夏南牛SOX6基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.07.006

摘要/Abstract

摘要： 【目的】克隆夏南牛SRY-box转录因子6（SRY-box transcription factor 6，SOX6）基因CDS区序列并进行生物信息学分析，探究SOX6基因在不同月龄夏南牛不同组织及牛原代骨骼肌细胞分化过程中的表达规律。【方法】以夏南牛腿肌组织cDNA为模板，PCR扩增SOX6基因CDS区序列，与其他物种进行相似性比对及系统进化树构建，运用在线软件对SOX6蛋白进行生物信息学分析；采用实时荧光定量PCR方法分别检测SOX6基因在夏南牛犊牛（0月龄）、青年牛（12月龄）和成年牛（24月龄）不同组织以及牛原代骨骼肌细胞不同分化时期的表达情况。【结果】夏南牛SOX6基因CDS区序列全长2 526 bp，编码841个氨基酸。系统进化树结果表明，夏南牛与牦牛亲缘关系最近，与鸡亲缘关系最远。SOX6蛋白为弱碱性、亲水性蛋白，分子质量为93.35 ku，定位于细胞核中，不含跨膜结构与信号肽。SOX6蛋白二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成，占比分别为41.26%、45.54%、8.09%和5.11%。蛋白互作分析表明，SOX6蛋白与Ⅱ型胶原α1（COL2A1）、软骨蛋白聚糖（ACAN）、羧基端结合蛋白2（CTBP2）和叉头框J3（FOXJ3）等蛋白间存在相互作用。实时荧光定量PCR结果显示，夏南牛SOX6基因在不同月龄夏南牛腿肌组织中相对表达量均最高，在脾脏中表达量最低；在12月龄腿肌组织中表达量显著高于0、24月龄（P<0.05）。随牛原代骨骼肌细胞分化，SOX6基因表达量逐渐升高，第6天时SOX6基因表达量显著高于0、2、4和8 d （P<0.05）。【结论】本研究成功克隆了夏南牛SOX6基因CDS序列，其编码蛋白为不稳定、弱碱性、亲水性蛋白，在不同月龄腿肌组织中表达量均显著高于其他组织，分化6 d牛原代骨骼肌细胞中表达量显著高于其他时期。本试验结果为进一步研究SOX6基因在夏南牛肌肉发育过程中的调控作用提供理论依据。

关键词: 夏南牛; SOX6基因; 克隆; 生物信息学; 组织表达

Abstract: 【Objective】 This study was aimed to clone the CDS sequence of RY-box transcription factor 6 (SOX6) gene in Xiannan cattle and carry out bioinformatics analysis, and explore the expression rules of SOX6 in different tissues of Xiannan cattle at different months and the differentiation of bovine primary skeletal muscle cells.【Method】 The CDS sequence of SOX6 gene was amplified by PCR using the cDNA template of leg muscle in Xianan cattle. Sequence similarity alignment and phylogentic tree contruction were carried out with other species, and the bioinformatics analysis of SOX6 protein was performed by the online software. The relative expression of SOX6 gene in different tissues of calves (0 month old), young cattle (12 months old) and adult cattle (24 months old) and different differentiation stages of bovine primary skeletal muscle cells were analyzed by Real-time quantitative PCR.【Result】 The CDS region of SOX6 gene in Xianan cattle was 2 526 bp in length, which encoded 841 amino acids.The results of phylogenetic tree showed that Xianan cattle had the closest relationship with yak and the farthest relationship with chicken.SOX6 protein was a weakly alkaline and hydrophilic protein with a molecular weight of 93.35 ku, which located in the nucleus without transmembrane structure and signal peptide.The secondary structure of SOX6 protein was mainly composed of alpha-helix, random coil, extended chain and beta-turn, accounting for 41.26%, 45.54%, 8.09% and 5.11%, respectively.Protein interaction analysis showed that SOX6 had interaction with type Ⅱ collagen α1 (COL2A1), chondroproteoglycan (ACAN), carboxy-terminal-binding protein 2 (CTBP2), and forkhead box 3(FOXJ3).The relative expression of SOX6 gene in leg muscle of Xianan cattle was the highest, and the expression in lung and spleen was the lowest.The expression level of leg muscle in 12 months old was significantly higher than that in 0 and 24 months old cattle (P<0.05).The expression of SOX6 gene increased gradually with the differentiation of bovine primary skeletal muscle cells, and the expression level of SOX6 gene on days 6 was significantly higher than that on days 0, 2, 4 and 8 (P<0.05).【Conclusion】 The CDS sequence of SOX6 gene in Xiannan cattle was successfully cloned, and its encoded protein was unstable, weakly alkaline and hydrophilic.The expression of SOX6 gene was significantly higher in leg muscle of different months than other tissues, and the expression in bovine primary skeletal muscle cells on days 6 of differentiation was significantly higher than that at other periods.The results provided a theoretical basis for further study on the regulatory role of SOX6 gene in the muscle development of Xiannan cattle.

Key words: Xianan cattle; SOX6 gene; cloning; bioinformatics; tissue expression

中图分类号:

邢珊珊, 李鑫, 余磊, 许会芬, 蔡含芳, 李明. 夏南牛SOX6基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(7): 2651-2660.

XING Shanshan, LI Xin, YU Lei, XU Huifen, CAI Hanfang, LI Ming. Cloning, Bioinformatics Analysis and Tissue Expression of SOX6 Gene in Xianan Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2651-2660.

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