牦牛PRLHR基因的克隆、序列分析及组织表达研究

试验旨在对牦牛催乳素释放激素受体（prolactin releasing hormone receptor，PRLHR）基因进行克隆、序列分析及组织表达研究。采集5头母牦牛和5头母黄牛的下丘脑、脑垂体前叶、卵巢、输卵管和子宫组织，采用RT-PCR技术扩增得到PRLHR基因cDNA全长，通过生物信息学方法分析该基因编码蛋白的生物信息学特征，利用实时荧光定量PCR技术测定PRLHR基因在牦牛及黄牛各组织中的表达量。结果显示，牦牛PRLHR基因序列长1 625 bp，其中CDS区1 113 bp、5'-UTR 22 bp和3'-UTR 490 bp，编码370个氨基酸，与黄牛、水牛、绵羊、猪和人的核苷酸序列有较高的同源性，在进化过程中十分保守；牦牛PRLHR为不稳定疏水蛋白，无信号肽，存在7个跨膜结构域；有13个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点和4个酪氨酸磷酸化位点；有3个N-糖基化位点和10个O-糖基化位点；蛋白二级结构中α-螺旋、无规则卷曲、延伸链和β-转角分别为49.19%、31.89%、15.68%和3.24%；蛋白质三级结构预测显示，牦牛PRLHR蛋白具有GPCRs超级家族中PrRP家族的典型结构域。实时荧光定量PCR结果表明，PRLHR基因在牦牛输卵管组织中的表达量显著高于其他组织（P<0.05）；在牦牛下丘脑、脑垂体前叶、子宫和输卵管组织中的表达量极显著高于黄牛（P<0.01）。试验成功克隆得到牦牛PRLHR基因序列，并对其进行了生物信息学和组织表达特性分析，为进一步研究PRLHR基因在牦牛繁殖活动中的调控作用奠定了基础。

Cloning,Sequence Analysis and Tissue Expression of PRLHR Gene in Yak

This study was aimed to clone,analyze sequence and determine the tissue expression of PRLHR gene in yak.The hypothalamus,anterior pituitary,ovary,oviduct and uterus tissues of 5 female yaks and 5 female cattle were collected.The full-length cDNA sequence of PRLHR gene in yak was amplified by RT-PCR techniques,and the characteristics of PRLHR protein in yak was analyzed by bioinformatics methods.The expression of PRLHR gene in yak and cattle were detected by Real-time quantitative PCR.The results showed that the sequence of PRLHR gene in yak was 1 625 bp,which contained 1 113 bp of the CDS region,22 bp of 5'-UTR and 490 bp of 3'-UTR,encoding 370 amino acids.The CDS was high homology with Bos taurus,Bubalus bubalis,Ovis aries,Sus scrofa and Homo sapiens,indicating it was conservative in the evolutionary course.PRLHR protein in yak was an unstable hydrophobic protein with no signal peptide,but had 7 transmembrane domains.There were 13 serine phosphorylation sites,6 threonine phosphorylation sites and 4 tyrosine phosphorylation sites.It was predicted that there were 3 N-glycosylation sites and 10 O-glycosylation sites.The secondary structure of PRLHR protein in yak contained alpha helix (49.19%),random coil (31.89%),extended chain (15.68%) and beta turn (3.24%).The tertiary structure of PRLHR protein in yak had a typical domain of PrRP family in GPCRs superfamily.The results of Real-time quantitative PCR showed that the expression of PRLHR gene in oviduct of yak was significantly higher than that of other tissues (P<0.05).The expression of PRLHR gene in hypothalamus,anterior pituitary,uterus and oviduct tissues of yak was extremely significantly higher than that of cattle (P<0.01).In conclusion,cloning,sequence analysis and tissue expression of PRLHR gene in yak were successfully carried out,which provided a foundation for the further study on the regulatory role of PRLHR gene in yak reproduction.