小尾寒羊SPARCL1基因编码区克隆及表达分析

doi:10.16431/j.cnki.1671-7236.2019.12.002

摘要/Abstract

摘要： 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，SPARCL1）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SPARCL1基因序列设计引物，应用PCR技术扩增SPARCL1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SPARCL1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SPARCL1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SPARCL1基因CDS 1 962 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；SPARCL1基因具有信息肽切割位点，为分泌蛋白；小尾寒羊SPARCL1基因序列与NCBI中绵羊序列同源性为99.80%，SPARCL1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。SPARCL1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SPARCL1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SPARCL1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。

关键词: 小尾寒羊; SPARCL1基因; 编码区; 克隆; 组织; 表达差异

Abstract: 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 SPARCL1 gene in sheep published in GenBank,and the SPARCL1 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 SPARCL1 gene was obtained.The sequence and protein structure were analyzed by bioinformatics software.The expression of SPARCL1 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 SPARCL1 gene CDS was 1 962 bp,encoding 653 amino acids;The molecular weight was 74.39 ku,the theoretical isoelectric point was 4.64,which was a hydrophilic protein;SPARCL1 gene had a information peptide cleavage site and it was a secreted protein;The homology of SPARCL1 gene sequence in Small-tail Han sheep was 99.80% with that of sheep provided by NCBI,and there were 4 mutation sites of SPARCL1 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 SPARCL1 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 SPARCL1 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 SPARCL1 gene and exploring its possible role of fat metabolism in Small-tail Han sheep.

Key words: Small-tail Han sheep; SPARCL1 gene; coding region; cloning; tissue; expression difference

中图分类号:

肖成, 金海国, 魏天, 高一, 于永生, 张立春, 马惠海, 曹阳. 小尾寒羊SPARCL1基因编码区克隆及表达分析[J]. 中国畜牧兽医, 2019, 46(12): 3466-3474.

XIAO Cheng, JIN Haiguo, WEI Tian, GAO Yi, YU Yongsheng, ZHANG Lichun, MA Huihai, CAO Yang. Cloning and Expression Analysis of SPARCL1 Gene Coding Region in Small-tail Han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3466-3474.

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