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中国沙漠  2018, Vol. 38 Issue (3): 584-591    DOI: 10.7522/j.issn.1000-694X.2017.00031
生物与土壤     
东方百合(Lilium)杂交品种索邦病程相关蛋白(PR1)基因的克隆与原核表达
王 乐1,2, 杨 柳1,2, 郭志鸿1, 张玉宝1, 王亚军1, 杨 果1, 谢忠奎1
1.中国科学院西北生态环境资源研究院 皋兰农业与生态综合研究站, 甘肃 兰州 730000
2.中国科学院大学, 北京 100049
Cloning and Prokaryotic Expression of Pathogenesis-related protein 1 Gene from Oriental Hybrid Lily Sorbonne
Wang Le1,2, Yang Liu1,2, Guo Zhihong1, Zhang Yubao1, Wang Yajun1, Yang Guo1, Xie Zhongkui1
1.Gaolan Station of the Agricultural and Ecological Experiment, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
2.University of Chinese Academy of Sciences, Beijing 100049, China
 全文: PDF 
摘要: 病程相关蛋白(PR)广泛参与植物对生物和非生物胁迫的应答。采用cDNA末端快速克隆技术,从东方百合(Lilium)杂交品种索邦中克隆了PR1基因,并命名为LhSorPR1LhSorPR1基因cDNA全长870 bp,5’非翻译区和3’非翻译区的长度分别为47 bp和274 bp。该基因的开放阅读框为549 bp,编码182个氨基酸。成熟的LhSorPR1蛋白(不含信号肽)分子量为18.19 kDa,等电点为5.69。采用同源建模方式构建的LhSorPR1蛋白三维结构与其模板番茄PR1蛋白p14a具有较高的相似性,说明百合PR1蛋白在结构和功能上与其他植物中的PR1蛋白具有较强的保守性。构建pCold II-LhSorPR1原核表达,经IPTG诱导后,目的蛋白在大肠杆菌中以包涵体形式成功表达。使用免疫印迹方法对表达的LhSorPR1重组蛋白进行了验证,并对带有His标签的重组蛋白成功进行了纯化。
关键词: 百合(Lilium) 抗病性 病程相关蛋白 同源建模 原核表达    
Abstract: Pathogenesis-related (PR) proteins are widely involved in plant defense against both biotic and abiotic stresses. In the present study, we characterized a PR1 protein from the oriental hyrid lily cultivar Sorbonne via rapid amplification of cDNA ends (RACE), and designated as LhSorPR1. The cDNA full-length of LhSorPR1 isolated was 870 bp, consisting a 5' untranslate region (UTR) of 47 bp and a 3' UTR of 274 bp. The predicted open reading frame (ORF) of LhSorPR1 was 549 bp, encoded a protein of 182 amino acids. The mature LhSorPR1 (devoid of a signal peptide) had a theoretical isoelectric point of 5.69 and a calculated molecular weight of 18.19 kDa. The three dimentional (3D) model of LhSorPR1 was bulit by homology modelling, and the bulit model showed high similarity to its template p14a. The similarity in 3D strucutures indicated the conservation of protein function of PR1 proteins in plants. The pCold II-LhSorPR1 recombinant vector was constructed to express LhSorPR1 in E.coli, and the His6-tagged recombinant protein was expressed as inclusion bodies in the insoluble fraction under IPTG induction. The His6-labeled recombinant LhSorPR1 protein was verified by immunoblot, and purified by chromatography.
Key words: lily    disease resistance    pathogenesis-related protein    homology modelling    prokaryotic expression
收稿日期: 2016-12-29 出版日期: 2018-06-14
ZTFLH:  Q943  
基金资助:

国家自然科学基金项目(31101190);

宁夏农业综合开发科技推广项目(NTKJ2015-05-01);

中国科学院“百人计划”项目(Y629721002);

中国科学院关键技术人才项目(2016-65);

中国博士后科学基金项目 (2015M580894);

2014年中国科学院"西部之光"重点项目

作者简介: 王乐(1986—),男,内蒙古阿拉善人,博士研究生,主要从事植物抗逆分子生物学研究。E-mail: lelenmg@163.com
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王 乐
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引用本文:

王 乐, 杨 柳, 郭志鸿, 张玉宝, 王亚军, 杨 果, 谢忠奎. 东方百合(Lilium)杂交品种索邦病程相关蛋白(PR1)基因的克隆与原核表达[J]. 中国沙漠, 2018, 38(3): 584-591.

Wang Le, Yang Liu, Guo Zhihong, Zhang Yubao, Wang Yajun, Yang Guo, Xie Zhongkui. Cloning and Prokaryotic Expression of Pathogenesis-related protein 1 Gene from Oriental Hybrid Lily Sorbonne. Journal of Desert Research, 2018, 38(3): 584-591.

链接本文:

http://www.desert.ac.cn/CN/10.7522/j.issn.1000-694X.2017.00031        http://www.desert.ac.cn/CN/Y2018/V38/I3/584

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