努比亚山羊Myf6基因的克隆及组织表达研究

doi:10.16431/j.cnki.1671-7236.2021.11.018

摘要/Abstract

摘要： 研究旨在克隆努比亚山羊生肌因子6（Myf6）基因，并对其进行生物信息学分析，检测Myf6基因在努比亚山羊不同组织的表达差异以及努比亚山羊和隆林山羊背最长肌组织的表达差异。以努比亚山羊背最长肌组织cDNA为模板，PCR扩增获得Myf6基因的完整编码区（CDS）后进行菌液验证，并进行序列相似性比对以及系统进化树构建；分析Myf6蛋白理化性质并预测其亲/疏水性，预测蛋白二级结构和三级结构。采用实时荧光定量PCR检测Myf6基因在努比亚山羊心脏、肝脏、脾脏、肾脏、背最长肌、皮下脂肪、瘤胃以及隆林山羊背最长肌中的表达。结果显示，努比亚山羊Myf6基因CDS序列长729 bp，编码242个氨基酸。努比亚山羊Myf6氨基酸序列与绵羊、黄牛、猪、驴、人、小鼠的相似性分别为99.3%、98.8%、94.1%、93.8%、93.4%和89.0%。蛋白理化性质分析结果表明，山羊Myf6蛋白的分子质量为26.98 ku，为不稳定酸性蛋白；亲疏水性分析发现，该蛋白为亲水蛋白；跨膜结构和信号肽预测结果发现，该蛋白不含有跨膜结构和信号肽，不属于跨膜蛋白和分泌蛋白；二级结构预测结果显示，该蛋白主要由无规则卷曲（49.59%）构成，其次为α-螺旋（33.88%）、延伸链（9.50%）和β-转角（7.02%），三级结构预测结果与二级结构一致。实时荧光定量PCR结果显示，Myf6基因在努比亚山羊背最长肌的表达量最高，显著高于其他组织（P<0.05），在肾脏的表达量最低，但与心脏、肝脏、脾脏、皮下脂肪、瘤胃等差异均不显著（P>0.05）；对比努比亚山羊和隆林山羊背最长肌的Myf6基因的表达情况发现，努比亚山羊的表达量显著高于隆林山羊（P<0.05）。试验为进一步揭示Myf6基因在肌肉分化中的作用提供了参考。

关键词: 山羊; Myf6基因; 克隆; 组织表达

Abstract: The research was aimed to clone myogenic factor 6 (Myf6) gene in Nubian goat and conducted bioinformatics analysis on it. The expression difference of Myf6 gene in different tissues of Nubian goat and the expression difference of longissimus muscle tissue of Nubian goat and Longlin goat were detected. Using the cDNA from the longissimus dorsi muscle tissue of Nubian goat as a template, PCR amplified the complete coding region (CDS) of the Myf6 gene and then verified the bacterial solution. Sequence similarity alignment and phylogenetic tree construction were carried out. The physicochemical properties of Myf6 protein were analyzed and its hydrophilicity/hydrophobicity was predicted. The protein secondary structure and tertiary structure were predicted. The expression of Myf6 gene in heart, liver, spleen, kidney, longissimus dorsi muscle, subcutaneous fat, rumen and Longlin goat longissimus dorsi muscle were detected by Real-time quantitative PCR. The results showed that the CDS sequence of Myf6 gene in Nubian goat was 729 bp and encoded 242 amino acids. The similarity of Myf6 amino acid sequence between Nubian goat and Ovis aries, Bos taurus, Sus scrofa, Equus asinus, Homo sapiens and Mus musculus were 99.3%, 98.8%, 94.1%, 93.8%, 93.4% and 89.0%, respectively. The analysis of the physical and chemical properties of the protein showed that the molecular weight of the goat Myf6 protein was 26.98 ku, which was an unstable acidic protein. The hydrophilic and hydrophobic analysis found that the protein was a hydrophilic protein. The transmembrane structure and signal peptide prediction results showed that the protein did not contain transmembrane structure and signal peptide, and was not a transmembrane protein and secreted protein. The prediction results of secondary structure showed that the protein was mainly composed of random coil (49.59%), followed by alpha helix (33.88%), extended chain (9.50%) and beta turn (7.02%). The prediction results of the tertiary structure were consistent with those of the secondary structure. The results of Real-time quantitative PCR showed that the expression of Myf6 gene in the longissimus dorsi muscle of Nubian goat was the highest, significantly higher than that of other tissues (P<0.05), and the expression in the kidney was the lowest, but there was no significant difference with heart, liver, spleen, subcutaneous fat and rumen (P>0.05). Compared the expression of Myf6 gene in the longissimus dorsi muscle of Nubian goat and Longlin goat, the expression level of Nubian goat was significantly higher than that of Longlin goat (P<0.05). The experiment provided a reference for further revealing the role of Myf6 gene in muscle differentiation.

Key words: goat; Myf6 gene; cloning; tissue expression

中图分类号:

S827

宋颖, 张叁保, 申玉建, 胡艳, 张瑜, 邹菊红, 徐建建, 连子童, 邹剑伟, 韦英明, 郑自华, 蒋钦杨. 努比亚山羊Myf6基因的克隆及组织表达研究[J]. 中国畜牧兽医, 2021, 48(11): 4084-4093.

SONG Ying, ZHANG Sanbao, SHEN Yujian, HU Yan, ZHANG Yu, ZOU Juhong, XU Jianjian, LIAN Zitong, ZOU Jianwei, WEI Yingming, ZHENG Zihua, JIANG Qinyang. Cloning and Tissue Expression of Myf6 Gene in Nubian Goat[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4084-4093.

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