团头鲂AGRP和NPY基因cDNA克隆及其在饥饿与再摄食状态下的表达分析  

Molecular cloning of AGRP and NPY and their expression analysis during fasting and refeeding in juvenile blunt snout bream(Megalobrama amblycephala)

作　　者：孙盛明 苏艳莉[2] 潘方艳 戈贤平 SUN Shengming;SU Yanli;PAN Fangyan;GE Xianping(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;Wuxi Fishery College,Nanjing Agricultural University,Wuxi 214081,China;Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China)

机构地区：[1]上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海201306 [2]南京农业大学无锡渔业学院,江苏无锡214081 [3]中国水产科学研究院淡水渔业研究中心,江苏无锡214081

出　　处：《水产学报》2020年第11期1777-1791,共15页Journal of Fisheries of China

基　　金：江苏省自然科学基金(BK20151103)。

摘　　要：为研究刺鼠相关蛋白(AGRP)和神经肽Y(NPY)在团头鲂幼鱼应答饥饿与再摄食过程中所起的调控作用,本研究采用RACE PCR技术首次克隆了团头鲂AGRP和NPY基因全长cDNA序列,通过设置正常对照组(control,体质量3%持续投喂4周)、饥饿再投喂组(F/refeeding,饥饿2周+3%再投喂2周)、饥饿过量投喂组(F/excessive refeeding,饥饿2周+8%再投喂2周)和饥饿组(fasted,持续饥饿4周)4个处理组,采用qRT-PCR技术分析团头鲂在脑和肠道组织中这2种基因在饥饿再投喂过程中的表达变化特征。结果显示:①组织学分析表明,持续饥饿组肠道黏膜下层出现明显的白细胞浸润现象。②团头鲂AGRP和NPY基因cDNA序列全长分别为770和557 bp,其中有5′非编码区77和101 bp以及3′非编码区315和120 bp,开放阅读框为378和336 bp,共编码了125个氨基酸和111个氨基酸。系统进化树分析表明,团头鲂的AGRP和NPY分别与其他鲤科鱼类聚为一支,具有最近的亲缘关系。③qRT-PCR分析显示,AGRP和NPY基因在所有检测组织中均有表达,二者均在脑组织中表达量最高,其次是肝脏和肠道。④饥饿再投喂处理对团头鲂脑和肠道中AGRP和NPY基因表达影响显著,饥饿组团头鲂脑和肠道组织AGRP基因表达量均呈先升后降趋势,第28天,饥饿再投喂组能够显著上调团头鲂AGRP基因在脑组织中的表达,但饥饿过量投喂组能够显著上调AGRP基因在肠道中的表达量。持续饥饿组团头鲂脑和肠道组织NPY基因表达呈先上升后下降趋势,且饥饿组脑和肠道组织中NPY表达量在28 d均显著高于其余3组。研究表明,AGRP和NPY基因在团头鲂摄食调控中发挥重要作用,且在脑和肠道组织中具有不同表达模式。Agouti-related protein(AGRP)and neuropeptide Y(NPY)are important factors involved in the regulation of feeding in both mammals and fish.In this study,the full-length cDNA sequences of AGRP and NPY genes of Megalobrama amblycephala were cloned by RACE technology for the first time,to investigate the expression patterns of AGRP and NPY in different tissues and different physiological conditions of fasting and refeeding in M.amblycephala by Quantitative Real-time PCR technology.Four hundred and eighty juvenile blunt snout breams with an average body weight of(5.30±0.01)g were randomly divided into 4 groups with 3 replicates in each group,i.e.the control group(the daily feeding experiment diet at 3%of the weight),the F/refeeding group(fasted from 1d to 14d,refed from 15d to 28d and the daily feeding experiment diet at 3%of the weight),the F/excessive refeeding group(fasted from 1d to 14d,refed from 15d to 28d and the daily feeding experiment diet at 8%of the weight),the fasted group(fasted for 28 d).The results showed as follows:①The results of histological analysis showed that the epithelium columnar cells of intestine in the fasted group displayed obviously leukocyte infiltration in the submucosa.②The full length cDNA of M.amblycephala AGRP and NPY are respectively 770 bp and 557 bp,consisting of a 5′untranslated region of 77bp and 101 bp,a 3′untranslated region of 315 bp and 120 bp,and an open reading frame of 378 bp and 336 bp,encoding 125 amino acid and 111 amino acids.Based on similarity comparison,AGRP is relatively conservative and has the highest similarity to AGRP in Cyprinus carpio,and the phylogenetic tree analysis indicated that AGRP and NPY of M.amblycephala is associated with AGRP and NPY in other fishes,respectively.③Quantitative Real-time PCR analysis showed that AGRP and NPY were both expressed in all tested tissues with highest expression in brain and then liver and intestine.④There were significantly differences in AGRP and NPY mRNA of fish during fasting and refeeding.The expression

关 键 词：团头鲂 刺鼠相关蛋白 神经肽Y 饥饿 基因表达 

分 类 号：Q786[生物学—分子生物学] S965.1[农业科学—水产养殖]