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2025年4月3日 星期四
  2015, Vol. 23 Issue (10): 1318-1326    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
苎麻谷胱甘肽还原酶基因(BnGR1)的克隆和表达分析
朱守晶1,余伟林2,石朝燕2,揭雨成2
1. 湖南农业大学
2. 湖南农业大学 苎麻研究所
Cloning and Expression Analysis of Glutathione Reductase Gene (BnGR1) from Ramie (Boehmeria nivea L.)
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摘要 谷胱甘肽还原酶(glutathione reductase, GR)是植物体内一种重要的抗氧化酶类。本研究根据苎麻(Boehmeria nivea (L.) Gaud.)转录组中的相关片段,采用RACE技术克隆到了GR基因的全长cDNA序列,命名为BnGR1(GenBank登录号: KF747758)。该基因的cDNA全长为1 977 bp,开放读码框为1 494 bp,编码497个氨基酸,推测其蛋白相对分子量为53.7 kD,理论等电点为5.68。氨基酸序列分析表明,该蛋白具有吡啶核苷酸-二硫化物氧化还原酶classⅠ活性位点、烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADP)结合位点、黄素腺嘌呤二核苷酸(flavin adenine dinucletide, FAD)结合位点结合位点、氧化型谷胱甘肽(L-glutathione oxidized, GSSG)结合位点和胞质GR特殊结构域,与可可(Theobroma cacao)的GR蛋白(EOY05332)相似性最高,达到88%。此外,本研究成功构建了原核表达载体pGEX-4T-BnGR1,经异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside, IPTG)诱导获得了53.7 kD左右蛋白,与理论值一致。qRT-PCR表达分析表明,该基因在苎麻的根、茎、茎尖和叶片中均有表达,其中在成熟叶中表达量最高,茎中表达量最低;同时,受CdCl2、外源脱落酸(abscisic acid, ABA)和水杨酸(salicylic acid, SA)的诱导上调表达。BnGR1可能与苎麻抗逆机制密切相关,研究结果为探寻苎麻对重金属Cd2+的耐受分子机理提供基础资料。
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朱守晶
余伟林
石朝燕
揭雨成
关键词 苎麻谷胱甘肽还原酶(GR)原核表达胁迫处理荧光定量PCR    
Abstract:Glutathione reductase (GR) is one of the key enzymes in the plant antioxidant system. In this study, a full-length cDNA sequence designated as BnGR1 was cloned by RACE methods based on the relative unigene sequence in ramie (Boehmeria nivea (L.) Gaud.) transcriptome sequencing. The full-length cDNA sequence (GenBank accession: KF747758) of BnGR1 was 1 977, and the ORF was 1 494 bp, which encoded 497 amino acids with predicted pI of 5.68 and molecule weight of 53.7 kD, respectively. BnGR1 contained a pyridine nucleotide-disulphide oxidoreductases classⅠ active site, a nicotinamide adenine dinucleotide phosphate (NADP) binding site, a flavin adenine dinucletide (FAD) binding site, two L-glutathione oxidized (GSSG) binding sites and a special structure domain of cytosolic GR, respectively. Homology comparison analysis showed that the deduced BnGR1 amino acid sequence shared a 88% homology with GR gene (EOY05332) in cocoa (Theobroma cacao). Prokaryotic expression vector pGEX-4T-BnGR1 was established and transformed into Escherichia coli BL21 after isopropyl-β-d-thiogalactoside (IPTG) induction, which showed a successful gene expression. The expression pattern analysis carried out by quantitative Real-time PCR indicated that BnGR1 expressed in root, stem, stem apex and leaves, with the highest expression level in mature leaves and the lowest in stem. The BnGR1 gene was up-regulated by CdCl2, abscisic acid (ABA) and salicylic acid (SA) treatments. The results indicated that BnGR1 gene might play an important role in response to abiotic stresses of ramie, which provide the basic data for exploring the ramie resistance molecular mechanism of heavy metals Cd2+.
Key wordsRamie    Glutathione reductase (GR)    Prokaryotic expression    Abiotic stress    Quantitative Real-time PCR
收稿日期: 2014-12-01      出版日期: 2015-07-24
基金资助:国家自然科学基金项目;湖南省科技厅重点项目;湖南省科技厅重点项目;湖南省科技厅重点项目;国家科技支撑计划
通讯作者: 揭雨成     E-mail: ibfcjyc@vip.sina.com
引用本文:   
朱守晶 余伟林 石朝燕 揭雨成. 苎麻谷胱甘肽还原酶基因(BnGR1)的克隆和表达分析[J]. , 2015, 23(10): 1318-1326.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I10/1318
 
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