策勒黑羊ERα基因克隆、生物信息学分析及卵巢组织表达研究

Cloning and Bioinformatics of ERαGene and Its Expression of Ovary Tissue in Cele Black Sheep

【目的】克隆策勒黑羊雌激素受体α(estrogen receptorα,ERα)序列并进行生物信息学分析,同时测定策勒黑羊卵巢中ERα基因的表达情况。【方法】利用RT-PCR和TA克隆方法获得策勒黑羊ERα基因序列,并采用在线软件对其进行生物信息学分析。采用实时荧光定量PCR方法检测ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d母羊卵巢中的表达情况。【结果】成功克隆获得策勒黑羊ERα基因序列,长1791 bp,编码596个氨基酸,与山羊和牛的相似性最高。生物信息学分析显示,ERα蛋白分子式为C_(2930)H_(4600)N_(822)O_(862)S_(41),理论等电点(pI)为7.32,不稳定指数为50.33,为不稳定亲水性蛋白;存在28个O-糖基化位点和54个磷酸化位点,不存在N-糖基化位点;主要定位在细胞核中,不具备跨膜结构;二级结构主要为α-螺旋及无规则卷曲。实时荧光定量PCR检测显示,ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d的卵巢中均有表达,与卵泡期相比,黄体期ERα基因表达量极显著下降(P<0.01),妊娠30 d ERα基因表达量极显著升高(P<0.01)。【结论】策勒黑羊ERα基因序列长1791 bp,编码596个氨基酸,为亲水蛋白,二级结构以α-螺旋和无规则卷曲为主。ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d的卵巢中均有表达。本研究结果为进一步探究ERα基因在策勒黑羊卵巢卵泡发育、黄体形成和妊娠中发挥的作用提供参考。

【Objective】The aim of this experiment was to clone the estrogen receptorα(ERα)gene sequence in Cele Black sheep,and bioinformatics analysis was carried out.At the same time,the expression of ERαgene in ovary of Cele Black sheep were measured.【Method】The ERαgene sequence of Cele Black sheep was obtained by RT-PCR and TA cloning,and bioinformatics analysis was carried out by online softwares.The expression of ERαgene in ovary of Cele Black sheep at follicular phase,luteal phase and 30 days of pregnancy were detected by Real-time quantitative PCR.【Result】The length of ERαgene sequence in Cele Black sheep was 1791 bp,and encoded 596 amino acids,with the highest similarity with Capra hircus and Bos taurus.Bioinformatics analysis results showed that the molecular formula of the protein was C_(2930)H_(4600)N_(822)O_(862)S_(41),the theoretical isoelectric point(pI)was 7.32,the instability index was 50.33,which was an unstable hydrophilic protein.There were 28 O-glycosylation sites and 54 phosphorylation sites,but no N-glycosylation sites.ERαprotein was mainly located in nucleus and there was no transmembrane structure.The secondary structure of ERαprotein was mainly alpha helix and random coil.Real-time quantitative PCR showed that ERαgene in Cele Black sheep was expressed in ovary at follicular phase,luteal phase and 30 days of pregnancy,compared with follicular phase,the expression of ERαgene in Cele Black sheep was decreased extremely significantly at luteal phase(P<0.01),and increased extremely significantly at 30 days of pregnancy(P<0.01).【Conclusion】The length of ERαgene sequence in Cele Black sheep was 1791 bp,and encoded 596 amino acids,which was a hydrophilic protein.The secondary structure of ERαprotein was mainly alpha helix and random coil.ERαgene in Cele Black sheep was expressed in ovaries at follicular phase,luteal phase and 30 days of pregnancy.The results provided a basis for further exploration of ERαgene which played a role in ovarian follicle development,corpus luteum formation and pregnancy of Cele Black sheep.