斜带石斑鱼阿片黑素促皮质素原全长cDNA克隆及序列分析

CLONING AND SEQUENCE ANALYSIS OF PROOPIOMELANOCORTIN (POMC) IN ORANGE-SPOTTED GROUPER,EPINEPHELUS COIOIDES

通过构建斜带石斑鱼垂体cDNA文库和随机测序 ,克隆了其阿片黑素促皮质素原 (Proopiomelanocortin ,POMC)全长cDNA。斜带石斑鱼POMC全长cDNA为 86 3bp(含Poly(A) ) ,编码的POMC多肽前体为 2 19aa。氨基酸序列比较分析表明 :斜带石斑鱼POMC前体包含有促肾上腺皮质激素 (Adrenocorticotropin ,ACTH) ,α 促黑素 (α melanocytestim ulatinghormone ,α MSH) ,β 促黑素 ( β MSH) ,γ 促脂素 (γ lipotrophichormone,γ LPH) ,β 内啡肽 ( β endorphin)等 ,但缺失了γ 促黑素 (γ MSH)和大部分连接区。斜带石斑鱼POMC与哺乳动物POMC的同源性为 39%— 4 2 %左右 ,与鸟类的同源性为 4 2 %左右 ,与两栖类的同源性为 36 %— 4 1% ,与其他鱼类POMC的同源性为 38%— 77%。斜带石斑鱼和罗非鱼的POMC的ACTH功能区都为 4 0个氨基酸残基 ,而其他脊椎动物为

Proopiomelanocortin(POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropin (ACTH), melanocyte stimulating hormone (MSH),and β endorphin, and they are producded by cell specific processing of POMC in pituitary. These peptides are known to be involved in stress responses and energy metabolism in vertebrates. It has been shown that POMC molecule is present in all vertebrates examined, and of highest diversities in fish. This paper was aimed to characterize the cDNA sequence of POMC in the orange spotted grouper Epinephelus coioides , a protogynous hermaphroditic marine fish of high economic values. The pituitary cDNA library of Epinephelus coioides was constructed from total RNA using SMART TM cDNA Library Construction Kit (Clontech, USA). The full length cDNA encoding Epinephelus coioides POMC was obtained by random sequencing the pituitary cDNA library. Seventy clones were randomly picked and sequenced, and five of them were found to be Epinephelus coioides POMC cDNA by BlastN analysis. There was no sequence heterogeneity found among the five POMC cDNA clones. The full length cDNA sequence of Epinephelus coioides POMC obtained spanned 863bp (including poly (A) tail),with a 5 UTR of 138 bp, a3 UTR of 68 bp, and an open reading frame encoding a protein of 219 amino acids with a putative signal peptide of 21 amino acid residues. The signal for the addition of Poly (A) tail is located 30 nucleotides upstream of the Poly (A) sequence. Sequence homology analysis at amino acid level indicates that Epinephelus coioides POMC contains ACTH (S 90 M 129 ),α MSH(S 90 V 102 ),β MSH(D 167 S 183 ),γ lipotrophic hormone (γ LPH)(R 132 E 164 ),β endorphin regions (Y 186 Q 219 ),but lacks the γ MSH and most of the joining peptide regions which was further confirmed by RT PCR analysis of pituitary total RNA. Epinephelus coioides POMC shares 39% 42% identities with mammalian POMC, about 42% with avian POMC, 36% 41% with amphibian POMC, 38%-77% with other fish POMC. The high homology was found in ACTH,α MSH, β MSH, and β endorphin regions of vertebrate POMCs, but not in γ LPH region. The ACTH region of Epinephelus coioides POMC shares 90% homology with that of Oreochromis mossambicus , 82% with Cyprinus carpio ,87% with Oncorhynchus mykiss ,85% with Lepisosteus osseus ,82% with Acipenser transmontanus ,65% with Squalus acanthias ,55% with Dasyatis akajei , 70% with Protopterus annectens ,80% with Xenopus laevis , 77.5% with Gallus gallus , and 75% with Bos taurus respectively. The α MSH region of Epinephelus coioides POMC shares 100% homology with those of Oreochromis mossambicus , Cyprinus carpio, Oncorhynchus mykiss, Lepisosteus osseus, Acipenser transmontanus, Protopterus annectens, Gallus gallus and Bos taurus and 92% homology with those of Squalus acanthias, Dasyatis akajei and Xenopus laevis respectively. The β MSH region of Epinephelus coioides POMC shares 88% homology with that of Oreochromis mossambicus , 76% with Cyprinus carpio ,82% with Oncorhynchus mykiss ,64.7% with Lepisosteus osseus ,70.6% with Acipenser transmontanus, 47.1% with Squalus acanthias ,41.7% with Dasyatis akajei , 58.8% with Protopterus annectens , 58.8% with Xenopus laevis , and 64.7% with Gallus gallus and Bos taurus respectively. The β endorphin region of Epinephelus coioides POMC shares 82% homology with that of Oreochromis mossambicus ,76.5% with Cyprinus carpio ,88% with Oncorhynchus mykiss ,82% with Lepisosteus osseus ,76% with Acipenser transmontanus ,73% with Squalus acanthias ,70% with Dasyatis akajei and Protopterus annectens ,52.9% with Gallus gallus ,and 55.8% with Xenopus laevis and Bos taurus respectively. The γ LPH region of the Epinephelus coioides POMC did not show significant similarities with those of other vertebrates. The POMC of Epineph