广灵驴ADSL基因克隆、序列分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2021.08.001

摘要/Abstract

摘要： 本研究旨在对广灵驴腺苷琥珀酸裂解酶（adenylosuccinatelyase，ADSL）基因进行克隆及生物信息学分析，并检测其在不同组织中的表达情况，为探究ADSL基因在广灵驴肌苷酸合成及风味形成中的作用机制提供理论参考。根据GenBank中公布的马（登录号：XM_001917207.5）、牛（登录号：NM_001102377.2）、猪（登录号：GU249574.1）等物种的ADSL基因mRNA序列，通过Primer Premier 3.0在线工具设计同源引物，RT-PCR法扩增并克隆ADSL基因序列，对编码序列进行结构与功能分析，最后用实时荧光定量PCR检测ADSL基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏、背最长肌组织中的表达水平。结果显示，广灵驴ADSL基因CDS长1 473 bp，编码490个氨基酸，提交至NCBI，登录号：MW037837，其核苷酸序列与马、牛、双峰驼、猪、绵羊、人、小鼠的相似性分别为99.5%、90.8%、92.3%、90.4%、90.7%、90.5%和86.0%。进化树分析结果表明，广灵驴与马的种属关系最近，与小鼠的亲缘关系最远。ADSL蛋白分子质量为55.44 ku，等电点为6.52，平均疏水指数为-0.243，是一种不稳定的酸性亲水蛋白。ADSL蛋白有41个磷酸化修饰位点，6个糖基化修饰位点，没有信号肽和跨膜结构，有1个卷曲螺旋。ADSL蛋白主要定位在细胞质，α-螺旋（68.98%）是主要的二级结构。ADSL基因在广灵驴6个组织中都有表达，其中肺脏中的表达量最高，显著高于其他组织（P<0.05），其次是心脏和肝脏，脾脏、肾脏和背最长肌中的表达量最低。本研究结果为今后探究ADSL基因在广灵驴肌苷酸合成及风味形成的分子机制奠定基础。

关键词: 广灵驴; ADSL基因; 克隆; 生物信息学分析; 组织表达

Abstract: The purpose of this study was to clone and analyze of adenylosuccinatelyase(ADSL) gene in Guangling donkey by bioinformatics, detect its expression in different tissues, and provide theoretical reference for exploring the action mechanism of ADSL gene in inosine monophosphate and flavor formation of Guangling donkey.Homologous primers were designed by Primer Premier 3.0 according to the mRNA sequences of ADSL gene in Equus caballus (accession No.:XM_001917207.5), Bos taurus (accession No.:NM_001102377.2) and Sus scrofa (accession No.:GU249574.1), and other species published in GenBank.The CDS sequence of ADSL gene in Guangling donkey was amplified with RT-PCR and cloned.The encoding sequence of ADSL gene was analyzed, and the expression of ADSL gene in heart, liver, spleen, lungs, kidney, longissimus dorsi muscle and subcutaneous fat of Guangling donkey were detected by Real-time quantitative PCR.The CDS of ADSL gene in Guangling donkey consisted of nucleotides of 1 473 bp, encoding 490 amino acids.It was submitted to NCBI, and obtain the accession No.:MW037837.The nucleotide sequence of ADSL gene showed 99.5%, 90.8%, 92.3%, 90.4%, 90.7%, 90.5% and 86.0% identity with that of Equus caballus, Bos taurus, Camelus bactrianus, Sus scrofa, Ovis aries, Homo sapiens and Mus musculus, respectively.Phylogenetic tree results revealed that Guangling donkey was the most closely related to Equus caballus and the farthest related to Mus musculus.The ADSL protein, with molecular weight of 55.44 ku, isoelectric point of 6.52, and grand average of hydrophobicity of -0.243, was an unstable acidic hydrophilic protein.There were 41 phosphorylation modification sites, 6 glycosylation modification sites, and a coiled helix in ADSL protein, with no signal peptide and transmembrane structure, it was mainly located in the cytoplasmic.The secondary structure of ADSL protein was mainly of 68.98% alpha helix.ADSL gene was expressed in 6 tissues, among which the expression in lung was the highest, which was significantly higher than that in other tissues(P<0.05), followed by heart and liver, and the lowest in spleen, kidney and longissimus dorsi muscle.The experiment results provided a solid theoretical basis for further exploring the role of ADSL gene in the molecular mechanism of inosine monophosphate synthesis and flavor formation in Guangling donkey.

Key words: Guangling donkey; ADSL gene; cloning; bioinformatics analysis; tissue expression

中图分类号:

关家伟, 孙瑜彤, 邱丽霞, 李武峰, 杜敏. 广灵驴ADSL基因克隆、序列分析及组织表达研究[J]. 中国畜牧兽医, 2021, 48(8): 2685-2694.

GUAN Jiawei, SUN Yutong, QIU Lixia, LI Wufeng, DU Min. Cloning,Sequence Analysis and Tissue Expression of ADSL Gene in Guangling Donkey[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2685-2694.

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