藏猪MYL1基因的克隆及生物信息学分析

Cloning and bioinformatics analysis of MYL1 gene in Tibetan pigs

为了克隆藏猪肌球蛋白轻链1 (myosin light chain 1, MYL1)基因编码区序列,并对其进行生物信息学分析,试验根据NCBI网站GenBank中公布的野猪MYL1基因mRNA序列(登录号为NM;14374.2)设计特异性引物,以提取的藏猪背最长肌组织总RNA反转录得到的cDNA为模板,对藏猪MYL1基因编码区序列进行PCR扩增,将目的基因进行测序,应用生物信息学分析软件对藏猪MYL1基因编码区序列的同源性、蛋白质的结构和功能(氨基酸组成、一般理化性质、疏水性、二级结构及三级结构、磷酸化位点、跨膜螺旋结构和信号肽亚细胞定位及蛋白网络互作)进行分析。结果表明:藏猪MYL1基因编码区序列全长为453 bp;藏猪与野猪的亲缘关系最近,与斑马鱼的亲缘关系最远;藏猪MYL1基因编码区序列与野猪、牛、智人、马、绵羊、犬、穴兔、家鼠、鸡和斑马鱼的核苷酸序列相似性分别为100%、92.1%、91.4%、91.2%、91.2%、90.5%、90.3%、89.2%、80.6%和68.2%;藏猪MYL1蛋白存在19种氨基酸,由150个氨基酸组成;藏猪MYL1蛋白分子质量为16 657.86 u,分子式为C;H;N;O;S;,脂肪系数为79.33,理论等电点pI为4.63,不稳定系数为37.81,负电荷氨基酸残基(Asp+Glu)总数为26个,正电荷氨基酸残基(Arg+Lys)总数为15个,理论半衰期为30 h,为亲水蛋白;二级结构中α螺旋占比最大(58.67%),其次为无规则卷曲(24.67%),β转角(8.67%)和延伸链(8.00%)占比较小;三级结构与二级结构预测结果相符合;氨基酸序列中共有16个磷酸化位点,其中有8个丝氨酸磷酸化位点,6个苏氨酸磷酸化位点,2个酪氨酸磷酸化位点;无跨膜螺旋结构,为非跨膜蛋白;无信号肽,为非分泌蛋白;主要定位于细胞质(47.8%)中,在细胞核约占26.1%,在细胞骨架约占8.3%,在内质网、高尔基体、分泌系统的囊泡、液泡中均约占4.3%;与TNNC2蛋白及MYH1、MYH3、MYH7等蛋白之间存在互作网络关系,与TNNC2蛋白相关系数高达0.999,与肌球蛋白重链家族MYH1、MYH3和MYH7蛋白相关系数分别为0.979,0.975和0.971,与MYBPC1和TPM1蛋白相关系数均为0.969,与TPM2和TNNI3蛋白相关系数均为0.965。说明藏猪MYL1基因可能具有较强大的生物学功能。

In order to clone the coding region of myosin light chain 1(MYL1) gene and analyze it bioinformatics.Specific primers were designed according to the mRNA sequence of boar MYL1 gene published on GenBank in NCBI website(registration number: NM;14374.2). PCR was performed on the encoding region of Tibetan pig MYL1 gene using cDNA reverse transcription of total RNA extracted from Longissimus dorsi tissue as template.Target purpose gene was sequenced, and Tibetan pig MYL1 gene coding sequence homology, protein structure and function(amino acids, the general physical and chemical properties, hydrophobicity and secondary structure and tertiary structure, phosphorylation sites, transmembrane helical structure and subcellular localization signal peptide and protein interactions) was analyzed by using bioinformatics analysis software.The results showed that the full length of MYL1 gene sequence was 453 bp.The Tibetan pig had the closest genetic relationship with wild boar and the farthest with zebrafish.The nucleotide sequences of MYL1 gene of Tibetan pig were 100%, 92.1%, 91.4%, 91.2%, 91.2%, 90.5%, 90.3%, 89.2%, 80.6% and 68.2%, similar to those of wild boar, ox, Homo sapiens, horse, sheep, dog, cave rabbit, house rat, chicken and zebrafish, respectively.There were 19 kinds of amino acids in the coding region of Tibetan pig MYL1 gene, which was composed of 150 amino acids.The molecular weight of Tibetan pig MYL1 protein was 16 657.86 u, the molecular formula is C;H;N;O;S;, the fat coefficient was 79.33, the theoretical isoelectric point pI was 4.63, the instability coefficient is 37.81, and the total number of negatively charged amino acid residues(Asp + Glu) was 26.The total number of positively charged amino acid residues(Arg + Lys) was 15, and the theoretical half-life was 30 h.MYL1 protein was a hydrophilic protein.The proportion of α helix was the largest(58.67%), followed by random curl(24.67%), β rotation(8.67%) and extended chain(8.00%).The predicted results of tertiary structure and secondary structure agree well.There were 16 phosphorylation sites in amino acid sequence, including eight serine phosphorylation sites, six threonine phosphorylation sites and two tyrosine phosphorylation sites.It had no transmembrane helix structure and was a non-transmembrane protein;no signal peptide, non-secretory protein;mainly located in the cytoplasm(47.8%), in the nucleus about 26.1%, in the cytoskeleton about 8.3%, in the endoplasmic reticulum, golgi apparatus, secretory system vesicles, vacuoles about 4.3%.There was an interaction network relationship with TNNC2 protein, MYH1 protein, MYH3 protein, MYH7 protein, and the correlation coefficient with TNNC2 protein was 0.999, the correlation coefficient with myosin heavy chain family MYH1 protein, MYH3 protein, MYH7 protein was 0.979, 0.975 and 0.971, respectively.The correlation coefficient between MYBPC1 protein and TPM1 protein was 0.969.The correlation coefficient between MYL1 protein and TPM2 protein and TNNI3 protein was 0.965.These results suggest that MYL1 gene of Tibetan pigs may have strong biological functions.