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.
郭婷婷, 孟繁星, 黎明, 王日昕. 大弹涂鱼肝型GP基因的克隆及其在氨氮胁迫下的响应[J]. 农业生物技术学报, 2020, 28(3): 514-529.
GUO Ting-Ting, MENG Fan-Xing, LI Ming, WANG Ri-Xin. Cloning of Liver GP Gene and Its Responses to Ammonia Nitrogen Stress in Great Blue Spotted Mudskipper (Boleophthalmus pectinirostris). 农业生物技术学报, 2020, 28(3): 514-529.
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