槟榔江水牛Dmrt7基因克隆及分子特征、组织表达谱分析
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摘要
【目的】Dmrt7基因是Dmrt基因家族成员之一,影响公畜精子形成。迄今,有关水牛精子发生的遗传基础的研究很少,本研究旨在对槟榔江水牛Dmrt7基因完整CDS区进行克隆和组织差异表达分析。【方法】采用RT-PCR和功能生物信息学方法。【结果】槟榔江水牛Dmrt7基因完整编码区序列长1 113 bp,编码370个氨基酸;Dmrt7包含DM和DMRT-like两个保守结构域,无信号肽,无跨膜结构,为核内蛋白。同源性分析显示,水牛与其他牛科物种Dmrt7氨基酸序列相似度达95%以上。在检测的10个组织中,Dmrt7基因仅在水牛睾丸组织中表达且高表达。在第6外显子发现1个SNP位点(c.665A>G),引起了p.Q222R的替换,此位点的替换对Dmrt7蛋白功能无影响。【结论】有助于了解水牛Dmrt7基因的基本功能。
[Purposes]As one of the members of Dmrt gene family,Dmrt7 gene affects the spermiogenesis of male animals.So far,few studies have been done related to the genetic basis of buffalo spermiogenesis.In this study,by employed Binglangjiang water buffalo as research object,the completeCDS sequence of water buffalo Dmrt7 gene was cloned,and its sequence characterization,tissue differential expression were assayed and analysed.[Methods]RT-PCR and functional bioinformatics methods were used.[Results] The results showed that the CDS of Binglangjiang buffalo Dmrt7 gene is 1 113 bp in length,which encodes a polypeptide composed of 370 amino acids.Buffalo Dmrt7 contains two conserved domains,viz.a DM and a DMRT-like domain.This protein was predicted a nuclear protein without signal peptide and transmembrane domain.Homology analysis showed that the amino acid sequence similarity of Dmrt7 between buffalo and other bovine species was more than 95%.Among 10 different tissues of adult buffalo,Dmrt7 gene was detected to only express in the testis of water buffalo,and its expression level was dominantly high.The polymorphism c.665 A > G was found in exon 6 of CDS of buffalo Dmrt7 gene which leads to a p.Q222R change.But this substitution was presumed to have no functional effect on buffalo Dmrt7 protein.[Conclusion] The results of this study will provide fundamental insights into the function of buffalo Dmrt7 gene.
[Purposes]As one of the members of Dmrt gene family,Dmrt7 gene affects the spermiogenesis of male animals.So far,few studies have been done related to the genetic basis of buffalo spermiogenesis.In this study,by employed Binglangjiang water buffalo as research object,the completeCDS sequence of water buffalo Dmrt7 gene was cloned,and its sequence characterization,tissue differential expression were assayed and analysed.[Methods]RT-PCR and functional bioinformatics methods were used.[Results] The results showed that the CDS of Binglangjiang buffalo Dmrt7 gene is 1 113 bp in length,which encodes a polypeptide composed of 370 amino acids.Buffalo Dmrt7 contains two conserved domains,viz.a DM and a DMRT-like domain.This protein was predicted a nuclear protein without signal peptide and transmembrane domain.Homology analysis showed that the amino acid sequence similarity of Dmrt7 between buffalo and other bovine species was more than 95%.Among 10 different tissues of adult buffalo,Dmrt7 gene was detected to only express in the testis of water buffalo,and its expression level was dominantly high.The polymorphism c.665 A > G was found in exon 6 of CDS of buffalo Dmrt7 gene which leads to a p.Q222R change.But this substitution was presumed to have no functional effect on buffalo Dmrt7 protein.[Conclusion] The results of this study will provide fundamental insights into the function of buffalo Dmrt7 gene.
引文
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[7]金帅,郭宪,包鹏甲,等.牦牛和犏牛Dmrt7基因序列分析及其在睾丸组织中的表达水平[J]中国农业科学,2013,46(5):1036.
[8]KAWAMATA M,INOUE H,NISHIMORI K.Male-specific function of Dmrt7 by sexually dimorphic translation in mouse testis[J].Sexual Development,2007,1(5):297.DOI:10.1159/000108932.
[9]淮亚红,许尚忠.牛Dmrt7基因的c DNA克隆及遗传变异[J].安徽农业科学,2009,37(30):14607.DOI:10.13989/j.cnki.0517-6611.2009.30.139.
[10]MURPHY M W,ZARKOWER D,BARDWELL V J.Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA[J].BMC Molecular Biology,2007,8(1):58.DOI:10.1186/1471-2199-8-58.
[11]NARENDRA U,ZHU L Y,LI B R,et al.Sex-specific gene regulation.The Doublesex DM motif is a bipartite DNA-binding domain[J].Journal of Biological Chemistry,2002,277(45):43463.DOI:10.1074/jbc.M204616200.
[12]KURAMOCHI-MIYAGAWA S,WATANABE T,GOTOH K,et al.DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes[J].Genes and Development,2008,22(7):908.DOI:10.1101/gad.1640708.
[13]KURAMOCHI-MIYAGAWA S,WATANABE T,GOTOH K,et al.MVH in piRNA processing and gene silencing of retrotransposons[J].Genes and Development,2010,24(9):887.DOI:10.1101/gad.1902110.
[14]DOBSON M J,PEARLMAN R E,KARAISKAKIS A,et al.Synaptonemal complex proteins:occurrence,epitope mapping and chromosome disjunction[J].Journal of Cell Science,1994,107(10):2749.
[15]MOENS P B,KOLAS N K,TARSOUNAS M,et al.The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNADNA interactions without reciprocal recombination[J].Journal of Cell Science,2002,115(8):1611.
[16]KIM S,NAMEKAWA S H,NISWANDER L M,et al.A mammal-specific doublesex homolog associates with male sex chromatin and is required for male meiosis[J].PLo S Genetics,2007,3(4):e62.DOI:10.1371/journal.pgen.0030062.
[17]YAN P,XIANG L,GUO X,et al.The low expression of Dmrt7 is associated with spermatogenic arrest in cattleyak[J].Molecular Biology Reports,2014,41(11):7255.DOI:10.1007/s11033-014-3611-x.
[2]VEITH A,KLATTIG J,DETTAI A,et al.Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse[J].Genomics,2006,88(2):185.DOI:10.1016/j.ygeno.2006.01.003.
[3]KAWAMATA M,NISHIMORI K.Mice deficient in Dmrt7 show infertility with spermatogenic arrest at pachytene stage[J].Febs Letters,2006,580(27):6442.DOI:10.1016/j.febslet.2006.10.066.
[4]ZHU L Y,WILKEN J,PHILLIPS N B,et al.Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers[J].Genes and Development,2000,14(14):1750.DOI:10.1101/gad.14.14.1750.
[5]DATE S,NOZAWA O,INOUE H,et al.Impairment of pachytene spermatogenesis in Dmrt7 deficient mice,possibly causing meiotic arrest[J].Bioscience Biotechnology and Biochemistry,2012,76(9):1621.DOI:10.1271/bbb.120024.
[6]淮亚红,许尚忠,姬爱国,等.牛Dmrt7基因c DNA的克隆和生物信息学分析[J].西北农林科技大学学报(自然科学版),2008,36(3):13.DOI:10.13207/j.cnki.jnwafu.2008.03.032.
[7]金帅,郭宪,包鹏甲,等.牦牛和犏牛Dmrt7基因序列分析及其在睾丸组织中的表达水平[J]中国农业科学,2013,46(5):1036.
[8]KAWAMATA M,INOUE H,NISHIMORI K.Male-specific function of Dmrt7 by sexually dimorphic translation in mouse testis[J].Sexual Development,2007,1(5):297.DOI:10.1159/000108932.
[9]淮亚红,许尚忠.牛Dmrt7基因的c DNA克隆及遗传变异[J].安徽农业科学,2009,37(30):14607.DOI:10.13989/j.cnki.0517-6611.2009.30.139.
[10]MURPHY M W,ZARKOWER D,BARDWELL V J.Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA[J].BMC Molecular Biology,2007,8(1):58.DOI:10.1186/1471-2199-8-58.
[11]NARENDRA U,ZHU L Y,LI B R,et al.Sex-specific gene regulation.The Doublesex DM motif is a bipartite DNA-binding domain[J].Journal of Biological Chemistry,2002,277(45):43463.DOI:10.1074/jbc.M204616200.
[12]KURAMOCHI-MIYAGAWA S,WATANABE T,GOTOH K,et al.DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes[J].Genes and Development,2008,22(7):908.DOI:10.1101/gad.1640708.
[13]KURAMOCHI-MIYAGAWA S,WATANABE T,GOTOH K,et al.MVH in piRNA processing and gene silencing of retrotransposons[J].Genes and Development,2010,24(9):887.DOI:10.1101/gad.1902110.
[14]DOBSON M J,PEARLMAN R E,KARAISKAKIS A,et al.Synaptonemal complex proteins:occurrence,epitope mapping and chromosome disjunction[J].Journal of Cell Science,1994,107(10):2749.
[15]MOENS P B,KOLAS N K,TARSOUNAS M,et al.The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNADNA interactions without reciprocal recombination[J].Journal of Cell Science,2002,115(8):1611.
[16]KIM S,NAMEKAWA S H,NISWANDER L M,et al.A mammal-specific doublesex homolog associates with male sex chromatin and is required for male meiosis[J].PLo S Genetics,2007,3(4):e62.DOI:10.1371/journal.pgen.0030062.
[17]YAN P,XIANG L,GUO X,et al.The low expression of Dmrt7 is associated with spermatogenic arrest in cattleyak[J].Molecular Biology Reports,2014,41(11):7255.DOI:10.1007/s11033-014-3611-x.