赤水乌骨鸡PITX1基因克隆、生物信息学及组织表达分析

doi:10.16431/j.cnki.1671-7236.2023.10.003

摘要/Abstract

摘要： 【目的】克隆赤水乌骨鸡成对样同源结构域转录因子1(paired-like homeodomain transcription factor 1,PITX1)基因并进行生物信息学分析,检测PITX1基因在赤水乌骨鸡不同组织中的表达水平,为探究PITX1基因在鸡肌肉生长发育中的生物学功能提供材料。【方法】以赤水乌骨鸡胸肌组织cDNA为模板,克隆PITX1基因CDS区序列并测序,利用在线软件对所获序列进行比对及生物信息学分析,利用实时荧光定量PCR检测PITX1基因在赤水乌骨鸡心脏、肝脏、脾脏、肾脏、肺脏、胸肌和腿肌组织中的表达量。【结果】赤水乌骨鸡PITX1基因CDS区长909 bp,存在5处同义突变,编码302个氨基酸。赤水乌骨鸡PITX1基因氨基酸序列与原鸡、绿头鸭、日本鹌鹑、兔、小鼠的相似性分别为100%、99.7%、99.0%、85.0%和84.4%,与人、牛和野猪的相似性均为85.7%。系统进化树分析表明,赤水乌骨鸡和原鸡的亲缘关系最近,与人的亲缘关系最远。赤水乌骨鸡PITX1蛋白属于亲水性蛋白,无跨膜结构和信号肽,有47个潜在磷酸化位点、1个N-糖基化修饰位点和15个O-糖基化修饰位点,主要分布在细胞核中。PITX1蛋白二级结构由无规则卷曲、α-螺旋、延伸链和β-转角组成,三级结构预测结果与其一致。蛋白互作分析结果显示,PITX1蛋白可能与BARX1、NKX2-8、POU1F1、EGR1、NR5A1、CTNNB1、TBX5和TBX4蛋白存在相互作用。实时荧光定量PCR结果显示,PITX1基因在赤水乌骨鸡各组织中均有表达,其中在肺脏中的表达量最高,其次是肝脏和腿肌,在胸肌中的表达量最低。【结论】赤水乌骨鸡PITX1基因CDS区长909 bp,编码302个氨基酸,为亲水性蛋白,主要分布在细胞核中。PITX1基因在赤水乌骨鸡各组织中均有表达,且在肺脏中表达量最高。试验结果为进一步研究鸡PITX1基因功能提供参考。

关键词: 赤水乌骨鸡; PITX1基因; 生物信息学; 表达

Abstract: 【Objective】 The purpose of this study was to clone the paired-like homeodomain transcription factor 1 (PITX1) gene in Chishui Black bone chickens and carry out bioinformatics analysis,and detect the expression of PITX1 gene in different tissues of Chishui Black bone chickens,so as to provide the materials for biological function of PITX1 gene in muscle growth and development of chickens.【Method】 The sequence of PITX1 gene CDS in Chishui Black bone chickens was cloned and sequenced using breast muscle cDNA as template.The alignment and bioinformatics analysis were carried out by online softwares.The expressions of PITX1 gene in heart,liver,spleen,kidney,lung,breast and leg muscles in Chishui Black bone chickens were detected by Real-time quantitative PCR.【Result】 The full length sequence of PITX1 gene CDS in Chishui Black bone chickens was 909 bp,which had 5 synonymous mutations,and encoding 302 amino acids.The amino acid sequence similarity of PITX1 gene in Chishui Black bone chickens were 100%,99.7%,99.0%,85.0% and 84.4% with Gallus gallus,Anas platyrhynchos,Coturnix japonica, Oryctolagus cuniculus and Mus musculus,respectively,and the sequence similarity with Homo sapiens,Bos taurus and Sus scrofa all were 85.7%.The results of phylogenetic tree analysis showed that Chishui Black bone chickens was the closest to Gallus gallus and the furthest to Homo sapiens.PITX1 protein of Chishui Black bone chickens was a hydrophilic protein,which had 47 phosphorylation sites,1 N-glycosylation modification site and 15 O-glycosylation modification sites,without transmembrane domain and signal peptide,and mainly distributed in nucleus.The secondary structure was composed of random coil,alpha helix,extended chain and beta turn,the tertiary structure prediction was consistent with it.Protein interaction prediction analysis indicated that PITX1 might be interacted with BARX1,NKX2-8,POU1F1,EGR1,NR5A1,CTNNB1,TBX5 and TBX4.Real-time quantitative PCR results revealed that PITX1 gene was expressed in different tissues of Chishui Black bone chickens,and the highest expression was found in lung,followed were liver and leg muscle,but that was the lowest in breast muscle.【Conclusion】 The sequence of PITX1 gene CDS in Chishui Black bone chickens was 909 bp,and encoding 302 amino acids,that was a hydrophilic protein,and mainly distributed in nucleus.PITX1 gene was expressed in all tissues of Chishui Black bone chickens,which was the highest in lung.The results provided a reference for further study on the function of PITX1 gene in chickens.

Key words: Chishui Black bone chickens; PITX1 gene; bioinformatics; expression

中图分类号:

吴芸, 欧正淼, 李秋玉, 陈粉粉, 闫吉美. 赤水乌骨鸡PITX1基因克隆、生物信息学及组织表达分析[J]. 中国畜牧兽医, 2023, 50(10): 3896-3905.

WU Yun, OU Zhengmiao, LI Qiuyu, CHEN Fenfen, YAN Jimei. Cloning,Bioinformatics and Tissue Expression Analysis of PITX1 Gene in Chishui Black Bone Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 3896-3905.

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