中华鳖GnRH 1基因cDNA克隆及表达特征
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摘要
为了解GnRH基因在中华鳖(Pelodiscus sinensis)性腺和胚胎发育过程中的表达特征,采用cDNA末端快速扩增(RACE)技术从中华鳖全脑中获得与生长生殖调控密切相关的GnRH 1基因全长cDNA,并运用实时荧光定量PCR(qRT-PCR)技术检测GnRH 1在成鳖不同组织和胚胎发育时期的表达水平。结果显示:中华鳖GnRH 1基因cDNA全长546 bp,其中5′非编码区(5′UTR)99 bp,3′非编码区(3′UTR)168 bp,开放阅读框(ORF)279 bp,编码92个氨基酸,分子质量为10.23 ku,理论等电点pI为5.65,具有N端信号肽(1～23 aa)、核心十肽区域(24～33 aa)、断裂位点GKR(34～36 aa)及相关肽区域(37～92 aa),符合GnRH蛋白典型结构特征。系统进化树结果显示,中华鳖GnRH 1基因和绿海龟(Chelonia mydas)、墨西哥箱龟(Terrapene carolina mexicana)及西部锦龟(Chrysemys picta bellii)GnRH 1基因聚为一支。qRT-PCR结果表明,GnRH 1基因在中华鳖雌雄个体的8个组织中均有表达,在脑和性腺组织中高表达,且具有性别差异,雄性中华鳖中的表达显著高于雌性(P<0.05);在10个胚胎发育时期均表达,且随发育时间的后移,表达量显著增加,在第16期达到峰值。GnRH 1基因可能在中华鳖生长及性腺分化中具有重要作用。
Gonadotropin releasing hormone(GnRH)is an important regulator of reproductive development in animals.The GnRH 1 cDNA were cloned by rapid amplification cDNA ends(RACE)and the expression characteristics of the gene by Quantitative real-time PCR(qRT-PCR)were analyzed to analyze features and functions of the GnRH 1 gene in the gonad developmen of Pelodiscus sinensis.The results showed that the full length of GnRH 1 cDNA sequence consisted of 546 bp,which contained 99 bp 5′ untranslated region(UTR),169 bp 3′ UTR,and an open reading frame of 279 bp,encoded a protein with 92 amino acids.GnRH 1 included a putative signal peptide(1～23 aa),a core decapeptide region(24～33 aa),a cleavage site GKR(34～36 aa)and GnRH associated peptide(GAP)(37～92 aa).Phylogenetic analysis showed that the GnRH 1 gene was clustered in the same subgroup with the reptile.qRT-PCR results indicated that the GnRH 1 gene were expressed widely in all 8 tissues in adults,the highest expression level was detected in the brain and gonad.The expression pattern exhibited sexual dimorphism.The expression level of GnRH 1 were significantly higher in male(P<0.05).The highest expression was found in the sixteenth period of embryo development.Results indicated that the GnRH 1 gene might play an important role in the growth and gonadal development of P.sinensis.
Gonadotropin releasing hormone(GnRH)is an important regulator of reproductive development in animals.The GnRH 1 cDNA were cloned by rapid amplification cDNA ends(RACE)and the expression characteristics of the gene by Quantitative real-time PCR(qRT-PCR)were analyzed to analyze features and functions of the GnRH 1 gene in the gonad developmen of Pelodiscus sinensis.The results showed that the full length of GnRH 1 cDNA sequence consisted of 546 bp,which contained 99 bp 5′ untranslated region(UTR),169 bp 3′ UTR,and an open reading frame of 279 bp,encoded a protein with 92 amino acids.GnRH 1 included a putative signal peptide(1～23 aa),a core decapeptide region(24～33 aa),a cleavage site GKR(34～36 aa)and GnRH associated peptide(GAP)(37～92 aa).Phylogenetic analysis showed that the GnRH 1 gene was clustered in the same subgroup with the reptile.qRT-PCR results indicated that the GnRH 1 gene were expressed widely in all 8 tissues in adults,the highest expression level was detected in the brain and gonad.The expression pattern exhibited sexual dimorphism.The expression level of GnRH 1 were significantly higher in male(P<0.05).The highest expression was found in the sixteenth period of embryo development.Results indicated that the GnRH 1 gene might play an important role in the growth and gonadal development of P.sinensis.
引文
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[12] Zhang T,Zhang G X,Han K P.Molecular cloning and characterization,and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken [J].Genet Mol Res,2015,14:2831-2849.
[13] Ohkubo M,Aranishi F,Shimizu A.Molecular cloning and brain distribution of three types of gonadotropin‐releasing hormone from mummichog Fundulus heteroclitus [J].J Fish Biol,2010,76(2):379-394.
[14] Amano M,Takahashi A,Yamanome T,et al.Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder [J].Gen Comp Endocrinol,2002,126(3):319-325.
[15]柳学周,徐永江,廖梅杰,等.圆斑星鲽促性腺激素释放激素基因克隆及表达特性[J].中国水产科学,2013,20(01):12-24.
[16]于久翔.美洲鲥鱼GnRH基因的克隆与表达分析[D].辽宁大连:大连海洋大学,2017:1-51.
[17]周晓苏.许氏平鲉和半滑舌鳎促性腺激素释放激素(GnRH)基因的克隆和表达分析[D].山东青岛:中国海洋大学,2012:1-105.
[18] Swapna I,Sudhakumari C C,Sakai F,et al.Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia,Oreochromis niloticus during early development [J].J Exp Zool Part A:Ecol Genet Physiol,2008,309A(7):419-426.
[19]马细兰,张勇,周立斌,等.LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响[J].水生生物学报,2013,37(1):42-47.
[20]邓利,林浩然.腹腔注射LHRH-A对黑鲷生长激素及其受体的影响[J].深圳大学学报(理工版),2003,(2):60-65.
[21]林信伟,林浩然,张庆.促性腺激素释放激素类似物促进鱼类生长激素分泌和生长[J].水产学报,1993,(4):282-288.
[22]李慧君.中华鳖性腺分化及温度对其影响[D].河北保定:河北大学,2012:1-47.
[23] Powell J F,Zohar Y,Elizur A,et al.Three forms of gonadotropin-releasing hormone characterized from brains of one species [J].Proc Natl Acad Sci USA,1994,91(25):12081-12085.
[24] Andersson E,Fjelldal P G,Klenke U,et al.Three forms of GnRH in the brain and pituitary of the turbot,Scophthalmus maximus:immunological characterization and seasonal variation [J].Comp Biochem Physiol Part B,2001,129(2):551-558.
[25] Holland M C,Hassin S,Zohar Y.Seasonal fluctuations in pituitary levels of the three forms of gonadotropin-releasing hormone in striped bass,Morone saxatilis(Teleostei),during juvenile and pubertal development [J].J Endocrinol,2001,169(3):527-538.
[2] Bromage N,Porter M,Randall C.The environmental regulation of maturation in farmed finfish with special reference to the role of photoperiod and melatonin [J].Aquaculture,2001,197(1-4):63-98.
[3] Shahjahan M,Hamabata T,Motohashi E,et al.Differential expression of three types of gonadotropin-releasing hormone genes during the spawning season in grass puffer,Takifugu niphobles [J].Gen Comp Endocrinol,2010,167(1):153-163.
[4] Herve T.Evolution of the gonadotropin-releasing hormone(GnRH)gene family in relation to vertebrate tetraploidizations [J].Gen Comp Endocrinol,2011,170(3):575-581.
[5] Fernald R D,White R B.Gonadotropin-releasing hormone genes:phylogeny,structure,and functions [J].Front Neuroendocrinol,1999,20(3):224-240.
[6] Okubo K,Nagahama Y.Structural and functional evolution of gonadotropin-releasing hormone in vertebrates [J].Acta Physiol,2008,193(1):3-15.
[7]田儒品,张海琪,许晓军,等.中华鳖(Pelodiscus sinensis)MITF基因的生物信息学及表达差异研究[J].海洋与湖沼,2018,49(03):662-670.
[8]农业部渔业渔政管理局.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017:11.
[9]梁宏伟,曹力欢,李翔,等.三个不同品系中华鳖形态差异分析[J].淡水渔业,2017,47(4):91-96.
[10] Tokita M,Kuratani S.Normal embryonic stages of the Chinese softshelled turtle Pelodiscus sinensis(Trionychidae)[J].Zool Sci,2001,18(5):705-715.
[11]刘文婷,张贝贝,张耀红,等.中华鳖实时定量PCR内参基因的筛选与评价[J].内蒙古大学学报(自然科学版),2017,48(05):531-537.
[12] Zhang T,Zhang G X,Han K P.Molecular cloning and characterization,and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken [J].Genet Mol Res,2015,14:2831-2849.
[13] Ohkubo M,Aranishi F,Shimizu A.Molecular cloning and brain distribution of three types of gonadotropin‐releasing hormone from mummichog Fundulus heteroclitus [J].J Fish Biol,2010,76(2):379-394.
[14] Amano M,Takahashi A,Yamanome T,et al.Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder [J].Gen Comp Endocrinol,2002,126(3):319-325.
[15]柳学周,徐永江,廖梅杰,等.圆斑星鲽促性腺激素释放激素基因克隆及表达特性[J].中国水产科学,2013,20(01):12-24.
[16]于久翔.美洲鲥鱼GnRH基因的克隆与表达分析[D].辽宁大连:大连海洋大学,2017:1-51.
[17]周晓苏.许氏平鲉和半滑舌鳎促性腺激素释放激素(GnRH)基因的克隆和表达分析[D].山东青岛:中国海洋大学,2012:1-105.
[18] Swapna I,Sudhakumari C C,Sakai F,et al.Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia,Oreochromis niloticus during early development [J].J Exp Zool Part A:Ecol Genet Physiol,2008,309A(7):419-426.
[19]马细兰,张勇,周立斌,等.LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响[J].水生生物学报,2013,37(1):42-47.
[20]邓利,林浩然.腹腔注射LHRH-A对黑鲷生长激素及其受体的影响[J].深圳大学学报(理工版),2003,(2):60-65.
[21]林信伟,林浩然,张庆.促性腺激素释放激素类似物促进鱼类生长激素分泌和生长[J].水产学报,1993,(4):282-288.
[22]李慧君.中华鳖性腺分化及温度对其影响[D].河北保定:河北大学,2012:1-47.
[23] Powell J F,Zohar Y,Elizur A,et al.Three forms of gonadotropin-releasing hormone characterized from brains of one species [J].Proc Natl Acad Sci USA,1994,91(25):12081-12085.
[24] Andersson E,Fjelldal P G,Klenke U,et al.Three forms of GnRH in the brain and pituitary of the turbot,Scophthalmus maximus:immunological characterization and seasonal variation [J].Comp Biochem Physiol Part B,2001,129(2):551-558.
[25] Holland M C,Hassin S,Zohar Y.Seasonal fluctuations in pituitary levels of the three forms of gonadotropin-releasing hormone in striped bass,Morone saxatilis(Teleostei),during juvenile and pubertal development [J].J Endocrinol,2001,169(3):527-538.