西农萨能奶山羊INSIG-1编码区的克隆、序列特征分析和组织表达

Cloning,sequence characters and tissue expression pattern of INSIG-1 in Saanen Dairy Goat

【目的】克隆西农萨能奶山羊胰岛素诱导基因1(INSIG-1)编码区,并对其进行序列特征分析和组织表达规律研究。【方法】提取西农萨能奶山羊总RNA,反转录cDNA后进行PCR反应,对克隆得到的INSIG-1序列进行生物信息学分析。用实时荧光定量PCR(RT-qPCR)检测INSIG-1在皮下脂肪、胸部肌肉、乳腺、肝脏、肾脏、心脏、肺、脾脏、瘤胃、小肠10个组织中的表达情况。【结果】获得西农萨能奶山羊INSIG-1基因1 014bp序列(Gen-Bank登录号JQ665439),其中编码区序列长度为831bp,编码276个氨基酸。西农萨能奶山羊INSIG-1编码区与牛(GenBank登录号NM_001077909)的亲缘关系最近,相似性达97%,编码氨基酸序列的相似性达90%,而与小鼠(GenBank登录号NM_025836)的亲缘关系较远。预测INSIG-1蛋白质分子量为29.70ku,理论等电点为8.99;IN-SIG-1蛋白质具有5个跨膜螺旋结构,且有较强的疏水性,不含信号肽。RT-qPCR检测结果表明,在西农萨能奶山羊10个组织中INSIG-1均有表达,其中在肝脏中的相对表达量最高,皮下脂肪、胸部肌肉、肺次之,在心脏中的相对表达量最低。【结论】克隆得到了西农萨能奶山羊的INSIG-1基因,并探明了其组织表达规律。

The objectives of this experiment were to clone insulin-induced gene 1（INSIG1）sequence of Saanen Dairy Goat from Northwest AF University,and to analyze the sequence characterization and quantitative tissues expression.【Method】The coding sequence region was cloned from goats mammary gland total RNA by PCR reaction with primers designed based on bovine INSIG-1,and sequence characterization was performed by biology information analyzing software.INSIG-1 gene expression patterns in ten tissues（subcutaneous fat,muscles,mammary gland,liver,kidney,heart,lung,spleen,rumen and small intestine）were conducted using real-time fluorescence quantitative PCR（RT-qPCR）.【Result】The experiment obtained a 1 014bp sequence（GenBank,accession number JQ665439）,which contained a coding sequence region of 831bp and 276 amino acids with molecular weight of 29.70ku and pI of 8.99.The genetic distance analysis showed that goat had the closest relationship with bovine（Bos taurus）.Nucleotides and amino acid sequences alignment showed both the nucleotides and amino acid of goat INSIG-1 had the highest similarity score（97%and 90%,respectively） with bovine in GenBank.Protein structure analysis of goat INSIG-1 protein showed that INSIG-1 contained five transmembranehelices and hydrophobicity analysis showed that the protein was hydrophilic.INSIG-1 expression patterns were examined by RT-qPCR,and the results showed that the liver had the highest expression level,followed by fat,and mammary gland while heart had the lowest expression level.【Conclusion】This study cloned originally INSIG-1 gene coding sequence region of Saanen Dairy Goat from Northwest AF University,and found the gene expression patterns.