类乌齐牦牛ACTA1基因克隆、分子特性及差异表达分析

Cloning, Molecular Characteristics and Differential Expression Pattern of ACTA1 Gene in Yak (Bos grunniens)

【目的】骨骼肌α肌动蛋白1(actin alpha1,ACTA1)主要参与骨骼肌纤维的发育,在骨骼肌活动中发挥重要作用。本试验主要扩增类乌齐牦牛ACTA1基因的cDNA序列,检测ACTA1基因在类乌齐牦牛不同组织中mRNA水平的表达规律。【方法】采用RT-PCR方法扩增并克隆类乌齐牦牛ACTA1基因CDS区;通过在线软件对其一级结构、二级结构、三级结构进行生物信息学分析;利用核苷酸序列及氨基酸序列进行同源性和系统进化树分析;应用RT-qPCR技术检测ACTA1基因在类乌齐牦牛不同组织中的表达模式。【结果】类乌齐牦牛ACTA1基因编码区全长1134bp,结构稳定,共编码377个氨基酸。生物信息学分析发现,类乌齐牦牛ACTA1基因编码的蛋白质是一种结构较稳定、带负电的亲水性蛋白,二、三级结构以α-螺旋为主,包含2个明显的跨膜区域,无信号肽,属胞内蛋白。保守结构域中含有明显的NDB区域,蛋白结构高度保守。同源性分析表明,类乌齐牦牛与水牛ACTA1基因的核苷酸及氨基酸同源性均较高。系统进化树分析显示,类乌齐牦牛与水牛亲缘关系最近,黄牛次之。实时荧光定量PCR结果显示,ACTA1基因在臀肌和大脑高表达。【结论】获得类乌齐牦牛ACTA1基因的CDS区全长1134bp,ACTA1基因在类乌齐牦牛肌肉和大脑组织中显著表达,为进一步研究分析ACTA1基因在调控牦牛肌肉发育及机制等方面奠定基础。

【Objective】Skeletal muscle actin alpha 1 (ACTA1) is mainly involved in the development of skeletal muscle fibers and plays an important role in skeletal muscle activities. The purpose of our experiment was to amplify the cDNA sequence of the ACTA 1 gene of the Leiwuqi yak and detect the expression of the mRNA level of the ACTA 1 gene in different tissues of Leiwuqi yak.【Method】 RT-PCR was used to amplify and clone the CDS of ACTA1 gene of Leiwuqi yak;The bioinformatics analysis of its primary structure,secondary structure and tertiary structure was carried out through online software;The amino acid sequence was analyzed by homology and phylogenetic tree;RT-qPCR was used to detect the expression of ACTA1 gene in different tissues of Leiwuqi yak.【Result】The coding region of the ACTA1 gene of the Leiwuqi yak was 1134 bp in length and stable in structure,encoding 377 amino acids total. Bioinformatics analysis found that the protein encoded by the ACTA1 gene of the Leiwuqi yak was a structurally stable negatively charged hydrophilic protein. The second and third-order structures were dominated by α-helix and contain two transmembranes region,no signal peptide. ACTA1 protein is an intracellular protein. The conserved domain contains a NDB region,and the highly conserved homology analysis of the protein structure indicates that the nucleotide and amino acid homology of the ACTA1 gene of the Leiwuqi yak and the buffalo were higher. The phylogenetic tree analysis showed that the Leiwuqi yak had the closest relationship with the buffalo,and the cattle is followed. Real-time PCR results showed that the ACTA1 gene was highly expressed in the gluteal muscles and brain.【Conclusion】The CDS region of the ACTA1 gene of the Leiwuqi yak was 1134 bp in length,and ACTA1 gene was significantly expressed in the muscle and brain tissues of the Leiwuqi yak,which laid a foundation for further research on the regulation of the ACTA1 gene in regulating the muscle development and mechanism of yak.