研究简报 Short Communications

胞质型磷脂酶A2基因克隆及其表达量随大黄鱼仔稚鱼生长发育的变化

展开
  • 中国海洋大学, 农业部水产动物营养与饲料重点实验室, 海水养殖教育部重点实验室, 青岛 266003
冯硕恒(1988-),男,山东潍坊人,硕士研究生,研究方向为水产动物营养与饲料。E-mail:bdwgl@126.com

收稿日期: 2014-04-14

  网络出版日期: 2014-09-10

基金资助

国家自然科学基金“大黄鱼仔稚鱼磷脂营养代谢研究”(31172425)

Cytosolic Phospholipase A2 Gene: Cloning and Expression Level Change with Growth Development of Large Yellow Croaker (Larmichthys crocea) Larvae

Expand
  • Key Laboratory of Mariculture of Ministry of Education, Key Laboratory of Aquaculture Nutrition and Feed of Ministry of Agriculture, Ocean University of China, Qingdao 266003, China

Received date: 2014-04-14

  Online published: 2014-09-10

摘要

利用同源克隆技术和cDNA末端快速扩增(RACE)技术克隆大黄鱼胞质型磷脂酶A2cPLA2)基因的cDNA全长。此外,应用实时定量PCR法检测不同日龄大黄鱼仔稚鱼cPLA2基因的表达量。序列分析结果表明:cPLA2基因全长2 550 bp(GenBank登录号:KF006240.1),其中5'端非编码区176 bp,开放阅读框2 169 bp,3'端非编码区205 bp,共编码723个氨基酸。系统进化树分析结果表明:克隆所得大黄鱼cPLA2基因与其他鱼类的cPLA2基因亲缘关系较近,与哺乳动物的亲缘关系较远。定量检测结果表明:大黄鱼仔稚鱼cPLA2基因的表达量随日龄的增加为先显著升高(1、3、7日龄vs.15日龄,P<0.05),在15日龄时达到最高值,随后显著下降(15日龄vs.19日龄,P<0.05),之后趋于平稳。由此可知,大黄鱼从仔稚鱼到幼鱼早期的变形过程中,磷脂分解代谢的关键酶cPLA2基因的表达量呈现有规律的变化,这可能对机体保持体内磷脂的动态平衡、维护细胞膜的流动性具有重要的意义。大黄鱼仔稚鱼cPLA2基因表达量的变化趋势在一定程度上可以衡量大黄鱼消化系统的发育情况。

本文引用格式

冯硕恒, 谢奉军, 蔡佐楠, 麦康森, 艾庆辉 . 胞质型磷脂酶A2基因克隆及其表达量随大黄鱼仔稚鱼生长发育的变化[J]. 动物营养学报, 2014 , 26(9) : 2883 -2891 . DOI: 10.3969/j.issn.1006-267x.2014.09.053

Abstract

In this present study, the full length of cytosolic phospholipase A2 (cPLA2) cDNA from large yellow croaker (Larmichthys crocea) was obtained by homology-based cloning and rapid amplification of cDNA ends (RACE) techniques. And, the expression level of cPLA2 gene of large yellow croaker larvae at different days of age was measured by real-time quantitative PCR method. Sequence analysis results showed that the full-length of cPLA2 cDNA (GenBank accession No.: KF006240.1) was 2 660 bp, which consists of 2 169 bp in size with an open-reading frame (ORF) encoding 723 amino acids, a 176 bp 5'-untranslated region (5'UTR) and a 205 bp 3'-untranslated region (3'UTR). Phylogenetic tree analysis results showed that the cPLA2 gene of large yellow croaker had a closer relationship with other fish species, but had a distant relationship with mammalian. Quantitative determination results showed that with the days of age of large yellow croaker increasing, the expression level of cPLA2 gene was significantly increased at first (1, 3 and 7 days of age vs. 15 days of age, P<0.05) and the 15 days of age had the highest value, then significantly decreased (15 days of age vs. 19 days of age, P<0.05), maintained stable until the end of the experiment. In conclusion, the key enzyme of phospholipid catabolism-cPLA2 gene expression level show a regular variation during the deformation process from larvae to juvenile of large yellow croaker, which may has an important significance for keeping the dynamic equilibrium of phospholipids and maintaining the fluidity of membrane. The change trend of cPLA2 gene expression level of large yellow croaker larvae can estimate the development of digestive system of large yellow croaker in a certain degree.

参考文献

[1] FAGONE P,JACKOWSKI S.Phosphatidylcholine and the CDP-choline cycle[J].Biochimica et BiophysicaActa:Molecular and Cell Biology of Lipids,2013,1831(3):523-532.  

[2] FRASER A J,GAMBLE J C,SARGENT J R.Changes in lipid content,lipid class composition and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua)[J].Marine Biology,1988,99(3):307-313.  

[3] RAINUZZO J R,REITAN K I,JØ RGENSEN L.Comparative study on the fatty acid and lipid composition of four marine fish larvae[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,103(1):21-26.  

[4] TOCHER D R,FRASER A J,SARGENT J R,etal.Fatty acid composition of phospholipids and neutral lipids during embryonic and early larval development in Atlantic herring (Clupea harengus,L.)[J].Lipids,1985,20(2):69-74.  

[5] TOCHER D R,BENDIKSEN E Å,CAMPBELL P J,etal.The role of phospholipids in nutrition and metabolism of teleost fish[J].Aquaculture,2008,280(1):21-34.

[6] POSTON H A.Effect of body size on growth,survival,and chemical composition of Atlantic salmon fed soy lecithin and choline[J].The Progressive Fish-Culturist,1990,52(4):226-230.  

[7] CAHU C,GUILLAUME J C,STÉPHAN G,etal.Influence of phospholipid and highly unsaturated fatty acids on spawning rate and egg and tissue composition in Penaeus vannamei fed semi-purified diets[J].Aquaculture,1994,126(1/2):159-170.

[8] DAPRÀ F,GEURDEN I,CORRAZE G,etal.Physiological and molecular responses to dietary phospholipids vary between fry and early juvenile stages of rainbow trout (Oncorhynchus mykiss)[J].Aquaculture,2011,319(3/4):377-384.

[9] GEURDEN I,COUTTEAU P,SORGELOOS P.Effect of a dietary phospholipid supplementation on growth and fatty acid composition of European sea bass (Dicentrarchus labrax L.) and turbot (Scophthal musmaximus L.) juveniles from weaning onwards[J].Fish Physiology and Biochemistry,1997,16(4):259-272.  

[10] HARADA K.Relationships between structure and feeding attraction activity of certain L-amino acids and lecithin in aquatic animals[J].Nippon Suisan Gakkaishi,1987,53(12):2243-2247.  

[11] KANAZAWA A.Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish[J].Aquaculture,1997,155(1/2/3/4):129-134.

[12] SEOKA M,KURATA M,TAMAGAWA R,etal.Dietary supplementation of salmon roe phospholipid enhances the growth and survival of Pacific bluefin tuna Thunnus orientalis larvae and juveniles[J].Aquaculture,2008,275(1/2/3/4):225-234.

[13] ZHAO J Z,AI Q H,MAI K S,etal.Effects of dietary phospholipids on survival,growth,digestive enzymes and stress resistance of large yellow croaker,Larmichthys crocea larvae[J].Aquaculture,2013,410/411:122-128.

[14] HENDERSON R J,TOCHER D R.The lipid composition and biochemistry of freshwater fish[J].Progress in Lipid Research,1987,26(4):281-347.  

[15] SOUPENE E,FYRST H,KUYPERS F A.Mammalianacyl-CoA:lysophosphatidylcholine acyltransferase enzymes[J].Proceedings of the National Academy of Sciencesof the United States of America,2008,105(1):88-93.  

[16] BURKE J E,DENNIS E A.Phospholipase A2 structure/function,mechanism,and signaling[J].Journal of Lipid Research,2009,50(Suppl.):S237-S242.

[17] ZAMBONINO I J L,CAHU C L.Ontogeny of the gastrointestinal tract of marine fish larvae[J].Comparative Biochemistry and Physiology Part C:Toxicology & Pharmacology,2001,130(4):477-487.  

[18] ONO H,IIJIMA N.Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream,Pagrus major[J].Fish Physiology and Biochemistry,1998,18(2):135-147.  

[19] SAELE Ø,NORDGREEN A,OLSVIK P A,etal.Characterisation and expression of secretory phospholipase A2 groupⅠB during ontogeny of Atlantic cod (Gadus morhua)[J].British Journal of Nutrition,2011,105(2):228-237.  

[20] FUJIKAWA Y,SHIMOKAWA M,SATOH F,etal.Ontogeny of gene expression of group IB phospholipase A2 isoforms in the red sea bream,Pagrus (Chrysophrys) major[J].Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology,2012,161(2):185-192.  

[21] MAI K,YU H,MA H,etal.A histological study on the development of the digestive system of Pseudosciaena crocea larvae and juveniles[J].Journal of Fish Biology,2005,67(4):1094-1106.  

[22] XIE F J,AI Q H,MAI K S,etal.Dietary lysine requirement of large yellow croaker (Pseudosciaena crocea,Richardson 1846) larvae[J].Aquaculture Research,2012,43(6):917-928.  

[23] LI W J,AI Q H,MAI K S,etal.Effects of dietary amino acid patterns on growth and protein metabolism of large yellow croaker (Larimichthys crocea) larvae[J].Aquaculture,2013,406/407:1-8.

[24] XIE F J,AI Q H,MAI K S,etal.The optimal feeding frequency of large yellow croaker (Pseudosciaena crocea,Richardson) larvae[J].Aquaculture,2011,311(1/2/3/4):162-167.

[25] AI Q H,ZHAO J Z,MAI K S,etal.Optimal dietary lipid level for large yellow croaker (Pseudosciaena crocea) larvae[J].Aquaculture Nutrition,2008,14(6):515-522.  

[26] ZUO R,AI Q,MAI K,etal.Effects of dietary n-3 highly unsaturated fatty acids on growth,nonspecificimmunity,expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans)[J].Fish & Shellfish Immunology,2012,32(2):249-258.  

[27] THOMPSON J D,HIGGINS D G,GIBSON T J.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice[J].Nucleic Acids Research,1994,22(22):4673-4680.  

[28] TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis (MEGA) software version 4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599.  

[29] KUMAR S,TAMURA K,JAKOBSEN I B,et al.MEGA2:molecular evolutionary genetics analysis software[J].Bioinformatics,2001,17(12):1244-1245.  

[30] YAO C L,KONG P,WANG Z Y,etal.Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker,Pseudosciaena crocea[J].Fish &Shellfish Immunology,2008,25(5):648-656.  

[31] UOZUMI N,SHIMIZU T.Roles for cytosolic phospholipase A2 α as revealed by gene-targeted mice[J].Prostaglandins & Other Lipid Mediators,2002,68:59-69.

[32] HSU Y H,BURKE J E,STEPHENS D L,etal.Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry[J].Journal of Biological Chemistry,2008,283(15):9820-9827.  

[33] LEE L K,BRYANT K J,BOUVERET R,etal.Selectiveinhibition of human group ⅡA-secreted phospholipase A2 (hGⅡA) signaling reveals arachidonicacid metabolism is associated with colocalization of hGⅡA to vimentin in rheumatoid synoviocytes[J].Journal of Biological Chemistry,2013,288(21):15269-15279.  

[34] MANSFELD J,GEBAUER S,DATHE K,etal.Secretory phospholipase A2 from Arabidopsis thaliana:insights into the three-dimensional structure and the amino acids involved in catalysis[J].Biochemistry,2006,45(18):5687-5694.  
文章导航

/