贵州白山羊肌细胞生成素基因克隆与生物信息学分析

doi:10.16431/j.cnki.1671-7236.2024.03.004

摘要/Abstract

摘要： 【目的】克隆贵州白山羊肌细胞生成素(myogenin)基因并对其结构特性进行生物信息学分析,为进一步探索该基因调控山羊肌肉发育的分子机制提供重要参考依据。【方法】以贵州白山羊血液基因组DNA为模板,设计4对特异性引物,运用PCR扩增、Sanger直接测序及序列拼接方法获得贵州白山羊myogenin基因完整编码区核苷酸序列,应用BioEdit 7.0、NetPhos 3.1、Conserved Domains Search、SMART、Motif Scan等生物信息学软件分别分析该基因核苷酸与氨基酸序列的组成、编码蛋白磷酸化位点、关键结构域和功能模体等结构特性,通过DNAStar Lasergene、DNAMAN 8.0和Mega 5.0软件分别进行15个偶蹄目物种myogenin基因编码氨基酸序列相似性比对及系统进化树构建。【结果】贵州白山羊myogenin基因序列全长2 424 bp(获得GenBank登录号:HZ53289),由3个外显子、2个内含子、部分5'-UTR和3'-UTR构成,长度分别为471、82、122、765、589、135和260 bp,编码区长度为675 bp,共编码224个氨基酸。贵州白山羊myogenin蛋白第1—86位氨基酸为生肌决定因子(myogenic determining factors,MyoD)家族特有碱性结构域(Basic domain),第81—139位氨基酸为螺旋-环-螺旋(helix-loop-helix,H-L-H)结构域,第160—170位氨基酸为低复杂度结构域,存在21个磷酸化位点和核定位信号蛋白等功能模体结构。氨基酸序列比对表明,6个半胱氨酸残基、1个核定位信号和1个低复杂度结构域在15个偶蹄目物种完全保守。myogenin基因编码氨基酸序列相似性与系统进化分析均显示,贵州白山羊与波尔山羊、湖羊和马可波罗盘羊的亲缘关系最近。【结论】本研究成功克隆得到贵州白山羊myogenin基因全长序列,大小为2 424 bp,编码224个氨基酸,myogenin蛋白包含Basic domain和H-L-H结构域。该结果可为myogenin基因调控山羊肌肉发育的分子机制研究提供理论参考。

关键词: 贵州白山羊; myogenin基因; 克隆; 分子系统进化; 生物信息学

Abstract: 【Objective】 This study was aimed to clone myogenin gene in Guizhou White goat and analyze the structure characteristics by bioinformatics,so as to provide an important reference basis for further exploring molecular regulation mechanism of muscle development of goat.【Method】 The blood genomic DNA of Guizhou White goat was used as template,four pairs of specific primers were designed,and the complete coding sequence of myogenin gene in Guizhou White goat was obtained by PCR amplification,Sanger sequencing and sequence assembly method.The molecular structure characteristics of myogenin gene,such as composition of nucleotide and amino acid sequence,phosphorylation sites,key domains and functional motifs were analyzed by BioEdit 7.0,NetPhos 3.1,Conserved Domains Search,SMART and Motif Scan softwares,respectively.The sequence similarity comparison of myogenin gene among 15 Artiodactyla species and phylogenetic tree construction were carried out by DNAStar Lasergene,DNAMAN 8.0 and Mega 5.0 softwares.【Result】 The length of myogenin gene sequence in Guizhou White goat was 2 424 bp(obtain GenBank accession No.:HZ53289),which was consisted of 3 exons,2 introns and partial of 5'-UTR and 3'-UTR,and was 471,82,122,765,589,135 and 260 bp in length,respectively.The length of coding region was 675 bp,which was encoded 224 amino acids.The deduced amino acid sequence analysis showed that 1-86 amino acids were the unique basic domain of myogenic determining factors family,81-139 amino acids were the helix-loop-helix DNA binding domain,and 160-170 amino acids were the low complexity regions.There were 21 phosphorylation sites and functional motif structure such as nuclear localization signal protein.Amino acid sequence alignment showed that six cysteine residues,one nuclear localization signal protein and one low complexity region were completely conserved among 15 Artiodactyla species.Sequence similarity and molecular phylogenetic analysis all showed that Guizhou White goat myogenin gene had the closest genetic relationship with Boer goat,Hu sheep and Marco Polo sheep.【Conclusion】 The myogenin gene of Guizhou White goat was successfully cloned,with a size of 2 424 bp in length and encoding 224 amino acids,which contained the Basic and H-L-H domains.These results could provide theoretical reference for further exploring the molecular regulation mechanism of muscle growth and development of goat.

Key words: Guizhou White goat; myogenin gene; cloning; molecular phylogenetic evolution; bioinformatics

中图分类号:

S827
S813.3

宋兴超, 孟金柱, 赵园园, 吴震洋, 安清明. 贵州白山羊肌细胞生成素基因克隆与生物信息学分析[J]. 中国畜牧兽医, 2024, 51(3): 926-935.

SONG Xingchao, MENG Jinzhu, ZHAO Yuanyuan, WU Zhenyang, AN Qingming. Cloning and Bioinformatics Analysis of myogenin Gene in Guizhou White Goat[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 926-935.

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