牦牛TNFAIP6基因克隆及其在卵巢活动中的时序表达

doi:10.13560/j.cnki.biotech.bull.1985.2019-0596

摘要/Abstract

摘要：

旨在探讨牦牛TNFAIP6基因的序列特征,比较分析其在牦牛不同组织以及发情周期卵巢中的时序表达差异性。采集健康雌性牦牛的心脏、肺脏、脾脏、肾脏、肝脏、子宫、小肠、胃、肌肉和不同发情期的卵巢组织,提取各组织总RNA和总蛋白,通过RT-PCR技术克隆获得牦牛TNFAIP6基因序列并对其进行生物信息学分析,用半定量PCR检测其在牦牛不同组织中的表达水平,Western blot和qRT-PCR法分别检测其在不同发情期牦牛卵巢中的蛋白和mRNA表达水平,并进行统计学分析。克隆获得牦牛TNFAIP6基因CDS区全长830 bp,共编码279个氨基酸。蛋白质分析显示,TNFAIP6蛋白为亲水酸性稳定蛋白,无跨膜结构但有信号肽,其中包含Link和CUB两个结构域,其二级结构和三级结构主要由无规卷曲和α-螺旋组成。通过同源性比对分析,发现牦牛TNFAIP6基因与野牦牛和黄牛的同源性较高。半定量PCR结果显示,TNFAIP6基因在牦牛各组织中均有表达,其中在卵巢、子宫、脾脏和肌肉组织中表达显著高于其他组织。qRT-PCR结果显示,TNFAIP6基因在卵泡期卵巢中的表达水平极显著高于其他两个时期（P<0.01）,而红体期与黄体期的表达水平差异不显著（P>0.05）。Western blot结果与qRT-PCR结果基本一致。提示TNFAIP6基因可能参与牦牛卵巢活动调控,为进一步探讨TNFAIP6基因在牦牛卵巢活动中的作用机制提供基础资料。

关键词: 牦牛, 卵巢, TNFAIP6, 时序表达

Abstract:

The aims of this study are to investigate the sequence characteristics of yak（Bos grunniens））TNFAIP6 gene and to analyze its temporal expression differences in different tissues and ovary at different stages of estrous cycle. Total RNA and protein from the heart,lung,spleen,kidney,liver,uterus,small intestine,gastric,muscle,and ovary tissues at different stages of estrous of healthy female yaks were extracted. The coding sequences of TNFAIP6 gene were cloned by RT-PCR and bioinformatics analysis of it was conducted. The expression level of TNFAIP6 gene in different tissues was detected by semi-quantitative PCR. Furthermore,the expression levels of protein and mRNA in yak ovary at different stages of estrous cycle were detected by Western blot and qRT-PCR. The collected data were statistically analyzed. The results showed that the CDS region of yak TNFAIP6 gene was 830 bp,encoding 279 amino acids. The analysis of proteins indicated that TNFAIP6 was hydrophilic and acidic-stable protein,had non-transmembrane domain and signal peptide,including Link and CUB structural domains. The secondary and tertiary structure of it was mainly composed of random coil and α-helix. Through homology alignment,the yak TNFAIP6 had high homology with cattle and wild yak. TNFAIP6 gene of yak from semi-quantitative PCR was widely expressed in various tissues and its expression in ovary,uterus,spleen and muscle was relatively higher than in other tissues. The expression level of TNFAIP6 in the follicular stage was significantly higher than that in the other two stages in ovary（P<0.01）,while there was no significant difference between the corpus luteum stage and red body stage（P>0.05）. The results of Western blot were basically consistent with qRT-PCR. In sum,the TNFAIP6 gene may be involved in the reproductive action of yak ovary,and the result provides basic reference in exploring the action mechanism of TNFAIP6 in the activity of yak ovary.

Key words: yak, ovary, TNFAIP6, temporal expression

马鸿程, 熊显荣, 穆松银, 海卓, 秦文昌, 李键. 牦牛TNFAIP6基因克隆及其在卵巢活动中的时序表达[J]. 生物技术通报, 2019, 35(11): 46-54.

MA Hong-cheng, XIONG Xian-rong, MU Song-yin, HAI Zhuo, QIN Wen-chang, LI Jian. Cloning of Yak TNFAIP6 Gene and Its Temporal Expression in Ovarian Activity[J]. Biotechnology Bulletin, 2019, 35(11): 46-54.

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