| 猪TRIM3基因克隆、生物信息学及组织表达分析 |
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| 引用本文： | 张航,沙惠阳,李华玮,黄良宗,赵孟孟.猪TRIM3基因克隆、生物信息学及组织表达分析[J].中国畜牧兽医,2022,49(7):2474-2483. |
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| 作者姓名： | 张航 沙惠阳 李华玮 黄良宗 赵孟孟 |
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| 作者单位： | 1. 佛山科学技术学院生命科学与工程学院, 佛山 528231;2. 河南牧业经济学院食品与生物工程学院, 郑州 450046;3. 佛山科学技术学院附属教学动物医院, 佛山 528231 |
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| 基金项目： | 国家自然科学基金项目（31902279、31902284） |
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| 摘 要： | 【目的】克隆大白猪三基序结合蛋白3（tripartite motif-containing 3，TRIM3）基因，并对其进行生物信息学和组织表达分析。【方法】采用PCR技术扩增并克隆大白猪TRIM3基因CDS全长序列，连接pMD18-T载体并转化大肠杆菌DH5α感受态细胞，通过蓝白斑筛选阳性克隆，菌液PCR鉴定后测序，与不同物种TRIM3基因序列比对并构建系统进化树；应用多种在线软件对其编码蛋白进行生物信息学分析，并利用实时荧光定量PCR方法检测TRIM3基因在大白猪不同组织中的相对表达量。【结果】大白猪TRIM3基因CDS序列全长2 235 bp，编码744个氨基酸。相似性和遗传进化分析结果显示，大白猪与野猪的相似性最高，达99.7%，与鸭的相似性最低，为75.1%；大白猪TRIM3基因与野猪先聚为一类，与牛和山羊亲缘关系较近。生物信息学分析显示，大白猪TRIM3蛋白分子质量为80.58 ku，理论等电点（pI）为8.32，不稳定系数为40.85，为亲水性蛋白，但不是分泌蛋白，无糖基化位点，预测其有60个磷酸化位点，主要存在于细胞质内；在TRIM3蛋白二级结构中以无规则卷曲为主，占41.67%，三级结构模型预测结果与二级结构一致。组织表达分析表明，大白猪TRIM3基因在心脏、肝脏、脾脏、肺脏、肾脏、肌肉、气管、结肠中均有分布，肺脏中表达量最多且显著高于其他组织（P＜0.05）。【结论】本研究成功克隆大白猪TRIM3基因CDS全长序列，并进行了生物信息学和组织表达分析，为进一步研究大白猪TRIM3蛋白的免疫学功能提供理论依据，对探究大白猪TRIM3基因参与先天性免疫和抗病毒感染分子机制具有重要意义。
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| 关 键 词： | 大白猪 TRIM3基因 克隆 表达分析 |
| 收稿时间： | 2021-12-23 |
Cloning,Bioinformatics and Tissue Expression Analysis of TRIM3 Gene in Swine |
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| ZHANG Hang,SHA Huiyang,LI Huawei,HUANG Liangzong,ZHAO Mengmeng.Cloning,Bioinformatics and Tissue Expression Analysis of TRIM3 Gene in Swine[J].China Animal Husbandry & Veterinary Medicine,2022,49(7):2474-2483. |
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| Authors: | ZHANG Hang SHA Huiyang LI Huawei HUANG Liangzong ZHAO Mengmeng |
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| Institution: | 1. College of Life Science and Engineering, Foshan University, Foshan 528231, China;2. College of Food and Bioengineering, Henan University of Animal Husbandry and Economics, Zhengzhou 450046, China;3. Veterinary Teaching Hospital, Foshan University, Foshan 528231, China |
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| Abstract: | 【Objective】 Tripartite motif-containing 3 (TRIM3) gene of Large White pigs was cloned and analyzed by bioinformatics and tissue expression.【Method】 The full-length CDS sequence of TRIM3 gene in Large White pigs was amplified by PCR technology and cloned,pMD18-T vector was ligated and Escherichia coli DH5α competent cells were transformed,positive clones were screened by blue and white spots,sequenced after PCR identification of bacterial fluid,compared with TRIM3 gene sequences of different species and constructed phylogenetic tree,and bioinformatics analysis of its coding proteins was applied to a variety of online software.The relative expression of TRIM3 gene in different tissues of Large White pigs was detected by Real-time quantitative PCR.【Result】 The CDS sequence of TRIM3 gene in Large White pigs was 2 235 bp in total length and encoded 744 amino acids.Similarity and genetic evolution analysis results showed that the similarity between Large White pigs and Sus scrofa was the highest,up to 99.7%,and the similarity with Anas platyrhynchos was the lowest (75.1%).And the TRIM3 gene of Large White pigs was first clustered with Sus scrofa,and the relationship with Bos indicus and Capra hircus were closer.Bioinformatics analysis showed that the molecular mass of TRIM3 protein in Large White pigs was 80.58 ku,the theoretical isoelectric point (pI) was 8.32,and the instability coefficient was 40.85,which was a hydrophilic protein but not a secretory protein,and there were no glycosylation sites,which were predicted to have 60 phosphorylation sites,mainly in the cytoplasm;In the secondary structure of TRIM3 protein,random coil was the mainstay,accounting for 41.67%,and the prediction results of the tertiary structure model were consistent with the secondary structure.Tissue expression analysis showed that the TRIM3 gene in Large White pigs was distributed in heart,liver,spleen,lung,kidney,muscle,trachea and colon,and the expression in lung was the highest, and significantly higher than that in other tissues (P＜0.05).【Conclusion】 In this study,the full-length CDS sequence of TRIM3 gene in Large White pigs was successfully cloned and analyzed by bioinformatics and tissue expression,which provided a theoretical basis for the further study of the immunological function of TRIM3 protein in Large White pigs,and was of great significance to explore the molecular mechanism of TRIM3 gene in innate immunity and antiviral infection. |
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| Keywords: | Large White pigs TRIM3 gene cloning expression analysis |
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