山羊子宫中LHR基因片段的克隆及半定量RT-PCR检测方法的建立

Cloning and establishment of the semi-quantitative RT-PCR system of LHR partial gene of Goat's Uterus

【目的】进一步研究处于发情周期不同阶段的济宁青山羊子宫中黄体生成素受体（LHR）mRNA表达量的变化，为探讨其对山羊生殖内分泌机制的作用规律提供理论依据。【方法】以GAPDH为内参照基因，从济宁青山羊的子宫组织中提取总RNA，用RT—PCR方法扩增目的基因，进行克隆测序，并对半定量RT—PCR反应体系进行了优化。【结果】获得了济宁青山羊LHR mRNA的部分序列，长度约为286bp，与已报道的GenBank中羊的I。HRmRNA（序列号为AF379199）41～326bp序列同源性为100％。以此基因片段的阳性克隆为基础优化的半定量RT—PCR体系为：LHR基因的适宜循环数为30个，内参照GAPDH基因的循环数为26个，Mg^2＋浓度均为1．5mmol／L。【结论】克隆了山羊LHRmRNA的部分序列，并建立了优化的半定量RT—PCR体系。

[Objective] Luteinizing hormone receptor （LHR） gene mRNA was amplified using RT PCR to further study the different levels of LHR mRNA expression in the goat uterus during the various estrous cycle phases and research the possible role of the reproductive endocrine secretion of goat. [Method] Using GAPDH as inner control,total RNA was extracted from uterus of Jining gray goat. [Result] Sequenced analysis suggested that this fragment was partial sequence of LHR gene. The gene homology of fragment obtained in this study compared with that of reported LHR mRNA （AF379199） of Capra hircus was 100~. Cycle numbers of PCR system and the concentration of MgCle was optimized and analyzed. [Conclusion] The goat＇s partial LHR mRNA gene was successfully cloned in present study and the optimized semi-quantitative RT-PCR system was established.