美仁牦牛FABP7基因克隆、生物信息学及表达分析

Cloning,bioinformatics and expression analysis of FABP7 gene in Meiren yak

【目的】为探究牦牛脂肪酸结合蛋白7基因(Fatty Acid Binding Protein 7,FABP7)的结构与功能。【方法】以美仁牦牛为试验对象,克隆FABP7基因的编码区(Coding sequence CDS)以及检测该基因在不同组织(心脏、肝脏、脾脏、肺脏、肾脏、睾丸、肌肉、脂肪)中的表达差异。以美仁牦牛心脏组织cDNA为模板,利用RT-PCR技术克隆牦牛FABP7基因的CDS区序列,并使用多种软件和在线工具进行生物信息学分析,利用qPCR技术检测FABP7基因在美仁牦牛8个组织中的表达情况。【结果】美仁牦牛FABP7基因编码区全长为399 bp,编码132个氨基酸,已上传至美国国家生物技术信息中心(NCBI)GenBank数据库中,获得基因序列号为OQ730164;分析牦牛FABP7蛋白显示,不稳定指数为26.27、理论等电点为5.38,脂肪系数73.71、总平均亲水指数-0.387,属于稳定的亲水性蛋白质,FABP7蛋白中存在1个N-糖基化潜在位点和14个潜在的磷酸化位点,该蛋白没有信号肽区域,不存在跨膜结构;在氨基酸组分中缬氨酸含量最高(11.4%),通过亚细胞定位发现,美仁牦牛FABP7基因分布在细胞质、细胞核、线粒体中;通过预测蛋白二、三级结构可知,蛋白质的高级结构主要由无规则卷曲和延伸链构成。同源性比对发现,美仁牦牛和牦牛、牛的亲缘关系最近(100%),与蜥蜴的最远(91.67%),FABP7基因与FABP2、FABP4、FABP5、FABP6以及APOA4等相关蛋白相互作用,对机体的脂质代谢具有重要的调控作用;qPCR结果显示,美仁牦牛的8个组织中FABP7基因的表达量存在明显的差异性,在心脏组织中的表达量最高,显著高于其他组织(P<0.05),在脾脏组织中低度表达。【结论】本试验成功克隆了美仁牦牛FABP7基因的编码区,且该基因在美仁牦牛心脏组织中表达最高,可为FABP7基因在牦牛后续的研究中提供理论依据。

【Objective】The study was conducted to explore the structure and function of Fatty Acid Binding Protein 7(FABP7)gene of yak,with Meiren yak as the experimental object,cloning the coding sequence CDS of FABP7 gene and detecting the difference in gene expression in different tissues(heart,liver,spleen,lung,kid‐ney,testis,muscle,fat).【Method】Using the cDNA of heart tissue as template,the CDS sequence of FABP7 gene was cloned by RT-PCR,and bioinformatics analysis was carried out by various software and online tools.The expression of FABP7 gene in 8 tissues of Meiren yak was detected by qPCR.【Result】The FABP7 coding region of Meiren yak was 399 bp,encoding 132 amino acids,which had been uploaded to the GenBank database of the National Center for Biotechnology Information(NCBI)and obtained the sequence number of OQ730164.The analysis of FABP7 protein showed that it belonged to the stable hydrophilic protein,with the instability in‐dex of 26.27,the theoretical isoelectric point of 5.38,the fat coefficient of 73.71,and with the total average hydro‐philic index of-0.387.FABP7 protein had 1 potential N-glycosylation site and 14 potential phosphorylation sites,and had no signal peptide region and transmembrane structure.Among the amino acids,the content of Valine was the highest(11.4%).According to subcellular mapping,FABP7 gene was distributed in cytoplasm,nucleus and mitochondria of Meiren yak.By predicting the secondary and tertiary structures of proteins,it was showed that the higher structure of proteins was mainly composed of random coiled and extended chains.The homology analysis showed that Meren yak had the closest relationship with Bos mutus and Cattle(100%),and the furthest relation‐ship with lizard(91.67%).FABP7 gene interacted with the related proteins such as FABP2,FABP4,FABP5,FABP6 and APOA4,which played an important role in regulating lipid metabolism in the body.The results of qPCR showed that FABP7 gene expression was significantly different in 8 tissues of Meiren yak,and the highest expression occurred in heart tissue,which was significantly higher than other tissues(P<0.05),and the lower ex‐pression occurred in spleen tissue.【Conclusion】In this experiment,the coding region of FABP7 gene was cloned successfully,and the expression of FABP7 gene was the highest in the heart tissue of Meiren yak,which could lay a theoretical basis for the subsequent research of FABP7 gene in yak.