Abstract: 【Objective】 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 of CsGR 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 of CsGRs. The bioinformatic characteristics of the cloned CsGRs were analyzed using online service. The expression profiles of CsGRs 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. For CsGR2 isolating, 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. CsGR1 and CsGR2 were submitted to GenBank with accession number KF906411 and KF418080, respectively. The molecular weights of CsGR1 and CsGR2 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 that CsGR1 might localize in cytoplasm without chloroplast transit peptides (cTP), whereas CsGR2, 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 that CsGR1 had more than 80% similarity with cytosolic-GRs and less than 60% similarity with chloroplastic-GRs, whereas CsGR2 shared over 70% identity with other chloroplastic-GRs and about 50% similarity with cytosolic-GRs. CsGR1 and CsGR2 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 and CsGR2 could be clustered into cytosolic-GRs and chloroplastic-GRs, respectively, and they had the closest genetic relationship with Vitis 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, CsGR1 encodes a putative cytosolic isoform, and CsGR2 belongs 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. And CsGR2 displayed the opposite expression pattern in tissues compared to CsGR1. Analysis of the expression patterns in response to abiotic stress revealed that CsGR1 and CsGR2 were down-regulated by ABA treatement in leaves and the suppression level of CsGR2 was higher than CsGR1. Furthermore, the expression of CsGR1 was repressed by cold (4℃) stress, whereas CsGR2 could be gradually induced with the extension of treatment time. Similarly, under salt stress, the expression of CsGR1 was suppressed, and CsGR2 was up-regulated after 24 h treatment. Under PEG treatment, both of CsGRs 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, two CsGRs 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.

Key words: tea plant (Camellia sinensis) , glutathione reductase (GR) , abiotic stress , expression analysis