鲤脂蛋白脂肪酶基因特性、表达分布、重组蛋白获得及酶活性比较  

Characteristics,gene expression,recombinant protein expression and enzyme activity of lipoprotein lipase in Cyprinus carpio

作　　者：王钰婧 冯文荣 徐逾鑫 李建林[1,2] 苏胜彦[1,2] 俞菊华[1,2] 唐永凯[1,2] WANG Yujing;FENG Wenrong;XU Yuxin;LI Jianlin;SU Shengyan;YU Juhua;TANG Yongkai(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214128,China;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture and Rural Affairs,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China)

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214128 [2]中国水产科学研究院淡水渔业研究中心,农业农村部淡水渔业与种质资源利用重点实验室,江苏无锡214081

出　　处：《水产学报》2023年第10期208-224,共17页Journal of Fisheries of China

基　　金：国家大宗淡水鱼产业技术体系专项(CARS-45):国家重点研发计划(2022YFF0608203);中国水产科学研究院基本科研业务费(2020TD37)。

摘　　要：为研究鲤脂蛋白脂肪酶(CcLPLs)的基因特征、时空表达分布及酶活性,实验利用基因组同源搜索获取鲤CcLPLs同源基因并分析其序列特征;通过荧光定量PCR(qPCR)方法对CcLPLs在不同组织的表达进行分析;采用原核表达系统获取CcLPLs重组蛋白,并使用对硝基苯酚法测定各重组蛋白的酶活性。结果显示,鲤基因组中挖掘到5个CcLPLs基因(CcLPLA1a、CcLPLA1b、CcLPLA2a、CcLPLA2b*和CcLPLBa),经验证,CcLPLA2b*是假基因,共线性分析显示,鱼类特有基因组加倍过程中出现基因丢失的现象,而鲤特有的基因组加倍致使鲤存在5个CcLPLs。CcLPLA1a和CcLPLA1b核苷酸序列和氨基酸序列相同,同源性分析显示,CcLPLBa与CcLPLA1s的同源性为64%,与CcLPLA2a同源性为50.8%。qPCR结果显示,CcLPLs的表达量在肝脏、心脏、脂肪、肌肉、脑、肠道和脾脏中依次降低,在各个组织中,各基因表达量从高到底依次为CcLPLA1s、CcLPLA2a和CcLPLBa。鲤正常投喂、饥饿及再投喂状态下CcLPLA1s和CcLPLA2a在肝脏、肌肉和脂肪组织中的表达结果显示,饥饿状态下,CcLPLA1s和CcLPLA2a在肝脏中的表达量高于正常投喂组,而在肌肉和脂肪中低于正常投喂组;再投喂后,CcLPLA1s和CcLPLA2a在肝脏中表达水平降至正常投喂组,而在肌肉和脂肪中表现为先升高后降低的趋势。通过构建具有促溶效果的原核表达载体,分别获得了原核表达重组蛋白Skp-CcLPLs和SlyDCcLPLs,酶活性测定结果显示,重组蛋白其脂蛋白脂肪酶活性从高到低依次为CcLPLA1a、CcLPLBa和CcLPLA2a,最适pH均为8.0,发挥最大活性的NaCl浓度为0.6 mol/L。本研究探讨了鲤CcLPLs同源基因在进化中的表现,对CcLPLA1s、CcLPLA2a和CcLPLBa的时空表达进行了分析,测定了投喂和饥饿对CcLPLA1s、CcLPLA2a表达的影响,揭示鲤饥饿胁迫下脂质代谢及响应对策,为控制鲤脂肪含量提供靶点,成功进行重组蛋白的原核表达并测定了其酶活性,为鱼类脂蛋白脂肪�Lipoprotein lipase(LPL)is a key enzyme in fat hydrolysis.In order to study the gene characteristics,temporal and spatial expression distribution and enzyme activity of Cyprinus carpio LPLs(CcLPLs),the homologous genes of CcLPLs were obtained by homology searches through C.carpio genome and the sequence characteristics were analyzed.The expression analysis of CcLPLs in different tissues was carried out by qPCR.CcLPLs recombinant protein were obtained by prokaryotic expression system,and the enzyme activity of recombinant proteins was determined by p-nitrophenol method.The results are as follows.Five CcLPLs homologous genes(CcLPLAla,CcLPLA1b,CcLPLA2a,CcLPLA2b*and CcLPLBa)were excavated from the carp genome,of which CcLPLA2b*is a pseudogene.The collinearity analysis showed that gene loss occurred during the doubling of the fish-specific genome,while the doubling of the carp-specific genome resulted in the presence of five CcLPLs in carp.By Homology analysis,CcLPLBa shared 64%indentity with CcLPLAls and 50.8%with CcLPLA2a.qPCR showed that the expression of CcLPLs decreased by degrees in liver,heart,fat,muscle,brain,intestine and spleen.In each tissue,the expression of CcLPLAls was significantly higher than that of CcLPLA2a,and the expression of CcLPLA2a was significantly higher than that of CcLPLBa.The expression levels of CcLPLA1s and CcLPLA2a in liver,muscle and adipose tissues under normal feeding,starvation and refeeding conditions were determined by qPCR.The results showed that the expression levels of CcLPLA1s and CcLPLA2a in liver under starvation were significantly higher than those in the normal feeding group,while the expression levels in muscle and adipose tissue were opposite to those in liver.After refeeding,the expression levels of CcLPLAls and CcLPLA2a were restored as normal feeding group in the three tissues.Using E.coli expression system,Skp-CcLPLs and SlyDCcLPLs recombinant proteins were obtained.The enzymatic activity of each recombinant protein was determined by the p-nitrophenol method,and the r

关 键 词：鲤 脂蛋白脂肪酶 基因表达 原核表达 脂蛋白脂肪酶活性 

分 类 号：S963.1[农业科学—水产养殖]