 谷氨酸脱氢酶 氨氮胁迫 基因克隆 组织表达 |
| 修稿时间： | 2016/11/9 0:00:00 |
Molecular cloning and expression analysis of glutamate dehydrogenase (GDH) in Penaeus monodon under ammonia nitrogen stress |
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| ZHOU Falin,CHEN Jinsong,HUANG Jianhu,YANG Qibin,QIU Lihu,MA Zhenhu,JIANG Shigui.Molecular cloning and expression analysis of glutamate dehydrogenase (GDH) in Penaeus monodon under ammonia nitrogen stress[J].Journal of Fishery Sciences of China,2016,23(6):1236-1246. |
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| Authors: | ZHOU Falin CHEN Jinsong HUANG Jianhu YANG Qibin QIU Lihu MA Zhenhu JIANG Shigui |
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| Institution: | 1. South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China;2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China |
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| Abstract: | Glutamate dehydrogenase (GDH) is found widely in many plants, animals and microorganisms as a mi-tochondrial enzyme, and it is the key enzyme in amino acid catabolism. It is distributed extensively in animal tis-sues such as the hepatopancreas, kidney and brain. GDH activity is very strong and it may catalyze the glutamic acid oxidative deamination reaction. To explore the function of the GDH gene during ammonia nitrogen metabo-lism of black tiger shrimps (Penaeus monodon), the complete cDNA sequence of GDH fromP. monodon(denoted PmGDH) was obtained by rapid amplification of cDNA ends. The full length of the sequence ofPmGDH was 2386 bp containing a 5′UTR of 21 bp and a 3′UTR of 688 bp, and the length of the open reading frame (ORF) was 1677 bp encoding 558 amino acids. The predicted molecular mass of the amino acid (aa) sequence was 61.837 kD with an estimated pI of 6.57, and there was a poly A with 27 bp. In common with the GDH of other animals, the struc-ture of thePmGDH protein contained two conservative domains, ELFV dehydrog N and NAD bind 1 Glu DH. There were 37 phosphorylation sites and three glycosylation sites in this protein. Through multiple sequence alignment and phylogenetic tree analysis, it was concluded that the homology and similarity betweenPmGDH and the GDH ofLitopenaeus vannameiwas the highest. Analysis of the tissue expression pattern ofPmGDH showed that thePmGDH mRNA was expressed in all tissues tested, including lymphoid tissue, ovary, eyestalk nerve, brain, stomach, muscle, intestines, thoracic nerve, hemolymph, hepatopancreas and gill. The highest levels were found in muscle, the next highest in the eyestalk and the lowest levels in the hemolymph. To study the functions ofPmGDH inP. monodon under conditions of ammonia nitrogen stress, the hepatopancreas and gill were sampled at 6 h, 12 h, 24 h, 48 h, 72 h and 96 h after exposure to different concentrations of ammonia nitrogen. The expression of PmGDHin the hepatopancreas and gill was significantly different compared with the control group (P<0.05), and the expression levels differed between hepatopancreas and gill. The results showed thatPmGDHmay play an im-portant role in shrimp ammonia metabolism and may be involved in responses to acute ammonia stress. |
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| Keywords: | Penaeus monodon GDH ammonia nitrogen stress gene clone tissue expression |
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