| 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 |
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| 引用本文： | 王钰婧,冯文荣,徐逾鑫,李建林,苏胜彦,俞菊华,唐永凯.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[J].水产学报,2023,47(10):109618-109618. |
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| 作者姓名： | 王钰婧 冯文荣 徐逾鑫 李建林 苏胜彦 俞菊华 唐永凯 |
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| 作者单位： | 南京农业大学无锡渔业学院;中国水产科学研究院淡水渔业研究中心,南京农业大学无锡渔业学院,南京农业大学无锡渔业学院,南京农业大学无锡渔业学院,南京农业大学无锡渔业学院,南京农业大学无锡渔业学院,南京农业大学无锡渔业学院 |
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| 基金项目： | 国家大宗淡水鱼产业技术体系（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) |
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| 摘 要： | 为研究鲤脂蛋白脂肪酶(Cc LPLs)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、...
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| 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 |
| 收稿时间： | 2022/10/11 0:00:00 |
| 修稿时间： | 2022/11/10 0:00:00 |
Characteristics, gene expression, recombinant protein expression and enzyme activity of lipoprotein lipase in Cyprinus carpio |
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| WANG Yujing,FENG Wenrong,XU Yuxin,LI Jianlin,SU Shengyan,YU Juhu,TANG Yongkai.Characteristics, gene expression, recombinant protein expression and enzyme activity of lipoprotein lipase in Cyprinus carpio[J].Journal of Fisheries of China,2023,47(10):109618-109618. |
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| Authors: | WANG Yujing FENG Wenrong XU Yuxin LI Jianlin SU Shengyan YU Juhu TANG Yongkai |
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| Institution: | Wuxi Fisheries College,Nanjing Agricultural University;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;China,Wuxi Fisheries College,Nanjing Agricultural University,Wuxi Fisheries College,Nanjing Agricultural University,Wuxi Fisheries College,Nanjing Agricultural University,Wuxi Fisheries College,Nanjing Agricultural University,Wuxi Fisheries College,Nanjing Agricultural University,Wuxi Fisheries College,Nanjing Agricultural University |
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| Abstract: | 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 (CcLPLA1a, 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 CcLPLA1s 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 CcLPLA1s 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 CcLPLA1s and CcLPLA2a were restored as normal feeding group in the three tissues. Using E. coli expression system, Skp-CcLPLs and SlyD-CcLPLs recombinant proteins were obtained. The enzymatic activity of each recombinant protein was determined by the p-nitrophenol method, and the results were (24.12 ±0.96), (22.66 ±0.46), (21.48 ±0.47), (21.13 ±0.46), (18.07 ±0.39) and (16.49 ±0.31) U/g for SlyD-CcLPLA1a, Skp-CcLPLA1a, Skp-CcLPLBa, Skp-CcLPLA2a, SlyD-CcLPLBa and SlyD-CcLPLA2a, respectively. Regardless of Skp or SlyD tags in the recombinant proteins, the enzymatic activities of CcLPLs from high to low were CcLPLA1a, CcLPLBa and CcLPLA2a. Determination of effect of pH and NaCl concentration on enzyme activity showed the optimal reaction condition of pH was 8.0 and the NaCl concentration that exerted the maximum activity was 0.6 mol/L. In conclusion, we explored the evolutionary expression of CcLPLs homologous genes, analyzed the temporal and spatial expression of CcLPLA1s, CcLPLA2a and CcLPLBa. The effects of starvation and refeeding on the expression of CcLPLA1s and CcLPLA2a were studied, and the results revealed the lipid metabolism and response countermeasures under starvation stress, and provided a targrt for controlling the fat content in C. carpio. The prokaryotic expressions of the recombinant proteins were successfully carried out and the enzymatic activites were determined, which provided a reference for the study of fish lipoprotein lipase. |
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| Keywords: | Cyprinus carpio lipoprotein lipase gene expression recombinant protein expression in prokaryotes lipoprotein lipase activity |
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