陆川猪<i>MyoD1</i>基因克隆及组织表达分析

doi:10.7668/hbnxb.20194657

摘要/Abstract

摘要：

为探究陆川猪骨骼肌生长发育过程中MyoD1基因所起作用,对陆川猪成肌细胞决定基因1(MyoD1)进行克隆,展开生物信息学分析,对其在陆川猪不同组织中的表达进行分析,以NCBI上已公布的野猪MyoD1基因序列为模板,进行特异性引物设计,克隆陆川猪MyoD1基因CDS区,并与NCBI已公布的野猪、牛、绵羊、人、小鼠、大鼠基因序列进行相似性比对,由此构建系统进化树,通过在线软件开展生物信息学分析,而后使用实时荧光定量PCR检测在陆川猪不同组织中MyoD1基因的相对表达量。陆川猪MyoD1基因CDS区全长960 bp,共编码319个氨基酸,碱基突变共存在5处,与野猪、牛、绵羊、人类、小鼠、大鼠的MyoD1基因CDS序列相似性分别为99.2%,93.0%,92.3%,90.0%,84.3%,84.0%,其中与野猪相似性最高,表明二者亲缘关系最为接近;陆川猪MyoD1基因原子总数为4 680,蛋白的分子质量为33.99 ku,分子式为C₁₄₅₇H₂₂₉₆N₄₄₂O₄₇₁S₁₄;不稳定系数为63.87,表明其缺乏稳定性;等电点为5.63,属于酸性蛋白;不存在跨膜结构,存在35处磷酸化位点及1处糖基化位点;蛋白二级结构以无规则卷曲为主,占比60.69%。陆川猪MyoD1基因在背最长肌中的表达量最高,且显著高于其他组织,表达量最低的组织为肾脏。陆川猪各组织中均有MyoD1基因表达,且主要在背最长肌中表达,由此推测,MyoD1基因在陆川猪肌肉生长发育过程中可能起到至关重要的作用。

关键词: 陆川猪, MyoD1基因, 生物信息学分析, 基因克隆, 组织表达

Abstract:

Luchuan pig myoblast determining gene 1(MyoD1)was cloned,bioinformatics analysis was performed,and its expression was analyzed in different tissues of Luchuan pigs,to explore the role of MyoD1 gene in the growth and development of skeletal muscle.Published MyoD1 gene of wild boar sequence on NCBI as the template,designed specific primer,cloned the CDS region of MyoD1,and compared the similarity with wild boar,cattle,sheep,human,mouse and rat gene sequence,constructed system tree for bioinformatics analysis by online software.Then,the relative expression of MyoD1 gene in different tissues of Luchuan pigs by RT-PCR.The CDS of MyoD1 gene in Luchuan pig was 960 bp,encoding 319 amino acids,five base mutations.The similarities of MyoD1 genes between Luchuan pig and wild boar,cattle,sheep,human,mouse and rats were 99.2%,93.0%,92.3%,90.0%,84.3% and 84.0%,respectively.Total number of MyoD1 gene atoms in Luchuan pigs was 4 680,molecular mass of the protein was 33.99 ku,molecular formula was C₁₄₅₇H₂₂₉₆N₄₄₂O₄₇₁S₁₄;the instability coefficient was 63.87,indicating a lack of stability;isoelectric point was 5.63,which belongs to the acidic protein;no transmembrane structure existed,found 35 phosphorylation sites and 1 glycosylation site;protein secondary structure was dominated by an irregular coil,which accounting for 60.69%.The MyoD1 gene in Luchuan pigs had the highest expression in the longest dorsal muscle,which was significantly higher than in other tissues,while kidney had the lowest expression.MyoD1 gene was expressed in all tissues of Luchuan pigs and mainly in the longest dorsal muscle,we speculated that MyoD1 gene might play a crucial role in the muscle growth and development of Luchuan pigs.

Key words: Luchuan pig, MyoD1 gene, Bioinformatics analysis, Gene cloning, Organizational expression

高泽仁, 潘鹏丞, 徐文文, 陈宝剑, 刘明君, 关志惠, 谢炳坤, 覃兆鲜. 陆川猪MyoD1基因克隆及组织表达分析[J]. 华北农学报, 2024, 39(2): 168-173. doi: 10.7668/hbnxb.20194657.

GAO Zeren, PAN Pengcheng, XU Wenwen, CHEN Baojian, LIU Mingjun, GUAN Zhihui, XIE Bingkun, QIN Zhaoxian. Cloning and Tissue Expression Analysis of MyoD1 Gene in Luchuan Pig[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 168-173. doi: 10.7668/hbnxb.20194657.

http://www.hbnxb.net/CN/Y2024/V39/I2/168

图/表 12

表1 引物信息

Tab.1 Primer information

基因 Genes	引物序列(5'—3') Primer sequence(5'—3')	片段大小/bp Product length
MyoD1-Fa	ATGGAGCTGCTGTCGCCA	1 006
MyoD1-Ra	TCAGAGCACCTGGTAGATAGGGTT
MyoD1-Fb	GTGCAAACGCAAGACCACTA	128
MyoD1-Rb	CGCTGATTCGGGTTGCTAGA
GAPDH-F	CCCGTTCGACAGATAGCCGTA	83
GAPDH-R	AATCCGTTCACTCCGACCT

图1 MyoD1基因CDS序列扩增条带

Fig.1 The CDS sequence amplified production of the MyoD1 gene

图2 陆川猪与野猪MyoD1蛋白质序列比对

Fig.2 The protein sequence alignment of MyoD1 between Luchuan pig and wild boar

图3 不同物种MyoD1基因CDS序列同源比对结果

Fig.3 Sequence homology alignment of the MyoD1 genes in different species

图4 MyoD1基因系统发育进化树

Fig.4 Phylogenetic evolutionary tree of the MyoD1 gene

图5 MyoD1蛋白亲水性结果

Fig.5 The hydrophilic results of MyoD1 protein

图6 陆川猪MyoD1蛋白信号肽预测

Fig.6 MyoD1 protein signal peptide prediction in Luchuan pigs

图7 陆川猪MyoD1蛋白跨膜结构预测

Fig.7 Transmembrane structure prediction of MyoD1 protein in Luchuan pigs

图8 陆川猪MyoD1蛋白磷酸化位点预测

Fig.8 Prediction of phosphorylation sites of MyoD1 protein in Luchuan pigs

图9 陆川猪MyoD1蛋白糖基化位点预测

Fig.9 Prediction of the glycosylation sites of MyoD1 protein in Luchuan pigs

图10 陆川猪MyoD1蛋白二级结构预测

Fig.10 Secondary structure prediction of MyoD1 protein in Luchuan pigs

图11 MyoD1基因组织表达谱不同小写字母代表不同组织间有显著差异(P<0.05)。

Fig.11 Tissue expression profiles of the MyoD1 gene Different lowercase letters represent significant differences in different tissue(P<0.05).

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