无量山乌骨鸡HMOX1基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.11.007

摘要/Abstract

摘要： 【目的】扩增无量山乌骨鸡血红素加氧酶1（heme oxygenase 1，HMOX1）基因CDS区并进行生物信息学分析，检测其组织表达特征，为后续该基因的功能研究奠定基础。【方法】以无量山乌骨鸡皮下脂肪组织cDNA为模板，PCR扩增HMOX1基因，连接PESI-T载体后进行测序分析，使用DNAMAN软件对所获序列进行翻译并与原鸡进行序列比对；使用BLAST、Mega 7.0、SOPMA、ProtParam和ExPASy等在线软件进行相似性比对、系统进化树构建、编码蛋白的理化性质及蛋白结构预测；利用STRING软件预测HMOX1互作蛋白；通过实时荧光定量PCR检测HMOX1基因在无量山乌骨鸡不同组织中的表达水平。【结果】无量山乌骨鸡HMOX1基因CDS区长为891 bp，编码296个氨基酸，与原鸡HMOX1基因（登录号：NM_205344.1）核苷酸序列相比存在3处突变，其中2处为同义突变，第217 bp处G>C为错义突变，导致第73位的天冬氨酸突变为甘氨酸。HMOX1基因与原鸡和日本鹌鹑的氨基酸序列相似性较高（99.6%和95.0%），与马的相似性最低（60.2%）。系统进化树分析发现，无量山乌骨鸡与原鸡和日本鹌鹑亲缘关系较近，与非禽类的亲缘关系较远。HMOX1蛋白具有不稳定性和亲水性，主要定位在细胞质中，不存在信号肽裂解位点，属于非分泌蛋白；存在1个跨膜螺旋结构，共有47个磷酸化修饰位点；二级结构以α-螺旋为主。HMOX1蛋白与HMOX2、BLVRA、HEPH、CP、COX10等10个蛋白可能存在相互作用。组织表达分析发现，HMOX1基因在无量山乌骨鸡各组织中均有表达，其中在肝脏中表达量最高，在心脏中表达量最低。【结论】无量山乌骨鸡HMOX1基因CDS区长为891 bp，编码296个氨基酸；预测其与10个蛋白存在相互作用；HMOX1基因在无量山乌骨鸡肝脏组织中高表达。研究结果为进一步探究HMOX1基因在鸡脂代谢中的作用及功能提供了理论依据。

关键词: 无量山乌骨鸡; HMOX1基因; 克隆; 生物信息学; 组织表达

Abstract: 【Objective】 The purpose of this study was to amplify the CDS region of heme oxygenase 1 (HMOX1) gene in Wuliangshan Sooty chickens and carry out the bioinformatics analysis, and detect the tissue expression characteristics of HMOX1 gene, so as to lay a foundation for the subsequent functional research of HMOX1 gene.【Method】 The HMOX1 gene was amplified by PCR using the adipose tissue cDNA in Wuliangshan Sooty chickens as the template, and the PESI-T vector was connected for sequencing analysis.The obtained sequence was translated by DNAMAN software, and the sequence was compared with that of Gallus gallus.BLAST, Mega 7.0, SOPMA, ProtParam and ExPASy were used to carry out the similarity alignment, phylogenetic tree construction and the prediction of physicochemical properties and protein structure.STRING software was used to predict HMOX1 interacting proteins.The expression of HMOX1 gene in different tissues of Wuliangshan Sooty chickens were detected by Real-time quantitative PCR.【Result】 The length of CDS region of HMOX1 gene in Wuliangshan Sooty chickens was 891 bp, encoding 296 amino acids.Three mutations were found in the nucleotide of HMOX1 gene with that of Gallus gallus (accession No.:NM_205344.1), two synonymous mutations, G>C at 217 bp was a missense mutation, resulting in aspartic acid mutation at 73 to glycine.The higher amino acid similarity of HMOX1 gene were 99.6% and 95.0% with Gallus gallus and Coturnix japonica, respectively, and the lowest was 60.2% with Equus caballus.Phylogenetic tree analysis showed that Wuliangshan Sooty chickens had the closer genetic relationship with Gallus gallus and Coturnix japonica, and was far away from non poultry. HMOX1 protein was unstable and hydrophilic, was mainly located in cytoplasm, and there was no signal peptide cleavage site, it was a non secreted protein.There were 1 transmembrane helix structure and 47 phosphorylation sites.The secondary structure of HMOX1 protein was mainly alpha helix.HMOX1 protein might interact with 10 proteins such as HMOX2, BLVRA, HEPH, CP and COX10.The tissue expression analysis showed that HMOX1 gene was expressed in all tissues of Wuliangshan Sooty chickens, with the highest expression in liver and the lowest expression in heart.【Conclusion】 The CDS length of HMOX1 gene in Wuliangshan Sooty chickens was 891 bp, encoding 296 amino acids, predicting it interacted with 10 proteins.HMOX1 gene was highly expressed in liver.The results provided a theoretical basis of informatics for further exploring the role of HMOX1 gene in chicken fat deposition.

Key words: Wuliangshan Sooty chickens; HMOX1 gene; cloning; bioinformatics; tissue expression

中图分类号:

S831.2

殴小嫚, 刘明欢, 吴芸, 郭爱伟, 李秋玉, 陈粉粉. 无量山乌骨鸡HMOX1基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(11): 4381-4391.

OU Xiaoman, LIU Minghuan, WU Yun, GUO Aiwei, LI Qiuyu, CHEN Fenfen. Cloning, Bioinformatics and Tissue Expression Analysis of HMOX1 Gene in Wuliangshan Sooty Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4381-4391.

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