中国美利奴成母羊促甲状腺激素受体基因的克隆与组织表达

Cloning and Expression of Thyroid- stimulating Hormone Receptor Gene in Sheep

【目的】探讨绵羊TSHR基因序列及组织表达特征,为进一步研究TSHR在绵羊季节性繁殖中作用的分子机理奠定基础。【方法】利用RT-PCR技术克隆绵羊TSHR基因,并对序列进行生物信息学分析;采用半定量RT-PCR技术检测TSHR在绵羊不同组织的表达情况。【结果】以中国美利奴羊卵巢组织cDNA为模板,获得了3 276 bp的TSHR cDNA序列,包括全部编码区序列(Coding Sequence,CDS)长2 295 bp,编码764个氨基酸。TSHR蛋白结构经预测发现存在多个磷酸化位点和蛋白激酶C磷酸化位点。半定量RT-PCR结果显示,绵羊TSHR基因在所检测组织中均有表达,其中在下丘脑、松果体和垂体中呈高丰度表达,在甲状腺、子宫等组织呈中等丰度表达。【结论】从绵羊卵巢组织中克隆到TSHR基因并进行了序列分析;TSHR基因在绵羊各组织中广谱表达,但在下丘脑、松果体、垂体、甲状腺中表达水平较高,提示TSHR在绵羊季节性繁殖等重要生理活动的神经内分泌调节过程中发挥重要作用。

[Objective]To clone and obtain expression characteristic of thyroid-stimulating hormone receptor （TSHR） gene in sheep.[Method] TSHR gene was cloned from sheep ovarian cDNA by RT-PCR and the sequence was analyzed by the means of bioinformatics.TSHR gene expression profiles in different tissues of sheep were detected by semi-quantitative RT-PCR.[Result] The whole cDNA of sheep TSHR was obtained,the nucleotide sequence was 3 276 bp long and contained an open reading frame encoding a protein of 764 amino acids.Multiple phosphorylation sites and protein kinase C phosphorylation sites were forecasted in sheep TSHR protein structure.Semi-quantitative RT-PCR showed that expression of TSHR gene was found in both detected tissues,there were significant differences between the expression levels of the organization.TSHR gene transcripts were highly abundant in hypothalamus,pituitary gland,pineal gland and thyroid gland.[Conclusions] TSHR gene was cloned from sheep ovarian cDNA.TSHR gene was expressed widely in many tissues tested but its level was higher in hypothalamus,pituitary gland,pineal gland and thyroid gland than in others.These results suggested that the TSHR may play an important role in the control of seasonal reproductive in sheep.