陆川猪ACOX1基因不同亚型克隆及其表达差异分析
Gening cloning and bioinformatics analysis of different subtypes of ACOX1 gene in Luchuan pigs
陈钊 1潘鹏丞 1潘星辰 1陈少梅 1覃兆鲜 1吴永绍 1覃倩 1农素群 1谢炳坤 1陈宝剑1
作者信息
- 1. 广西农业职业技术大学,广西南宁 530005
- 折叠
摘要
[目的]克隆陆川猪脂酰辅酶A氧化酶1(Acyl-CoA oxidase 1,ACOX1)基因不同亚型,并进行生物信息学分析,分析ACOX1基因及亚型在不同品种猪背最长肌中表达情况,为研究ACOX1基因在陆川猪机体内的功能作用提供参考依据.[方法]根据GenBank中ACOX1基因序列(NM_001101028.1)及所克隆获得陆川猪ACOX1基因序列编码区(CDS),设计特异性扩增和验证引物,采用TRIzol法提取0月龄陆川猪肝脏及8月龄陆川猪、杜洛克、杜陆猪、陆杜猪背最长肌的总RNA,反转录合成cDNA,以0月龄陆川猪肝脏cDNA为模板进行克隆和验证,获得陆川猪ACOX1基因不同亚型序列,并利用各在线工具进行生物信息学分析,利用实时荧光定量PCR检测ACOX1基因及亚型在背最长肌组织中的表达情况.[结果]陆川猪ACOX1基因存在2种亚型结构,NCBI登录号OQ701687和OQ701688;2种亚型CDS长度均为1986 bp,共编码661个氨基酸残基,2种异构体主要是第270～429位氨基酸进行选择性剪切,陆川猪ACOX1-I型编码蛋白分子量为74786.50 Da,理论等电点(pI)为7.36,ACOX1-II型编码蛋白分子量为74670.49 Da,pI为8.17;ACOX1-I型蛋白中α-螺旋占47.96%,β-转角占10.14%,延伸链占14.52%,无规则卷曲占27.38%,ACOX1-II型蛋白中α-螺旋占47.66%,β-转角占10.89%,延伸链占14.37%,无规则卷曲占27.08%,陆川猪ACOX1蛋白不存在跨膜结构.ACOX1-I基因在陆川猪背最长肌中的相对表达量最高,杜洛克猪中的相对表达量最低,4个品种猪之间差异不显著(P>0.05),ACOX1-II基因在陆川猪和杜洛克猪背最长肌中的相对表达量显著高于杜陆猪和陆杜猪(P<0.05).[结论]陆川猪ACOX1基因存在2种亚型(ACOX1-I和ACOX1-II),ACOX1基因及其亚型在不同品种猪中的表达存在明显差异.
Abstract
[Objective]This study was aimed to clone different isoforms of Acyl-Coa oxidase 1(Acyl-CoA oxidase 1,ACOX1)gene in Luchuan pig and conduct bioinformatics analysis,analyze the expression of ACOX1 gene and its iso-forms in longissimus dorsi muscle of different breeds of pigs.So as to provide the basics for further exploring the regula-tory roles of ACOX1 gene in Luchuan pigs.[Method]Based on pig ACOX1 gene sequence published in GenBank(Acces-sion No(NM_001101028:1)and the CDS sequence of the Luchuan pig ACOX1 gene,The specific primers and validation primers were designed to amplify ACOX1 gene from Luchuan pig using RT-PCR technique.Total RNA was extracted from the liver of Luchuan pig aged 0 months and the longissimus dorsi muscle of Luchuan pig,Duroc,Dulu and Ludu pigs aged 8 months by TRIzol method.The relative expression of ACOX1 gene and its subtypes in longissimus dorsi muscle of Lu-chuan pig,Duroc,Dulu pig and Luchuan pig were detected by Real-time quantitative PCR.[Result]There are two subtype structures of ACOX1 gene in Luchuan pigs,the GenBank accession numbers were OQ701687 and OQ701688.the full Length sequence of ACOX1 gene CDS in in Luchuan pigs were 1986 bp,encoding 661 amino acids,the two isomers mainly undergo selective cleavage between sequences between positions 270 to 429,The molecular weight of Luchuan pig ACOX1-I was 74786.5 Da,and its theoretical isoelectric point(pI)was 7.36;The molecular weight of ACOX1-II was 74670.49 Da,and its theoretical isoelectric point(pI)was 8.17.The secondary structure of ACOX1-I protein In Luchuan pig was composed of Alpha helix(47.96%),Beta turn(10.14%),extended Chain(14.52%)and random coil(27.38%).The secondary structure of ACOX1-II protein in Luchuan pig was composed of Alpha helix(47.66%),Beta turn(10.89%),ex-tended Chain(14.37%)and random coil(27.08%).The ACOX1 protein of Luchuan pig had no transmembrane domain.The expression of ACOX1-I gene in the longissimus dorsi muscle of Luchuan pigs was the highest,but the difference was not significant(P>0.05).The expression of ACOX1-II gene in the longissimus dorsi muscle of Luchuan pig and Duroc pigs were significantly higher than other breeds(P<0.05).[Conclusion]Two different subtypes of ACOX1 gene in Lu-chuan pig were successfully cloned,,the expression of ACOX1 gene and its subtypes in the longissimus dorsi muscle of different pig breeds were significant differences.
关键词
陆川猪/ACOX1基因/亚型/生物信息学分析/表达分析Key words
Luchuan pig/ACOX1gene/isoforms/bioinformatics analysis/expression analysis引用本文复制引用
基金项目
广西重点研发计划项目(桂科AB21196060)
中央引导地方科技发展专项(桂科ZY21195052)
广西农业科技自筹经费项目(Z2022119)
广西农业科技自筹经费项目(Z2022111)
出版年
2023
国家科技期刊平台
NSTL
万方数据