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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(Gen Bank登录号:KF747758)。该基因的cDNA全长为1 977 bp,开放读码框为1 494bp,编码497个氨基酸,推测其蛋白相对分子量为53.7 k D,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 k D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。
Glutathione reductase (GR) is an important antioxidant enzyme in plants. In this study, we cloned the full-length cDNA sequence of GR gene by RACE technique and named BnGR1 (Gen Bank accession number: KF747758) based on the related fragments of the ramie (Boehmeria nivea (L.) Gaud.) Transcriptome. The full-length cDNA of this gene was 1 977 bp with an open reading frame of 1 494 bp encoding a protein of 497 amino acids with a predicted relative molecular mass of 53.7 kD and a theoretical isoelectric point of 5.68. Amino acid sequence analysis showed that this protein has the class I active site of pyridine nucleotide disulfide oxidoreductase, nicotinamide adenine dinucleotide phosphate (NADP) binding site, flavin adenine dinucleotide (FAD) binding site binding site, glutathione oxidized (GSSG) binding site and cytoplasmic GR specific domain, and the combination of the GR of theobroma cacao The protein (EOY05332) had the highest similarity of 88%. In addition, the prokaryotic expression vector pGEX-4T-BnGR1 was successfully constructed in this study, and about 53.7 kD protein was induced by isopropyl-β-d-thiogalactoside (IPTG) Consistent with the theoretical value. qRT-PCR analysis showed that the gene was expressed in roots, stems, shoot tips and leaves of ramie, and the highest expression was found in mature leaves and the lowest in stems. Meanwhile, the expression of CdCl2, exogenous ABA abscisic acid, ABA) and salicylic acid (SA). BnGR1 may be closely related to the ramie resistance mechanism. The results provide the basic information for exploring the molecular mechanism of tolerance of ramie to heavy metal Cd2 +.