生物技术通报 ›› 2022, Vol. 38 ›› Issue (6): 279-290.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1152

• 研究报告 • 上一篇    下一篇

牦牛TGF-β1基因克隆及在雌性生殖系统主要器官中的表达定位

王楠(), 张瑞, 潘阳阳, 何翃宏, 王靖雷, 崔燕, 余四九()   

  1. 甘肃农业大学动物医学院/甘肃省牛羊胚胎工程技术研究中心,兰州 730070
  • 收稿日期:2021-09-07 出版日期:2022-06-26 发布日期:2022-07-11
  • 作者简介:王楠,女,硕士研究生,研究方向:动物生殖生理;E-mail: 594302635@qq.com
  • 基金资助:
    国家自然科学基金项目(31972760);甘肃省杰出青年基金(20JR10RA561);甘肃省教育厅产业支撑引导项目(2019C-03);国家自然科学基金地区项目(32160859)

Cloning of Bos grunniens TGF-β1 Gene and Its Expression in Major Organs of Female Reproductive System

WANG Nan(), ZHANG Rui, PAN Yang-yang, HE Hong-hong, WANG Jing-lei, CUI Yan, YU Si-jiu()   

  1. College of Veterinary Medicine/Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat,Gansu Agricultural University,Lanzhou 730070
  • Received:2021-09-07 Published:2022-06-26 Online:2022-07-11

摘要:

转化生长因子β1(transforming growth factor beta1,TGF-β1)是一种多功能的生长与分化因子,广泛调控机体的多个生理病理过程,具有重要的生物学功能和广阔的应用前景。试验采集牦牛发情期和妊娠期卵巢、输卵管和子宫组织,克隆牦牛TGF-β1基因,利用实时荧光定量PCR(qRT-PCR)、免疫组织化学(immunohistochemistry,IHC)、蛋白质免疫印迹(Western blot,WB)等方法,对牦牛TGF-β1在基因和蛋白水平上进行定量分析。结果显示,牦牛TGF-β1基因(GenBank No.MZ004937)高度保守,牦牛TGF-β1核苷酸序列与普通牛(Bos taurus)序列相比,存在差异,所编码氨基酸由丙氨酸突变为甘氨酸。与普通牛(Bos taurus)、瘤牛×普通牛(Bos indicus×Bos taurus)亲缘关系最近,与犬(Canine)亲缘关系最远,其编码的蛋白为稳定的亲水性膜蛋白。牦牛TGF-β1在卵泡期、黄体期、妊娠期的卵巢、输卵管和子宫组织中均有表达,在卵巢中,妊娠期的表达量显著高于卵泡期和黄体期(P<0.05);在输卵管中,TGF-β1基因妊娠期表达量极显著高于卵泡期和黄体期(P<0.01);在子宫中,卵泡期和妊娠期表达显著高于黄体期(P<0.05)。IHC结果显示,牦牛TGF-β1主要在卵巢生殖上皮、卵泡膜、卵泡颗粒层、黄体细胞、输卵管黏膜上皮细胞、子宫腺(UG)和子宫内膜细胞中表达。研究结果为进一步探索TGF-β1参与牦牛生殖生理的分子机制提供基础数据,以期为探讨高原哺乳动物对高寒环境的适应性提供理论依据。

关键词: 牦牛, TGF-β1, 基因克隆, 生物信息学分析, 表达

Abstract:

Transforming growth factor β1(TGF-β1)is a multifunctional growth and differentiation factor,which regulates many physiological and pathological processes of the body,and has important biological functions and broad application prospects. In this study,we collected ovarian,oviductal and uterine tissues from yaks during estrus and pregnancy,cloned the yak TGF-β1 gene,and performed the quantitative analysis of yak TGF-β1 at the gene and protein levels using quantitative real-time fluorescence PCR(qRT-PCR),immunohistochemistry(IHC),and protein immunoblotting(Western blot). The results showed that TGF-β1 gene(GenBank No.MZ004937)was highly conserved,and the nucleotide sequence differed from the TGF-β1 nucleotide sequence of Bos taurus,and the encoded amino acid was mutated from alanine to glycine. It was most closely related to Bos taurus,Bos indicus×Bos taurus,and most distantly related to Canine. The encoded protein was a stably hydrophilic transmembrane protein. TGF-β1 was expressed in ovary,oviduct and uterus tissues of yak during follicular phase,luteal phase and gestation phase. In ovary,the expression of TGF-β1 during gestation phase was significantly higher than that during follicular phase and luteal phase(P<0.05). In oviduct,the expression of TGF-β1 gene in gestation phase was significantly higher than that in follicular and luteal phases(P<0.01). In utero,the expression in follicular phase and gestation phase was significantly higher than that in luteal phase(P<0.05). Immunohistochemistry(IHC)results indicated that TGF-β1 was mainly expressed in ovarian reproductive epithelium,follicular membrane,granular layer of follicle,luteal cells,tubal mucosal epithelial cells,uterine gland(UG)and endometrium cells. The results will provide basic data for further exploring the molecular mechanism of TGF-β1 in reproductive physiology of yaks,and provide theoretical basis for exploring the adaptability of plateau mammals to alpine environment.

Key words: yak, TGF-β1, gene cloning, bioinformatics analysis, expression