牦牛CRH基因克隆及其在生殖轴中的表达研究

为了探讨促肾上腺皮质激素释放激素（corticotropin-releasing hormone，CRH）基因在牦牛繁殖中的调控作用，试验分别采集5头成年母牦牛和5头成年母黄牛的下丘脑、脑垂体前叶、输卵管、卵巢和子宫等组织，通过RT-PCR技术及生物信息学软件对牦牛CRH基因编码区进行扩增、克隆及序列分析，并构建系统进化树；采用实时荧光定量PCR法检测CRH基因的组织表达。结果表明，牦牛CRH基因编码区全长573 bp，编码190个氨基酸。牦牛与黄牛、猫、鸡、人、小鼠、大鼠和猪的CRH基因核苷酸同源性分别为99.8%、43.3%、36.1%、46.2%、39.0%、38.5%和56.4%。系统进化树表明，牦牛与黄牛亲缘关系最近，并与其他物种类聚，符合物种进化规律。CRH蛋白分子式为C₉₂₀H₁₅₀₃N₂₇₉O₂₆₃S₃，分子质量为20776.95 u，理论等电点为10.95，其氨基酸组成中亮氨酸（L）、脯氨酸（P）、丙氨酸（A）和精氨酸（R）所占比例较高，分别为15.8%、12.6%、12.1%和10.5%。CRH蛋白为不稳定亲水蛋白，存在信号肽和跨膜结构。CRH蛋白二级结构中α-螺旋、β-转角、延伸链和无规则卷曲分别占41.05%、1.05%、8.42%和49.48%，三级结构分析结果与其相一致。CRH基因mRNA在牦牛下丘脑中表达量最高，显著高于其他组织（P<0.05），在子宫中表达量最低。牦牛脑垂体前叶CRH基因mRNA表达量极显著高于黄牛（P<0.01），在卵巢、输卵管中表达量显著高于黄牛（P<0.05），而两个品种在下丘脑、子宫中的表达量差异不显著（P>0.05）。提示CRH基因在牦牛繁殖调控中具有重要作用。

Cloning and Expression of Corticotropin-releasing Hormone (CRH) Gene in the Reproductive Axis of Yaks (Bos grunniens)

In order to investigate the regulation of corticotropin-releasing hormone (CRH) gene in reproduction of yak, tissue samples of hypothalamus, anterior pituitary, oviduct, ovaries and uterus were collected from 5 adult female yaks and cattle, respectively.The yak CRH gene was amplified, cloned and sequenced by RT-PCR and bioinformatics software, and phylogenetic tree was constructed.The mRNA expression in different tissues was detected by Real-time PCR.The results showed that the coding region of CRH gene in yak was 573 bp, encoding a 190 amino acid protein.The nucleotide homology of CRH gene in yak (Bos grunniens) and cattle (Bos taurus), Felis catus, Gallus gallus, Homo sapiens, Mus musculus, Rattus norvegicus and Sus scrofa was 99.8%, 43.3%, 36.1%, 46.2%, 39.0%, 38.5% and 56.4%, respectively.Phylogenetic tree results showed that yak and cattle had the closest relationship and clustered with other species, which accorded with the evolution law of species.The formula of CRH protein was C₉₂₀H₁₅₀₃N₂₇₉O₂₆₃S₃, the molecular weight and theoretical isoelectric point were 20 776.95 u and 10.95, respectively.CRH protein contained a high proportion of leucine (15.8%), proline (12.6%), alanine (12.1%) and arginine (10.5%).CRH protein was a hydrophilicity protein which contained a signal peptide and a transmembrane region.The secondary structure prediction results showed that alpha helix, beta turn, extended chain and random coil accounted for 41.05%, 1.05%, 8.42% and 49.48%, respectively, which was consistent with the tertiary structure analysis results.The expression of CRH gene mRNA in hypothalamus and uterus of yak was the highest and the lowest, respectively, and the expression of CRH gene mRNA in hypothalamus was significantly higher than that in other tissues (P<0.05).The expression of CRH gene mRNA in anterior pituitary, ovary and fallopian tube of yak were extremely significantly or significantly higher than that of cattle (P<0.01;P<0.05), but there was no significant difference in hypothalamus and uterus between two breeds (P>0.05).These results suggested that CRH gene played an important role in reproductive regulation of yak.