【目的】研究金钱鱼(Scatophagus argus)Dmrt4(Doublesex and Mab-3 related transcription factors 4)基因在性腺和胚胎发育中的表达。【方法】克隆金钱鱼Dmrt4,采用逆转录PCR检测Dmrt4的组织表达,用实时荧光定量PCR检测Dmrt4在性腺和...【目的】研究金钱鱼(Scatophagus argus)Dmrt4(Doublesex and Mab-3 related transcription factors 4)基因在性腺和胚胎发育中的表达。【方法】克隆金钱鱼Dmrt4,采用逆转录PCR检测Dmrt4的组织表达,用实时荧光定量PCR检测Dmrt4在性腺和胚胎发育过程中的表达。【结果与结论】金钱鱼Dmrt4的开放阅读框(ORF)全长1 260 bp,编码蛋白全序列为419个氨基酸。金钱鱼Dmrt4氨基酸序列含有DM和DMA结构域。金钱鱼Dmrt4与欧洲海鲈(Dicentrarchus labrax)和大黄鱼(Larimichthys crocea)的同源性最高,为87.6%;与非洲爪蟾(Xenopus laevis)的同源性最低,为47.1%。金钱鱼Dmrt4与其他鲈形目鱼类在系统进化树上聚为一支。金钱鱼Dmrt4在精巢、卵巢和鳃中高表达,在Ⅲ和Ⅳ期精巢中的表达显著高于V期精巢和Ⅱ、Ⅲ和Ⅳ期卵巢。胚胎发育过程中Dmrt4均有表达,原肠胚的表达水平最高。展开更多
Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood.A single gene known to control somatic sex determination,the DM-domain containing(Doublesex...Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood.A single gene known to control somatic sex determination,the DM-domain containing(Doublesex/Mab-3 DNA-binding motif)gene,is highly conserved across species.Vertebrate DMRT1(DM-related transcription factor 1)expression occurs predominantly in the testis.Here,however,isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4(DM-related transcription factor 4)and DMRT1 by BLAST,respectively.Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure,function,and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination,the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression.After purification and cleavage,DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols.Consequently,it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity.The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard.DMRT1 was expressed only in testis,whereas DMRT4 was over expressed in the ovary,but in both female and male,a slight expression in the brain was also detected.Statistical analysis showed that in the brain,mean DMRT4 mRNA levels in female were significantly higher than in male.Meanwhile,the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry.It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis.Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.展开更多
The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players ...The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players in male gonad differentiation in mammals, in P. fucata sex diff erentiation and sexual development. Pf-Dmrt4 exhibits features typical of the D mrt family, and displays significant homologies to the DMRT4 cluster. Pf-Dmrt4 mRNA expression in the gonads during a gametogenic cycle, measured by quantitative polymerase chain reaction, was maximal in mature individuals. P f-Dmrt4 expression, demonstrated by in situ hybridization, was localized in the spermatozoa, spermatids, oocytes and vitellogenic oocytes. Knockdown of Pf-Dmrt4 with double-stranded RNA resulted in decreased mRNA expression levels. And Pf-Dmrt4-dsRNA-injected groups showed spawning-stage male gonads, with ruptured follicles and released spermatozoa. Our results enhance the understanding of sex determination and differentiation in P. fucata and suggest that Pf-Dmrt4 could be involved in male gonadal development, and maintenance of male gonadal function.展开更多
基金supported by the National Basic Research Program of China(No.2004CB117401)Chinese National Programsfor High Technology Research and Development(No.2004AA243060).
文摘Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood.A single gene known to control somatic sex determination,the DM-domain containing(Doublesex/Mab-3 DNA-binding motif)gene,is highly conserved across species.Vertebrate DMRT1(DM-related transcription factor 1)expression occurs predominantly in the testis.Here,however,isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4(DM-related transcription factor 4)and DMRT1 by BLAST,respectively.Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure,function,and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination,the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression.After purification and cleavage,DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols.Consequently,it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity.The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard.DMRT1 was expressed only in testis,whereas DMRT4 was over expressed in the ovary,but in both female and male,a slight expression in the brain was also detected.Statistical analysis showed that in the brain,mean DMRT4 mRNA levels in female were significantly higher than in male.Meanwhile,the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry.It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis.Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.
基金Supported by the Earmarked Fund for Modern Agro-industry Technology Research System(No.CARS-49)the Marine Fishery Science and Technology Development Project of Guangdong Province,China(Nos.Z2014012,Z2015014)the Science and Technology Planning Project of Guangdong Province,China(No.2014B030301064)
文摘The mechanisms of sex determination and sex differentiation in the pearl oyster P inctada fucata are currently poorly understood. We therefore investigated the roles of orthologs of the D mrt gene family, key players in male gonad differentiation in mammals, in P. fucata sex diff erentiation and sexual development. Pf-Dmrt4 exhibits features typical of the D mrt family, and displays significant homologies to the DMRT4 cluster. Pf-Dmrt4 mRNA expression in the gonads during a gametogenic cycle, measured by quantitative polymerase chain reaction, was maximal in mature individuals. P f-Dmrt4 expression, demonstrated by in situ hybridization, was localized in the spermatozoa, spermatids, oocytes and vitellogenic oocytes. Knockdown of Pf-Dmrt4 with double-stranded RNA resulted in decreased mRNA expression levels. And Pf-Dmrt4-dsRNA-injected groups showed spawning-stage male gonads, with ruptured follicles and released spermatozoa. Our results enhance the understanding of sex determination and differentiation in P. fucata and suggest that Pf-Dmrt4 could be involved in male gonadal development, and maintenance of male gonadal function.
