大口黑鲈脂代谢相关基因NPY、UCP2、LPL、HL克隆与分子进化分析
MOLECULAR CLONING AND EVOLUTIONAL ANALYSIS OF NPY,UCP2,LPL AND HL GENE OF LARGEMOUTH BASS(MICROPTERUS SALMOIDES)
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摘要: 采用RT-PCR和RACE方法克隆了大口黑鲈NPY基因cDNA全序列及UCP2、LPL、HL基因cDNA核心片段。序列分析结果表明,大口黑鲈NPY基因cDNA全序列长664 bp,其中5′端非翻译区(5′-UTR)长53 bp,3′端非翻译区(3′-UTR)长311 bp,开放阅读框(ORF)长300 bp,编码99个氨基酸,即前体NPY。大口黑鲈前体NPY包括三个部分,28个氨基酸组成的信号肽、36氨基酸组成的成熟NPY以及32个氨基酸组成的由Gly-Lys-Arg指示的NPY C端肽(CPON)。大口黑鲈UCP2、LPL、HL基因cDNA核心片段长度分别为737 bp、509 bp和666 bp,各自编码245个氨基酸、169个氨基酸和222个氨基酸。将4个基因的氨基酸序列分别与其他物种的氨基酸序列进行同源性比较,并通过MEGA3构建系统树,对这4个脂代谢相关基因的分子进化特征进行了探讨。Abstract: Largemouth bass(Micropterus salmoides) is a piscivorous teleost fish,found only in the fresh waters of North America.It is a good game fish in the United States,and a highly valued cultivated species in China.In order to study its lipid metabolism as well as its evolutional relationship with other percoids,we used RT-PCR method to get the partial cDNA sequences of NPY,UCP2,LPL and HL genes,and deduced their corresponding amino acid sequences.Rapid amplification of the cDNA ends(RACE) PCR was used to amplify the 3′-and 5′-end of NPY cDNA.The complete NPY cDNA sequence of largemouth bass was 664bp in length,composed of an open reading frame(ORF) of 300bp,encoding a protein of 99 amino acid residues.A search of the BLAST neuropeptide database revealed that the deduced neuropeptide sequence of largemouth bass NPY was very similar to the NPY of other species,such as European sea bass(Dicentrarchus labrax,93﹪ identity),orange-spotted grouper(Epinephelus coioides,91﹪ identity),house mouse(Mus musculus,86﹪ identity),and human(Homo sapiens,87﹪ identity).The obtained UCP2 core sequence was 737bp in length,which contains an open reading frame coding 245 amino acids.The length of the obtained LPL core sequence was 509bp,coding 169 amino acids.The length of the obtained HL core sequence was 666bp,coding 222 amino acids.We made the amino acid sequence comparisons among the four obtained genes of largemouth bass and other species,and got four phylogenetic trees by NJ method using software MEGA3.The phylogenetic tree built on the basis of conservative NPY gene,showed a right relationship of fish with mollusk,amphibia,aves and mammals,but it could not present accurate relationship of fish species.The phylogenetic tree built by less conservative UCP2 gene showed that largemouth bass and other percoids were farther away from mammals than the more primitive cyprinids.In order to make a further determination of the evolutional position of largemouth bass,we used the least conservative genes(LPL and HL) of the four to build phylogenetic trees,and found that largemouth bass was the closest to Chinese perch(Siniperca chuatsi),which was distributed only in the fresh waters of East Asia.These results support the close relationship of largemouth bass and Chinese perch.
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Keywords:
- Largemouth bass /
- NPY /
- UCP2 /
- LPL /
- HL /
- Molecular cloning /
- Phylogenetic analysis
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[1] Yang F. Animal nutrition ( second edition). Beijing: China Ag2riculture Press. 2003, 76-88 [杨凤. 动物营养学(第二版).北京: 中国农业出版社. 2003, 76-88]
[2] Feng J, J ia G. Studies on the fatty liver diseases resulted fromdifferent lip id levels in sciaenop s ocellatus diets [J]. ActaHydrobiologica S inica, 2005, 29 (1) : 61-64 [冯健, 贾刚. 饵料中不同脂肪水平诱导红姑鱼脂肪肝病的研究. 水生生物学报, 2005, 29 (1) : 61-64]
[3] Feng J, Qin ZB. Effects of four dietary lip ids on the performanceof growth and body composition in pacific salmon, oncorhynchuy [J]. Acta Hydrobiologica S inica, 2006, 30 (3) : 256-261 [冯健, 覃志彪. 4 种不同脂肪源对太平洋鲑生长和体组成的影响. 水生生物学报, 2006, 30 (3) : 256-261]
[4] Allen JM,Adrian T E, Tatemoto K, et al. Two novel related pep2tides, neuropep tide Y (NPY) and pep tide YY ( PYY) inhibitthe contraction of the electrically stimulated mouse vas deferens [J]. N europeptides, 1982, 3 (2) : 71-77
[5] Inui A. Neuropep tide Y feeding recep tors: are multip le subtypesinvolved [J] ? Trend Pharm acol Sci, 1999, 20 (2) : 43-46
[6] Lopez2PatinoM A, Guijarro A I, Isorna E, et al. Neuropep tide Yhas a stimulatory action on feeding in goldfish (Carassius aura2tus) [J]. Eur J Pham acol, 1999, 377: 147-153
[7] Narnaware Y K, Peyon P P, Lin X, et al. Regulation of food in2take by neuropep tide Y (NPY) in goldfish [J]. Am J PhysiolRegul Integr Com p Physiol, 2000, 279 (3) : 1025-1034
[8] Fleury C, Neverova M, Collins S, et al. Uncoup ling p rotein22: anovel gene linked to obesity and hyperinsulinemia [J]. N at Gen2et, 1997, 15 (3) : 269-272
[9] Zhou Y T, ShimabukuroM, Koyama K, et al. Induction by lep tinof uncoup ling p rotein22 and enzymes of fatty acid oxidation [J].Proc N atl Acad Sci USA, 1997, 94 (12) : 6386-6390
[10] Auwerx J,Leroy P, Schoonjans K. Lipoprotein lipase: recent contri2butions from molecular biology [J]. Crit Rev Clin Lab Sci, 1992,29 (3-4) : 243-268
[11] Zechner R. The tissue2specific exp ression of lipop rotein lipase:imp lications for energy and lipop rotein metabolism [J]. CurrOpin L ipidol, 1997, 8 (2) : 77-88
[12] Kirchgessner T G, Chuat J C, Heinzmann C, et al. Organization ofthe human lipop rotein lipase gene and evolution of the lipase genefamily [J]. Proc N atl Acad Sci USA, 1989, 86 ( 24 ) :9647-9651
[13] Dugi K A,Vaisman B L, SakaiN, et al. Adenovirus2mediated ex2p ression of hepatic lipase in LCAT transgenic mice [J]. L ipidRes, 1997, 38 (9) : 1822-1832
[14] Marques2Vidal P, Azema C, Collet X, et al. Hepatic lipase p ro2motes the up take of HDL esterified cholesterol by the perfused ratliver: a study using reconstituted HDL particles of defined phos2pholip id composition [J]. L ipid Res, 1994, 35 (3) : 373-384
[15] LiaoW Q, Liang X F, Wang L, et al. Molecular cloning andcharacterization of alpha2class glutathione S2transferase gene fromthe liver of silver carp, bighead carp, and other major Chinesefreshwater fishes [J]. B iochem M ol Toxicol, 2006, 20 ( 3 ) :114-126
[16] M Bienengraeber, K S Echtay, M Klingenberg. H + transport byuncoup ling p rotein (UCP21 ) is dependent on a histidine pair,absent in UCP22 and UCP23 [J]. B iochem istry, 1998, 37: 3-8
[17] Nagase I, Yoshida T, SaitoM. Up2regulation of uncoup ling p ro2teins by beta2adrenergic stimulation in L6 myotubes [J]. FEBSLett, 2001, 494 (3) : 175-180
[18] BossO, Hagen T, LowellB B. Uncoup ling p roteins 2 and 3: po2tential regulators ofmitochondrial energymetabolism [J]. D iabe2tes, 2000, 49 (2) : 143-156
[19] Li L X, Skorpen F, Egeberg K. Uncoup ling p rotein22 partici2pates in cellular defense against oxidative stress in clonal beta2cells [J]. B iochem B iophys Res Comm un, 2001, 282 (1) : 273-277
[20] Klingenberg M, Echtay K S. Uncoup ling p roteins: the issuesfrom a biochemist point of view [J]. B iochim B iophys Acta,2001, 1504 (1) : 128-143
[21] Liang X F, Lin X T, Huang F, et al. The liver uncoup ling p ro2tein 2 gene of the red sea bream ( Pagrusm ajor) with reference toits physiological function [J]. Acta Zoologica S inica, 2003, 49(1) : 110-117 [梁旭方, 林小涛, 黄芬, 尾形博. 真鲷肝脏解偶联蛋白2 (UCP2) 基因及其功能的探讨. 动物学报,2003, 49 (1) : 110-117]
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