中国大鲵Cyp51基因特征与性腺表达分析
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摘要
采用RT-PCR法从大鲵(Andrias davidianus)性腺中分离获得Cyp51基因全长cDNA 1 975 bp, 5′端非编码区135 bp, 3′端非编码区331 bp,开放阅读框(ORF)1 509 bp,编码503氨基酸。生物信息学分析显示,Cyp51基因编码蛋白的二级结构含有50.20%的α-螺旋,9.96%的延伸链,3.59%的β-转角,36.25%的无规则卷曲。系统进化树显示,大鲵与两栖类中的热带爪蟾和非洲爪蟾同源性最高,与其它物种也有较高的同源性。qRT-PCR分析结果显示,Cyp51基因在卵巢、肝、肾等组织表达水平显著性高于其它各组织;性腺发育不同时期表达结果显示,Cyp51基因在1-5Y卵巢中表达显著性高于精巢,在6Y时无显著性差异;在雌性与伪雌性性腺中表达水平显著高于雄性及伪雄性性腺。结果表明Cyp51基因对大鲵的性腺分化,尤其是卵巢发育有着重要作用。
Full length of the Cyp51 cDNA sequence was isolated employing RT-PCR technology with total of 1 975 bp containing 5′UTR 135 bp noncoding region, 3′UTR 331 bp noncoding region and open reading frame(ORF) 1 509 bp encoding 503 aa. Bioinformatics analysis displayed that CYP51 protein secondary structure included 50.20% α-helix, 9.96% extension chain, 3.59% beta-turn and 36.25% random coil. The phylogenetic tree showed that Andrias davidianus had the highest homology with Xenopus laevis and X. tropicalis in amphibians and extensive homology with other species. The expression of Cyp51 exhibited significantly high expression level, which was observed in the ovary, liver and kidney. The low expressions were observed in other tissues. The expression pattern in developing gonad showed that Cyp51 had a significant higher level in ovary than that in testis during 1-5 Y, however, there was no significant difference in 6 Y. Additionally, Cyp51 expression level was significantly higher in ovary of normal or sex reversal female individuals than that in testis of normal or sex reversal male individuals. The current results indicated that Cyp51 plays a vital role in gonadal differentiation in Chinese giant salamander, especially in ovary development.
Full length of the Cyp51 cDNA sequence was isolated employing RT-PCR technology with total of 1 975 bp containing 5′UTR 135 bp noncoding region, 3′UTR 331 bp noncoding region and open reading frame(ORF) 1 509 bp encoding 503 aa. Bioinformatics analysis displayed that CYP51 protein secondary structure included 50.20% α-helix, 9.96% extension chain, 3.59% beta-turn and 36.25% random coil. The phylogenetic tree showed that Andrias davidianus had the highest homology with Xenopus laevis and X. tropicalis in amphibians and extensive homology with other species. The expression of Cyp51 exhibited significantly high expression level, which was observed in the ovary, liver and kidney. The low expressions were observed in other tissues. The expression pattern in developing gonad showed that Cyp51 had a significant higher level in ovary than that in testis during 1-5 Y, however, there was no significant difference in 6 Y. Additionally, Cyp51 expression level was significantly higher in ovary of normal or sex reversal female individuals than that in testis of normal or sex reversal male individuals. The current results indicated that Cyp51 plays a vital role in gonadal differentiation in Chinese giant salamander, especially in ovary development.
引文
[1]Lepesheva G I,Waterman M R.Sterol 14 alpha-demethylase cytochrome P450(CYP51),a P450 in all biological kingdoms [J].Biochim Biophys Acta,2007,1770(3):467-477.
[2] Lepesheva G I,Waterman M R.Structural basis for conservation in the CYP51 family [J].Biochim Biophys Acta,2011,1814(1):88-93.
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[5] Kahn R A,Bak S,Olsen C E,et al.Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14alpha-demethylase from Sorghum bicolor(L.) Moench [J].J Biol Chem,1996,271(51):32944-32950.
[6] Bellamine A,Mangla A T,Nes W D,et al.Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis [J].Proc Natl Acad Sci U S A,1999,96(16):8937-8942.
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[9] Byskov A G,Andersen C Y,Nordholm L,et al.Chemical structure of sterols that activate oocyte meiosis [J].Nature,1995,374(6522):559-562.
[10] Ning G,Ouyang H,Wang S,et al.3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation [J].Mol Endocrinol,2008,22(7):1682-1694.
[11] Zhang H,Xu B,Xie H,et al.Lanosterol metabolic product(s) is involved in primordial folliculogenesis and establishment of primordial folliclepool in mouse fetal ovary [J].Mol Reprod Dev,2009,76(5):514-521.
[12] Xie H,Xia G,Byskov A G,et al.Roles of gonadotropins and meiosis-activating sterols in meiotic resumption of cultured follicle-enclosed mouse oocytes [J].Mol Cell Endocrinol,2004,218(1-2):155-163.
[13]肖汉兵,刘鉴毅,林锡芝,等.养殖条件下大鲵性腺周年变化的研究[J].淡水渔业,1995,(3):9-11.
[14]方耀林,张燕,杨焱清,等.大鲵遗传多样性分析[J].淡水渔业,2006,36(6):8-11.
[15] Hu Q M,Tian H F,Li W,et al.Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome [J].Mol Genet Genomics,2019,294(2):287-299.
[16] Debeljak N,Fink M,Rozman D.Many facets of mammalian lanosterol 14alpha-demethylase from the evolutionarily conserved cytochrome P450 family CYP51 [J].Arch Biochem Biophys,2003,409(1):159-171.
[17] Nitahara Y,Kishimoto K,Yabusaki Y,et al.The amino acid residues affecting the activity and azole susceptibility of rat CYP51(sterol 14-demethylase P450) [J].J Biochem,2001,129(5):761-768.
[18] Munro S,Nichols B J.The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins [J].Curr Biol,1999,9(7):377-380.
[19] Horinouchi S,Kito M,Nishiyama M,et al.Primary structure of AfsR,a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2) [J].Gene,1990,95(1):49-56.
[20] Wang F,Shen Y,Song X,et al.cDNA cloning,genomic structure and expression analysis of the bovine lanosterol 14α-Demethylase(CYP51) in Gonads [J].Biol Pharm Bull,2006,29(3):430-436.
[21] Kojima M,Morozumi T,Onishi A,et al.Structure of the pig sterol 14a-demethylase(CYP51) gene and its expression in the testis and other tissues [J].J Biochem(Tokyo),2000,127(5):805-811.
[22] Debeljak N,Horvat S,Vouk K,et al.Characterization of the mouse lanosterol 14α-demethylase(CYP51),a new member of the evolutionarily most conserved cytochrome P450 family [J].Arch Biochem Biophys,2000,379(1):37-45.
[23] Kojima M,Tagami T,Degawa M.Cloning of chicken lanosterol 14alpha-demethylase(CYP51) cDNA:discovery of a testis-specific CYP51 transcript [J].Comp Biochem Physiol A Mol Integr Physiol,2006,145(3):383-389.
[24] Morrison A M,Goldstone J V,Lamb D C,et al.Identification,modeling and ligand affinity of early deuterostome CYP51s,and functional characterization of recombinant zebrafish sterol 14α-demethylase [J].Biochim Biophys Acta,2014,1840(6):1825-1836.
[25] Keber R,Acimovic J,Majdic G,et al.Male germ cell-specific knockout of cholesterogenic cytochrome P450 lanosterol 14α-demethylase(Cyp51) [J].J Lipid Res,2013,54(6):1653-1661.
[26] Wang C,Xu B,Zhou B,et al.Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro [J].J Lipid Res,2009,50(11):2164-2172.
[27]陈孝红.斑马鱼几种CYP基因在性腺不同发育时期的表达研究[D].大连:大连海洋大学,2014.
[2] Lepesheva G I,Waterman M R.Structural basis for conservation in the CYP51 family [J].Biochim Biophys Acta,2011,1814(1):88-93.
[3] Aoyama Y,Funae Y,Noshiro M,et al.Occurrence of a P450 showing high homology to yeast lannosterol 14-demethylase(P450 14DM) in the rat liver [J].Biochem Biophys Res Commun,1994,201(3):1320-1326.
[4] Aoyama Y,Noshiro M,Gotoh O,et al.Sterol 14-demethylase P450(P45014DM) is one of the most ancient and conserved P450 species [J].J Biochem,1996,119(5):926-933.
[5] Kahn R A,Bak S,Olsen C E,et al.Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14alpha-demethylase from Sorghum bicolor(L.) Moench [J].J Biol Chem,1996,271(51):32944-32950.
[6] Bellamine A,Mangla A T,Nes W D,et al.Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis [J].Proc Natl Acad Sci U S A,1999,96(16):8937-8942.
[7] Yoshida Y,Noshiro M,Aoyama Y,et al.P450(CYP51),the most conserved and gene structures.Structural and evolutionary studies on sterol 14-demethylase P450 monooxygenase:II.Evolutionary analysis of protein [J].J Biochem,1997,122(6):1122-1128.
[8] Xia G L,Byskov A G,Andersen C Y.Cumulus cells secrete a meiosis-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate [J].Mol Reprod Dev,1994,39(1):17-24.
[9] Byskov A G,Andersen C Y,Nordholm L,et al.Chemical structure of sterols that activate oocyte meiosis [J].Nature,1995,374(6522):559-562.
[10] Ning G,Ouyang H,Wang S,et al.3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation [J].Mol Endocrinol,2008,22(7):1682-1694.
[11] Zhang H,Xu B,Xie H,et al.Lanosterol metabolic product(s) is involved in primordial folliculogenesis and establishment of primordial folliclepool in mouse fetal ovary [J].Mol Reprod Dev,2009,76(5):514-521.
[12] Xie H,Xia G,Byskov A G,et al.Roles of gonadotropins and meiosis-activating sterols in meiotic resumption of cultured follicle-enclosed mouse oocytes [J].Mol Cell Endocrinol,2004,218(1-2):155-163.
[13]肖汉兵,刘鉴毅,林锡芝,等.养殖条件下大鲵性腺周年变化的研究[J].淡水渔业,1995,(3):9-11.
[14]方耀林,张燕,杨焱清,等.大鲵遗传多样性分析[J].淡水渔业,2006,36(6):8-11.
[15] Hu Q M,Tian H F,Li W,et al.Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome [J].Mol Genet Genomics,2019,294(2):287-299.
[16] Debeljak N,Fink M,Rozman D.Many facets of mammalian lanosterol 14alpha-demethylase from the evolutionarily conserved cytochrome P450 family CYP51 [J].Arch Biochem Biophys,2003,409(1):159-171.
[17] Nitahara Y,Kishimoto K,Yabusaki Y,et al.The amino acid residues affecting the activity and azole susceptibility of rat CYP51(sterol 14-demethylase P450) [J].J Biochem,2001,129(5):761-768.
[18] Munro S,Nichols B J.The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins [J].Curr Biol,1999,9(7):377-380.
[19] Horinouchi S,Kito M,Nishiyama M,et al.Primary structure of AfsR,a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2) [J].Gene,1990,95(1):49-56.
[20] Wang F,Shen Y,Song X,et al.cDNA cloning,genomic structure and expression analysis of the bovine lanosterol 14α-Demethylase(CYP51) in Gonads [J].Biol Pharm Bull,2006,29(3):430-436.
[21] Kojima M,Morozumi T,Onishi A,et al.Structure of the pig sterol 14a-demethylase(CYP51) gene and its expression in the testis and other tissues [J].J Biochem(Tokyo),2000,127(5):805-811.
[22] Debeljak N,Horvat S,Vouk K,et al.Characterization of the mouse lanosterol 14α-demethylase(CYP51),a new member of the evolutionarily most conserved cytochrome P450 family [J].Arch Biochem Biophys,2000,379(1):37-45.
[23] Kojima M,Tagami T,Degawa M.Cloning of chicken lanosterol 14alpha-demethylase(CYP51) cDNA:discovery of a testis-specific CYP51 transcript [J].Comp Biochem Physiol A Mol Integr Physiol,2006,145(3):383-389.
[24] Morrison A M,Goldstone J V,Lamb D C,et al.Identification,modeling and ligand affinity of early deuterostome CYP51s,and functional characterization of recombinant zebrafish sterol 14α-demethylase [J].Biochim Biophys Acta,2014,1840(6):1825-1836.
[25] Keber R,Acimovic J,Majdic G,et al.Male germ cell-specific knockout of cholesterogenic cytochrome P450 lanosterol 14α-demethylase(Cyp51) [J].J Lipid Res,2013,54(6):1653-1661.
[26] Wang C,Xu B,Zhou B,et al.Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro [J].J Lipid Res,2009,50(11):2164-2172.
[27]陈孝红.斑马鱼几种CYP基因在性腺不同发育时期的表达研究[D].大连:大连海洋大学,2014.