Isolation And Charaterization Of Genes Responding To Salt Stress From Zoysia Japonica

Soil salinization is a growing problem for plant growth in China, so it’s urgent to improve plant drought-resistant and salt-tolerant ability to adapt to this situation. Zoysia japonica is one of the most popular warm-season-type turfgrass and is widely used for its salinity tolerance. In this study, four genes responding to salt stress were cloned based on the Zoysia transcriptome database under the salt stress condition, three(ZjERF10、ZjRAP2-11、ZjERF7) of them were cloned by rapid amplification of cDNA ends (RACE) method and the other one (ZjCCS) was cloned by homologous cloning We characterized the four genes, respectively, hoping to provide certain theoretical basis and applicable genetic resourses in future breeding strateges.The main results are listed as follows:The cDNA structure of ZjERF10 is composed of one open-reading frame(ORF) which encodes 145 amino acid residues,117-bp-length 5’UTR and 215-bp-length 3’UTR. Subcellular localization showed that the genes encodes a non-nuclear-localized protein. Bioinformatics analysis showed that the protein encoded by ZjERF10 is an unstable, hydrophobic and acidic protein,which contains transmembrane helices but not signal peptide. qRT-PCR results showed that ZjERF10 was induced by abiotic stress and its expression pattern in leaf was different from in root under salt stress. Compared with wild type, the ZjERF10 overexpression lines were much less fertilized which resulted a significant reduction in seeds setting rate. ZjERF10 OE lines had an increase in seeds germination rate,and were shorter and more tolerant to salinity than wild type during vegetative stage. Therefore, we speculated that ZjERF10 gene might be a negative regulator in plant growth and development but a positive regulator in plant responding to abiotic stress.The cDNA structure of ZjRAP2-11 is composed of one open-readiing frame(ORF) which encodes 305 amino acid residues,92-bp-length 5’UTR and 262-bp-length 3’UTR.The cDNA structure of ZjERF7 is composed of one open-readiing frame(ORF) which encodes 86 amino acid residues, 157-bp-length 5’UTR and 159-bp-length 3’UTR.Subcellular localization showed that ZjRAP2-11 encodes a nuclear-localized protein and ZjERF7 encodes a protein which located in both nuclear and cytoplasm. Bioinformatics analysis showed that the proteins encoded by ZjRAP2-11 and ZjERF7 are both unstable, hydrophobic and acidic, however,ZjRAP2-11 has no transmembrane helices but ZjERF7 has transmembrane helices. qRT-PCR results showed that ZjRAP2-11 and ZjERF7 were both induced by abiotic stress,but ZjRAP2-11 was not much influenced by air dry, and the expression pattern was different in leaf and root for ZjRAP2-11 and ZjERF1 when responding to salt stress. Therefore,it is a hypothesis that ZjRAP2-11 and ZjERF1 may play a role for zoysia in response to abiotic stress.Copper chaperone for superoxide dismutase gene from Zoysiajaponica(ZjCCS) was cloned and its expression level was analyzed for elucidating its role in response to salt stress, drought stress and heavy-metal stress. ZjCCS gene was isolated by RT-PCR, containing a 924-nucleotides-long open reading frame (ORF) with two translation initiation sites, which encoded a protein of 307 amino acid residues. Bioinformatics analysis showed that the protein encoded by ZjCCS with the signal peptide and transmembrane helices was hydrophobic, stable and acidic protein, located in the chloroplast. Homology researches with the deduced amino residues indicated that ZjCCS gene had a high similarity to other plant CCS genes, and the predicted ZjCCS polypeptide contained 3 distinct domains. qRT-PCR showed that ZjCCS mRNA levels were much induced by salt stress and peaked at 4h. Drought stress and heavy-metal stress didn’t show a significant influence on the ZjCCS mRNA level compared to salt stress, which suggested that ZjCCS might play a major role when zoysia japonica responding to salt stress.