摘要斑马鱼(Danio rerio)中蝶啶代谢通路调控蝶啶色素合成，而GTP环化水解酶1基因(GTP cyclohydrolase 1, Gch1)是该代谢通路的限速酶。锦鲤(Cyprinus carpio var. koi)是一种被广泛养殖的观赏鱼，有多种的体色组合模式。为了研究锦鲤Gch1基因的功能、保守性及在不同体色的表达模式，本研究组装鲤(Cyprinus carpio)高通量转录组数据，并依此扩增获得锦鲤Gch1全长cDNA序列(GenBank登录号: KP056544)。该基因编码一个由251个氨基酸组成的蛋白。结构域分析发现，该蛋白含有GTP-cyclohydrolaseⅠ 功能域。Gene Ontology注释发现，该蛋白具有水解酶活性分子功能，并参与细胞内含氮化合物代谢和细胞内氨基酸代谢过程。多物种Gch1同源性比较发现，该基因较为保守。适应性进化分析发现，锦鲤与斑马鱼Gch1的非同义替换率(the number of nonsynonymous substitutions per non-synonymous site, Ka)与同义替换率(the number of synonymous substitutions per synonymous site, Ks)比值为0.06，说明该基因在进化中受到强烈负选择压。研究结果表明，锦鲤Gch1 参与蝶啶代谢通路；Gch1在红皮的表达水平显著高于白皮的(P＜0.05)，提示该基因高表达可能导致体色变红。本研究首次阐明Gch1在锦鲤红白体色间的表达差异，为进一步研究Gch1在锦鲤其他体色变化的作用机理提供了基础信息。另外，本研究还揭示Gch1在脊椎动物中的保守性，为研究其他观赏鱼类的体色变化机制提供借鉴。
Abstract：The pteridine pathway regulates the synthesis of yellow to reddish pteridine pigments in zebrafish (Danio rerio). GTP cyclohydrolase 1 (Gch1) is found as the rate-limiting enzyme of this pathway. Koi carp (Cyprinus carpio var. koi) is one of the most widespread cultured ornamental fish with various color patterns. In order to study the function, conservation and expression pattern of Gch1 in different colors of Koi carp, the present study amplified the full-length Gch1 cDNA (GenBank No. KP056544) based on the assembled transcriptome of common carp (Cyprinus carpio). The protein consistsed of 251 amino acids with a conserved GTP-cyclohydrolaseⅠdomain. Gene Ontology annotation indicated that Gch1 was involved in the hydrolase activity, nitrogen compound metabolic process and amino acid metabolic process. Homolog search found it's high conservation across vertebrates. Adaptive evolution analysis revealed that the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) of Gch1 between Koi carp and zebrafish was only 0.06, indicating that it was under negative selection. These results indicated that Gch1 participated in the pteridine pathway in Koi carp. Differential expression analysis revealed significantly higher level (P＜0.05) of Gch1 in red skin than in white skin, indicating that the high expression of Gch1 maight turn the skin color to red. This study revealed the different expression of Gch1 between red and white skin in Koi carp and found the conservation of Gch1 across vertebrates, which may be helpful to the study of pigmentation mechanism in other fish.
[1] Brown ME.The Physiology of Fishes: Behavior[M], Academic Press, 1957.
[2]Mellgren EM, Johnson SL.The evolution of morphological complexity in zebrafish stripes[J].TRENDS in Genetics, 2002, 18(3):128-34
[3] Braasch I, Brunet F, Volff J-N, Schartl M.Pigmentation pathway evolution after whole-genome duplication in fish.[J].Genome biology and evolution, 2009, (1):479-493
[4]Ceol CJ, Houvras Y, White RM, Zon LI.Melanoma biology and the promise of zebrafish[J].Zebrafish, 2008, 5(4):247-55
[5]Ziegler I.The pteridine pathway in zebrafish: regulation and specification during the determination of neural crest cell‐fate[J].Pigment cell research, 2003, 16(3):172-82
[6]Hoekstra H.Genetics,development and evolution of adaptive pigmentation in vertebrates[J].Heredity, 2006, 97(3):222-34
[7]Ziegler I, McDonaldo T, Hesslinger C, Pelletier I, Boyle P.Development of the pteridine pathway in the zebrafish,Danio rerio[J].Journal of Biological Chemistry, 2000, 275(25):18926-18932
[8]Henze M, Rempeters G, Anders F.Pteridines in the skin of xiphophorine fish ( Poeciliidae )[J].Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1977, 56(1):35-46
[9]Kim H, Kim K, Yim J.Biosynthesis of drosopterins,the red eye pigments of Drosophila melanogaster[J].IUBMB life, 2013, 65(4):334-340
[10]Crabtree MJ, Channon KM.Synthesis and recycling of tetrahydrobiopterin in endothelial function and vascular disease[J].Nitric Oxide, 2011, 25(2):81-88
[11]Chaisson MJ, Pevzner PA.Short read fragment assembly of bacterial genomes[J].Genome research, 2008, 18(2):324-330
[12]Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al.Primer3—new capabilities and interfaces[J].Nucleic acids research, 2012, 40(15):e115-e115
[13]Solovyev V, Kosarev P, Seledsov I, Vorobyev D.Automatic annotation of eukaryotic genes,pseudogenes and promoters[J].Genome Biol, 2006, 7(Suppl 1):S10.1-S10.2
[14]Geourjon C, Deleage G.SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Computer applications in the biosciences: CABIOS, 1995, 11(6):681-684
[15]Zdobnov EM, Apweiler R.InterProScan–an integration platform for the signature-recognition methods in InterPro[J].Bioinformatics, 2001, 17(9):847-848
[16]Hulo N, Bairoch A, Bulliard V, Cerutti L, De Castro E, Langendijk-Genevaux PS, et al.The PROSITE database[J].Nucleic acids research, 2006, 34(suppl 1):D227-D230
[17]Kent WJ.BLAT—the BLAST-like alignment tool[J].Genome research, 2002, 12(4):656-664
[18]Suyama M, Torrents D, Bork P.PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments[J].Nucleic acids research, 2006, 34(suppl 2):W609-W612
[19]Schmittgen TD, Livak KJ.Analyzing real-time PCR data by the comparative CT method[J].Nature protocols, 2008, 3(6):1101-1108
[20]Conesa A, G?tz S, García-Gómez JM, Terol J, Talón M, Robles M.Blast2GO: a universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics, 2005, 21(18):3674-3676
[21]Serova L, Nankova B, Rivkin M, Kvetnansky R, Sabban EL.Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells[J].Molecular brain research, 1997, 48(2):251-258
[22]Adlam D, Herring N, Douglas G, De Bono JP, Li D, Danson EJ, et al.Regulation of β-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin[J].Cardiovascular research, 2012, 93(4):694-701
[23] McNeill E, Crabtree MJ, Sahgal N, Patel J, Chuaiphichai S, Iqbal AJ, et al.Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation[J].Free Radical Biology and Medicine, 2014, :-
