大弹涂鱼肝型GP基因的克隆及其在氨氮胁迫下的响应  

Cloning of Liver GP Gene and Its Responses to Ammonia Nitrogen Stress in Great Blue Spotted Mudskipper(Boleophthalmus pectinirostris)

作　　者：郭婷婷[1] 孟繁星 黎明[1] 王日昕[1] GUO Ting-Ting;MENG Fan-Xing;LI Ming;WANG Ri-Xin(School of Marine Sciences,Ningbo University,Ningbo 315211,China)

机构地区：[1]宁波大学海洋学院,宁波315211

出　　处：《农业生物技术学报》2020年第3期514-529,共16页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31472279)。

摘　　要：随着集约化水产养殖的迅速发展,氨氮在养殖水体中的积累会影响鱼类糖原代谢,严重的甚至造成鱼体死亡。为了研究氨氮胁迫对大弹涂鱼(Boleophthalmus pectinirostris)糖原动员能力的影响及肝型糖原磷酸化酶(glycogen phosphorylase,GP)基因序列特征,本研究以大弹涂鱼为研究对象,对照组生活在10‰海水中,实验组进行为期72 h的氨氮胁迫(10‰海水+8 mmol/L氯化铵)。对大弹涂鱼血糖、糖原含量、糖原合成酶、肝型GP序列和酶活及氨氮胁迫对基因表达的影响进行了研究,结果显示,实验组大弹涂鱼血糖水平比对照组显著升高(P<0.05),肝糖原含量比对照组显著下降(P<0.05),肌糖原、鳃糖原含量无显著变化,表明大弹涂鱼在氨氮胁迫下,主要动员肝糖原而非肌糖原或鳃糖原,以维持体内葡萄糖稳态。基因表达及酶活性分析显示,实验组大弹涂鱼的肝型GP活性及表达量显著升高(P<0.05),肝糖原合成酶活性及表达量无显著变化。表明氨氮胁迫下,肝糖原的分解代谢增加,肝糖原合成代谢无显著变化。本研究克隆了大弹涂鱼的肝型GP基因(登录号为:XM020932431.1,XM020935772.1)。GP基因的ORF长为2544 bp,编码847个氨基酸,AT含量为44.54%,蛋白质的理论等电点为5.94,分子量为97 kD,含有1个磷酸化酶吡哆醛磷酸酯附着位点(EA-[SC]-Gx-[GS]-xMKx(2)-[LM]-N)、8个N-糖基化位点和11个蛋白激酶磷酸化位点,其中一种是环磷酸腺苷(cyclic adenosine monophosphate,cAMP)和环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)依赖性蛋白激酶磷酸化位点。大弹涂鱼肝型GP基因与人类(Homo sapiens)的同源序列相似性为80.36%,与其他鱼类的同源序列相似性为81.11%~86.89%,表明大弹涂鱼肝型GP基因具有高度序列保守性。分子进化研究在大弹涂鱼GP基因中检测到3个正选择位点(686G,715N,807G),表明大弹涂鱼GP基因出现了独特的适应性进化。本研究为进一步研究大弹涂鱼肝�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 and72 h and their m RNA abundances at 1,3,12,24,48 and 72 h were significantly increased under ammonianitrogen stress(P<0.05),while liver glycogen synthase(GS)activities and their m RNA abundances showed no significant change,indicating increased liver glycogen catabolism,rather than anabolism,under ammonianitrogen stress.Liver glycogen,not the gill glycogen,is the main carbohydrate mobilization site for great bluespotted mudskippers to cope with ammonia-nitrogen stress.In this conditi

关 键 词：大弹涂鱼 糖原含量 糖原合成酶 糖原磷酸化酶(GP) 氨氮胁迫 

分 类 号：S917.4[农业科学—水产科学]