绵羊IRS1基因CDS区克隆、序列分析及其组织表达研究

CDS Cloning,Sequence Analysis and Tissue Expression of IRS1 Gene in Sheep

【目的】克隆绵羊IRS1基因CDS区,并研究其序列特征和组织表达。【方法】选择健康、3岁、处于第4胎的泌乳高峰期(产后第3周)小尾寒羊和空怀期小尾寒羊母羊各3只作为研究对象,利用克隆测序技术获得绵羊IRS1基因的CDS序列,用生物信息学分析其编码的氨基酸序列特征,用RT-qPCR检测IRS1基因在肝脏、心脏、背最长肌、脾脏、肺脏、卵巢、肾脏和乳腺组织中的表达模式。【结果】绵羊IRS1基因的CDS全长为3708 bp,编码1235个氨基酸,蛋白分子量为130462.80,等电点为8.99,不稳定指数为73.55,预测该蛋白为不稳定、碱性亲水性蛋白。蛋白互作分析表明,绵羊IRS1可以与胰岛素样生长因子1受体(IGFR1)、磷酸肌醇-3-激酶调节亚基1(PIK3R1)、丝裂原活化蛋白激酶8(MAPK8)、丝裂原活化蛋白激酶10(MAPK10)和生长因子受体结合蛋白2(GRB2)相互作用,KEGG分析发现IRS1主要参与了PI3K/AKT和MAPK 2个信号通路。RT-qPCR分析表明,IRS1基因在小尾寒羊各组织中均表达,并且表现出明显的组织特异性,它在背最长肌中的表达量最高,其次是肝脏和心脏,在肺脏、肾脏、卵巢和乳腺组织中表达量较低,在脾脏中弱表达。同时,绵羊IRS1基因也表现出明显的时序表达性,在泌乳高峰期小尾寒羊中,IRS1基因在乳腺组织中的表达量是空怀期乳腺组织中表达量的2.77倍(P<0.05)。但在除乳腺和脾脏外的其他6个组织中,该基因在空怀期中的表达量均显著高于泌乳高峰期中的表达量(P<0.05)。【结论】克隆得到绵羊IRS1基因完整的CDS序列,它全长3708 bp,编码1235个氨基酸。IRS1在小尾寒羊各组织中广泛表达,并且表达出明显的组织特异性和时序特异性。

[Objective]This CDS study dealt with cloning of IRS1 gene in sheep and investigation into its sequence characteristics and tissue expression.[Method]Three healthy,3-year-old small-tailed han sheep ewes during the peak lactation(3 weeks after delivery)in the fourth fetus and another three ewes from the empty period were selected as the research objects.The CDS sequence of sheep IRS1 gene was obtained by cloning and sequencing,and the amino acid sequence encoded by IRS1 gene was analyzed by bioinformatics.The expression patterns of IRS1 gene in liver,heart,longissimus dorsi muscle,spleen,lung,ovary,kidney and breast were detected by RT-qPCR.[Results]The CDS of sheep IRS1 gene was 3708 bp,encoding 1235 amino acids.The molecular weight of protein was 130462.80,the isoelectric point was 8.99,and the instability index was 73.55.The protein was predicted to be unstable and alkaline hydrophilic.Protein interaction analysis showed that sheep IRS1 could interact with insulin-like growth factor receptor1(IGFR1),phosphate inositol 3-kinase regulatory 1(PIK3R1),mitogen-activated protein kinase 8(MAPK8),mitogen-activated protein kinase 10(MAPK10)and growth factor receptor-binding protein 2(GRB2).KEGG analysis showed that IRS1 was mainly involved in PI3K/AKT and MAPK signaling pathways.RT-qPCR analysis showed that IRS1 gene was expressed in all tissues of small-tailed han sheep and showed obvious tissue specificity.The expression of IRS1 gene was the highest in longissimus dorsi muscle,followed by that in liver and heart;low expression in lung,kidney,ovary and breast tissue;weak expression in spleen.At the same time,IRS1 gene showed obvious timing specificity in small-tailed han sheep.During the peak lactation period,the expression of IRS1 gene in breast tissue was 2.77 times of that during the empty period(P<0.05),but in 6 other tissues,the expression of this gene during the empty period was significantly higher than that during the peak lactation period(P<0.05)except fot that in breast and spleen.[Conclusion]In this study,the complete CDS sequence of sheep IRS1 gene,was successfully cloned for the first time.It was 3708 bp in length and encoded 1235 amino acids.IRS1 was widely expressed in all tissues of small-tailed han sheep,and it showed obvious tissue specificity and timing specificity.