牦牛FSHR基因克隆及其在生殖轴中的表达研究

本研究旨在阐明牦牛促卵泡素受体(follicle stimulating hormone receptor,FSHR)基因CDS序列及其在牦牛生殖轴中表达的特点,为探讨其在牦牛繁殖活动中的调控作用奠定基础。试验采集卵泡期的牦牛与黄牛下丘脑、脑垂体前叶、卵巢、输卵管及子宫组织,通过RT-PCR技术对牦牛FSHR基因cDNA进行扩增、克隆与序列分析;采用实时荧光定量PCR法检测FSHR基因在牦牛与黄牛中的组织表达差异。结果显示,牦牛FSHR基因编码区全长2 088bp,编码695个氨基酸,蛋白质分子式为C6378H10670N2088O2637S576,分子质量为177 263.85u,理论等电点(pI)为4.88,与黄牛、绵羊、山羊和猪氨基酸序列同源性较高(91.50%～99.38%)。FSHR蛋白为酸性不稳定疏水蛋白,存在信号肽与8个跨膜结构;二级结构由延伸链(15.54%)、α-螺旋(42.30%)、β-转角(1.44%)和无规则卷曲(40.72%)组成;系统进化树表明,牦牛与黄牛亲缘关系最近;实时荧光定量PCR结果显示,FSHR基因在黄牛、牦牛检测组织中均有表达,牦牛子宫中表达量显著或极显著高于除卵巢外的其他组织(P<0.05;P<0.01),而黄牛卵巢中表达量显著或极显著高于其他组织(P<0.05;P<0.01);黄牛卵巢中表达量极显著高于牦牛(P<0.01)。说明FSHR基因在动物进化中较为保守,其在牦牛卵巢中表达量低可能影响到牦牛繁殖机能。

Cloning and Expression Analysis in Reproductive Axis of Yak FSHR Gene

The aim of this study was to investigate the potential role of follicle stimulating hormone receptor (FSHR) in regulating yak reproduction by clarifying its CDS sequence and expression characteristics in the reproductive axis of yak (Bos grunniens).The hypothalamus,anterior pituitary,ovary,oviduct and uterus of yak and cattle (Bos taurus) were collected during the follicular phase.The yak FSHR gene was amplified and cloned by RT-PCR,and its mRNA expression levels in different tissues was detected by Real-time PCR.The results showed that the FSHR coding region of the yak was 2 088 bp,encoding 695 amino acids.The formula of FSHR protein was C₆₃₇₈H₁₀₆₇₀N₂₀₈₈O₂₆₃₇S₅₇₆,the molecular weight of FSHR protein was 177 263.85 u,and the theoretical isoelectric point was 4.88.Yak FSHR amino acids shared high homology with cattle,sheep,goat and wild boars (91.50% to 99.38%).The FSHR protein was an acid labile hydrophobin protein with 8 transmembrane structures and signal peptides;The secondary structure consisted of extended chain (15.54%),α-helix (42.30%),β-turn (1.44%) and random coil (40.72%).The phylogenetic tree showed the genetic relationship was the closest between yak and cattle.The Real-time PCR results showed that FSHR gene expressed in all tissues examined.Its expression in yak uterus was significantly or extremely significantly higher than other tissues (except ovary)(P<0.05;P<0.01),while it was significantly of extremely significantly higher in ovary of cattle than other tissues (P<0.05;P<0.01).Its expression level in cattle ovary was extremely significantly higher than yak (P<0.01).These data indicated that FSHR gene was relatively conservative in the course of animal evolution,and its lower expression in yak ovary possibly affected yak reproduction.