山羊DLK1基因分子克隆、生物信息学分析及表达研究

Molecular Cloning,Bioinformatics Analysis and Expression of DLK1 Gene in Goat

本研究旨在克隆山羊DLK1基因,预测编码蛋白的结构与功能,同时研究该基因在山羊组织器官中的表达规律及其与肌内脂肪含量的相关性。以简州大耳羊为试验动物,采用RT-PCR克隆DLK1基因序列,结合生物信息学方法分析其生物学特性,荧光定量检测DLK1mRNA表达情况,并将表达量与肌内脂肪含量进行相关性分析。结果显示,克隆得到山羊DLK1长度为1 137bp,开放阅读框(ORF)长度为927bp(GenBank登录号:KP686197),编码308个氨基酸,山羊核苷酸序列和氨基酸序列与牛的相似性最高(97%),系统进化树分析表明山羊与牛亲缘关系最近;生物信息学预测显示,该蛋白属于不稳定酸性疏水蛋白;有8个磷酸化位点和1个N-糖基化位点;亚细胞定位于内质网(44.4%)、高尔基体(33.3%)和质膜(22.2%),属于分泌跨膜蛋白;具有5个表皮生长因子结构域和2个表皮生长因子家族特有的保守结构域EGF-CA;预测二级结构由12.99%β-折叠和87.01%无规则卷曲组成的非常规蛋白。荧光定量PCR结果显示,DLK1基因在山羊不同组织中都存在表达,其在肾中表达水平最高,在脂肪组织中表达水平最低;DLK1基因在羔羊背最长肌组织中表达水平最高,高于育成羊和成年羊,但差异不显著(P>0.05);相关分析显示,山羊背最长肌、腿肌、臂三头肌中DLK1mRNA表达与肌内脂肪含量分别呈显著负相关(R=-0.6,P<0.05)、无显著正相关(R=0.2,P>0.05)和无显著负相关(R=-0.2,P>0.05)。研究结果显示,DLK1可能对山羊肌内脂肪沉积起着重要作用。本结果为进一步研究DLK1在肌内脂肪沉积过程中的作用提供了参考。

The present study aimed to clone DLK1 gene of goat,predict the structure and function of peptone encoded by DLK1 gene,further investigate its expression regulation in organs or tissues of goat and analyze the correlation between DLK1 mRNA expression and intramuscular fat（IMF）content in muscles.Jianzhou Da＇er goat was used as experimental animals and the RTPCR technique was employed to amplify the DLK1 gene.The biological characteristics of this gene were analyzed by bioinformatics,and the tissue expression specificity as well as the development stage expression specificity of this gene were analyzed using the fluorescence quantitativePCR.The results showed that the DLK1 cDNA was 1 137 bp in length（GenBank accession No.：KP686197）,927 bp for ORF and 308 amino acids for protein encoded.The nucleotide sequence and the deduced amino acids sequence of goat DLK1 shared 97% homology with the DLK1 of cattle.Accordingly,the close genetic relationship between goat and cattle was indicated by the phylogenetic tree analysis.The DLK1 protein was an unstable,acidity and hydrophobicity protein.There were 8phosphorylation sites and 1 N-glycosylation site within the DLK1 protein.This protein was located in endoplasmic reticulum（44.4%）,golgi（33.3%）and plasma membrane（22.2%）,respectively through the sub-cellular level prediction.It was a secretory transmembrance protein.It had 5domains of the EGF family,which had 2EGF-CA of EGF family conservative domains.Moreover,the secondary structure of the DLK1 protein was composed of 87.01% random coil and12.99%β-sheet,indicating an unconventional protein.The results of real-time PCR exhibited that the DLK1 gene was expressed in various tissues at different levels.The mRNA expression level of the DLK1 gene was highest in kidney and lowest in fat.The mRNA expression level of the DLK1 gene in longissimus dorsi of lamb was not significant higher than that in longissimus dorsi of youth and adult goat（P〉0.05）.The results of correlation analysis showed that the mRNA expression level of the DLK1 gene in longissimus dorsi was correlated negatively with IMF content（R=-0.6,P〈0.05）,in leg was not correlated positively with IMF content（R=0.2,P〉0.05）,in arm triceps was not correlated negatively with IMF content（R=-0.2,P〈0.05）.The results suggest that the DLK1 gene may play an important role in the IMF deposition in goat,which would lay a foundation for further studies about the function of DLK1 gene in IMF deposition.