有性生殖是多细胞动物物种繁衍的普遍方式。本研究以大型淡水贝类中国圆田螺(Cipangopaludina chinensis)为例,从全基因组层面鉴定与中国圆田螺性别分化相关的Dmrt(double-sex and mab-3 relatated transcription factor)、Sox(sry-rela...有性生殖是多细胞动物物种繁衍的普遍方式。本研究以大型淡水贝类中国圆田螺(Cipangopaludina chinensis)为例,从全基因组层面鉴定与中国圆田螺性别分化相关的Dmrt(double-sex and mab-3 relatated transcription factor)、Sox(sry-related high mobility group box)和Wnt(wingless-type mmtv integration site family)基因家族并分析其特点、演化及在性腺中的表达模式。结果显示中国圆田螺CchDmrt、CchSox和CchWnt基因家族分别含有3个、5个和10个成员,主要分布于2号、3号、5号、7号和9号染色体上。选取腹足纲代表性物种构建系统进化树,结果显示Dmrt、Sox和Wnt各亚家族分布聚类在一起,其中中国圆田螺与铜锈环棱螺(Bellamya aeruginosa)亲缘关系最近。3个基因家族中,CchSox10、CchWnt5和CchWnt7受到正选择作用,相对进化速率较快。三维结构分析显示CchDmrt和CchWnt基因家族编码的蛋白质较为保守,而CchSox基因家族编码的蛋白质之间则存在一定差异性,提示其存在功能差异。雌雄性腺转录组表达分析显示,CchSox5、CchWnt2和CchWnt16在精巢中的表达量高于卵巢的,并形成差异表达基因,而CchDmrt4、CchSox2、CchSox10、CchSox12和CchFoxl2基因则相反,说明不同家族成员在性腺发育中的作用不同。选取差异表达基因进行实时定量PCR(real-time quantitative PCR,RT-qPCR)验证,结果与转录组结果一致。本研究鉴定了中国圆田螺Dmrt、Sox和Wnt等性别决定相关基因,为其功能研究和中国圆田螺遗传育种研究提供基础资料。展开更多
Dmrt(double-sex and mab-3 related transcription factor)基因家族编码的蛋白质是一类转录因子,具有保守的DM(double-sex/mab-3)结构域。在鱼类生长发育和进化中,Dmrt在多种生理过程尤其是性别决定和性腺发育方面起到至关重要的作用...Dmrt(double-sex and mab-3 related transcription factor)基因家族编码的蛋白质是一类转录因子,具有保守的DM(double-sex/mab-3)结构域。在鱼类生长发育和进化中,Dmrt在多种生理过程尤其是性别决定和性腺发育方面起到至关重要的作用。弓背青鳉(Oryzias curvinotus)和日本青鳉(O.latipes)均具有XX/XY性别决定系统和雄性决定基因dmrt1-bY/dmy(DM domain gene on Y chromosome),但是Dmrt基因家族在弓背青鳉中的作用尚不清楚。本研究利用生物信息学方法对弓背青鳉Dmrt基因家族的系统进化关系、基因结构、保守基序、保守结构域以及在不同组织中基因的表达水平进行分析,用实时定量PCR(real-time quantitative PCR,RT-qPCR)方法验证转录组信息。结果显示,弓背青鳉湛江群体中鉴定出Ocu_ZJ_dmrt1、Ocu_ZJ_dmy、Ocu_ZJ_dmrt2a、Ocu_ZJ_dmrt2b.1、Ocu_ZJ_dmrt2b.2、Ocu_ZJ_dmrt3、Ocu_ZJ_dmrtA1和Ocu_ZJ_dmrtA2,弓背青鳉三亚群体中鉴定出Ocu_SY_dmrt1、Ocu_SY_dmrt2a、Ocu_SY_dmrt2b、Ocu_SY_dmrt3、Ocu_SY_dmrtA1和Ocu_SY_dmrtA2。保守结构域分析显示,14个Dmrt蛋白质序列均具有DM保守结构域。利用弓背青鳉湛江群体胚胎发育转录组以及湛江和三亚群体雌雄性腺转录组数据对Dmrt基因家族进行表达水平分析,结果显示,Dmrt基因家族在胚胎各个发育时期表达不一,在两个群体的性腺中均有表达,其中Ocu_ZJ_dmy、Ocu_ZJ_dmrt1和Ocu_SY_dmrt1在雄性高表达,Ocu_ZJ_dmrt2a和Ocu_SY_dmrt2a在雌性性腺表达更高,三亚群体未检测到dmy的表达。通过RT-qPCR验证了11个差异表达基因(differentially expressed genes,DEGs)的RNA-seq数据的可靠性。本研究为青鳉属物种Dmrt基因家族的研究提供必要的基础数据,为更好地认识Dmrt基因家族在弓背青鳉性别决定和性腺、胚胎发育中的作用提供参考。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 （Doubl...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
脊椎动物的Dmrt(double-sex and mab-3related transcription factor)是指与果蝇Dsx基因和线虫Mab-3基因同源的基因家族。本文阐述了Dmrt家族中8个成员的基因和蛋白结构特征,并通过分析Dmrt在各类脊椎动物发育中的表达显示,Dmrt家族主...脊椎动物的Dmrt(double-sex and mab-3related transcription factor)是指与果蝇Dsx基因和线虫Mab-3基因同源的基因家族。本文阐述了Dmrt家族中8个成员的基因和蛋白结构特征,并通过分析Dmrt在各类脊椎动物发育中的表达显示,Dmrt家族主要参与性别决定和性腺分化,也参与某些非性腺组织器官的发育:Dmrt1主要参与精巢分化,也与卵巢发育有关;Dmrt2主要参与体节的发生、分化以及非对称器官的形成;Dmrt3,4,5可共同参与或依次调控嗅器和脑的发育;Dmrt7为雄性配子发生所必须;Dmrt8属雄性偏向表达基因。Dmrt1在各脊椎动物的遗传性别决定通路中的位置和作用位点不同。展开更多
Dmrt(Double sex and Mab-3 related transcription factor)基因是一类含有高度保守的DM(Double sex and Mab-3)结构域的基因家族,该基因家族的很多成员都参与了性别决定和分化过程的调控。目前已研究了很多物种的Dmrt基因,但对于具有...Dmrt(Double sex and Mab-3 related transcription factor)基因是一类含有高度保守的DM(Double sex and Mab-3)结构域的基因家族,该基因家族的很多成员都参与了性别决定和分化过程的调控。目前已研究了很多物种的Dmrt基因,但对于具有重要进化地位和经济价值的贝类,却鲜见相关报道。Dmrt5作为Dmrt基因家族的重要成员,是否也参与了动物的性别决定与分化的调控,至今为止没有定论。采用RACE-PCR技术,从马氏珠母贝(Pinctadamatensii,Dunker)精巢的SMARTcDNA中克隆了Dmrt5基因的全长cDNA序列。同源性比对显示,pmDmrt5编码的氨基酸序列与海胆、线虫、青鳉、斑马鱼、爪蟾和小鼠的Dmrt5基因的一致性分别为17.2%、26.7%、24.8%、18.7%、20.8%和15.8%。虽然各个物种间同源性并不高,但他们的DM结构域是高度保守的。RT-PCR结果表明,pmDm-rt5基因在雄性性腺中的表达量显著高于鳃、外套膜、闭壳肌、消化盲囊和雌性性腺;在早期雄性性腺、成熟期雄性性腺中的表达量显著高于性腺发育其他时期。这意味着pmDmrt5基因可能参与了马氏珠母贝性别发育的调控。展开更多
文摘有性生殖是多细胞动物物种繁衍的普遍方式。本研究以大型淡水贝类中国圆田螺(Cipangopaludina chinensis)为例,从全基因组层面鉴定与中国圆田螺性别分化相关的Dmrt(double-sex and mab-3 relatated transcription factor)、Sox(sry-related high mobility group box)和Wnt(wingless-type mmtv integration site family)基因家族并分析其特点、演化及在性腺中的表达模式。结果显示中国圆田螺CchDmrt、CchSox和CchWnt基因家族分别含有3个、5个和10个成员,主要分布于2号、3号、5号、7号和9号染色体上。选取腹足纲代表性物种构建系统进化树,结果显示Dmrt、Sox和Wnt各亚家族分布聚类在一起,其中中国圆田螺与铜锈环棱螺(Bellamya aeruginosa)亲缘关系最近。3个基因家族中,CchSox10、CchWnt5和CchWnt7受到正选择作用,相对进化速率较快。三维结构分析显示CchDmrt和CchWnt基因家族编码的蛋白质较为保守,而CchSox基因家族编码的蛋白质之间则存在一定差异性,提示其存在功能差异。雌雄性腺转录组表达分析显示,CchSox5、CchWnt2和CchWnt16在精巢中的表达量高于卵巢的,并形成差异表达基因,而CchDmrt4、CchSox2、CchSox10、CchSox12和CchFoxl2基因则相反,说明不同家族成员在性腺发育中的作用不同。选取差异表达基因进行实时定量PCR(real-time quantitative PCR,RT-qPCR)验证,结果与转录组结果一致。本研究鉴定了中国圆田螺Dmrt、Sox和Wnt等性别决定相关基因,为其功能研究和中国圆田螺遗传育种研究提供基础资料。
文摘Dmrt(double-sex and mab-3 related transcription factor)基因家族编码的蛋白质是一类转录因子,具有保守的DM(double-sex/mab-3)结构域。在鱼类生长发育和进化中,Dmrt在多种生理过程尤其是性别决定和性腺发育方面起到至关重要的作用。弓背青鳉(Oryzias curvinotus)和日本青鳉(O.latipes)均具有XX/XY性别决定系统和雄性决定基因dmrt1-bY/dmy(DM domain gene on Y chromosome),但是Dmrt基因家族在弓背青鳉中的作用尚不清楚。本研究利用生物信息学方法对弓背青鳉Dmrt基因家族的系统进化关系、基因结构、保守基序、保守结构域以及在不同组织中基因的表达水平进行分析,用实时定量PCR(real-time quantitative PCR,RT-qPCR)方法验证转录组信息。结果显示,弓背青鳉湛江群体中鉴定出Ocu_ZJ_dmrt1、Ocu_ZJ_dmy、Ocu_ZJ_dmrt2a、Ocu_ZJ_dmrt2b.1、Ocu_ZJ_dmrt2b.2、Ocu_ZJ_dmrt3、Ocu_ZJ_dmrtA1和Ocu_ZJ_dmrtA2,弓背青鳉三亚群体中鉴定出Ocu_SY_dmrt1、Ocu_SY_dmrt2a、Ocu_SY_dmrt2b、Ocu_SY_dmrt3、Ocu_SY_dmrtA1和Ocu_SY_dmrtA2。保守结构域分析显示,14个Dmrt蛋白质序列均具有DM保守结构域。利用弓背青鳉湛江群体胚胎发育转录组以及湛江和三亚群体雌雄性腺转录组数据对Dmrt基因家族进行表达水平分析,结果显示,Dmrt基因家族在胚胎各个发育时期表达不一,在两个群体的性腺中均有表达,其中Ocu_ZJ_dmy、Ocu_ZJ_dmrt1和Ocu_SY_dmrt1在雄性高表达,Ocu_ZJ_dmrt2a和Ocu_SY_dmrt2a在雌性性腺表达更高,三亚群体未检测到dmy的表达。通过RT-qPCR验证了11个差异表达基因(differentially expressed genes,DEGs)的RNA-seq数据的可靠性。本研究为青鳉属物种Dmrt基因家族的研究提供必要的基础数据,为更好地认识Dmrt基因家族在弓背青鳉性别决定和性腺、胚胎发育中的作用提供参考。
基金This work was 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.
文摘脊椎动物的Dmrt(double-sex and mab-3related transcription factor)是指与果蝇Dsx基因和线虫Mab-3基因同源的基因家族。本文阐述了Dmrt家族中8个成员的基因和蛋白结构特征,并通过分析Dmrt在各类脊椎动物发育中的表达显示,Dmrt家族主要参与性别决定和性腺分化,也参与某些非性腺组织器官的发育:Dmrt1主要参与精巢分化,也与卵巢发育有关;Dmrt2主要参与体节的发生、分化以及非对称器官的形成;Dmrt3,4,5可共同参与或依次调控嗅器和脑的发育;Dmrt7为雄性配子发生所必须;Dmrt8属雄性偏向表达基因。Dmrt1在各脊椎动物的遗传性别决定通路中的位置和作用位点不同。
文摘Dmrt(Double sex and Mab-3 related transcription factor)基因是一类含有高度保守的DM(Double sex and Mab-3)结构域的基因家族,该基因家族的很多成员都参与了性别决定和分化过程的调控。目前已研究了很多物种的Dmrt基因,但对于具有重要进化地位和经济价值的贝类,却鲜见相关报道。Dmrt5作为Dmrt基因家族的重要成员,是否也参与了动物的性别决定与分化的调控,至今为止没有定论。采用RACE-PCR技术,从马氏珠母贝(Pinctadamatensii,Dunker)精巢的SMARTcDNA中克隆了Dmrt5基因的全长cDNA序列。同源性比对显示,pmDmrt5编码的氨基酸序列与海胆、线虫、青鳉、斑马鱼、爪蟾和小鼠的Dmrt5基因的一致性分别为17.2%、26.7%、24.8%、18.7%、20.8%和15.8%。虽然各个物种间同源性并不高,但他们的DM结构域是高度保守的。RT-PCR结果表明,pmDm-rt5基因在雄性性腺中的表达量显著高于鳃、外套膜、闭壳肌、消化盲囊和雌性性腺;在早期雄性性腺、成熟期雄性性腺中的表达量显著高于性腺发育其他时期。这意味着pmDmrt5基因可能参与了马氏珠母贝性别发育的调控。
