Identification And Characterization Of ZjCSD1 Gene And Its Copper Chaperone ZjCCS Gene In Zoysia Japonica

The copper/zinc-superoxide dismutase(CSD)is a key enzyme involved in plant responding to abiotic stresses.The content and activity of CSD in plants is closely related to stress tolerance.The CSD genes have been cloned and identified in a variety of plants,but there are few reports about CSD genes in Zoysia japonica.In this study,ZjCSD1 gene was isolated from the transcriptome database of Zoysia Japonica by homologous cloning.It encoded a protein of 152 amino acid residues.Bioinformatics analysis revealed that the protein encoded by ZjCSD1 gene was stable,hydrophobic,acidic and fat soluble.With typical Cu2+and Zn2+binding sites,ZjCSD1 belonged to the plant SOD super family.Homology analysis with the deduced amino residues indicated that ZjCSD1 had a closer relationship with CSD from Setaria italica and Zea mays.Subcellular localization showed that the protein was located in the cytoplasm.The expression profiles of ZjCSD1 in different tissues and under different stress treatments were also investigated,qRT-PCR experiments showed that ZjCSD1 was expressed in root,stem and leaf,among which leaf was the highest.Also,ZjCSDl mRNA levels were up-regulated by salt,drought and Cd2+stress and down-regulated by Pb2+stress.These results suggested that ZjCSD1 responded to drought,salt and heavy metal stress tolerance of Zoysia japonica.Besides,ZjCSD1 was overexpressed in rice by Agrobacterium-mediated transformation,and 102 transgenic rice lines were successfully obtained and identified.The phenotype of transgenic progeny is to be explored.The activation of CSD is mainly dependent on its copper chaperone.The copper chaperone for superoxide dismutase(CCS)is an intracellular copper transporter that delivers copper ions to CSD by protein-protein interactions,thereby activating CSD.In this paper,based on the previous studies of our laboratory,75 bases before the ORF of ZjCCS and 3 types of 3'-UTR with different length were obtained by RACE.It was also found that ZjCCS could be transcribed from two different transcription initiation sites.Subcellular localization analysis showed that full length ZjCCS protein was located in the chloroplast,while the ZjCCS protein without transit peptide was located in the cytoplasm.The expression proflile of ZjCCS in different tissues of Zoysia japonica was investigated by q-PCR,which shows that ZjCCS was expressed in root,stem and leaf with different levels.Among them,the expression level in leaf is the highest,while the level in root is the lowest.Besides,ZjCCS was overexpressed in rice by Agrobacterium-mediated transformation,and 30 transgenic rice lines were successfully obtained and identified.The phenotype of transgenic progeny is to be explored.Furthermore,it was proved that ZjCCS without the chloroplast transit peptide could interact with the ZjCSD1 protein in the cytoplasm,and the 160?286th amino acids of ZjCCS are essential in the process of interaction.In conclusion,the ZjCSDI and ZjCCS genes from Zoysia japonica were isolated and studied in this paper.It's helpful to further understand the role of CSD and CCS genes in stress tolerance of plants and also provide theory evidence for the molecular mechanism of CCS activating CSD in Zoysia japonica.