豚鼠发情周期不同时期的子宫LHR mRNA差异表达研究

摘要

[目的] 克隆豚鼠子官黄体生成素受体(LHR)基因片段并分析其进化关系,研究豚鼠子宫LHR mRNA在发情周期不同时期的表达特性.[方法] 以豚鼠子宫总RNA为模板,根据小鼠、大鼠、猪LHR保守区设计引物,RT-PCR扩增LHR基因片段.筛选阳性克隆测序,采用DNAMAN软件分析比对同源性.以β-actin为分子内参,采用优化的半定量RT-PCR技术,测定豚鼠发情周期不同时期的子官组织LHR mRNA表达量.[结果] 测序得到686 bp的剪接变异体l和缺失75 bp 4号外显子的剪接变异体2.686 bp序列与人类LHR mRNA序列同源性为83.8％,与牛、猪及绵羊LHR mRNA序列同源性分别为84.6％、84.2％及83.6％,而与大鼠和小鼠LHRmRNA序列同源性则分别高达93.7％和99.4％.LHR mRNA在豚鼠整个发情周期的子宫中都有表达,发情周期第0天的表达水平为0.635±0.0194,第4天为0.617±0.099,均显著低于第8天的表达水平(P＜0.05),随之在第12天升至1.218±0.049.[结论] 克隆的片段为豚鼠LHR基因的部分序列,其mRNA存在着剪接变异体.豚鼠子官LHR mRNA在发情周期第0、4天表达水平较低,第12天升至最高,即发情周期不同时期的LHR mRNA表达呈现明显的规律性,该结果为探究哺乳动物非性腺组织中LHR的功能及生殖内分泌调控机制提供了理论依据.%[Objective] This paper is to clone luteinizing hormone receptor (LHR) fragment from uterine tissue of guinea pig and to analyze evolutionary relationships and the expression characteristics of LHR mRNA during estrous cycle. [Method] Total RNA extracted from the uterus of guinea pig and primers designed according to the conservative sequence of pig, rat and mice was used for RT-PCR to amplify LHR fragment. Positive clones were identified and sequenced. The DNAMAN software was applied to analyze the homology. Β-actin was used as an inner control and an optimal semi-quantitative RT-PCR method was established to analyze LHR mRNA expression levels in the uterus during 4 estrous cycle phases. [Result] Sequencing analysis indicated that there were two kinds of spliced variants: One was the 686 bp fragment and another was 611 bp with a deletion of 75 bp in exon 4. The 686 bp fragment was 83.8% identical to the reported human LHR mRNA, 84.6% to the cattle, 84.2% to the pig, 83.6% to the sheep, 93.7% to the rat, and 99.4% to the mice of LHR mRNA. The uterine LHR mRNA expression was different during 4 phases of the estrous cycle. The levels on day 0(0.635 ± 0.019) and day 4(0.617 ± 0.099) were both significantly lower than that on day 8(.P<0.05), then it rose to the highest on day 12(1.218 ± 0.049) of the cycle. [ Conclusion ] The fragment cloned was a partial sequence of LHR, and the mRNA alternatively spliced variant often existed. The expression level of LHR mRNA in the uterine decreased on days 0 and 4, and increased on day 12. The results have provided a theoretical basis for exploring the functions of LHR in non-gonad tissues ofmammals, and meanwhile provided molecular data for further researches in regulation of reproductive endocrinology in other mammals.