陆川猪ANKRD1基因克隆及其表达差异分析

Gene cloning and differential expression analysis of ANKRD1 gene in Luchuan pig

【目的】克隆陆川猪心肌锚蛋白重复域1基因(ANKRD1),并进行生物信息学及组织表达谱分析,为研究ANKRD1在陆川猪机体内的功能作用提供参考依据。【方法】根据NCBI已公布的野猪ANKRD1基因序列(NM_213922.1)设计特异性引物,采用TRIzol法提取陆川猪心脏、肝脏、脾脏、肺脏、肾脏、背最长肌和皮下脂肪的总RNA,反转录合成cDNA,并以此为模板进行ANKRD1基因克隆,通过MegAlign、Protaram、Protscale、MHMM Server和SignalP等在线分析软件进行生物信息学分析,最后以实时荧光定量PCR检测ANKRD1基因在陆川猪各组织中的表达情况。【结果】陆川猪ANKRD1基因蛋白编码区(CDS)序列全长960 bp,编码319个氨基酸残基,与NCBI已公布野猪ANKRD1基因(NM_213922.1)的CDS序列存在4处碱基突变,但均为同义突变,二者的ANKRD1氨基酸序列同源性为99.6%。陆川猪ANKRD1基因编码蛋白分子量为36125.70 Da,理论等电点(pI)为7.09,属于稳定蛋白,其二级结构中α-螺旋占46.39%、无规则卷曲占39.81%、β-转角占9.09%、延伸链占4.70%;陆川猪ANKRD1蛋白不存在跨膜结构,也无信号肽,有多个磷酸化位点。陆川猪ANKRD1基因在其心脏、肝脏、脾脏、肺脏、肾脏、背最长肌和皮下脂肪等7个组织中均有表达,其中以心脏中的相对表达量最高,显著高于在其他组织中的相对表达量(P<0.05,下同),在脾脏中的相对表达量最低,显著低于在心脏、肝脏、肺脏和背最长肌中的相对表达量。【结论】ANKRD1基因在陆川猪的心脏、肝脏、脾脏、肺脏、肾脏、背最长肌和皮下脂肪等组织中均有表达,且存在明显差异,故推测ANKRD1基因在不同组织中发挥不同作用。

【Objective】The ankyrin repeat domain 1 gene(ANKRD1)of Luchuan pig was cloned and analyzed by bioinformatics and tissue expression profile,the purpose of this study was to provide a reference for studying the function of ANKRD1 in Luchuan pig.【Method】The specific primers were designed according to the ANKRD1 gene sequence(NM_213922.1)published by NCBI.The total RNA of heart,liver,spleen,lung,kidney,longissimus dorsi and subcutaneous fat of Luchuan pig was extracted by TRIzol method,the cDNA was synthesized by reverse transcription,and the ANKRD1 gene was cloned using this template.The biological information was analyzed by online analysis software such as MegALign,Protaram,Protscale,MHMM Server and SignalP.Finally,the expression ofANKRD1 gene in various tissues of Luchuan pig was detected by real-time fluorescent quantitative PCR.【Result】The protein coding region(CDS)of ANKRD1 gene of Luchuan pig was 960 bp in length and encoded 319 amino acid residues.There were four base mutations between Luchuan pigANKRD1 gene and the CDS sequence of wild boarANKRD1 gene(NM_213922.1)published by NCBI,but all of them were synonymous mutations.The homology of ANKRD1 amino acid sequence between Luchuan pig and wild boar was 99.6%.The molecular weight and theoretical isoelectric point(pI)of ANKRD1 gene of Lu-chuan pig were 36125.70 Da and 7.09 respectively,which was a stable protein.α-helix accounted for 46.39%,random coil accountedfor 39.81%,β-turn accounted for 9.09%,and extended strand accounted for 4.70%.The ANKRD1 protein of Luchuan pig had no transmembrane structure and signal peptide,and had multiple phosphorylation sites.The results showed thatANKRD1 gene was expressed in seven tissues including heart,liver,spleen,lung,kidney,longissimus dorsi and subcutaneous fat.The relative expression level of ANKRD1 in heart was the highest,which was significantly higher than that in other tissues(P<0.05,the same below),and the relative expression level in spleen was the lowest,which was significantly lower than that in heart,liver,lung and longissimus dorsi.【Conclusion】ANKRD1 gene is expressed in heart,liver,spleen,lung,kidney,longissimus dorsi and subcutaneous fat of Luchuan pigs,and there are obvious differences.It is speculated thatANKRD1 gene plays a different role in different tissues.