秦川牛Snail2基因扩增、序列特征及表达特性分析

doi:10.16431/j.cnki.1671-7236.2021.09.005

摘要/Abstract

摘要： 试验旨在扩增秦川牛Snail2基因，构建Snail2基因在其各组织不同发育阶段及脂肪细胞不同分化时期的时空表达规律，为进一步研究Snail2基因在牛脂肪沉积中的功能及调控作用奠定基础。以秦川牛为研究对象，经PCR扩增得到Snail2基因CDS区序列，运用生物信息学软件对其功能结构进行预测，同时采用实时荧光定量PCR检测Snail2基因在新生牛和成年牛各组织及脂肪细胞成脂分化过程中的时空表达谱。结果显示，秦川牛Snail2基因编码序列全长为952 bp。不同物种系统进化树结果表明，牛Snail2蛋白在家牛、水牛、山羊、绵羊中高度保守。磷酸化位点分析发现，存在41个潜在磷酸化位点，其中周期蛋白依赖性激酶1（cyclin dependent kinase 1，CDK1）、CDK5、CKⅡ、CKⅠ等多个细胞周期相关激酶参与了Snail2蛋白的磷酸化修饰。蛋白结构域预测发现，牛、人和鼠等8个物种中有8个相似Motif。Snail2蛋白二级结构主要由不规则卷曲构成，二、三级结构预测结果一致。Snail2基因启动子区序列分析发现1个CpG岛及E2F、C/EBPα（CCAAT/enhancer binding proteins alpha）、AP2和Sp1等与脂肪生成相关的转录因子结合位点。实时荧光定量PCR结果显示，Snail2基因在牛脂肪组织中呈现较高表达水平，且随着脂肪细胞成脂分化和牛生长发育过程均呈现上升趋势，表明Snail2基因在牛脂肪沉积过程中发挥重要作用。研究结果为进一步揭示Snail2基因通过影响脂肪细胞增殖和分化进而影响肉牛脂肪沉积作用机制奠定基础。

关键词: 秦川牛; Snail2基因; 生物信息学; 时空表达谱

Abstract: The purpose of this experiment was to amplify the Snail2 gene of Qinchuan cattle, and construct the temporal and spatial expression pattern of Snail2 gene in different developmental stages of tissues and different differentiation stages of adipocytes, which laid a foundation for further study on the function and regulation of Snail2 gene in bovine fat deposition. Qinchuan cattle were taken as the research object, the CDS region sequence of Snail2 gene was amplified by PCR, and its functional structure was predicted by bioinformatics softwares. At the same time, Real-time quantitative PCR was used to detect the temporal and spatial expression profile of Snail2 gene in the process of adipogenic differentiation of newborn and adult bovine tissues and adipocytes. The results showed that the full length of Snail2 gene coding sequence of Qinchuan cattle was 952 bp. The phylogenetic tree results of different species showed that bovine Snail2 protein was highly conserved in cattle, buffalo, goat and sheep. 41 potential phosphorylation sites were found by phosphorylation site analysis, in which cyclin dependent kinase 1 (CDK1), CDK5, CKⅡ, CKⅠ and other cell cycle-related kinases were involved in the phosphorylation of Snail2 protein. Protein domain prediction found that there were 8 similar Motif among 8 species including cattle, human and mouse, etc. The secondary structure of Snail2 protein was mainly composed of irregular curls, and the prediction results of secondary and tertiary structures were consistent. Sequence analysis of the promoter region of Snail2 gene revealed a CpG island and E2F, C/EBPα(CCAAT/enhancer binding proteins alpha), AP2 and Sp1 transcription factor binding sites related to adipogenesis. The results of Real-time quantitative PCR showed that Snail2 gene was highly expressed in bovine adipose tissue, and showed an upward trend with adipogenic differentiation and bovine growth and development, indicating that Snail2 gene played an important role in bovine fat deposition. The results laid a foundation for further revealing the mechanism of Snail2 gene affecting fat deposition in beef cattle by affecting the proliferation and differentiation of adipocytes.

Key words: Qinchuan cattle; Snail2 gene; bioinformatics; temporal and spatial expression pattern

中图分类号:

S823

田媛, 龙凤, 李安奇, 陈嘉玥, 杨森, 昝林森, 成功. 秦川牛Snail2基因扩增、序列特征及表达特性分析[J]. 中国畜牧兽医, 2021, 48(9): 3147-3157.

TIAN Yuan, LONG Feng, LI Anqi, CHEN Jiayue, YANG Sen, ZAN Linsen, CHENG Gong. Amplification, Sequence Feature and Expression Pattern of Snail2 Gene in Qinchuan Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3147-3157.

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