鲤两种孕激素受体基因克隆、表达及比较分析
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摘要
孕激素受体(Progesterone receptor,Pgr)是介导孕激素调控鱼类性腺发育和生殖细胞成熟的受体。为研究鲤(Cyprinus carpio)2种Pgr时空表达模式和表达分化趋势,采用cDNA末端快速扩增方法克隆两种Pgr基因(Pgr1和Pgr2)。Pgr1和Pgr2的全长cDNA序列分别为2 713 bp和2 730 bp,均编码628个氨基酸。二者核酸和蛋白质一致性分别为91%和90%。这两个基因都含有锌指结构域和核激素受体域同源超家族两个功能域。进化分析将鲤Pgr1和鲫Pgr1聚在一起,鲤Pgr2和鲫Pgr2聚为一支;这两支再与斑马鱼Pgr汇成一支。鲤Pgr1和Pgr2的非同义替换率与同义替换率比值为0.360,表明Pgr1和Pgr2受负选择压力。实时荧光qPCR分析表明,Pgr1和Pgr2在性腺的表达显著高于其他组织,说明二者可能参与性腺发育过程。催产素诱导后,Pgr1和Pgr2在精液和卵细胞的表达量高于受精卵,表明这两种基因与鲤生殖细胞成熟相关。大多数状态下Pgr1表达量显著高于Pgr2,表明Pgr1是介导鲤性腺发育和生殖细胞成熟的主表达基因。
Progesterone receptor(Pgr)is a receptor mediating the regulation of progesterone on fish gonad development and germ cell maturation. To study the expression pattern and differential expression tendency of two common carp(Cyprinus carpio)Pgr genes,Pgr1 and Pgr2 were cloned using rapid amplification of cDNA ends. The full-length cDNA sequence lengths of Pgr1 and Pgr2 were 2 713 bp and 2730 bp,both of which encoded proteins of 628 amino acids. They had a nucleotide identity of 91% and a protein identity of 90%. They both contained two domains,zinc finger domain and nuclear hormone receptor domain homologous superfamily. In the phylogenetic analysis,common carp Pgr1 and goldfish Pgr1 were clustered together and common carp Pgr2 and goldfish Pgr2 were grouped together. These two branches were then clustered with zebrafish Pgr. The ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site between Pgr1 and Pgr2 was 0.360,suggesting that they were under negative selection. qRTPCR analysis demonstrated that Pgr1 and Pgr2 had higher expression levels in the gonads than in the other tissues,revealing that both genes might be associated with the gonad development of common carp. They had higher expression levels in sperms and oocytes than in embryos after induced by spawning drugs,indicating that they might be involved in germ cell maturation. In the most examined cases,significantly higher Pgr1 expression level was revealed compared with Pgr2,suggesting that Pgr1 plays the dominate role in common carp gonad development and germ cell maturation.
Progesterone receptor(Pgr)is a receptor mediating the regulation of progesterone on fish gonad development and germ cell maturation. To study the expression pattern and differential expression tendency of two common carp(Cyprinus carpio)Pgr genes,Pgr1 and Pgr2 were cloned using rapid amplification of cDNA ends. The full-length cDNA sequence lengths of Pgr1 and Pgr2 were 2 713 bp and 2730 bp,both of which encoded proteins of 628 amino acids. They had a nucleotide identity of 91% and a protein identity of 90%. They both contained two domains,zinc finger domain and nuclear hormone receptor domain homologous superfamily. In the phylogenetic analysis,common carp Pgr1 and goldfish Pgr1 were clustered together and common carp Pgr2 and goldfish Pgr2 were grouped together. These two branches were then clustered with zebrafish Pgr. The ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site between Pgr1 and Pgr2 was 0.360,suggesting that they were under negative selection. qRTPCR analysis demonstrated that Pgr1 and Pgr2 had higher expression levels in the gonads than in the other tissues,revealing that both genes might be associated with the gonad development of common carp. They had higher expression levels in sperms and oocytes than in embryos after induced by spawning drugs,indicating that they might be involved in germ cell maturation. In the most examined cases,significantly higher Pgr1 expression level was revealed compared with Pgr2,suggesting that Pgr1 plays the dominate role in common carp gonad development and germ cell maturation.
引文
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[34]Hanna RN, Daly SCJ, Pang Y, et al. Characterization and expression of the nuclear progestin receptor in zebrafish gonads and brain[J]. Biology of Reproduction, 2010, 82(1):112-122.
[2]Wang C, Croll RP. Effects of sex steroids on gonadal development andgenderdeterminationintheseascallop,Placopecten magellanicus[J]. Aquaculture, 2004, 238(1-4):483-498.
[3]Chen SX, et al. Cloning, pharmacological characterization, and expression analysis of Atlantic salmon(Salmo salar L.)nuclear progesterone receptor[J]. Reproduction, 2011, 11:491-500.
[4]Dequattro ZA, et al. Effects of progesterone on reproduction and embryonic development in the fathead minnow(Pimephales promelas)[J]. Environ Toxicol Chem, 2012, 4:851-856.
[5]Wang CL, Liu DT, Chen WT, et al. Progestin increases the expression of gonadotropins in pituitaries of male zebrafish[J]. J Endocrinol, 2016, 230(1):143-156.
[6]赖晓健,洪万树.介导鱼类孕激素快速、非基因作用的受体研究进展[J].中国水产科学, 2010, 17(1):182-191.
[7]Aranda A, Pascual A. Nuclear hormone receptors and gene expression[J]. Physiol Rev, 2001, 81(3):1269-1304.
[8]Hill KK, Roemer SC, Churchill MEA, et al. Structural and functional analysis of domains of the progesterone receptor[J]. Mol Cell Endocrinol, 2012, 348(2):418-429.
[9]Hanna RN, Zhu Y. Controls of meiotic signaling by membrane or nuclear progestin receptor in zebrafish follicle-enclosed oocytes[J]. Mol Cell Endocrinol, 2011, 337(1):80-88.
[10]Feng C, Xu S, Liu Y, et al. Progestin is important for testicular development of male turbot(Scophthalmus maximus)during the annual reproductive cycle through functionally distinct progestin receptors[J]. Fish Physiol Biochem, 2018, 44(1):35-48.
[11]史宝,李晓晓,柳学周,等.半滑舌鳎膜孕激素受体基因克隆与组织表达分析[J].渔业科学进展, 2014, 34(3):61-67.
[12]Fang XL, Wu LM, Yang LY, et al. Nuclear progestin receptor(Pgr)knockouts resulted in subfertility in male tilapia(Oreochromis niloticus)[J]. J Steroid Biochem Mol Biol, 2018, 182:62-71.
[13]Liu G, Luo F, Song Q, et al. Blocking of progestin action disrupts spermatogenesis in Nile tilapia(Oreochromis niloticus)[J]. J Mol Endocrinol, 2014, 53(1):57-70.
[14]Lien S, et al. The atlantic salmon genome provides insights into rediploidization[J]. Nature, 2016, 533:200-205.
[15]Wang JT, Li JT, Zhang XF, et al. Transcriptome analysis reveals the time of the fourth round of genome duplication in common carp(Cyprinus carpio)[J]. BMC Genomics, 2012, 13(1):96.
[16]Larhammar D, Risinger C. Molecular genetic aspects of tetraploidy in the common carp Cyprinus carpio[J]. Mol Phylogenet Evol,1994, 3(1):59-68.
[17]Ma Z, Yu Y, Tang S, et al. Differential modulation of expression of nuclear receptor mediated genes by tris(2-butoxyethyl)phosphate(TBOEP)on early life stages of zebrafish(Danio rerio)[J]. Aquatic Toxicology, 2015, 169:196-203.
[18]Ogiwara K, Takahashi T. Involvement of the nuclear progestin receptor in LH-induced expression of membrane type 2-matrix metalloproteinase required for follicle rupture during ovulation in the medaka, Oryzias latipes[J]. Mol Cell Endocrinol, 2017,450:54-63.
[19]Chauvigne F, Parhi J, Olle J, et al. Dual estrogenic regulation of the nuclear progestin receptor and spermatogonial renewal during gilthead seabream(Sparus aurata)spermatogenesis[J]. Comp Biochem Physiol A Mol Integr Physiol, 2017, 206:36-46.
[20]Li M, Sun Y, Zhao J, et al. A tandem duplicate of anti-mullerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in nile tilapia, Oreochromis niloticus[J].PLoS Genetics, 2015, 11(11):e1005678.
[21]Chen S, et al. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle[J]. Nat Genet, 2014, 46(3):253-260.
[22]张哲,卢毅,詹俊阁,等.红白锦鲤的人工催产及胚胎发育研究[J].经济动物学报, 2018, 22(1):24-29.
[23]Hu B, Jin J, et al. GSDS 2. 0:an upgraded gene feature visualization server[J]. Bioinformatics, 2015, 31(8):1296-1297.
[24]Finn RD, et al. InterPro in 2017-beyond protein family and domain annotations[J]. Nucleic Acids Res, 2016, 45:D190-D199.
[25]Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2. 0[J]. Bioinformatics, 2007, 23(21):2947-2948.
[26]Kumar S, Stecher G, Tamura K. MEGA7:Molecular evolutionary genetics analysis version 7. 0 for bigger datasets[J]. Mol Biol Evol, 2016, 33(7):1870-1874.
[27]Suyama M, Torrents D, Bork P. PAL2NAL:robust conversion of protein sequence alignments into the corresponding codon alignments[J]. Nucleic Acids Res, 2006, 34:W609-612.
[28]Li XM, Song YN, Xiao GB, et al. Gene expression variations of redwhite skin coloration in common carp(Cyprinus carpio)[J]. Int J Mol Sci, 2015, 16(9):21310-21329.
[29]Schmittgen TD. Analyzing real-time PCR data by the comparative CT method[J]. Nat Protoc, 2008, 3(6):1101-1108.
[30]Chen SX, Bogerd J, García-Lópezá, et al. Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor[J]. Biology of Reproduction, 2010, 82(1):171-181.
[31]Zapater C, et al. Alternative splicing of the nuclear progestin receptor in a perciform teleost generates novel mechanisms of dominant-negative transcriptional regulation[J]. Gen Comp Endocrinol, 2013, 182:24-40.
[32]Zhang YT, Liu DT, Zhu Y, et al. Cloning and olfactory expression of progestin receptors in the Chinese black sleeper Bostrichthys sinensis[J]. Gen Comp Endocrinol, 2016, 230-231:87-102.
[33]Morini M, Penaranda DS, Vilchez MC, et al. Nuclear and membrane progestin receptors in the European eel:Characterization and expression in vivo through spermatogenesis[J]. Comp Biochem Physiol A Mol Integr Physiol, 2017, 207:79-92.
[34]Hanna RN, Daly SCJ, Pang Y, et al. Characterization and expression of the nuclear progestin receptor in zebrafish gonads and brain[J]. Biology of Reproduction, 2010, 82(1):112-122.