锦鲤Δ6脂肪酸去饱和酶基因克隆及表达研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.023

摘要/Abstract

摘要： 旨在揭示变温鱼类合成HUFAs过程中的关键酶及其功能，以锦鲤(Cyprinus carpiokoi)为材料，通过RT-PCR扩增获得了一段长度为854 bp的Δ6脂肪酸去饱和酶基因的cDNA 序列片段，并对其进行了序列分析和蛋白产物结构预测。结果显示，该片段与南亚野鲮(Labeo rohita)Δ6脂肪酸去饱和酶基因有93.5%的同源性，编码的蛋白产物具有典型的Δ6脂肪酸去饱和酶结构特点，包含2个组氨酸保守区(HDFGH、HFQHH)和2个跨膜结构域。荧光定量PCR表明，Δ6脂肪酸去饱和酶在锦鲤肝脏中的表达量最高，在肠、脑和心脏中表达量依次降低，而在肌肉和鳃的表达量相对较低。幼鱼在不同培养水温下，各组织中该基因的表达量均随温度的下降而升高，以适应低温环境。

关键词: 锦鲤, 高不饱和脂肪酸, △, 6脂肪酸去饱和酶, 鱼类抗冻

Abstract: Highly unsaturated fatty acids(HUFAs)are important constituents of cell membrane, and the unsaturated degree of carbon chain is an important factor in resisting to low temperature, maintaining and regulating the fluidity of cell membrane. In order to discover the key enzymes of synthetizing HUAs and their functions, using koi carp(Cyprinus carpiokoi)as studying materials, a 854 bp cDNA of delta 6 fatty acid desaturase(Fad6)was amplified by RT-PCR, their sequences were analyzed and the structures of proteins were predicted. Results showed that the amplified fragment had great homology(93.5%)with that of rohu(Labeo rohita). The encoded protein possessed the features of typical Fad6, containing two histidine boxes(HDFGH and HFQHH)and two transmembrane regions. Real time PCR results demonstrated that Fad6 was expressed in all tissues, and the order of their expression levels was as follows:liver > intestine > brain > heart > muscle > gill. The expressions of the Fad6 gene in different tissues of koi carp larva, while exposing to different water temperatures, increased as water temperature decreasing for adapting to the lower temperature environment.

Key words: koi carp, highly unsaturated fatty acids, delta 6 fatty acid desaturase, fish antifreeze

张萍,薛伟伟,蒋雪薇,朱双,刘江东. 锦鲤Δ6脂肪酸去饱和酶基因克隆及表达研究[J]. 生物技术通报, 2015, 31(12): 158-166.

Zhang Ping, Xue Weiwei, Jiang Xuewei, Zhu Shuang, Liu Jiangdong. The Cloning and Gene Expression of Delta 6 Fatty Acid Desaturase of Koi Carp(Cyprinus carpiokoi)[J]. Biotechnology Bulletin, 2015, 31(12): 158-166.

参考文献

[1]Stoner AW, Ottmar ML, Copeman LA. Temperature effects on the molting, growth, and lipid composition of newly-settled red king crab[J]. Journal of Experimental Marine Biology and Ecology, 2010, 393(1-2):138-147.
[2]Xu YQ, Zhang YM, Ding ZK. Cloning and gene expression of delta 6 fatty acid desaturase cDNA of cobia(Rachycentron canadum)[J]. Journal of Fishery Sciences of China, 2010, 17(6):1183-1191.
[3]Lin P, Qi LW, Wang YD, et al. A review of advances in fatty acid desaturase on cold-resistance in plants[J]. Molecular Plant Breeding, 2006, 4(3):404-410.
[4]Wen SH, Li XF, Ju B, Wang CH. Sutdies on polyunsaturated fatty acid in microalgae[J]. Marine Science Bulletin, 2000, 19(4):86-91.
[5]Tang H, Pan ZX, Lu LZ, Wang JW. The function and expression regulation of elongases of very long chain fatty acids[J]. Chemistry of Life, 2009, 29(6):898-902.
[6]Tocher DR, Bell JG, McGhee F, et al. Effects of dietary lipid level and vegetable oil on fatty acid metabolism in Atlantic salmon(Salmo salar)over the entire production cycle[J]. Fish Physiology and Biochemistry, 2003, 29(3):193-209.
[7]Zeng SS, Jiang LM, Yuan DJ. Research and development of fatty acid desaturase[J]. Chinese Bulletin of Life Sciences, 2008, 20(5):816-821.
[8] Ren HT, Zhang GQ, Li JL, et al. Two Δ6-desaturase-like genes in common carp(Cyprinus carpio var. Jian):Structure characteriza-tion, mRNA expression, temperature and nutritional regulation[J]. Gene, 2013, 525:11-17.
[9] Brenner RR, Garda H, de Gómez Dumm IN, Pezzano H. Early effects of essential fatty acid deficiency on structure and enzymatic activity of rat liver microsomes[J]. Progress in Lipid Research, 1981, 20:315-321.
[10]Brenner RR. Nutritional and hormonal factors influencing desaturation of essential fatty acids in animals[J]. Progress in Lipid Research, 1981, 20:41-47.
[11]Zheng X, Seiliez I, Hastings N, et al. Characterization and comparison of fatty acyl △6 desaturase cDNAs from freshwater and marine teleost fish species[J]. Comp Biochem Physiol Part B, 2004, 139:269-279.
[12]Seiliez I, Panserat S, Kaushik S, et al. Cloning, tissue distribution and nutritional regulation of a △6-desaturase-like enzyme in rainbow trout[J]. Comp Biochem Physiol Part B, 2001, 130:83-93.
[13]Seiliez I, Panserat S, Corraze G, et al. Cloning and nutritional regulation of a △6-desaturase-like enzyme in the marine teleost gilthead seabream(Sparus aurata)[J]. Comp Biochem Physiol Part B, 2003, 135:449-460.
[14]Watson CA, Hill JE, Pouder DB. Species profile:Koi and goldfish[J]. Southern Regional Aquaculture Center, SRAC Publication, 2004, 7201:1-6.
[15]Tamadachi M. The cult of the Koi[M]. Neptune City:TFH Publications Inc, 1990:288-289.
[16]苏建通. 锦鲤的养殖与鉴赏[M]. 北京:中国农业出版社, 2011.
[17]Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Research, 2001, 29(9):2003-2007.
[18]Steel R, Torrie J. Principles and procedures of statistics:A biometrical approach[M]. New York:McGraw-Hill, 1980.
[19]Sprecher H, Luthria DL, Mohammed BS, et al. Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids[J]. Journal of Lipid Research, 1995, 36(12):2471-2477.
[20]Francis DS, Peters DJ, Turchini GM. Apparent in vivo delta-6 desaturase activity, efficiency, and affinity are affected by total dietary C18 PUFA in the freshwater fish Murray cod[J]. Journal of Agricultural and Food Chemistry, 2009, 57(10):4381-4390.
[21]Izquierdo MS, Robaina L, Juárez-Carrillo E, et al. Regulation of growth, fatty acid composition and delta-6 desaturase expression by dietary lipids in gilthead seabream larvae(Sparus aurata)[J]. Fish Physiology and Biochemistry, 2008, 34(2):117-127.
[22]Tocher DR, Fonseca-Madrigal J, Dick JR, et al. Effects of water temperature and diet containing palm oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes of rainbow trout(Oncorhynchus mykiss)[J]. Comp Biochem Physiol Part B, 2004, 137:49-63.
[23]Vagner M, Robin JH, Zambonino Infante JL, Person-Le Ruyet J. Combined effect of dietary HUFA level and temperature on sea bass(D. labrax)larvae development[J]. Aquaculture, 2007, 266:179-190.
[24]Vagner M, Zambonino Infante JL, Robin JH, Person-Le Ruyet J. Is it possible to influence European sea bass(Dicentrarchus labrax)juvenile metabolism by a nutritional conditioning during larval stage?[J]. Aquaculture, 2007, 267:165-174.
[25]Zheng X, King Z, Xu Y, et al. Physiological roles of fatty acyl desaturases and elongases in marine fish:characterisation of cDNAs of fatty acyl △6 desaturase and elovl5 elongase of cobia(Rachycentron canadum)[J]. Aquaculture, 2009, 290(1-2):122-131.
[26]Bell MV, Dick JR, Porter AE. Pyloric ceca are significant sites of newly synthesized 22:6n-3 in rainbow trout(Oncorhynchus mykiss)[J]. Lipids, 2003, 38(1):39-44.
[27]Tocher DR, Mourente G, Sargent JR. Metabolism of[1-¹⁴C]docosahexaenoate(22:6n-3), [1-¹⁴C]eicosapentaenoate(20:5n-3)and[1-¹⁴C]linolenate(18:3n-3)in brain cells from juvenile turbot Scophthalmus maximus[J]. Lipids, 1992, 27(7):494-499.
[28]Naughton JM. Supply of polyenoic fatty acids to the mammalian brain:the ease of conversion of the short-chain essential fatty acids to their longer chain polyunsaturated metabolites in liver, brain, placenta and blood[J]. International Journal of Biochemistry, 1981, 13(1):21-32.
[29]Ren HT, Yu JH, Xu P, et al. Cloning and expression of full length gene recoding for two △6 fatty acyl desaturases of common carp(Cyprinus carpio var. Jian)[J]. Chinese Journal of Biochemistry and Molecular Biology, 2012, 28(7):677-684.
[30]Bell JG, Tocher DR, Farndale BM, et al. The effect of dietary lipid on polyunsaturated fatty acid metabolism in Atlantic salmon(Sal-mo salar)undergoing parr-smolt transformation[J]. Lipids, 1997, 32:515-525.
[31]Tocher DR, Bell JG, Henderson RJ, et al. The effect of dietary linseed and rapeseed oils on polyunsaturated fatty acid metabolismin Atlantic salmon(Salmo salar)undergoing parr-smolt transformation[J]. Fish Physiology and Biochemistry, 2000, 23:59-73.
[32]Hagar AF, Hazel JR. Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout[J]. Comp Biochem Physiol Part B, 1985, 156:35-42.
[33]Ninno RE, de Torrengo MAP, Castuma JC, Brenner RR. Specificity of 5- and 6-fatty acid desaturases in rat and fish[J]. Biochim Biophys Acta, 1974, 360:124-133.
[34]de Mendoza D, Cronan JE. Thermal regulation of membrane fluidity in bacteria[J]. Trends in Biochemical Sciences, 1983, 8:49-52.
[35]de Mendoza D, Grau R, Cronan JE, et al. Biosynthesis and function of membrane lipids[M]// Losick R, Sonenshein AL, Hoch JA. Bacillus subtilis and other ram-positive bacteria:physiology, biochemistry and molecular biology. Washington:American Society for Microbiology Press, 1993:411-425.
[36] Russell NJ. Mechanisms of thermal adaptation in bacteria:bluep-rints for survival[J]. Trends in Biochemical Sciences, 1984, 3:108-112.
[37]Suutari M, Laakso S. Microbial fatty acids and thermal adaptation[J]. Critical Reviews in Microbiology, 1994, 20(4):285-328.
[38]de Mendoza D, Klages UA, Cronan JE, et al. Thermal regulation of membrane fluidity in Escherichia coli effects of overproduction of beta-ketoacyl-acyl carrier protein synthase I[J]. Journal of Biological Chemistry, 1983, 258(4):2098-2101.
[39]Fujii DK, Fulco AJ. Biosynthesis of unsaturated fatty acids in bacilli:hyperinduction and modulation of desaturase synthesis[J]. Journal of Biological Chemistry, 1977, 252:3660-3670.
[40]Fulco AJ. The biosynthesis of unsaturated fatty acids by bacilli, temperature induction of the desaturation reaction[J]. Journal of Biological Chemistry, 1969, 244:889-895.
[41]Boldwim J, Hochachra PW. Functional significance of isoenzymes in thermal acclimatization-acetylcholimesterase from trout brain[J]. Journal of Biological Chemistry, 1970, 116:883-887.
[42] Beneden R, Cashon RE, Powers DA. Inheritance of isocitrate dehy-drogenzse(Idh-A and Idlh-B), 6 phosphogluconate dehydrogen-ase(6 padh-A)and serum caterase polymorphisme[J]. Bioche-mical Genetics, 1981, 19:701-714.
[43]Davies PL, Hew CL. Biochemistry of fish antifreeze proteins[J]. The FASEB Journal, 1990, 4:2460-2468.
[44]Goldstein J, Pollite NS, Inouy M, et al. Major cold shock protein of Escherchia coli[J]. Proceedings of the National Academy of Sciences of the USA, 1990, 87:283-287.