牦牛RBM3的基因克隆及其在不同繁殖时期卵巢、输卵管、子宫中的表达定位

RBM3 Gene Cloning and Its Expression,Localization in Ovary,Fallopian Tube and Uterus at Different Reproductive Stages of Yak(Bos grunniens)

旨在研究RBM3在牦牛(Bos grunniens)生殖相关调控中的作用。本研究以卵泡期、黄体期和妊娠期4~6岁健康雌性牦牛各3头为采集对象,采集其卵巢、子宫和输卵管共9组试验样品,每组设置3个生物学重复。利用基因克隆技术克隆牦牛RBM3基因并进行生物信息学分析;利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和蛋白免疫印迹(Western blot,WB)分别在基因层面和蛋白层面检测RBM3在牦牛子宫、卵巢和输卵管中的相对表达量;利用免疫组织化学(immunocytochemistry,IHC)方法检测RBM3蛋白在试验样品中的分布定位。牦牛RBM3基因(GenBank No.:MF142258.1)被成功克隆并预测出二、三级结构,发现其与野牦牛亲缘性最近,基因编码区第51位、98位核苷酸不同于常见哺乳动物,生物信息学分析预测其编码的蛋白质为稳定的非跨膜蛋白。RBM3蛋白在本试验9组样品的表达量有如下差异:在卵巢中,黄体期显著高于卵泡期和妊娠期(P<0.05);输卵管中,卵泡期显著低于黄体期和妊娠期(P<0.05);子宫上,卵泡期显著高于黄体期和妊娠期(P<0.05)。免疫组织化学结果显示,RBM3在卵巢中的主要表达位置是卵泡膜、颗粒层和黄体细胞;在输卵管的表达位置是黏膜上皮;子宫上的表达主要以子宫内膜和子宫腺为主。本研究结果提示,RBM3可能参与牦牛个体发情周期和妊娠过程的调控以及妊娠识别等过程,为RBM3这一冷应激调节因子参与牦牛对高原环境的适应性生理机制方面的研究提供参考。

The study aimed to investigate the role of RBM3 in the reproductive-related regulation in yak(Bos grunniens).Three healthy female yaks aged 4-6 years old in different reproductive periods(follicular,luteal and pregnancy phases)were used in this study.Nine groups of experimental samples were collected from ovaries,uterus and fallopian tubes,and 3 biological replicates were set in each group.The RBM3 gene of yak was cloned by gene cloning technology and analyzed by bioinformatics.RBM3 relative expression levels of gene and protein in 9 groups of experimental samples were detected by quantitative real-time PCR(qRT-PCR)and Western blot(WB).The distribution of RBM3 protein in samples was detected by immunohistochemistry(IHC).RBM3 gene(GenBank No.:MF142258.1)of yak was cloned in this experiment,it had the closest relationship with wild yak(Bos mutus).Bioinformatics analysis showed that nucleotide No.51 and No.98 were different from several common mammals,and its secondary and tertiary structure was constructed.After prediction,the encoded protein was stable non-transmembrane protein.WB results showed that RBM3 was expressed in fallopian tube,ovary and uterus of yak,but the expression level at luteal phase was significantly higher than that at follicular and pregnancy phases(P<0.05).The expression in fallopian tube at follicular phase was significantly lower than that at luteal and pregnancy phases(P<0.05).The expression in uterus at follicular phase was significantly higher than that at luteal and pregnancy phases(P<0.05).Immunohistochemical results showed that the main expression sites of RBM3 in the ovary were theca follicle,granular layer and luteal cells,while the expression sites in the fallopian tube were epithelium mucosae.For the uterus,the expression sites was mainly in the endometrium and uterine glands.The results of this study suggest that RBM3 may be involved in the regulation of individual estrous cycle and pregnancy process in yak,as well as pregnancy recognition.It provides a reference for the research of RBM3,a cold stress regulatory factor,in the physiological mechanism of yak in cold and hypoxic environment at high altitude,and other related aspects.