大长杂交猪DJ-1基因克隆、生物信息学及表达分析

Cloning, Bioinformatics and Expression Analysis of DJ-1 Gene in Large White×Landrace Pigs

【目的】克隆大长杂交猪DJ-1基因CDS区并进行生物信息学分析,探讨DJ-1基因在猪脂肪组织中的表达规律,为后续探究该基因在猪脂肪沉积中的功能奠定基础。【方法】以大长杂交猪腹股沟皮下脂肪组织cDNA为模板,PCR扩增DJ-1基因CDS区,利用在线工具对DJ-1蛋白进行生物信息学分析;利用实时荧光定量PCR检测DJ-1基因在大长杂交猪、大白猪(瘦肉型猪)和莱芜猪(脂肪型猪)脂肪组织中的表达量,并比较表达差异。【结果】大长杂交猪DJ-1基因CDS区全长570 bp,编码189个氨基酸。系统进化树分析表明,大长杂交猪和牛亲缘关系最近,与斑马鱼亲缘关系最远。DJ-1蛋白分子质量为19.94 ku,是一种稳定的酸性、亲水性蛋白,且含有1个GAT_1超家族成员Thij结构域;无信号肽和跨膜结构域,具有21个磷酸化位点、4个N-糖基化修饰位点和1个O-糖基化修饰位点。DJ-1蛋白二级结构由α-螺旋、延伸链、β-转角、无规则卷曲组成,占比分别为42.86%、17.99%、7.94%和31.22%,三级结构预测结果与其一致。蛋白互作分析发现,DJ-1蛋白可能与SOD2、NDUFV2、SNCA、BCL2L1和MAP3K等蛋白存在相互作用。实时荧光定量PCR结果表明,DJ-1基因在大长杂交猪背膘、腹股沟脂肪和肾周脂肪组织中均有表达,但无显著差异(P>0.05);且在大白猪脂肪组织中的表达量显著或极显著低于莱芜猪(P<0.05;P<0.01)。【结论】本研究成功克隆了大长杂交猪DJ-1基因CDS区,其编码蛋白属稳定的酸性、亲水性蛋白,在莱芜猪脂肪组织中的表达量显著或极显著高于大白猪。本试验结果为进一步研究猪DJ-1基因功能提供理论参考。

【Objective】 The purpose of this study was to clone the CDS region of DJ-1 gene in Large White×Landrace pigs and analyzed by bioinformatics, explore the expression pattern of DJ-1 gene in porcine adipose tissue, and lay a foundation for further studies on the role of DJ-1 gene in porcine adipose deposition.【Method】 The CDS region of DJ-1 gene was amplified by PCR using the cDNA of inguinal subcutaneous adipose tissue in Large White×Landrace pigs as a template, and the bioinformatics analysis of DJ-1 protein was performed using online tools.Real-time quantitative PCR was used to measure the expression of DJ-1 gene in adipose tissue of Large White×Landrace pigs, Large White pigs(lean-type) and Laiwu pigs(obese-type),and the expression differences were compared.【Result】 The length of DJ-1 gene CDS in Large White×Landrace pigs was 570 bp, which coded 189 amino acids.According to the phylogenetic tree, Large White×Landrace pigs was most closely related to Bos taurus and most distantly related to Danio rerio.The molecular weight of DJ-1 protein was 19.94 ku, it was stable, acidic and hydrophilic protein, which had one conserved domain called GAT_1 superfamily Thij.The DJ-1 protein had 21 phosphorylation sites, 4 N-glycosylation modification sites and 1 O-glycosylation modification site, but no signal peptide and transmembrane domain.The secondary structure of DJ-1 protein was composed of alpha helix, extended chain, beta turn and random coil, the proportions were 42.86%,17.99%,7.94% and 31.22%,respectively, and the tertiary structure prediction results were consistent with them.Protein interaction analysis revealed that DJ-1 protein interacted with SOD2,NDUFV2,SNCA,BCL2 L1 and MAP3 K.Real-time quantitative PCR results showed that DJ-1 gene was generally expressed in backfat, inguinal fat and perirenal fat of Large White×Landrace pigs, there were no significant difference among them(P>0.05).The expression of DJ-1 gene in Large White pigs was significantly or extremely significantly lower than that of Laiwu pigs(P<0.05 or P<0.01).【Conclusion】 This study successfully cloned the CDS region of DJ-1 gene in Large White×Landrace pigs, and DJ-1 was a stable, acidic and hydrophilic protein.The expression of DJ-1 gene in Laiwu pigs was significantly or extremely significantly higher than that of Large White pigs. The results provided a theoretical reference for further study the function of porcine DJ-1 gene.