稀有鮈鲫雄激素受体基因全长cDNA的克隆和内分泌干扰物对其表达的影响
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摘要
雄激素(Androgen)在脊椎动物的性分化,性腺成熟和繁殖行为中扮演重要的角色。雄激素的作用通过与特定的核雄激素受体结合使其发生构象改变从而诱导相应基因的应答来发挥作用。雄激素受体是一种配体依赖型的转录因子,属于核受体超家族成员。它含有4个主要的结构域:N端高变异的转录激活域(TAD)、高度保守的DNA结合域(DBD)、铰链域、C端的高保守的配体结合域(LBD)。在本研究中,我们以稀有鮈鲫为研究对象,通过以RT-PCR和RACE法分离和克隆了稀有鮈鲫AR基因的cDNA全长并对其AR的mRNA组织差异表达进行研究,为进一步研究稀有鮈鲫由雄激素受体参与的重要的生理功能奠定基础。
随着人类的活动日益频繁,内分泌干扰物对动物和人类健康的影响日益受到关注,其类雌激素效应已成为研究热点。在本研究中通过类雌激素乙炔雌醇(EE2)、壬基酚(NP)和双酚A(BPA)作为类雌激素暴露的模型化合物,暴露稀有鮈鲫的幼鱼,使用实时定量(Real-time)PCR研究了稀有鮈鲫在内分泌干扰物暴露环境下幼鱼AR的mRNA的表达情况,所得结果和结论如下:
1.从精巢总RNA中克隆稀有鮈鲫AR基因的cDNA全长是根据分段克隆和RACE技术获得的。分别克隆了600bp、570bp、731bp、979bp四个片段和3’末端及5’末端两个片段146bp和492bp,将六个片段进行序列拼接首次完成了稀有鮈鲫AR基因共3130bp的cDNA序列全长(Genbank Accession No:GU226857)。ORF从104bp至2638bp,总长2535bp,共编码844个氨基酸残基。该cDNA序列5’末端非编码区有103bp,在3’末端的非编码区有492bp。
2.稀有鮈鲫推测的氨基酸与已知的硬骨鱼类的全长AR氨基酸序列进行BLASTP分析,稀有鮈鲫AR与黑头软口鲦和金鱼的相似性为86%。稀有鮈鲫的氨基酸序列分析表明它具有核激素受体所共有的三个主要功能域,它们分别TAD、DBD和LBD。其DBD和LBD在鲤科鱼类中表现出了很高的同源性,DBD同源性高达98%~99%,LBD同源性也达到了97%~98%,TAD同源性为75%~82%。
3.稀有鮈鲫雌雄成体AR基因在性腺、肝脏、肌肉、脑和肠组织中均有表达,雌性成体的AR基因在卵巢、肝、脑及肠组织中都有中等水平的表达,而在肌肉组织中的表达很弱,而在雄性成体中,AR基因在精巢和肝脏组织的表达量很高,而肌肉、脑和肠组织中AR基因表达较弱。
4. 0.01和0.1nmol/L的乙炔基雌二醇暴露3天后,能够分别不显著和显著地提高稀有鮈鲫幼鱼AR的mRNA表达,而1nmol/L的乙炔基雌二醇则对其表达有下调的趋势;0.01μmol/L壬基酚对其表达有显著下调,而0.1和1μmol/L壬基酚对其表达均有下调的趋势;0.1-10nmol/L的双酚A对其表达均有显著下调,因此不同种类内分泌干扰物及其不同暴露浓度对稀有鮈鲫AR的mRNA表达有不同影响。
Androgens play key roles in sex differentiation, gonad maturation and reproductive behaviors and their actions are generally mediated through androgen receptor (AR). Upon hormone binding, the AR dissociates from accessory proteins, translocates into the nucleus and dimerizes, thus stimulating transcription of androgen responsive genes. Androgen receptor is a ligand-activated transcription factor that belongs to a large family of nuclear receptors. This family of receptors share a molecular structure that consists four main functional domains.In the present study, RT-PCR and RACE (Rapid Amplification cDNA Ends) methods were used for the isolation of the cDNA of AR gene from testis of Gobiocypris rarus. We show for the first time in fish, different mRNA AR expression levels between males and females in five different tissues, suggesting that AR is involved in many physiological functions in fish.
With the frequency of human production and life, much attention has been focused on the potential health threats of endocrine disrupting chemicals capable of disrupting the endocrine systems of animal and human population. Research mostly focused on estrogenic compounds. In this report, we use EE2, NP and BPA as estrogenic model compounds ,after 3 days exposure of rare minnow juveniles using real-time PCR. We discuss whether AR gene can be sensitive as a molecular biomarker in assessing the potential impact of estrogenic compounds using this specie as a model system. The main results and conclusions are as follows:
1. RT-PCR and RACE methods were used for the isolation of the whole cDNA of AR gene from the testis of Gobiocypris rarus. We cloned cDNA fragments of 600bp, 570bp, 731bp, 979bp and 146bp in 3′end and 492 bp in 5′end. The full-length cDNA sequences of AR gene have been formed by joint for the first time, consisting of a 103bp 5’untranslated region (UTR), a 2535bp open reading frame (ORF) encoding 844 amino acid residues and a 492bp 3’UTR. The Genbank accession number is GU226857.
2. The deduced amino acid sequence of rare minnow AR was aligned with known full-length AR amino acid sequences from teleost fish fathead minnow and goldfish using Blastp. The identity of rare minnow AR was with fathead minnow and goldfish with 86%. Multiple amino acids sequence alignment indicated AR has three main functional domain, TAD, DBD and LBD. DBD and LBD are highly conserved among the cyprinid fish species, whose identity ranged from 98% to 99%. The identity of LBD is about 97% to 98%. TAD is the most variable domain, whose identity fluctuated from 75% to 82%.
3. AR mRNA expression was detected using semiquantitative RT-PCR in gonad, liver, brain, intestine, and muscle. AR mRNA was expressed at high levels in the testis and liver, low levels in the brain, intestine, muscle of male. However it was expressed at moderate levels in all the tissues except the muscle of female.
4. Exposure to 0.01 and 0.1 nmol/L ethynylestradiol (EE2) for 3 days caused nonsignificant and significant increase of AR mRNA expression in rare minnow juveniles, respectively. On the contrary, 1nmol/L EE2 caused nonsignificant decrease in its expression. 0.01μmol/L nonylphenol (NP) caused significant decrease of AR gene expression. While AR mRNA expression had downregulation tendency in both 0.1 and 1μmol/L NP exposed groups. Bisphenol (BPA) 0.1,1 and 10 nmol/L caused significant decrease of AR gene expression.Therefore AR gene expression was differentially modulated by various classes of EDCs and their different exposure concentrations.
随着人类的活动日益频繁,内分泌干扰物对动物和人类健康的影响日益受到关注,其类雌激素效应已成为研究热点。在本研究中通过类雌激素乙炔雌醇(EE2)、壬基酚(NP)和双酚A(BPA)作为类雌激素暴露的模型化合物,暴露稀有鮈鲫的幼鱼,使用实时定量(Real-time)PCR研究了稀有鮈鲫在内分泌干扰物暴露环境下幼鱼AR的mRNA的表达情况,所得结果和结论如下:
1.从精巢总RNA中克隆稀有鮈鲫AR基因的cDNA全长是根据分段克隆和RACE技术获得的。分别克隆了600bp、570bp、731bp、979bp四个片段和3’末端及5’末端两个片段146bp和492bp,将六个片段进行序列拼接首次完成了稀有鮈鲫AR基因共3130bp的cDNA序列全长(Genbank Accession No:GU226857)。ORF从104bp至2638bp,总长2535bp,共编码844个氨基酸残基。该cDNA序列5’末端非编码区有103bp,在3’末端的非编码区有492bp。
2.稀有鮈鲫推测的氨基酸与已知的硬骨鱼类的全长AR氨基酸序列进行BLASTP分析,稀有鮈鲫AR与黑头软口鲦和金鱼的相似性为86%。稀有鮈鲫的氨基酸序列分析表明它具有核激素受体所共有的三个主要功能域,它们分别TAD、DBD和LBD。其DBD和LBD在鲤科鱼类中表现出了很高的同源性,DBD同源性高达98%~99%,LBD同源性也达到了97%~98%,TAD同源性为75%~82%。
3.稀有鮈鲫雌雄成体AR基因在性腺、肝脏、肌肉、脑和肠组织中均有表达,雌性成体的AR基因在卵巢、肝、脑及肠组织中都有中等水平的表达,而在肌肉组织中的表达很弱,而在雄性成体中,AR基因在精巢和肝脏组织的表达量很高,而肌肉、脑和肠组织中AR基因表达较弱。
4. 0.01和0.1nmol/L的乙炔基雌二醇暴露3天后,能够分别不显著和显著地提高稀有鮈鲫幼鱼AR的mRNA表达,而1nmol/L的乙炔基雌二醇则对其表达有下调的趋势;0.01μmol/L壬基酚对其表达有显著下调,而0.1和1μmol/L壬基酚对其表达均有下调的趋势;0.1-10nmol/L的双酚A对其表达均有显著下调,因此不同种类内分泌干扰物及其不同暴露浓度对稀有鮈鲫AR的mRNA表达有不同影响。
Androgens play key roles in sex differentiation, gonad maturation and reproductive behaviors and their actions are generally mediated through androgen receptor (AR). Upon hormone binding, the AR dissociates from accessory proteins, translocates into the nucleus and dimerizes, thus stimulating transcription of androgen responsive genes. Androgen receptor is a ligand-activated transcription factor that belongs to a large family of nuclear receptors. This family of receptors share a molecular structure that consists four main functional domains.In the present study, RT-PCR and RACE (Rapid Amplification cDNA Ends) methods were used for the isolation of the cDNA of AR gene from testis of Gobiocypris rarus. We show for the first time in fish, different mRNA AR expression levels between males and females in five different tissues, suggesting that AR is involved in many physiological functions in fish.
With the frequency of human production and life, much attention has been focused on the potential health threats of endocrine disrupting chemicals capable of disrupting the endocrine systems of animal and human population. Research mostly focused on estrogenic compounds. In this report, we use EE2, NP and BPA as estrogenic model compounds ,after 3 days exposure of rare minnow juveniles using real-time PCR. We discuss whether AR gene can be sensitive as a molecular biomarker in assessing the potential impact of estrogenic compounds using this specie as a model system. The main results and conclusions are as follows:
1. RT-PCR and RACE methods were used for the isolation of the whole cDNA of AR gene from the testis of Gobiocypris rarus. We cloned cDNA fragments of 600bp, 570bp, 731bp, 979bp and 146bp in 3′end and 492 bp in 5′end. The full-length cDNA sequences of AR gene have been formed by joint for the first time, consisting of a 103bp 5’untranslated region (UTR), a 2535bp open reading frame (ORF) encoding 844 amino acid residues and a 492bp 3’UTR. The Genbank accession number is GU226857.
2. The deduced amino acid sequence of rare minnow AR was aligned with known full-length AR amino acid sequences from teleost fish fathead minnow and goldfish using Blastp. The identity of rare minnow AR was with fathead minnow and goldfish with 86%. Multiple amino acids sequence alignment indicated AR has three main functional domain, TAD, DBD and LBD. DBD and LBD are highly conserved among the cyprinid fish species, whose identity ranged from 98% to 99%. The identity of LBD is about 97% to 98%. TAD is the most variable domain, whose identity fluctuated from 75% to 82%.
3. AR mRNA expression was detected using semiquantitative RT-PCR in gonad, liver, brain, intestine, and muscle. AR mRNA was expressed at high levels in the testis and liver, low levels in the brain, intestine, muscle of male. However it was expressed at moderate levels in all the tissues except the muscle of female.
4. Exposure to 0.01 and 0.1 nmol/L ethynylestradiol (EE2) for 3 days caused nonsignificant and significant increase of AR mRNA expression in rare minnow juveniles, respectively. On the contrary, 1nmol/L EE2 caused nonsignificant decrease in its expression. 0.01μmol/L nonylphenol (NP) caused significant decrease of AR gene expression. While AR mRNA expression had downregulation tendency in both 0.1 and 1μmol/L NP exposed groups. Bisphenol (BPA) 0.1,1 and 10 nmol/L caused significant decrease of AR gene expression.Therefore AR gene expression was differentially modulated by various classes of EDCs and their different exposure concentrations.
引文
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