陆川猪MYL2基因生物信息学分析、真核表达载体构建及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.05.007

摘要/Abstract

摘要： 【目的】了解陆川猪肌球蛋白轻链2(myosin light chain 2,MYL2)基因CDS区序列及其编码蛋白的结构和功能,探究MYL2基因对肌肉生长发育的影响,为陆川猪的开发利用提供分子基础。【方法】采用RT-PCR技术扩增陆川猪MYL2基因CDS区,利用MegAlign软件对陆川猪MYL2基因与不同物种进行相似性比对和系统进化树构建,并通过生物信息学软件分析MYL2蛋白理化性质、疏水性和跨膜结构等;构建MYL2基因真核表达载体,利用脂质体法将重组质粒转染C2C12细胞并观察荧光;通过实时荧光定量PCR检测MYL2基因在陆川猪不同组织中的表达情况。【结果】陆川猪MYL2基因CDS区全长501 bp,编码166个氨基酸,与NCBI上公布的野猪MYL2基因相似性为99.6%,存在2处突变:156 bp处C突变为T,为同义突变;404 bp处T突变为C,使异亮氨酸(I)突变为苏氨酸(T)。生物信息学分析显示,MYL2蛋白原子总数为2 633个,分子质量为18.879 ku,理论等电点(pI)为4.83,不存在跨膜结构,无信号肽,为非分泌蛋白。MYL2蛋白有16个位点可能会被磷酸化,在第78位氨基酸有1个N-糖基化位点。亚细胞定位结果显示,MYL2蛋白主要存在于细胞质(56.5%)、细胞骨架(21.7%)、线粒体(8.7%)、细胞核(8.7%)和液泡(4.3%)中。MYL2蛋白二级结构主要由α-螺旋、无规则卷曲、β-转角和延伸链组成,占比分别为57.83%、29.52%、9.04%和3.61%。细胞试验结果显示,转染48 h后试验组和对照组均有荧光,与对照组相比,试验组MYL2基因表达量极显著升高(P＜0.01)。不同组织中MYL2基因表达量由高到低依次为心脏、背最长肌、肝脏、皮下脂肪、肺脏、脾脏和肾脏,且在背最长肌中的表达量显著高于除心脏外的其他组织(P＜0.05)。【结论】本试验成功克隆陆川猪MYL2基因CDS区,构建了真核表达载体和组织表达谱,并对其所编码蛋白的结构和功能进行了分析,为探究MYL2基因在肌肉生长发育中的作用及陆川猪的开发利用提供了分子依据。

关键词: 陆川猪; MYL2基因; 生物信息学; 真核表达载体

Abstract: 【Objective】 The pourse of this study was to investigate the structure and function of CDS region sequence of myosin light chain 2 (MYL2) gene and its encoded protein of Luchuan pigs,and to explore the effects of MYL2 gene on muscle growth and development,so as to provide molecular basis for the development and utilization of Landchuan pigs.【Method】 The CDS region of MYL2 gene in Luchuan pigs was amplified by RT-PCR.MegAlign software was used to compare MYL2 gene in Luchuan pigs with different species and construct phylogenetic tree.The physical and chemical properties,hydrophobicity and transmembrane structure of MYL2 protein were analyzed by bioinformatics software.The eukaryotic expression vector of MYL2 gene was constructure,the recombinant plasmid was transfected into C2C12 cells by the liposome method, and then the fluorescence was observed. Real-time quantitative PCR was used to detect the expression of MYL2 gene in different tissues of Luchuan pigs.【Result】 The results showed that the CDS region of MYL2 gene in Luchuan pigs was 501 bp length,encoded 166 amino acids,and the similarity was 99.6% with the wild boar MYL2 gene published in NCBI.There were two mutations,C mutates to T at 156 bp,which was synonymous mutation.Mutation from T to C at 404 bp caused amino acid mutation from isoleucine (I) to threonine (T).Bioinformatics analysis showed that the total number of MYL2 protein atoms was 2 633,the molecular mass was 18.879 ku,the theoretical isoelectric point (pI) was 4.83.There was no transmembrane structure,no signal peptide,and it was a non-secreted protein.The MYL2 protein had 16 sites that might be phosphorylated and 1 N-glycosylation site at the 78-position amino acid.The subcellular localization results showed that MYL2 protein was mainly found in cytoplasm (56.5%),cytoskeleton (21.7%), mitochondria (8.7%),nucleus (8.7%) and vacuoles (4.3%).The secondary structure of MYL2 protein was mainly composed of alpha helix,random coil,beta turn and extended chain,accounting for 57.83%,29.52%,9.04% and 3.61%,respectively.The results of cell tests showed that fluorescence was observed in both the experimental group and control group after 48 h.Compared with control group,the expression of MYL2 gene in experiment group was extremely significantly increased (P＜0.01).The expression of MYL2 gene in different tissues from high to low were heart,longissimus dorsi muscle,liver,subcutaneous fat,lung,spleen and kidney,and the expression in longissimus dorsi muscle was significantly higher than that in other tissues except heart (P＜0.05).【Conclusion】 In this study,the CDS region of MYL2 gene in Luchuan pigs was cloned successfully,the eukaryotic expression vector and tissue expression profile were constructed.The structure and function of the protein encoded by MYL2 gene were analyzed,which provided molecular basis for exploring the role of MYL2 gene in the growth and development of muscle and the development and utilization of Luchuan pigs.

Key words: Luchuan pigs; MYL2 gene; bioinformatics; eukaryotic expression vector

中图分类号:

潘鹏丞, 雷宗全, 李显, 胡湘云, 秦谦涛, 覃兆鲜, 关志惠, 陈宝剑, 谢炳坤. 陆川猪MYL2基因生物信息学分析、真核表达载体构建及组织表达研究[J]. 中国畜牧兽医, 2023, 50(5): 1796-1806.

PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.

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