金钱鱼Amhr2基因的克隆及表达分析
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摘要
【目的】Amh(anti-Müllerian hormone)与其特异的Ⅱ型受体Amhr2共同参与脊椎动物性腺发育过程,本研究旨在了解金钱鱼Amhr2在性腺发育中的作用。【方法】克隆金钱鱼(Scatophagus argus)Amhr2,采用逆转录PCR和实时荧光定量PCR分别检测Amhr2的组织分布及其卵巢发育过程中的表达。【结果】金钱鱼Amhr2的开放阅读框(ORF)全长为1 533 bp,编码510个氨基酸。氨基酸序列分析表明,金钱鱼Amhr2 N端有信号肽、跨膜螺旋区和配体结合结构域。金钱鱼Amhr2与欧洲海鲈(Dicentrarchus labrax)的相似性最高,为73.1%,与人类的相似性最低,为23.2%。金钱鱼与欧洲海鲈和大黄鱼(Larimichthys crocea)亲缘关系最近,与进化地位一致,说明金钱鱼Amhr2与其他物种具有一定保守性。金钱鱼Amhr2主要在性腺表达,且精巢表达高于卵巢。Amhr2在Ⅱ时相卵巢中的表达显著高于ⅡI和IV时相卵巢。【结论】金钱鱼Amhr2序列与鲈形目同源性高,在雌雄性腺均发挥作用。
【Objectives】The spotted scat(Scatophagus argus) Amh(anti-Müllerian hormone)and its specific type Ⅱ receptor Amhr2(anti-Müllerian hormone receptor type Ⅱ) are both involved in gonad development in the vertebrates. However, the gonadal function of Amhr2 is still deficient. 【Method】In this study, we have cloned spotted scat Amhr2,and anlyzed its tissue distribution and expression profiles at different ovary development stages by RT-PCR and quantitative Real-time quantitative q PCR, respectively. 【Result】The spotted scat Amhr2 full-length ORF is 1533 bp and encodes 510 amino acids. Sequence analysis revealed Amhr2 consists of a signal peptide, a transmembrane domain and a ligand banding domain. Multiple sequence alignment showed that Amhr2 was highly homologous to Amhr2 from Dicentrarchus labrax(73.1%), but was only slightly homologous to human(Homo sapiens)(23.2%) at amino acid level. Phylogenetic analysis revealed that Amhr2 are closest to Dicentrarchus labrax and Larimichthys crocea, which is consistent with the taxology indicating the conversation of Amhr2. In adult spotted scat, Amhr2 is mainly expressed in the gonads, and expressed relatively higher in the testis than the ovary. In female fish, Amhr2 was expressed significantly higher in phase Ⅱ ovary than those of phase ⅡI and IV ovaries. 【Conclusion】Spotted scat Amhr2 has a higher homology with that of the Perciformes, and may playsan important role in gonads development of the fish.
【Objectives】The spotted scat(Scatophagus argus) Amh(anti-Müllerian hormone)and its specific type Ⅱ receptor Amhr2(anti-Müllerian hormone receptor type Ⅱ) are both involved in gonad development in the vertebrates. However, the gonadal function of Amhr2 is still deficient. 【Method】In this study, we have cloned spotted scat Amhr2,and anlyzed its tissue distribution and expression profiles at different ovary development stages by RT-PCR and quantitative Real-time quantitative q PCR, respectively. 【Result】The spotted scat Amhr2 full-length ORF is 1533 bp and encodes 510 amino acids. Sequence analysis revealed Amhr2 consists of a signal peptide, a transmembrane domain and a ligand banding domain. Multiple sequence alignment showed that Amhr2 was highly homologous to Amhr2 from Dicentrarchus labrax(73.1%), but was only slightly homologous to human(Homo sapiens)(23.2%) at amino acid level. Phylogenetic analysis revealed that Amhr2 are closest to Dicentrarchus labrax and Larimichthys crocea, which is consistent with the taxology indicating the conversation of Amhr2. In adult spotted scat, Amhr2 is mainly expressed in the gonads, and expressed relatively higher in the testis than the ovary. In female fish, Amhr2 was expressed significantly higher in phase Ⅱ ovary than those of phase ⅡI and IV ovaries. 【Conclusion】Spotted scat Amhr2 has a higher homology with that of the Perciformes, and may playsan important role in gonads development of the fish.
引文
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[21]兰国宝,阎冰,廖思明,等.金钱鱼生物学研究及回顾[J].水产科学,2005,24(7):39-41.
[22]蔡泽平,胡家玮,王毅,金钱鱼早期发育的观察[J].热带海洋学报,2014,33(4):20-25.
[23]蔡泽平,王毅,胡家玮,等.金钱鱼的繁殖生物学及诱导产卵试验[J].热带海洋学报,2010,29(5):180-185.
[24]LI G L,ZHANG M Z,DENG,S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 m RNA expression in spotted scat Scatophagus argus[J].Journal of Fish Biology,2015,86(1):248-260.
[25]崔丹,刘志伟,刘南希,等.金钱鱼性腺发育及其组织结构观察[J].水产学报,2013,37(5):696-704.
[26]曾文刚,刘振浩,李红,等.金钱鱼抗缪勒氏管激素基因克隆及其在性腺发育不同时期m RNA表达水平的分析[J].水产学报,2015,39(11):1604-1612.
[27]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[28]CUI X F,ZHAO Y,CHEN H P,et al.Cloning,expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus[J].General and Comparative Endocrinology,2017,246:37-45.
[29]YANG W,CHEN H P,CUI X F,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus,(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[30]唐永凯,李建林,俞菊华,等.奥利亚罗非鱼AMH基因结构及其表达[J].水产学报,2009,33(3):379-388.
[31]赵九娥.Amhy/Amh及其受体AmhrⅡ对尼罗罗非鱼雄性性别的决定作用[D].重庆:西南大学,2015.
[32]CIMINO I,CASONI F,LIU X,et al.Novel role for anti-Müllerian hormone in the regulation of Gn RH neuron excitability and hormone secretion[J].Nature Communications,2016,7:10055.
[33]ROCHA A,ZANUY S,GóMEZ A.Conserved anti-Müllerian hormone:Anti-Müllerian hormone type-2receptor specific interaction and intracellular signaling in teleosts.[J].Biology of Reproduction,2016,94(6):1-13.
[2]BEHRINGER R R,CATE R L,FROELICK G J,et al.Abnormal sexual development in transgenic mice chronically expressing Müllerian inhibiting substance[J].Nature,1990,345(6271):167-170.
[3]BEHRINGER R R,FINEGOLD M J,CATE R L.Müllerian-inhibiting substance function during mammalian sexual development[J].Cell,1994,79(3):415-425.
[4]JAMIN S P,ARANGO N A,MISHINA Y,et al.Requirement of Bmpr1a for Müllerian duct regression during male sexual development[J].Nature Genetics,2002,32(3):408-410.
[5]JOSSO N,BELVILLE C,DI CLEMENTE,N,et al.AMH and AMH receptor defects in persistent Müllerian duct syndrome[J].Human Reproduction Update,2005,11(4):351-356.
[6]BAARENDS W M,HOOGERBRUGGE J W,POST M,et al.Anti-Müllerian hormone and Anti-Müllerian hormone type II receptor messenger ribonucleic acid expression during postnatal testis development and in the adult testis of the rat[J].Endocrinology,1995,136(12):5614-5622.
[7]TEIXEIRA J,HE W W,SHAH P C,et al.Developmental expression of a candidate Müllerian inhibiting substance type II receptor[J].Endocrinology,1996,137(1):160-165.
[8]JOSSO N,RACINE C,DI CLEMENTE N,et al.The role of anti-Müllerian hormone in gonadal development[J].Molecular and Cellular Endocrinology,1998,145(1/2):3-7.
[9]VIGIER B,FOREST M G,EYCHENNE B,et al.Anti-Müllerian hormone produces endocrine sex reversal of fetal ovaries[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(10):3684-3688.
[10]RONESS H,GAVISH Z,COHEN Y,et al.Ovarian follicle burnout:A universal phenomenon[J].Cell Cycle,2013,12(20):3245-3246.
[11]刘艳飞,施新颜,卢红梅,等.抗缪勒氏管激素及其在预测更年期应用的研究进展[J].中国卫生检验杂志,2016,26(15):2278-2280.
[12]KLüVER N,PFENNIG F,PALA I,et al.Differential expression of anti-Müllerian hormone(amh)and anti-Müllerian hormone receptor type II(amhr II)in the teleost Medaka[J].Developmental Dynamics,2007,236(1):271-281.
[13]WU G C,CHIU P C,LYU Y S,et al.The expression of amh and amhr2 is associated with the development of gonadal tissue and sex change in the protandrous black porgy,Acanthopagrus schlegeli[J].Biology of Reproduction 2010,83(3):443-453.
[14]SKAAR K S,NóBREGA R H,MAGARAKI A,et al.Proteolytically activated,recombinant Anti-Müllerianhormone inhibits androgen secretion,proliferation,and differentiation of spermatogonia in adult zebrafish testis organ cultures[J].Endocrinology,2011,152(9):3527-3540.
[15]HATTORI R S,MURAI Y,OURA M,et al.A Y-linked anti-Müllerian hormone duplication takes over a critical role in sex determination[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(8):2955-2959.
[16]LI M,SUN Y,ZHAO J,et al.A tandem duplicate of Anti-Müllerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in nile tilapia,Oreochromis niloticus[J].PLo S Genetics,2015,11(11):e1005678.
[17]BEJ D K,MIYOSHI K,HATTORI R S,et al.A duplicated,truncated amh gene is involved in male sex determination in an old world silverside[J].G3(Bethesda,Md.),2017,7(8):2489-2495.
[18]KAMIYA T,KAI W,TASUMI S,et al.A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish,Takifugu rubripes(Fugu)[J].PLo S Genetics,2012,8(7):e1002798.
[19]MORINAGA C,SAITO D,NAKAMURA S,et al.The hotei Mutation of Medaka in the anti-Müllerian Hormone Receptor Causes the Dysregulation of Germ Cell and Sexual Development[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(23):9691-9696.
[20]NAKAMURA S,WATAKABE I,NISHIMURA T,et al.Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka[J].Development,2012,139(13):2283-2287.
[21]兰国宝,阎冰,廖思明,等.金钱鱼生物学研究及回顾[J].水产科学,2005,24(7):39-41.
[22]蔡泽平,胡家玮,王毅,金钱鱼早期发育的观察[J].热带海洋学报,2014,33(4):20-25.
[23]蔡泽平,王毅,胡家玮,等.金钱鱼的繁殖生物学及诱导产卵试验[J].热带海洋学报,2010,29(5):180-185.
[24]LI G L,ZHANG M Z,DENG,S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 m RNA expression in spotted scat Scatophagus argus[J].Journal of Fish Biology,2015,86(1):248-260.
[25]崔丹,刘志伟,刘南希,等.金钱鱼性腺发育及其组织结构观察[J].水产学报,2013,37(5):696-704.
[26]曾文刚,刘振浩,李红,等.金钱鱼抗缪勒氏管激素基因克隆及其在性腺发育不同时期m RNA表达水平的分析[J].水产学报,2015,39(11):1604-1612.
[27]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[28]CUI X F,ZHAO Y,CHEN H P,et al.Cloning,expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus[J].General and Comparative Endocrinology,2017,246:37-45.
[29]YANG W,CHEN H P,CUI X F,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus,(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[30]唐永凯,李建林,俞菊华,等.奥利亚罗非鱼AMH基因结构及其表达[J].水产学报,2009,33(3):379-388.
[31]赵九娥.Amhy/Amh及其受体AmhrⅡ对尼罗罗非鱼雄性性别的决定作用[D].重庆:西南大学,2015.
[32]CIMINO I,CASONI F,LIU X,et al.Novel role for anti-Müllerian hormone in the regulation of Gn RH neuron excitability and hormone secretion[J].Nature Communications,2016,7:10055.
[33]ROCHA A,ZANUY S,GóMEZ A.Conserved anti-Müllerian hormone:Anti-Müllerian hormone type-2receptor specific interaction and intracellular signaling in teleosts.[J].Biology of Reproduction,2016,94(6):1-13.
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