茶树谷胱甘肽还原酶基因CsGRs的克隆与表达分析

摘要

The objectives of the present study were to clone the genes of glutathione reductase family (CsGRs) from tea plant (Camellia sinensis) and investigate their functions under different abiotic stresses.[Method] According to the sequences ofCsGR genes obtained from the transcriptome database of tea plant, specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) to clone the full-length sequences ofCsGRs. The bioinformatic characteristics of the clonedCsGRs were analyzed using online service. The expression profiles ofCsGRs in various tissues and in response to cold, drought, salt and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR). Spectrophotometer technique was employed to determine the content of glutathione (GSH) upon cold and drought stress.[Result]CsGR1 was isolated from tea plant using RT-PCR and had 1 827 bp in length containing a 1 482 bp open reading frame (ORF) encoded 493 amino acid residues. ForCsGR2isolating, 712 bp and 1 624 bp in length of 5′ and 3′ terminal sequences were amplified by RACE-PCR, respectively, after sequences assembling and verified by RT-PCR,CsGR2 with 2 282 bp in length containing a 1 698 bp ORF encoded polypeptide of 565 amino acids was obtained.CsGR1andCsGR2 were submitted to GenBank with accession number KF906411 and KF418080, respectively. The molecular weights ofCsGR1 andCsGR2 encoded protein were 53.9 and 61.0 kD, respectively, and both of them did not contain the signal peptide sites, indicating that they were not the secretory proteins. Subcellular localization prediction showed thatCsGR1 might localize in cytoplasm without chloroplast transit peptides (cTP), whereasCsGR2, containing a putative cTP of 71 amino acid residues at the N-terminal, was most likely to target to chloroplast. Comparison of sequences similarity with reported GRs showed thatCsGR1 had more than 80% similarity with cytosolic-GRs and less than 60% similarity with chloroplastic-GRs, whereasCsGR2 shared over 70% identity with other chloroplastic-GRs and about 50% similarity with cytosolic-GRs.CsGR1 andCsGR2 shared 63.4% and 49.9% sequence identity in nucleotide and amino acid, respectively, and had high similarity with each other in secondary structure of protein. Phylogenetic tree analysis showed that CsGR1 andCsGR2 could be clustered into cytosolic-GRs and chloroplastic-GRs, respectively, and they had the closest genetic relationship withVitis vinifera. The redox-active disulfide bridge, the glutathione-binding residues and the conserved arginine residues for NADPH binding were found in both of them. Hence,CsGR1encodes a putative cytosolic isoform, andCsGR2belongs to chloroplastic GR which dual-targeted to both chloroplasts and mitochondria. Transcript abundance of CsGR1 was higher in flowers and roots than that in leaves and stems. AndCsGR2 displayed the opposite expression pattern in tissues compared toCsGR1. Analysis of the expression patterns in response to abiotic stress revealed thatCsGR1 andCsGR2 were down-regulated by ABA treatement in leaves and the suppression level ofCsGR2 was higher than CsGR1. Furthermore, the expression ofCsGR1was repressed by cold (4℃) stress, whereasCsGR2 could be gradually induced with the extension of treatment time. Similarly, under salt stress, the expression ofCsGR1 was suppressed, andCsGR2 was up-regulated after 24 h treatment. Under PEG treatment, both ofCsGRs were up-regulated in leaves and down-regulated in roots. Analysis of the concentration of GSH showed that GSH content was gradually induced by cold and PEG treatment in leaves. Moreover, the GSH concentration could be returned to and kept at constant level after 48 h recovery in PEG treatment.[Conclusion] In this study, twoCsGRs were cloned from tea plant, and their expressions were regulated by ABA, cold (4℃), salt and PEG stress. The results demonstrated that GR might play a role in abiotic stresses tolerance of tea plant.%【目的】克隆谷胱甘肽还原酶基因CsGRs，研究CsGRs在茶树抵御不同逆境胁迫中的作用。【方法】在茶树转录组数据库中搜索茶树CsGRs，根据获得的基因片段，设计反转录PCR（RT-PCR）引物和RACE-PCR特异引物，从茶树中克隆 CsGRs的 cDNA 全长序列，并利用在线生物信息学软件对其进行分析。采用实时荧光定量 PCR （qRT-PCR）分析CsGRs在茶树不同组织间的表达差异及其在低温、干旱、高盐胁迫和ABA处理下的表达模式，利用分光光度计测定低温胁迫和干旱胁迫下叶片中还原型谷胱甘肽（GSH）的含量变化。【结果】RT-PCR克隆获得CsGR1的cDNA全长，其长度为1827 bp，包含1482 bp开放阅读框（ORF），编码493个氨基酸；RACE扩增获得712 bp和1624 bp的5′和3′末端序列，拼接并进行RT-PCR验证后得到CsGR2序列，全长2282 bp，包含1698 bp ORF，编码565个氨基酸；CsGR1和CsGR2的GenBank登录号分别为KF906411和KF418080。CsGR1和CsGR2编码的蛋白质分子量分别为53.9 kD和61.0 kD，无信号肽位点，均为非分泌性蛋白。亚细胞定位预测CsGR1主要定位在细胞质等亚细胞中，无叶绿体锚定信号肽位点；CsGR2中N-端的71个氨基酸残基具有叶绿体转运信号功能，主要定位在叶绿体上。序列相似性比较显示，CsGR1与其他植物中胞质GR的相似性均在80％以上，而与叶绿体GR的相似性低于60％；CsGR2与其他叶绿体GR的相似性70％以上，与胞质GR的相似性在50％左右。二者在核酸序列和氨基酸序列水平上分别有63.4％和49.9％的相似性，且蛋白质二级结构也具有较高的相似性。系统发育树显示，CsGR1与胞质GR聚为一类，而CsGR2与叶绿体GR聚在一起，且都与葡萄的亲缘关系最近。二者均含有氧化还原二硫键活性位点、谷胱甘肽结合位点以及 NADPH结合的 Arg保守位点等结构域。CsGR1为胞质GR，CsGR2为双向定位在叶绿体和线粒体上的叶绿体GR。CsGR1在花和根中表达量较高，在叶片和茎中表达量低；CsGR2在叶片和茎中的表达量比在根和花中的表达量高。在短时胁迫处理24 h过程中，成熟叶片在100μmol·L-1 ABA处理后，CsGR1和CsGR2的表达均被抑制，且CsGR2的抑制作用较显著；在4℃低温胁迫下， CsGR1的表达被抑制，而CsGR2的表达随处理时间延长逐渐被诱导；250 mmol·L-1 NaCl盐胁迫抑制CsGRs的表达，但胁迫24 h时CsGR2的表达被诱导。10%（w/v）PEG胁迫处理茶树8 h，叶片中的CsGRs均被诱导表达，且在复水48 h后CsGR1的表达被显著上调；根中CsGRs的表达在处理和复水48 h过程中均被抑制。随低温胁迫时间延长，茶树叶片中的GSH含量逐渐升高；干旱胁迫也能促进GSH在叶片中的积累，在复水48 h后又恢复到处理前的水平。【结论】克隆了2个茶树CsGRs，2个基因对4℃低温、NaCl盐、10％PEG和ABA处理均具有响应。推测CsGRs在茶树抵御逆境胁迫中起作用。