大弹涂鱼肝型<em>GP</em>基因的克隆及其在氨氮胁迫下的响应

doi:10.3969/j.issn.1674-7968.2020.03.014

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (9056 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract With the rapid development of intensive aquaculture, the accumulations of ammonia nitrogen in the culture water environment threaten the ability of ammonia excretion of fish and lead to oxidative damage and immune suppression, physiological structure and glycogen metabolism injured, even the death of fish. This study aims to explore the effects of ammonia-nitrogen stress on the glycogen mobilizing ability of great blue-spotted mudskippers (Boleophthalmus pectinirostris). The blood glucose levels, glycogen contents, glycogen synthase and phosphorylase activities as well as their gene expression levels of great blue-spotted mudskippers were investigated under ammonia-nitrogen stress. In this study, fish were accumulated in 10‰ seawater for 2 weeks 5 d and then transferred to 10‰ seawater (the control group) or to 10‰ seawater + 8 mmol/L ammonium chloride (the ammonia stress group) for 72 h. Under ammonia-nitrogen stress conditions, the blood glucose levels of great blue-spotted mudskippers at 1, 3, 6, 12, 48 and 72 h were significantly higher than those of the control group (P<0.05), and the liver glycogen contents at 1, 3, 6, 24, 48 and 72 h were significantly lower (P<0.05). While the contents of muscle glycogen and gill glycogen were not significantly changed, indicating that great blue-spotted mudskippers mainly mobilized their liver glycogens, rather than the muscle or gill glycogens, to maintain glucose homeostasis under ammonia-nitrogen stress. Gene expression and enzyme activity analysis showed that liver glycogen phosphorylase (GP) activities at 1, 3, 6, 12, 48 and 72 h and their mRNA abundances at 1, 3, 12, 24, 48 and 72 h were significantly increased under ammonia-nitrogen stress (P<0.05), while liver glycogen synthase (GS) activities and their mRNA abundances showed no significant change, indicating increased liver glycogen catabolism, rather than anabolism, under ammonia-nitrogen stress. Liver glycogen, not the gill glycogen, is the main carbohydrate mobilization site for great blue-spotted mudskippers to cope with ammonia-nitrogen stress. In this condition, great blue-spotted mudskippers mobilize the liver glycogen, mainly through accelerating the catabolism, rather than suppressing the anabolism, to maintain blood glucose homeostasis. The ORF of its liver GP (GenBank No. XM_020932431.1, XM_020935772.1) was 2 544 bp in length with 44.54% AT content, and encoded a peptide of 847 amino acids with molecular mass of 97 kD and a theoretical isoelectric point of 5.94. The liver glycogen phosphorylase of great blue-spotted mudskipper contained one phosphorylase pyridoxal-phosphate attachment site (consensus: E-A-[SC]-G-x-[GS]-x-M-K-x(2)-[LM]-N), 8 N-glycosylation sites (consensus: N-{P}-[ST]-{P}) and 11 protein kinase phosphorylation sites (consensus: [ST]-X-[RK]), one of which was cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinase phosphorylation site (consensus: [RK](2)-X-[ST]). This gene showed 80.36% in homologous similarity with human (Homo sapiens) and 81.11%~86.89% with other fish homologous, indicating the high conservation for GP genes. Molecular evolutionary analysis detected 3 positively selected sites (686G, 715N, 807G) on great blue-spotted mudskipper's liver GP gene, indicated the unique adaptive evolution for great blue-spotted mudskippers. This study enriches the molecular biological information of the liver type glycogen phosphorylase gene and glycogen metabolism for great blue-spotted mudskippers under ammonia-nitrogen stress.

Key words： Boleophthalmus pectinirostris Glycogen content Glycogen synthase Glycogen phosphorylase (GP) Ammonia-nitrogen stress

Received: 04 August 2019

ZTFLH:

S917.4

Corresponding Authors: * wrx_zjou@163.com

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	GUO Ting-Ting
	MENG Fan-Xing
	LI Ming
	WANG Ri-Xin

Cite this article:

GUO Ting-Ting,MENG Fan-Xing,LI Ming, et al. Cloning of Liver GP Gene and Its Responses to Ammonia Nitrogen Stress in Great Blue Spotted Mudskipper (Boleophthalmus pectinirostris)[J]. 农业生物技术学报, 2020, 28(3): 514-529.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2020.03.014 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2020/V28/I3/514

1 拜占春, 崔燕, 余四九, 等. 2019. HIF1α和Beclin1在成年牦牛脑组织中的表达与分布[J]. 农业生物技术学报, 27(10): 1878-1884.
(Bai Z C, Cui Y, Yu S J, et al.2019. Expression and distribution of HIF1α and Beclin1 in adult yak (Bos grunniens) brain tissues[J]. Journal of Agricultural Biotechnology, 27(10): 1878-1884.)
2 巩珊珊. 2017. 低温胁迫下东北林蛙GS、Akt2基因表达与分析[D]. 硕士学位论文, 东北林业大学, 导师: 肖向红, pp. 27-28.
(Gong S S.2017. Gene expression and analysis of GS and Akt2 in Rana dybowskii under low temperature stress[D]. Thesis for M.S., Northeast Forestry University, Supervisor: Xiao X H, pp. 27-28.)
3 韩雪晴, 高风英, 卢迈新, 等. 2019. 尼罗罗非鱼TRAF4基因的克隆、表达及功能分析[J]. 农业生物技术学报, 27(3): 381-392.
(Han X Q, Gao F Y, Lu M X, et al.2019. Cloning, expression and functional analysis of TRAF4 gene in nile tilapia (Oreochromis niloticus)[J]. Journal of Agricultural Biotechnology, 27(3): 381-392.)
4 黄厚见. 2012. 摄食水平、氨氮胁迫对梭鱼幼鱼生长的影响及其毒理效应研究[D]. 硕士学位论文, 上海海洋大学, 导师: 沈新强, pp. 5-9.
(Huang H J.2012. The effects of ammonia and ration size on growth and toxicological response of mullet, Liza haematocheila[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Shen X Q, pp. 5-9.)
5 纪利芹, 蒋克勇, 韩龙江, 等. 2014. 连续降温对大菱鲆成鱼代谢机能的影响[J]. 海洋科学, 38(5): 46-53.
(Ji L Q, Jiang K Y, Han L J, et al.2014. Effect of continuous cooling on metabolic function of adult Scophthalmus maximus L.[J]. Marine Sciences, 38(5): 46-53.)
6 李利. 2010. 低氧胁迫对日本沼虾呼吸代谢、能量代谢和抗氧化能力的影响[D]. 硕士学位论文, 河北大学, 导师: 管越强, pp. 27-28. (Li L. 2010. Effects of hypoxia on respiratory metabolism, energy metabolism and antioxidant capability of Macrobrachium nipponense[D]. Thesis for M. S., Hebei University, Supervisor: Guan Y Q, pp. 27-28.)
7 李秀峰, 肖向红, 柴龙会, 等2012. 低温胁迫对东北林蛙雄性成体GP、PEPCK比活性的影响[J]. 经济动物学报, 16(2): 67-70.
(Li X F, Xiao X H, Chai L H, et al.2012. Effect of low temperature stress on enzyme specific activity of GP and PEPCK in male Rana dybowskii[J]. Journal of Economic Animal, 16(2): 67-70.)
8 龙章强. 2008. 黑鲷(Acanthopagrus schlegeli)幼鱼对氨氮胁迫的生理响应及其维生素C的营养需求研究[D]. 硕士学位论文, 华东师范大学, 导师: 陈立侨, pp. 28-30.
(Long Z Q.2008. The physiological responses to ammonia stress and vitamin C requirement of juvenile black seabream (Aeanthopagrus schlegell)[D]. Thesis for M.S., East China Normal University, Supervisor: Chen L Q, pp. 28-30.)
9 马芳, 刘哲, 康玉军, 等. 2019. 虹鳟Hsp40家族基因鉴定及其热应激下的表达分析[J]. 农业生物技术学报, 27(10): 1782-1792.
(Ma F, Liu Z, Kang Y J, et al.2019. Identification of Hsp40 family gene and the expression analysis under heat stress in rainbow trout (Oncorhynchus mykiss)[J]. Journal of Agricultural Biotechnology, 27(10): 1782-1792.)
10 邢晓丹. 2018. 大弹涂鱼适应盐度胁迫的糖代谢模式及葡萄糖和二甲双胍的调控作用研究[D]. 硕士学位论文, 浙江海洋大学, 导师: 石戈, pp. 16-22.
(Xing X D.2018. Study on the glucose metabolism of Boleophthalmus pectinirosris adapted to salinity stress and regulation by glucose and metformin. Thesis for M.S., Zhejiang Ocean University, Supervisor: Shi G, pp. 16-22.)
11 王静涵. 2017. 低温胁迫下东北林蛙PHK和GP差异表达及组织内糖原含量变化[D]. 硕士学位论文, 东北林业大学, 导师: 肖向红, pp. 4-5.
(Wang J H.2017. Differential expression of PHK and GP and changes of glycogen content in tissues of Rana dybowskii under low temperature stress[D]. Thesis for M.S., Northeast Forestry University, Supervisor: Xiao X H, pp. 4-5.)
12 王林杰. 2010. 猪糖原合成酶(GS)与糖原合成酶激酶(GSK3)基因的分离克隆、表达分析及其功能研究[D]. 硕士学位论文, 华中农业大学, 导师: 熊远著, pp. 5-6.
(Wang L J.2010. Molecular characterization, expression patterns and functional study of the porcine GS and GSK3 genes[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Xiong Y Z, pp. 5-6.)
13 王瑞芳, 安晓萍, 齐景伟, 等. 2019. 达里湖瓦氏雅罗鱼生殖洄游过程中能量代谢和消化酶活性的变化[J]. 中国水产科学, 26(4): 703-712.
(Wang R F, An X P, Qi J W, et al.2019. Energy metabolism and digestive enzyme activity regulation in Amur Ide (Leuciscus waleckii) during spawning migration from Dali Nor Lake to Gongger River)[J]. Journal of Fishery Sciences of China, 26(4): 703-712.)
14 张赛赛, 姜欣彤, 王伟, 等. 2018. 四溴联苯醚慢性胁迫对大泷六线鱼血液生化指标的影响[J]. 中国渔业质量与标准, 8(3): 1-10.
(Zhang S S, Jiang X T, Wang W, et al.2018. Effects of tetra-brominated diphenyl ethers stress on the plasma biochemical index of Hexagrammos otakii[J]. Chinese Fishery Quality and Standards, 8(3): 1-10.)
15 赵娜. 2013. 糖原磷酸化酶基因在中国卤虫胚胎发育和温度胁迫过程中的表达模式[D]. 硕士学位论文, 辽宁师范大学, 导师: 侯林, pp. 41-44.
(Zhao N.2013. Expression pattern of glycogen phosphorylase gene during development and in response to temperature stress from brine shrimp, Artemia sinica[D]. Thesis for M.S., Liaoning Normal University, Supervisor: Hou L, pp. 41-44.)
16 Bacca H, Huvet A, Fabioux C, et al.2005. Molecular cloning and seasonal expression of oyster glycogen phosphorylase and glycogen synthase genes[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 140(4): 635-646.
17 Chang J C, Wu S M, Tseng Y C, et al.2007. Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater[J]. The Journal of Experimental Biology, 210(19): 3494-3504.
18 Chen Y J, Zhang T Y, Chen H Y, et al.2017. An evaluation of hepatic glucose metabolism at the transcription level for the omnivorous GIFT tilapia, Oreochromis niloticus, during postprandial nutritional status transition from anabolism to catabolism[J]. Aquaculture, 473: 375-382.
19 Ching B, Chew S F, Wong W P, et al.2009. Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddarti (mudskipper)[J]. Aquatic Toxicology, 95(3): 203-212.
20 Das P C, Ayyappan S, Jena J K, et al.2004. Acute toxicity of ammonia and its sub-lethal effects on selected haematological and enzymatic parameters of mrigal, Cirrhinus mrigala (Hamilton)[J]. Aquaculture Research, 35(2): 134-143.
21 Foss A, Imsland A K, Roth B, et al.2009. Effects of chronic and periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus)[J]. Aquaculture, 296(1-2): 45-50.
22 Grobler J M B, Wood C M.2018. The effects of high environmental ammonia on the structure of rainbow trout hierarchies and the physiology of the individuals therein[J]. Aquatic Toxicology, 195: 77-87.
23 Harmon K J, Bolinger M T, Rodnick K J.2011. Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout[J]. Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology, 158(4): 423-431.
24 Hong M, Chen L, Sun X, et al.2007. Metabolic and immune responses in Chinese mitten-handed crab (Eriocheir sinensis) juveniles exposed to elevated ammonia[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 145(3): 363-369.
25 Huang C Y, Lin H C, Lin C H.2015a. Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 179: 25-34.
26 Huang C Y, Lin H H, Lin C H, et al.2015b, The absence of ion-regulatory suppression in the gills of the aquatic air-breathing fish Trichogaster lalius during oxygen stress[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 179: 7-16.
27 Huelsenbeck J P, Ronquist F.2001. MRBAYES: Bayesian inference of phylogenetic trees[J]. Bioinformatics, 17(8): 754-755.
28 Ip Y K, Chew S F.2018. Air-breathing and excretory nitrogen metabolism in fishes[J]. Acta Histochemica, 120(7): 680-690.
29 Jiang D, Wu Y, Xing R, et al.2017. Effect of blood glucose level on acute stress response of grass carp Ctenopharyngodon Idella[J]. Fish Physiology and Biochemistry, 43: 1433-1442.
30 Kim J H, Park H J, Hwang I K, et al.2017. Toxic effects of juvenile sablefish, Anoplopoma fimbria by ammonia exposure at different water temperature[J]. Environmental Toxicology and Pharmacology, 54: 169-176.
31 Knoph M B, Thorud K.1996. Toxicity of ammonia to Atlantic Salmon (Salmo salar L.) in seawater-Effects on plasma osmolality, ion, ammonia, urea and glucose levels and hematologic parameters[J]. Comparative Biochemistry & Physiology Part A Physiology, 113(4): 375-381.
32 Lai J C, Cooper A J.1991. Neurotoxicity of ammonia and fatty acids: Differential inhibition of mitochondrial dehydrogenases by ammonia and fatty acyl coenzyme a derivatives[J]. Neurochemical Research, 16(7): 795-803.
33 Li H, Xu W, Jin J, et al.2018a. Effects of starvation on glucose and lipid metabolism in gibel carp (Carassius auratus gibelio var. CASⅢ)[J]. Aquaculture, 496: 166-175.
34 Li M, Wang X, Qi C, et al.2018b. Metabolic response of Nile Tilapia (Oreochromis niloticus) to acute and chronic hypoxia stress[J]. Aquaculture, 495: 187-195.
35 Li S, Sang C, Zhang J, et al.2018c. Effects of acute hyperglycemia stress on plasma glucose, glycogen content, and expressions of glycogen synthase and phosphorylase in hybrid grouper (Epinephelus fuscoguttatus ♀×E. lanceolatus ♂)[J]. Fish Physiology and Biochemistry, 44(4): 1185-1196.
36 Liang Z, Liu R, Zhao D, et al.2016. Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei)[J]. Fish & Shellfish Immunology, 54: 523-528.
37 Lin K, Hwang P K, Fletterick R J.1997. Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases[J]. Structure, 5(11): 1511-1523.
38 Lin Y S, Tsai S C, Lin H C, et al.2011. Changes of glycogen metabolism in the gills and hepatic tissue of tilapia (Oreochromis mossambicus) during short-term Cd exposure[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 154(4): 296-304.
39 Ma H J, Mou M M, Pu D C, et al.2019. Effect of dietary starch level on growth, metabolism enzyme and oxidative status of juvenile largemouth bass, Micropterus salmoides[J]. Aquaculture, 498: 482-487.
40 Mckenzie D J, Shingles A, Taylor E W.2003. Sub-lethal plasma ammonia accumulation and the exercise performance of salmonids[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 135(4): 515-526.
41 Miron D d S, Moraes B, Becker A G, et al.2008. Ammonia and pH effects on some metabolic parameters and gill histology of silver catfish, Rhamdia quelen (Heptapteridae)[J]. Aquaculture, 277(3-4): 192-196.
42 Mommsen T P, Vijayan M M, Moon T W.1999. Cortisol in teleosts: Dynamics, mechanisms of action, and metabolic Regulation[J]. Reviews in Fish Biology and Fisheries, 9(3): 211-268.
43 Polakof S, Míguez J M, Soengas J L.2007. Daily changes in parameters of energy metabolism in liver, white muscle, and gills of rainbow trout: Dependence on feeding[J]. Comparative Biochemistry & Physiology Part A: Molecular & Integrative Physiology, 147(2): 363-374.
44 Silva M J d S, Costa F F B d, Leme F P, et al.2018. Biological responses of Neotropical freshwater fish Lophiosilurus alexandri exposed to ammonia and nitrite[J]. Science of the Total Environment, 616-617: 1566-1575.
45 Sinha A K, Rasoloniriana R, Dasan A F, et al.2015. Interactive effect of high environmental ammonia and nutritional status on ecophysiological performance of European sea bass (Dicentrarchus labrax) acclimated to reduced seawater salinities[J]. Aquatic Toxicology, 160: 39-56.
46 Waitt A E, Liam R, Ransom B R, et al.2017. Emerging roles for glycogen in the CNS[J]. Frontiers in Molecular Neuroscience, 10: Ariticle 73.
47 Yang Z H.1997. PAML: A program package for phylogenetic analysis by maximum likelihood[J]. Cabios Applications Note, 13(5): 555-556.
48 Zhang Y S, Li F X, Yao C L.2019. Glycogen phosphorylase of shrimp (Litopenaeus vannamei): Structure, expression and anti-WSSV function[J]. Fish and Shellfish Immunology, 91: 275-283.