摘要
本研究对湖羊(Ovis aries)m^(6)A甲基转移酶基因METTL3进行克隆和生物信息学分析,以探究其分子特征和时空表达变化规律。以湖羊肌肉组织cDNA为模板,通过克隆获得METTL3基因编码序列(coding sequence,CDS)区,并使用生物信息学软件进行相似性比对、系统进化树构建和生物信息学分析;利用实时定量PCR(real time quantitative PCR)检测METTL3 mRNA在湖羊9个不同组织中的表达水平。结果显示,湖羊METTL3基因完整CDS区全长1739 bp,编码579个氨基酸。湖羊METTL3基因与山羊(Capra hircus)的亲缘关系最近。METTL3蛋白等电点为6.05,半衰期为30 h,结构不稳定,属于亲水性蛋白。METTL3具有一个MT-A70保守结构域,无信号肽和跨膜结构域。预测共有59个潜在磷酸化位点。METTL3蛋白高级结构由无规则卷曲(50.09%)、α-螺旋(33.85%)、延伸链(12.44%)和β-转角(3.62%)组成,且主要与METTL14、WTAP等参与m^(6)A甲基化过程的相关蛋白紧密联系。实时定量PCR结果显示,METTL3 mRNA在湖羊9个组织中均有表达,与背最长肌相比,肾脏、脾脏和皮下脂肪中的表达量更高(P<0.01)。本研究成功克隆获得METTL3基因CDS区全长序列,并初步研究了其在湖羊各个组织中的表达变化规律,为探索METTL3基因在哺乳动物生长发育过程中发挥的作用提供理论依据。
Cloning and bioinformatics analysis of m^(6)A methyltransferase gene METTL3 in Hu sheep(Ovis aries)were performed in this study to explore its molecular characteristics and spatiotemporal expression change rule.Using muscle cDNA of Hu sheep as template,the coding sequence(CDS)region of METTL3 gene was cloned,and the similarity alignment,phylogenetic tree construction and bioinformatics analysis were carried out by using bioinformatic software.Real time quantitative PCR analysis was performed to test METTL3 mRNA expression in 9 different tissues of Hu sheep.The results indicated that the complete CDS of the Hu sheep METTL3 gene was 1739 bp in length and encoded 579 amino acids.The genetic relationship of METTL3 gene between Hu sheep and goat(Capra hircus)was the closest.The isoelectric point of METTL3 protein was 6.05,with the half-life of 30 h,unstable and hydrophilic.METTL3 had a conserved MT-A70 domain without a signal peptide and transmembrane structural domains.A total of 59 potential phosphorylation sites were predicted.The advance structure of METTL3 protein was composed of irregularly coiled(50.09%),α-helix(33.85%),extended chain(12.44%),andβ-turn(3.62%),which was closely related to METTL14,WTAP and other related protein involved in the methylation process of m^(6)A.According to real time quantitative PCR results,the METTL3 mRNA was wildly expressed in all nine tissues of Hu sheep.Compared with longissimus dorsi,the expression of METTL3 mRNA was higher in kidney,spleen,and subcutaneous fat(P<0.01).This study successfully cloned and obtained the full-length sequence of the CDS of the METTL3 gene,and preliminarily studied its expression variety rule in different tissues of Hu sheep,which provided theoretical basis for studying the role of the METTL3 gene in mammalian growth and development.
作者
陈博雯
张丹
刘建斌
杨博辉
卢曾奎
CHEN Bowen;ZHANG Dan;LIU Jianbin;YANG Bohui;LU Zengkui(Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agricultural Sciences,Lanzhou,730050;Sheep Breeding Engineering Technology Research Center of Chinese Academy of Agricultural Sciences,Lanzhou,730050;Key Laboratory of Ministry of Agriculture and Rural Affairs for Animal Genetics and Breeding on Tibetan Plateau,Lanzhou,730050)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2023年第2期172-182,共11页
Genomics and Applied Biology
基金
国家自然科学基金项目(32002170)
甘肃省青年科技基金项目(20JR10RA024)
中国农业科学院科技创新工程项目(25-LZIHPS-07)共同资助
作者简介
共同通信作者:杨博辉,yangbohui@caas.cn;共同通信作者:卢曾奎,luzengkui@caas.cn
