亚洲黑熊(Ursus thibetanus)α-肌动蛋白ACTA1基因的克隆及序列分析

Cloning and sequence analysis of α-actin protein ACTA1 gene in Asiatic black bear (Ursus thibetanus)

为了解亚洲黑熊(Ursus thibetanus)的α-肌动蛋白ACTA1基因的结构特征和其编码蛋白功能,试验采用PCR技术从亚洲黑熊肌肉组织的总DNA和RNA中扩增α-肌动蛋白ACTA1基因,并对其基因表达和序列进行克隆、测序。采用ORF finder软件进行DNA序列ORF的查找和氨基酸序列的推定;采用Gen scan对结构基因序列进行分析;通过DNAMAN Version 6.0软件对基因编码序列和氨基酸序列进行比较;采用MEGA 7.0软件进行亲缘性比较;利用ExPASy软件预测分析了蛋白质功能位点和生化特性。结果表明:亚洲黑熊ACTA1基因的ORF为1 134 bp,编码377个氨基酸;ACTA1蛋白等电点为5.24,分子质量为42 ku,具有1个N-糖基化位点、10个N-肉豆蔻酰化位点、5个蛋白激酶C磷酸化位点、2个酪氨酸激酶磷酸化位点、4个酪蛋白激酶Ⅱ磷酸化位点,以及1个依赖cAMP和cGMP蛋白激酶磷酸化位点。亚洲黑熊和北极熊之间的ACTA1基因编码序列的同源性最高,达到86.1%。ACTA1蛋白相对保守且具有较高亲水性及较多的螺旋结构,并具有跨膜螺旋和多核苷酸的结合位点。说明ACTA1基因高度保守,ACTA1蛋白具有多种宏观及微观生物学功能,并参与了信号转导过程,在亚洲黑熊生长发育、生物调控及修复中起重要作用。

In order to understand the structural characteristics and protein function of α-actin protein ACTA1 gene in Asiatic black bear(Ursus thibetanus), the α-actin protein ACTA1 gene was amplified by PCR from the total DNA and RNA in the muscle tissue of the Asian black bear, and its gene and sequence were cloned and sequenced. ORF finder software was used to find the ORF of DNA sequence and to infer the amino acid sequence. Gen scan was used to analyze the structural gene sequences. The gene coding sequence and amino acid sequence were compared by DNAMAN Version 6.0 software. The genetic relationship was compared by using MEGA 7.0 software. The functional sites and biochemical characteristics of proteins were predicted and analyzed by using ExPASy software. The results showed that the ORF of ACTA1 gene of Asiatic black bear was 1 134 bp, encoding 377 amino acids. The isoelectric point of ACTA1 protein was 5.24, the molecular weight was 42 ku, with 1 N-glycosylation site, 10 N-myristoylation sites, 5 protein kinase C phosphorylation sites, 2 tyrosine kinase phosphorylation sites, 4 casein kinase II phosphorylation sites, and 1 cAMP-and cGMP-dependent protein kinase phosphorylation site. The homology of ACTA1 gene coding sequence between Asian black bears and Polar bears was the highest, reaching 86.1%. ACTA1 protein was relatively conservative, which had high hydrophilicity and more helix structure, and had binding sites of transmememal helix and polynucleotide. It indicated that ACTA1 gene was highly conserved in evolution, ACTA1 protein had a variety of macro and micro biological functions, and participated in the signal transduction process, which played an important role in growth, development, biological regulation and repair of Asiatic black bear.