白来航鸡半乳糖凝集素-1基因克隆、生物信息学及组织表达特性分析

Cloning, Bioinformatics and Tissue Expression Analysis of Galectin-1 Gene in White Leghorn Chickens

【目的】克隆白来航鸡半乳糖凝集素-1(galectin-1,Gal-1)基因,对其编码蛋白进行生物信息学分析,并检测其在不同组织中的表达情况,为进一步阐明其抗病毒功能提供科学依据。【方法】以鸡脾脏cDNA为模板,通过PCR扩增鸡Gal-1基因完整CDS区序列,并进行相似性比对及系统进化树构建;运用生物信息学软件对其编码蛋白的理化性质、亲/疏水性、跨膜区、信号肽、修饰结构、保守结构域及高级结构进行预测。利用实时荧光定量PCR检测Gal-1基因在白来航鸡心脏、肝脏、脾脏、肺脏、肾脏、脑、腺胃、肌胃、十二指肠、空肠、盲肠、直肠、胸肌和腿肌组织中的表达情况。【结果】白来航鸡Gal-1基因CDS区序列长度为408 bp,编码135个氨基酸。相似性比对结果表明,白来航鸡Gal-1基因核苷酸序列与火鸡、绿头鸭和珍珠鸟的相似性分别为97.1%、88.4%和82.2%;系统进化树结果表明,白来航鸡与火鸡亲缘关系最近。Gal-1蛋白分子质量为15.06 ku,理论等电点为6.57,不稳定系数为36.05,脂肪系数为74.30,平均亲水指数为-0.259。Gal-1蛋白无信号肽,不存在跨膜区;存在2个明显的亲水区,其编码蛋白较稳定,为亲水性蛋白。二级结构预测显示,Gal-1蛋白以无规则卷曲(45.93%)和延伸链(41.48%)为主,三级结构预测结果与二级结构一致。实时荧光定量PCR结果显示,Gal-1基因mRNA在白来航鸡组织中广泛表达,在肺脏中表达量最高,在脑中表达最低。【结论】本研究成功克隆了白来航鸡Gal-1基因CDS区序列,Gal-1基因在白来航鸡心脏、肝脏等14种组织中广泛表达,结果可为鸡Gal-1蛋白功能的深入研究提供参考。

【Objective】 This study was aimed to clone the galectin-1(Gal-1) gene in chickens, analyze its encoded protein by bioinformatics techniques, and detect its expression in different tissues in White Leghorn chickens, in order to provide scientific basis for further elucidating its in antiviral effect.【Method】 PCR was used to amplify the complete CDS region of Gal-1 gene with the spleen cDNA as template, and the similarity alignment and phylogenetic tree construction were carried out.Bioinformatics softwares were used to predict the physicochemical properties, hydrophilicity/hydrophobicity, transmembrane domain, signal peptide, modified structure, conserved domains and advanced structures of Gal-1 protein.Real-time quantitative PCR was used to detect the expression of Gal-1 gene in heart, liver, spleen, lung, kidney, brain, glandular stomach, muscular stomach, duodenum, jejunum, cecum, rectum, pectorales and leg muscles tissnes.【Result】 The length of CDS region of chicken Gal-1 gene was 408 bp, encoding 135 amino acids.The similarity alignment results showed that the similarity of Gal-1 gene nucleotide sequence of White Leghorn chickens with Meleagris gallopavo,Anas platyrhynchos and Taeniopygia guttata were 97.1%,88.4% and 82.2%,respectively.The phylogenetic tree analysis showed that White Leghorn chickens had the closest relationship with Meleagris gallopavo.The relative molecular weight of Gal-1 protein was 15.06 ku, the isoelectric point was 6.57,the instability coefficient was 36.05 and the lipid coefficient was 74.30,and the average hydrophilic index was ―0.259.Gal-1 protein had no signal peptide and no transmembrane domain, while had two obvious hydrophilic domains, and the coding protein was stable and hydrophilic.The prediction of secondary structure showed that the Gal-1 protein was dominated by random coil(45.93%) and extended chain(41.48%),and the prediction of tertiary structure was consistent with that of secondary structure.Real-time quantitative PCR results showed that Gal-1 gene mRNA was widely expressed in tissues of White Leghorn chickens, with the highest expression level in the lung and the lowest in the brain.【Conclusion】 In this study, the CDS region sequence of Gal-1 gene was successfully cloned, and it was widely expressed in 14 tissues such as heart and liver of White Leghorn chickens, which could provide references for further study on the function of Gal-1 protein in chickens.