绵羊RARG基因在发情不同阶段卵巢中的mRNA表达

doi:10.3864/j.issn.0578-1752.2014.15.016

摘要/Abstract

摘要： 【目的】分离鉴定绵羊视黄酸受体-γ（Retinoic acid receptor gamma，RARG）基因，分析该基因的分子特征，研究其在发情周期不同阶段卵巢中的表达差异。【方法】选取年龄和体况相近、健康的甘肃高山细毛羊周岁母羊8只，通过同期发情处理，根据卵巢生理状态，将其分为两组：黄体期组和卵泡期组，各4只，屠宰后，采集卵巢组织，以周岁母羊卵巢组织RNA为模板，采用RT-PCR技术扩增绵羊RARG基因并克隆到pMD18-T载体中进行测序验证。利用ORF Finder和DNAStar等生物信息学软件分析绵羊RARG基因的理化性质、同源性和预测结构域和功能。利用Q-PCR技术，检测绵羊RARG基因在母羊发情周期不同阶段卵巢组织中的相对表达量，用SPSS19.0软件进行差异显著性分析，探讨RARG基因对绵羊发情周期的调控机制。【结果】获得了绵羊RARG基因包含完整编码区序列在内的1 588 bp的绵羊RARG基因序列，并提交至NCBI，GenBank登录号为KF019682。该序列含有1个1 377 bp的完整开放阅读框（Open reading frame，ORF），编码458个氨基酸，其蛋白分子质量为50 955.8 D，理论等电点为7.45。绵羊RARG基因与牛、普通狨、家犬、仓鼠、家猫、人、小鼠、虎鲸、狒、家鼠、野猪、海牛、海豚和蟾蜍等14个物种的同源性分析，结果显示RARG基因在上述物种间高度保守，且与牛的亲缘关系较近，序列同源性最高。GO功能分析结果显示，绵羊RARG基因作为结构蛋白参与转录调控的几率最高，分别为0.272和0.223。在绵羊RARG基因编码的氨基酸序列上发现了2个功能结构域，分别是核激素受体c4锌指结构域（c4 zinc finger in nuclear hormone receptors，ZnF_C4）和激素受体配体结构域（ligand binding domain of hormone receptors，HOLI）。Q-PCR结果显示，绵羊RARG mRNA 在甘肃高山细毛羊卵泡期卵巢中的相对表达量显著高于黄体期（P<0.05）。【结论】绵羊RARG基因在物种间高度保守，含有ZnF_C4和HOLI等2个与转录调控相关的功能结构域，推测该基因可能作为一种重要的转录因子参与母羊发情周期调控。

关键词: 绵羊 , 发情周期 , 卵巢 , RARG基因 , mRNA表达

Abstract: 【Objective】The objective of this study is to identify ovine retinoic acid receptor gamma (RARG) gene and to discover the differential expression of ovine RARG in ovary at different estrous cycles. 【Method】 Eight one-year old Gansu alpine fine-wool sheep were chosen for estrus synchronization treatment and then divided into 2 groups based on the ovarian physiological situation: one is corpus luteum group and the other one is estrous group. RNA were isolated from the ovarian tissue and RT-PCR was used to clone the ovine RARG gene, bioinformation method was used to construct ovine RARG gene phylogenetic tree as well as the prediction of protein structure and potential function. Q-PCR was applied to discover the differential expression of ovine RARG in ovary at different estrous cycles. 【Result】 A 1 588 bp full-length ovine RARG cDNA was identified and contained a 1 377 bp predicted ORF (GenBank accession No: KF019682), which encoded 458 amino acids with a calculated molecular weight of 50.96 kDa and an estimated isoelectric point (pI) of 7.45. Phylogenetic tree analysis showed that the ovine RARG gene has a close relationship with Bos taurus’, and the gene sequence among these species contained high homologous. Protein structure prediction indicated that the ovine RARG protein contains two main domains: c4 zinc finger in nuclear hormone receptors(ZnF_C4), and ligand binding domain of hormone receptors (HOLI). Q-PCR result demonstrated that the expression of ovine RARG mRNA in estrous situation is higher than that in corpus luteum stage. 【Conclusion】 The ovine RARG gene may work as an important transcriptional factor involved in female ovine estrous cycle control.

Key words: sheep (Ovis aries) , estrous cycle , ovary , RARG , expression

王维民, 胡庭溪, 李发弟, 马友记, 樊红樱, 潘香羽. 绵羊RARG基因在发情不同阶段卵巢中的mRNA表达[J]. 中国农业科学, 2014, 47(15): 3069-3076.

WANG Wei-Min, HU Ting-Xi, LI Fa-Di, MA You-Ji, FAN Hong-Ying, PAN Xiang-Yu. Study on Differential Expression of Ovine RARG in Ovary at Different Estrous Cycles[J]. Scientia Agricultura Sinica, 2014, 47(15): 3069-3076.

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