新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建

doi:10.16431/j.cnki.1671-7236.2021.10.001

摘要/Abstract

摘要： 肌肉生长抑制素（MSTN）是骨骼肌发育的主要调控因子，为获得新西兰白兔MSTN基因序列及其表达规律，试验通过RT-PCR技术从新西兰白兔腿肌组织中扩增并克隆得到MSTN基因序列，利用在线网站对其进行生物信息学分析，并采用实时荧光定量PCR技术检测MSTN基因在新西兰白兔同一时期不同组织及不同时期腿肌组织中的表达量。结果显示，新西兰白兔MSTN基因CDS区序列长1 128 bp，编码375个氨基酸，其核苷酸序列与GenBank上公布的人、猪、马、小鼠、犬、牛和鸡的核苷酸序列相似性分别为95.9%、94.9%、94.9%、91.7%、91.5%、91.3%及84.2%。新西兰白兔MSTN蛋白属于酸性、亲水性分泌蛋白，且具有不稳定性，不含跨膜结构，含有1个信号肽，主要分布在内质网和线粒体中。MSTN蛋白二级结构和三级结构预测结果一致，以无规则卷曲为主，其次是α-螺旋、延伸链，为混合型蛋白。蛋白质互作关系预测发现，MSTN蛋白与PAX7、MYOG、IGF1、FST、Smad3及Smad2等与肌肉生长发育有关的蛋白之间存在相互作用。不同组织表达分析结果表明，新西兰白兔MSTN基因在心脏、肝脏、脾脏、肺脏、肾脏、腿肌、子宫和胃中均有表达，在腿肌中表达量最高，极显著高于其他组织（P<0.01），其次是肾脏，在肝脏中的表达量最低。不同时期腿肌组织中的表达量分析结果显示，新西兰白兔MSTN基因在180日龄腿肌组织中的表达量显著高于90和240日龄（P<0.05）。研究结果为深入探讨MSTN基因对家兔肌肉生长发育的调控机制奠定了基础。

关键词: 新西兰白兔; MSTN基因; 生物信息学分析; 实时荧光定量PCR; 表达

Abstract: Myostatin (MSTN) was a master regulator of skeletal muscle development. In order to obtain the sequence and expression pattern of MSTN gene in New Zealand White rabbits, the MSTN gene sequence was amplified and cloned from leg muscle tissue of New Zealand White rabbit by RT-PCR and then was analyzed by online bioinformatics software. The expression level of MSTN gene in different tissues and different periods of leg muscle was detected by Real-time quantitative PCR. The results showed that the CDS region of MSTN gene in New Zealand White rabbit was 1 128 bp in length, encoding 375 amino acids, and had 95.9%, 94.9%, 94.9%, 91.7%, 91.5%, 91.3% and 84.2% similarity with the nucleotide sequences of Homo sapiens, Sus scrofa, Equus caballus, Mus musculus, Canis lupus familiaris, Bos taurus, Gallus gallus published in GenBank, respectively. MSTN protein was an acidic and hydrophilic secretory protein, without transmembrane structure and contains a signal peptide. It mainly distributed in endoplasmic reticulum and mitochondria. The prediction results of secondary structure and tertiary structure of MSTN protein were consistent, which were mainly composed of random coil, followed by alpha helix and extended chain. It was a mixed protein. Protein interaction prediction found that MSTN protein interacted with PAX7, MYOG, IGF1, FST, Smad3 and Smad2 proteins related to muscle growth and development. Results of expression analysis in different tissues showed that MSTN gene was widely expressed in heart, liver, spleen, lung, kidney, leg muscle, uterus and stomach of New Zealand White rabbits. The expression level in leg muscle was the highest, which was extremely significantly higher than that in other tissues (P<0.01), followed by kidney, and the lowest expression in liver. Results of expression analysis in leg muscle tissue at different stages showed that the expression of MSTN gene in 180-day-old leg muscle tissue of New Zealand White rabbits was significantly higher than that in 90- and 240-day-old (P<0.05). The results laid a foundation for further study on the regulatory mechanism of MSTN gene on muscle growth and development in rabbits.

Key words: New Zealand White rabbits; MSTN gene; bioinformatics analysis; Real-time quantitative PCR; expression

中图分类号:

S829.1

姜懿轩, 王亚玲, 邢珊珊, 宋国华, 许会芬, 李明. 新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建[J]. 中国畜牧兽医, 2021, 48(10): 3501-3511.

JIANG Yixuan, WANG Yaling, XING Shanshan, SONG Guohua, XU Huifen, LI Ming. Cloning, Bioinformatics Analysis and Expression Profile Construction of MSTN Gene in New Zealand White Rabbits[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3501-3511.

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