猪蛋白质二硫化物异构酶A4基因克隆及密码子偏好性分析

doi:10.16431/j.cnki.1671-7236.2019.04.014

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (4): 1074-1085.doi: 10.16431/j.cnki.1671-7236.2019.04.014

猪蛋白质二硫化物异构酶A4基因克隆及密码子偏好性分析

张小丹¹, 牛熙^1,2, 许瑶^1,2, 阮亦麒³, 李大鹏¹, 冉雪琴³, 王嘉福^1,2,4, 吴娟¹, 杨镜¹, 田兰¹, 龙艳霞¹

1. 贵州大学农业生物工程研究院生命科学学院, 贵州省农业生物工程重点实验室, 山地植物资源保护与种质创新省部共建教育部重点实验室, 贵阳 550025;
2. 贵州省山地生态与农业生物工程2011协同创新中心, 贵阳 550025;
3. 贵州大学动物科学学院, 贵阳 550025;
4. 铜仁学院, 铜仁 554300

收稿日期:2018-08-29 出版日期:2019-04-20 发布日期:2019-04-22
通讯作者: 牛熙 E-mail:haifuhong@163.com
作者简介:张小丹(1996-),女,贵州遵义人,本科生,研究方向:动物生物化学与分子生物学,E-mail:1329931028@qq.com
基金资助:
贵州科技厅与贵州大学联合基金项目（黔科合LH字[2015]7673）；国家高技术研究发展计划（863计划）课题（2013-AA102503）；国家自然科学基金（31672390）；贵州省"百"层次创新型人才项目（黔科合人才2016-4012号）；贵州省农业攻关项目（黔科合支撑[2017]2585、黔科合支撑[2017]2587）；大学生创新创业训练计划项目（贵大（省）创字2017（025））

Cloning and Codon-preference Analysis of Protein Disulfide Isomerase A4 Gene in Pig

ZHANG Xiaodan¹, NIU Xi^1,2, XU Yao^1,2, RUAN Yiqi³, LI Dapeng¹, RAN Xueqin³, WANG Jiafu^1,2,4, WU Juan¹, YANG Jing¹, TIAN Lan¹, LONG Yanxia¹

1. The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education), Guizhou Key Lab of Agro-Bioengineering, Institute of Agro-Bioengineering, College of Life Sciences, Guizhou University, Guiyang 550025, China;
2. The 2011 Collaboration Innovation Center for Mountain Ecology and Agro-Bioengineering in Guizhou Province, Guiyang 550025, China;
3. College of Animal Sciences, Guizhou University, Guiyang 550025, China;
4. Tongren University, Tongren 554300, China

Received:2018-08-29 Online:2019-04-20 Published:2019-04-22

摘要/Abstract

摘要：

试验旨在研究猪蛋白质二硫化物异构酶（protein disulfide isomerase A4，PDIA4）基因的密码子使用特性及其与其他物种密码子使用的差异。试验以香猪子宫组织为试验材料，采用RT-PCR方法扩增获得猪PDIA4基因，参考从NCBI数据库中下载的18个不同物种PDIA4基因序列，对猪PDIA4基因的密码子使用特性及其与其他物种密码子的使用差异和进化关系进行分析，同时对猪PDIA4基因与4个模式生物密码子使用偏性进行比较。结果表明，猪PDIA4基因编码区长1 941 bp，共编码646个氨基酸。与NCBI数据库公布的野猪PDIA4基因（GenBank登录号：NM_001267834.1）的相似性达99%。猪PDIA4基因偏好使用G/C结尾的密码子，27种密码子为猪PDIA4基因的偏好密码子（RSCU>1），其中使用频率最高的前3个密码子分别为CTG（RSCU=3.69）、GCC（RSCU=2.31）和CGC（RSCU=2.29）。比较19个物种PDIA4基因发现，不同物种间PDIA4基因的有效密码子数（ENc）、密码子适应指数（CAI）、GC含量以及T、C、A、G、G+C在密码子第3位含量（T3s、C3s、A3s、G3s、GC3s）均存在差异，且密码子偏好以G/C结尾；聚类分析发现，基于CDS序列的系统进化树更符合19个物种的真实分类系统。在密码子使用频率上，猪PDIA4基因与小鼠基因组间密码子使用偏好程度更为接近，故小鼠更适合作为猪PDIA4基因的遗传转化和异源表达的受体系统。

关键词: 猪; PDIA4基因; 克隆; 密码子偏好性

Abstract:

The objective of the study was to explore the codon usage feature of protein disulfide isomerase A4 (PDIA4) gene in pig,the codon usage differences and evolutionary relationship between pig and other reference species.In this research,the coding region of the PDIA4 gene was amplified by RT-PCR using the uterus of pig as experimental material.The PDIA4 gene sequences of 18 species were downloaded as conferences from NCBI database.At the same time,the differences of codon usage bias between pig and four different kinds of model organisms were compared.The results showed that the complete coding region of PDIA4 gene of pig was 1 941 bp in length,which coded 646 amino acid residues.The results of molecular identification showed that the identity between PDIA4 gene cloned in this study with the known sequences of Sus scrofa in NCBI database (GenBank accession No.:NM_001267834.1) was 99%.The PDIA4 in pig preferred to use G-or C-ending codons and had 27 preferred codons (RSCU>1).Moreover,the three codons with the highest frequency were CTG (RSCU=3.69),GCC (RSCU=2.31) and CGC (RSCU=2.29),respectively.Comparing the codon usage feature of the PDIA4 gene among 19 species,there were significant differences in the related data and most of them tended to use G/C-ending codons.These related data included the effective number of codons (ENc),codon adaptation index (CAI),GC contents,contents of T,C,A,G and GC on the 3rd site (T3s,C3s,A3s,G3s and GC3s).Although there were differences between cluster analysis based on the relative synonymous codon usage and the results of phylogeny derived from coding region,and the molecular phylogenetic tree rooted in the PDIA4 CDS region was more consistent with the true classification system of the 19 species.The differences in codon usage frequency between pig PDIA4 gene and genome of Mus musculus were less than those in other three kinds model organisms' genome.Mus musculus might be the best receptors for genetic transformation and heterologous expression of PDIA4 gene in pig.

Key words: pig; PDIA4 gene; cloning; condon usage bias

中图分类号:

Q755

张小丹, 牛熙, 许瑶, 阮亦麒, 李大鹏, 冉雪琴, 王嘉福, 吴娟, 杨镜, 田兰, 龙艳霞. 猪蛋白质二硫化物异构酶A4基因克隆及密码子偏好性分析[J]. 《中国畜牧兽医》, 2019, 46(4): 1074-1085.

ZHANG Xiaodan, NIU Xi, XU Yao, RUAN Yiqi, LI Dapeng, RAN Xueqin, WANG Jiafu, WU Juan, YANG Jing, TIAN Lan, LONG Yanxia. Cloning and Codon-preference Analysis of Protein Disulfide Isomerase A4 Gene in Pig[J]. , 2019, 46(4): 1074-1085.

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猪蛋白质二硫化物异构酶A4基因克隆及密码子偏好性分析

Cloning and Codon-preference Analysis of Protein Disulfide Isomerase A4 Gene in Pig

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