陆川猪ACOX1基因不同亚型克隆及其表达差异分析  

09Cloning and expression differential analysis of different subtypes of ACOX1 gene in Luchuan pigs

作　　者：陈钊 潘鹏丞 潘星辰 陈少梅 覃兆鲜 吴永绍 覃倩 农素群 谢炳坤 陈宝剑 CHEN Zhao;PAN Peng-cheng;PAN Xing-chen;CHEN Shao-mei;QIN Zhao-xian;WU Yong-shao;QIN Qian;NONG Su-qun;XIE Bing-kun;CHEN Bao-jian(Guangxi Vocational University of Agriculture,Nanning,Guangxi 530005,China)

机构地区：[1]广西农业职业技术大学,广西南宁530005

出　　处：《南方农业学报》2023年第12期3739-3747,共9页Journal of Southern Agriculture

基　　金：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,Z2022111)。

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,进行生物信息学分析,并分析ACOX1基因及其亚型在不同品种猪背最长肌中的表达情况,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基因编码区(CDS)序列,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、杜洛克猪、杜陆猪和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基因不同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(ACOX1-Ⅰ和ACOX1-Ⅱ),NCBI登录号分别为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74786.50Da,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,该结果可为阐明ACOX1基因在陆川猪脂肪沉积及代谢方面的调控机制提供参考。【Objective】This study was aimed to clone different subtypes of acyl-coenzyme A oxidase 1(Acyl-CoA oxidase 1,ACOX1)gene in Luchuan pig and conduct bioinformatics analysis,analyze the expression of ACOX1 gene and its subtypes in longissimus dorsi muscle of different breeds of pigs.So as to provide reference for further exploring the functional roles of ACOX1 gene in Luchuan pigs.【Method】Based on pig ACOX1 gene sequence published in GenBank(NM_001101028.1)and the coding region(CDS)sequence of the Luchuan pig ACOX1 gene,the specific primers and validation primers were designed.Total RNA was extracted from the liver of Luchuan pig aged 0 month and the longissimus dorsi muscle of Luchuan pig,Duroc pig,Dulu pig and Ludu pig aged 8 months by TRIzol method,reverse transcription cDNA was synthesized,using 0-month-old Luchuan pig liver cDNA as template for cloning and validation,to obtain different subtypes of ACOX1 gene sequences of Luchuan pig,and bioinformatic analysis was performed using various online tools,and real-time fluorescent quantitative PCR was used to detect the expression of different subtypes of ACOX1 gene in longissimus dorsi muscle tissue.【Result】There were two subtypes of ACOX1 gene in Luchuan pigs(ACOX1-Ⅰand ACOX1-Ⅱ),the GenBank accession numbers were OQ701687 and OQ701688,the full length sequence of ACOX1 gene CDS in Luchuan pigs were 1986 bp,encoding 661 amino acids residues,the two isomers were mainly selective cleavage between sequences between positions 270 to 429.The molecular weight of Luchuan pig ACOX1-Ⅰencoded protein was 74786.50 Da,and its theoretical isoelectric point(pI)was 7.36,the molecular weight of ACOX1-Ⅱencoded protein was 74670.49 Da,and pI was 8.17.The secondary structure of ACOX1-Ⅰprotein In Luchuan pig was composed ofα-helix(47.96%),β-turn(10.14%),extended chain(14.52%)and random coil(27.38%).The secondary structure of ACOX1-Ⅱprotein in Luchuan pig was composed ofα-helix(47.66%),β-turn(10.89%),extended chain(14.37%)and random coil(27.08%).The ACOX1 prot

关 键 词：陆川猪 ACOX1基因 亚型 生物信息学分析 表达分析 

分 类 号：S828.89[农业科学—畜牧学]