西农萨能奶山羊SERPINA1基因克隆、生物信息学及组织表达分析

doi:10.16431/j.cnki.1671-7236.2020.07.001

摘要/Abstract

摘要： 本研究旨在克隆西农萨能奶山羊SERPINA1基因的CDS区，采用生物软件和在线预测工具进行生物信息学分析，通过实时荧光定量PCR技术检测SERPINA1基因在西农萨能奶山羊各组织间mRNA的表达水平。根据GenBank中山羊SERPINA1基因CDS区序列（登录号：XM_018066209.1），利用Primer Premier 5.0软件设计特异性引物，RT-PCR扩增目的基因，构建原核表达载体测序后对序列进行生物信息学分析；采集西农萨能奶山羊心脏、肝脏、脾脏、肺脏、乳腺、肾脏、肌肉、瘤胃和小肠组织，提取组织RNA，反转录为cDNA模板，设计特异性定量引物，进行实时荧光定量PCR，检测SERPINA1基因在不同组织中的表达差异。结果显示，西农萨能奶山羊SERPINA1基因CDS区全长1 326 bp，编码441个氨基酸；同源性比对分析显示，西农萨能奶山羊与山羊、绵羊、牛和小鼠SERPINA1基因核苷酸序列同源性分别为100%、98.6%、95.7%和71.6%，与山羊亲缘关系最近，其次是绵羊。SERPINA1蛋白分子质量为48.71 ku，等电点为5.71，为跨膜亲水蛋白；SERPINA1氨基酸序列分别有62个磷酸化位点，3个跨膜区结构。组织表达分析显示，SERPINA1基因在西农萨能奶山羊肝脏组织中显著高表达（P<0.05），其次是乳腺组织，在肺脏组织中表达量最低。研究结果为进一步探究SERPINA1基因在奶山羊乳蛋白合成代谢中的作用提供理论依据。

关键词: 西农萨能奶山羊; SERPINA1基因; 克隆; 生物信息学分析; 组织表达

Abstract: The objective of this study was to clone the CDS region of SERPINA1 gene in Xinong Saanen dairy goats (Capra hircus),conduct the bioinformatics analysis using biological software and online prediction tools,and detect the mRNA expression level of SERPINA1 gene in different tissues of Xinong Saanen dairy goats by Real-time quantitative PCR.Specific primers were designed by Primer Premier 5.0 according to the CDS sequence of SERPINA1 gene in Capra hircus (GenBank accession No.:XM_018066209.1),the SERPINA1 gene CDS was amplified by RT-PCR and analyzed by bioinformatics after prokaryotic expression vector was constructed.Collecting tissues such as heart,liver,spleen,lung,mammary gland,kidney,muscle,rumen and small intestine,mRNA was extracted from these tissues and reversed transcription as cDNA,specific primers were designed for Real-time quantitative PCR to detect the expression differences of SERPINA1 gene mRNA in different tissues.The results showed that the CDS region of SERPINA1 gene in Xinong Saanen dairy goats (Capra hircus) was 1 326 bp in length,encoding 441 amino acids.Homology alignment analysis showed that the nucleotide sequences of Xinong Saanen dairy goats with Capra hircus,Ovis aries,Bos taurus and Mus musculus were 100%,98.6%,95.7% and 71.6%,respectively,and the goat was the most closest species with dairy goat,the second was sheep.The SERPINA1 protein had the molecular weight of 48.71 ku,the isoelectric point was 5.71,and was a hydrophilic protein.Amino acid sequence of SERPINA1 had 62 phosphorylation sites,with 3 transmembrane structure.Tissue expression analysis showed that SERPINA1 gene was highly expressed in liver of Xinong Saanen dairy goats (P<0.05),followed by the mammary gland,and was the least expressed in lung.The results provided a theoretical basis for further exploration of the role of SERPINA1 gene in milk protein synthesis and metabolism of dairy goats.

Key words: Xinong Sannen dairy goats; SERPINA1 gene; cloning; bioinformatics analysis; tissue expression

中图分类号:

潘檀, 高梁嘉慧, 程菲, 罗军, 李聪. 西农萨能奶山羊SERPINA1基因克隆、生物信息学及组织表达分析[J]. 中国畜牧兽医, 2020, 47(7): 1971-1980.

PAN Tan, GAO Liangjiahui, CHENG Fei, LUO Jun, LI Cong. Cloning,Bioinformatics and Tissue Expression Analysis of SERPINA1 Gene in Xinong Saanen Dairy Goats (Capra hircus)[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 1971-1980.

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