草原红牛肝配蛋白A5基因克隆及其在不同组织中的表达研究

doi:10.16431/j.cnki.1671-7236.2021.06.009

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (6): 1967-1975.doi: 10.16431/j.cnki.1671-7236.2021.06.009

草原红牛肝配蛋白A5基因克隆及其在不同组织中的表达研究

薛佳佳^1,2, 秦立红², 曹阳², 高一², 刘理想², 李旭², 肖成², 王晶², 张国梁^2,3,4,5

1. 吉林农业大学动物科学技术学院, 长春 130000;
2. 吉林省农业科学院, 公主岭 130033;
3. 吉林坤成牧业科技发展有限公司, 公主岭 136100;
4. 农业农村部肉牛遗传育种重点实验室, 公主岭 136100;
5. 吉林省肉牛繁育及养殖技术科技创新中心, 公主岭 136100

收稿日期:2020-09-11 出版日期:2021-06-20 发布日期:2021-06-18
通讯作者: 张国梁 E-mail:zgl7777777@163.com
作者简介:薛佳佳(1994-),女,陕西榆林人,硕士生,研究方向:动物遗传育种与繁殖,E-mail:2439458062@qq.com
基金资助:
吉林省畜牧局肉用草原红牛培育（C02294007）；国家重点研发计划（2018YFD0501802）；国家肉牛牦牛产业技术体系（CARS-37）；吉林省重点科技研发项目（20180201039NY）；吉林省科技攻关计划（20160204006NY）；吉林省创新平台"优质草原红牛高效选育技术研究与应用"（20200602052ZP）；吉林省农业科技创新工程（C92072027）；吉林省农业科学院创新工程（CXGC2019DC005）

Cloning of Ephrin A5 Gene and Its Expressing in Different Tissues in Red Steppe

XUE Jiajia^1,2, QIN Lihong², CAO Yang², GAO Yi², LIU Lixiang², LI Xu², XIAO Cheng², WANG Jing², ZHANG Guoliang^2,3,4,5

1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130000, China;
2. Jilin Academy of Agricultural Sciences, Gongzhuling 130033, China;
3. Jilin Kuncheng Animal Husbandry Technology Development Co., Ltd, Gongzhuling 136100, China;
4. Key Laboratory of Beef Cattle Genetics and Breeding, Ministry of Agriculture and Rural Agriculture, Gongzhuling 136100, China;
5. Jilin Beef Cattle Breeding and Breeding Technology Innovation Center, Gongzhuling 136100, China

Received:2020-09-11 Online:2021-06-20 Published:2021-06-18

摘要/Abstract

摘要： 研究旨在克隆中国草原红牛肝配蛋白A5（ephrin A5，EFNA5）基因，并对其进行生物信息学分析，检测EFNA5基因在中国草原红牛不同组织中表达的差异。以中国草原红牛为研究对象，根据GenBank公布的牛EFNA5基因序列（登录号：NM_001076432.1）设计引物，PCR扩增获得EFNA5基因的完整编码区（CDS）序列后进行测序验证，利用分析软件进行序列相似性比对并构建系统进化树；分析对应的氨基酸序列蛋白理化特性并预测蛋白亚细胞定位、蛋白亲/疏水性和磷酸化位点；预测蛋白二级结构并构建蛋白三级结构模型；以实时荧光定量PCR法检测EFNA5基因在中国草原红牛各组织中的相对表达量。结果显示：中国草原红牛EFNA5基因与普通牛和美洲野牛相似性最高，分别为100%和99.8%，与羊、人的相似性分别为95.6%、97.2%，与海豚、鸡、马、猕猴、小鼠和猪的相似性较低，分别为83.6%、85.8%、84.5%、84.9%、84.1%、82.8%；EFNA5基因CDS大小为687 bp，编码228个氨基酸，EFNA5基因编码蛋白的分子式为C₁₁₈₃H₁₇₉₁N₃₁₅O₃₃₉S₁₃，总原子数为3 641，分子质量为26.266 ku，理论等电点为5.97，不稳定系数为49.66，总平均亲水性为-0.313。磷酸化位点预测分析发现，EFNA5蛋白共有27个磷酸化位点；亚细胞定位结果表明，EFNA5蛋白主要位于细胞质（26.1%）、分泌系统囊泡（27.1%）、细胞核（13.0%）、线粒体（13.0%）、质膜（8.7%）、内质网（4.3%）、高尔基体（4.3%）、细胞骨架（4.3%）及液泡（4.3%）中；二级结构主要由α-螺旋、β-转角、β-折叠和无规则卷曲组成，分别占18.9%、30.2%、26.4%和24.5%，与三级结构预测结果相符；EFNA5基因在中国草原红牛不同组织的相对表达量差异较大，在肾脏和胃组织中表达量较高，在肺脏组织中表达量最少。本研究结果可为进一步筛选草原红牛肉质候选基因提供参考。

关键词: 中国草原红牛; EFNA5基因; 克隆; 生物信息学分析; 表达差异

Abstract: The purpose of this study was to explore the gene function of ephrin A5(EFNA5) in Chinese Red steppe,and to detect the difference of EFNA5 gene expression in different tissues of Chinese Red steppe by bioinformatics analyzed.Primers were designed according to the predicted sequence of EFNA5 gene in GenBank (accession No.:NM_001076432.1).Verification of complete coding sequence of EFNA5 gene was obtained by PCR and sequencing.Using analysis software to carry out sequence similarity alignment and construct phylogenetic tree.The corresponding amino acid sequence was obtained.The physical and chemical property and subcellular structure was analysed by online prediction software.Protein hydrophilicity,hydrophobicity and phosphorylation sites were analysed.Protein secondary structure was predicted and protein tertiary structure model was constructed.EFNA5 gene expression in tissues of Chinese Red steppe was detected by Real-time quantitative PCR.The results showed that the EFNA5 gene of Chinese Red steppe had the highest similartiy with common cattle and American bison (100% and 99.8%),and with sheep and human were 95.6% and 97.2% respectively,and lower similartiy with dolphins,chickens,horses,rhesus monkeys,mice and pigs (83.6%,85.8%,84.5%,84.9%,84.1% and 82.8%,respectively).The CDS of EFNA5 gene was 687 bp,encoding 228 amino acids.The molecular formula of the protein was C₁₁₈₃H₁₇₉₁N₃₁₅O₃₃₉S₁₃,the molecular weight was 26.266 ku,the theoretical isoelectric point was 5.97.The protein instability index was 49.66,and the total average hydrophilicity was －0.313. Phosphorylation sites prediction analysis showed that there were 27 phosphorylation sites in EFNA5 protein.The results of subcellular localization showed that EFNA5 protein was mainly located in cytoplasm (26.1%),secretory system vesicles (27.1%),nucleus (13.0%),mitochondria (13.0%),plasma membrane (8.7%),endoplasmic reticulum (4.3%),golgi apparatus (4.3%),cytoskeleton (4.3%) and vacuole (4.3%).The main forms of secondary structure were α-helix (18.9%),β-turn (30.2%),β-fold (26.4%) and irregular crimp (24.5%),which were consistent with the prediction of tertiary structure.The expression of EFNA5 gene was the highest in kidney and stomach,and the least in lung.There was significant difference in different tissues of Chinese Red steppe (P<0.05).This study could provide reference for further investigation of the effects of EFNA5 gene on lipid metabolism in livestock and the screening of quality candidate genes in Red steppe.

Key words: Chinese Red steppe; EFNA5 gene; cloning; bioinformatics analysis; expression difference

中图分类号:

薛佳佳, 秦立红, 曹阳, 高一, 刘理想, 李旭, 肖成, 王晶, 张国梁. 草原红牛肝配蛋白A5基因克隆及其在不同组织中的表达研究[J]. 中国畜牧兽医, 2021, 48(6): 1967-1975.

XUE Jiajia, QIN Lihong, CAO Yang, GAO Yi, LIU Lixiang, LI Xu, XIAO Cheng, WANG Jing, ZHANG Guoliang. Cloning of Ephrin A5 Gene and Its Expressing in Different Tissues in Red Steppe[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1967-1975.

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草原红牛肝配蛋白A5基因克隆及其在不同组织中的表达研究

Cloning of Ephrin A5 Gene and Its Expressing in Different Tissues in Red Steppe

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