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
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摘要: 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83bp(含Poly(A)),编码的POMC多肽前体为219aa.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-melanocyte stimulating hormone,α-MSH),β-促黑素(β-MSH),γ-促脂素(γ-lipotrophic hormone, 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%-42%左右,与鸟类的同源性为42%左右,与两栖类的同源性为3%-41%,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40个氨基酸残基,而其他脊椎动物为39个氨基酸残基.Abstract: 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 SMARTTM 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 (S90- M129 ),α MSH(S90-V102 ),β-MSH(D167-S183 ),γ-lipotrophic hormone (γ-LPH)(R132-E164 ),β-endorphin regions (Y186-Q219 ),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 allus 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.
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Keywords:
- Epinephelus coioides Hamilton /
- Proopiomelanocortin /
- cDNA cloning
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[1] Wei P.The research progresses on the regulation of pituitary and extrapituitary POMC gene expression [J].Foreign Medical Sciences: Section of Endocrinology,2001,21(1):39-41[魏平.垂体内外POMC基因表达调控研究进展[J].国外医学内分泌学分册,2001,21(1):39-41][2] Arends R J, Vermeer H, Martens G J, et al. Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.)[J].Mol Cell Endocrinol, 1998,143:23-31[3] Pritchard L E, Turnbull A V, White A. Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signaling and obesity [J].J Endocrinol,2002,172:411-421[4] Alrubaian J, Danielson P, Fitzpatrick M, et al. Cloning of a second proopiomelanocortin cDNA from the pituitary of the sturgeon,Acipenser transmontanus[J].Peptides,1999,20:431:-436[5] Amemiya Y, Takahashi A, Suzuki N, et al. 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Sturgeon proopiomelanocortin has a remnant of γ-elanotropin[J].Biochem Biophys Res Comm,1997,230:452-456[12] Dores R M, Smith T R,Marra L E, et al. Deciphering post-tractional processing events in the pituitary of a neopterygian fish: cloning of a gar proopiomelanocortin cDNA [J]. Gen Comp Endocrinol,1997,107:401-413[13] Kitahara N, Nishizawa T, Iida K, et al. Absence of a γ-melanocyte-stimulating hormone sequence in proopiomelancortin mRNA of chum salmon,Oncorhynchus keta[J].Biochem Biophys Res Comm,1988,91B:365-370[14] Okuta A. Ando H, Ueda H, et al. Two types of cDNA encoding proopiomelanocortin of sockeye salmon,Oncorhynchus nerka[J].Zool Sci,1996,13:421-427[15] Salbert G, Chauveau I, Bonnec G, et al. One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides [J].Mol Endocrinol,1992,6:1605-1612[16] Heining J A, Keeley F W, Robson P, et al. The appearance of proopiomelanocortin early in vertebrate evolution: cloning and sequencing of POMC from a lamprey pituitary cDNA library [J].Gen Comp Endocrinol,1995,99:137-144[17] Takahashi A, Amemiya Y,Sarashi M, et al. Melanoctropin and corticotropin are encoded on two distinct genes in the lamprey, the earliest evolved extant vertebrate [J].Biochem Biophys Res Comm,1995,231:490-496[18] Takahashi A, Amemiya Y, Nozaki M, et al. Evolutionary significance of proopiomelanocortin in agnatha and chondrichthyes [J].Comp Biochem Physiol,2001,129B:283-289 Wei P.The research progresses on the regulation of pituitary and extrapituitary POMC gene expression [J].Foreign Medical Sciences: Section of Endocrinology,2001,21(1):39-41[魏平.垂体内外POMC基因表达调控研究进展[J].国外医学内分泌学分册,2001,21(1):39-41][2] Arends R J, Vermeer H, Martens G J, et al. Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.)[J].Mol Cell Endocrinol, 1998,143:23-31[3] Pritchard L E, Turnbull A V, White A. Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signaling and obesity [J].J Endocrinol,2002,172:411-421[4] Alrubaian J, Danielson P, Fitzpatrick M, et al. Cloning of a second proopiomelanocortin cDNA from the pituitary of the sturgeon,Acipenser transmontanus[J].Peptides,1999,20:431:-436[5] Amemiya Y, Takahashi A, Suzuki N, et al. A newly characterized melanotropin in proopiomelanocortin in pituitaries of an elasmobranch, squalus acanthias[J].Gen Comp Endocrinol,1999,114:387-395[6] Amemiya Y, Takahashi A, Suzuki N, et al. Molecular cloning of proopiomelanocortin cDNA from an elasmobranch, the stingray,Dasyatis akajei[J].Gen Comp Endocrinol,2000,118:105-112[7] Douglass J, Civelli O, Herbert E. Polyprotein gene expression:generation of diversity of neuroendocrine peptides [J].Ann Rev Biochem,1984,53:665-715[8] Hilario E, Lihrmann I, Vaudry H. Characterization of cDNA encoding proopiomelanocortin in the frog Rana ridibunda[J].Biochem Biophys Res Comm,1990,173:653-659[9] Martens G J M. Expression of two proopiomelanocortin genes in the pituitary of Xenopus laevis: complete structure of the two preprohormone [J].Nucl Acids Res,1986,14:3791-3798[10] Pan F, Cheng W C. Nucleotide sequence of bullfrog proopiomelanocortin cDNA[J].Nucl Acids Res,1989,17:5843[11] Amemiya Y, Takahashi A, Dores R M, et al. Sturgeon proopiomelanocortin has a remnant of γ-elanotropin[J].Biochem Biophys Res Comm,1997,230:452-456[12] Dores R M, Smith T R,Marra L E, et al. Deciphering post-tractional processing events in the pituitary of a neopterygian fish: cloning of a gar proopiomelanocortin cDNA [J]. Gen Comp Endocrinol,1997,107:401-413[13] Kitahara N, Nishizawa T, Iida K, et al. Absence of a γ-melanocyte-stimulating hormone sequence in proopiomelancortin mRNA of chum salmon,Oncorhynchus keta[J].Biochem Biophys Res Comm,1988,91B:365-370[14] Okuta A. Ando H, Ueda H, et al. Two types of cDNA encoding proopiomelanocortin of sockeye salmon,Oncorhynchus nerka[J].Zool Sci,1996,13:421-427[15] Salbert G, Chauveau I, Bonnec G, et al. One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides [J].Mol Endocrinol,1992,6:1605-1612[16] Heining J A, Keeley F W, Robson P, et al. The appearance of proopiomelanocortin early in vertebrate evolution: cloning and sequencing of POMC from a lamprey pituitary cDNA library [J].Gen Comp Endocrinol,1995,99:137-144[17] Takahashi A, Amemiya Y,Sarashi M, et al. Melanoctropin and corticotropin are encoded on two distinct genes in the lamprey, the earliest evolved extant vertebrate [J].Biochem Biophys Res Comm,1995,231:490-496[18] Takahashi A, Amemiya Y, Nozaki M, et al. Evolutionary significance of proopiomelanocortin in agnatha and chondrichthyes [J].Comp Biochem Physiol,2001,129B:283-289
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