小尾寒羊SPARCL1基因编码区克隆及表达分析

Cloning and Expression Analysis of SPARCL1 Gene Coding Region in Small-tail Han Sheep

为了探究小尾寒羊富含半胱氨酸酸性分泌蛋白类似物1(secreted protein acidic and rich in cysteine like 1,SPARCL 1)基因结构及其各组织间表达差异,试验提取小尾寒羊肝脏组织总RNA,根据GenBank中公布的绵羊SPARCL 1基因序列设计引物,应用PCR技术扩增SPARCL 1基因编码区(CDS)。将扩增产物连接到pMD18-T载体进行测序,获得小尾寒羊SPARCL 1基因完整CDS区序列信息,应用生物信息学软件分析序列及蛋白结构。以小尾寒羊心脏、肝脏、肌肉、胃、十二指肠、小肠组织mRNA为模板,通过实时定量荧光PCR技术检测SPARCL 1基因在小尾寒羊各组织间的表达差异。结果显示,试验成功获得小尾寒羊SPARCL 1基因CDS 1962 bp,编码653个氨基酸;分子质量为74.39 ku,理论等电点为4.64,为亲水性蛋白质;SPARCL 1基因具有信息肽切割位点,为分泌蛋白;小尾寒羊SPARCL 1基因序列与NCBI中绵羊序列同源性为99.80%,SPARCL 1基因CDS区出现4处突变位点,但未引起氨基酸改变;SPARCL1蛋白存在77个蛋白磷酸化位点和3个糖基化位点;SPARCL1蛋白表达预测主要定位在细胞质;SPARCL1蛋白二级结构中α-螺旋、β-折叠、β-转角和无规则卷曲分别占31.9%、6.4%、28.7%和33.0%,三级结构预测结果与其一致。SPARCL 1基因在小尾寒羊皮下脂肪组织中相对表达量明显高于其他组织。本研究成功克隆获得小尾寒羊SPARCL 1基因CDS区完整序列,并对其序列、蛋白理化特性、结构及各组织间表达差异进行了详细分析,为研究小尾寒羊SPARCL 1基因功能,探究其在小尾寒羊脂肪代谢过程中可能发挥的作用提供参考依据。

This study was aimed to explore the gene structure of cysteine-rich acidic and rich in cysteine-like 1(SPARCL1)and its expression differences in tissues of Small-tail Han sheep.The total RNA was extracted from the liver tissue of Small-tail Han sheep.Primers were designed based on the sequence of SPARCL 1 gene in sheep published in GenBank,and the SPARCL 1 gene codings region(CDS)was amplified by PCR.The amplified product was constructed into pMD18-T vector for sequencing,and the sequence information of the complete CDS of SPARCL 1 gene was obtained.The sequence and protein structure were analyzed by bioinformatics software.The expression of SPARCL 1 gene mRNA in different tissues(liver,muscle,stomach,duodenum and small intestine)of Small-tail Han sheep was detected by Real-time quantitative PCR.The results showed that the length of SPARCL 1 gene CDS was 1962 bp,encoding 653 amino acids;The molecular weight was 74.39 ku,the theoretical isoelectric point was 4.64,which was a hydrophilic protein;SPARCL 1 gene had a information peptide cleavage site and it was a secreted protein;The homology of SPARCL 1 gene sequence in Small-tail Han sheep was 99.80%with that of sheep provided by NCBI,and there were 4 mutation sites of SPARCL 1 gene CDS,but no amino acid changes were caused;There were 77 protein phosphorylation sites and 3 glycosylation sites of SPARCL1 protein.SPARCL1 protein expression was mainly localized in cytoplasm.The alpha helix,beta fold,beta turn and random coil of SPARCL1 protein in the secondary structure were 31.9%,6.4%,28.7%and 33.0%,respectively,the tertiary structure prediction result of SPARCL1 protein were consistent with the secondary structure.The relative expression of SPARCL 1 gene in subcutaneous adipose tissue of Small-tail Han sheep was significantly higher than that in other tissues.In this study,the complete CDS sequence of SPARCL 1 gene was successfully cloned,and its sequence components,protein physicochemical properties,structure and expression differences among tissues were analyzed,which provided a theoretical basis for studying the function of SPARCL 1 gene and exploring its possible role of fat metabolism in Small-tail Han sheep.