牦牛GNAQ基因的克隆及其在雌性生殖轴系中的表达  

Cloning of GNAQ Gene in Yak(Bos grunniens) and Its Expression in Female Reproductive Axis

作　　者：陈祎玮 褚敏[1,3] 张任政 谢建鹏 马兰花 潘和平 阎萍[1,3] CHEN Yi-Wei;CHU Min;ZHANG Ren-Zheng;XIE Jian-Peng;MALan-Hua;PAN He-Ping;YAN Ping(Lanzhou Institute of Husbandry and Pharmaceutical Sciences/Key Laboratory of Yak Breeding Engineering Gansu Province,Chinese Academy of Agricultural Sciences,Lanzhou 730050,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China;Key Laboratory ofAnimal Genetics and Breeding on Tibetan Plateau,Ministry ofAgriculture and RuralAffairs,Lanzhou 730050,China)

机构地区：[1]中国农业科学院兰州畜牧与兽药研究所/甘肃省牦牛繁育工程重点实验室,兰州730050 [2]西北民族大学生命科学与工程学院,兰州730030 [3]农业农村部青藏高原畜禽遗传育种重点实验室,兰州730050

出　　处：《农业生物技术学报》2023年第2期322-333,共12页Journal of Agricultural Biotechnology

基　　金：中国农业科学院创新工程项目(CAAS-ASTIP-2014-LIHPS-01);中国农业科学院基本科研业务费院级统筹任务(1610322020009);现代农业(肉牛牦牛)产业技术体系建设专项资金(CARS-37)。

摘　　要：鸟嘌呤核苷酸结合蛋白q多肽(G protein subunit alpha q, GNAQ)通过编码Gαq蛋白调节G蛋白偶联受体和介导下游信号通路,在营养代谢、繁殖调控以及细胞信号传导等生理过程中参与调节。为研究GNAQ在雌性牦牛(Bos grunniens)生殖轴系中的表达情况,本研究选取成年健康卵泡期雌性牦牛的下丘脑、垂体和卵巢组织,以牦牛卵巢cDNA为模板,采用逆转录PCR(reverse transcription-PCR, RT-PCR)技术克隆得到GNAQ基因完整的CDS序列,并进行生物信息学分析;以qRT-PCR、蛋白质免疫印迹(Western blot)和免疫组织化学(immunohistochemistry, IHC)方法对GNAQ基因和蛋白在牦牛下丘脑-垂体-卵巢生殖轴(hypothalamic-pituitary-ovarian axis, HPOA)的表达进行检测和定位。结果表明,牦牛GNAQ基因(GenBank No. OP811271)的CDS区全长1 080 bp,编码359个氨基酸;牦牛GNAQ基因与普通牛(B. taurus)同源性高,与小鼠(Mus musculus)同源性较低,在物种进化过程中高度保守。经qRT-PCR、Western blot检测发现,GNAQ基因和蛋白在牦牛HPOA轴的下丘脑、垂体以及卵巢中呈现不同程度的表达,在垂体中的表达量最高,在下丘脑中表达水平显著高于卵巢(P<0.05)。免疫组化结果显示,GNAQ蛋白在牦牛卵巢颗粒细胞呈强阳性表达,且主要表达于细胞质中,与亚细胞定位结果一致。本研究为深入探索GNAQ基因在牦牛生殖生理调控中的作用提供理论依据,为牦牛季节性发情调控规律及机制的研究提供参考。G protein subunit alpha Q(GNAQ) regulate G protein-coupled receptors and mediate downstream signaling pathways through encoding Gαq protein, which involves many physiological processes such as nutrient metabolism, reproduction regulation and cell signal transduction. This study aimed to explore the expression of GNAQ in reproductive axons of female yak(Bos grunniens). The samples of hypothalamus,pituitary and ovarian of healthy female yaks(adult) during follicular phase were collected. The CDS of GNAQ gene was cloned with yak ovarian cDNA as template by reverse transcription-PCR(RT-PCR) and it was analyzed by bioinformatics software. The hypothalamic-pituitary-ovarian reproductive axis(HPOA)expression characteristics of yak GNAQ gene and protein levels were detected by qRT-PCR, Western blot and immunohistochemistry(IHC).The results showed that the CDS of yak GNAQ gene(GenBank No. OP811271) was 1 080 bp in length and encoded 359 amino acids. GNAQ was close to amino acid homology of ordinary cattle(B. taurus), and distant to mouse(Mus musculus), and it was highly conserved in the evolution of the species. It was found that GNAQ gene and protein levels were expressed in hypothalamus, pituitary and ovary of yak by qRT-PCR and Western blot, and the highest expression level was found in pituitary, the expression level in hypothalamus was significantly higher than in ovary(P<0.05). Immunohistochemistry results showed that GNAQ protein was strongly positive in ovarian granulosa cells and was mainly expressed in cytoplasm, which was consistent with the prediction of subcellular localization. This study provides theoretical basis for further exploration of the role of GNAQ gene in reproductive physiology regulation of yak, which is helpful to study the regulation and mechanism of seasonal estrus in yak.

关 键 词：牦牛 鸟嘌呤核苷酸结合蛋白q多肽(GNAQ) 生殖轴 

分 类 号：S823.85[农业科学—畜牧学]