| Apple suffers from various adverse environmental stresses in its life cycle,It has serious impact on the yield and quality of apple.The calcium-dependent protein kinase(CDPK)is an important calcium receptors,which play a key role on stress perception and decoding process under intracellular calcium signalin during various developmental processes and during responses to biotic and abiotic stresses.And it has been reported that CDPKs participate in the process of plant response to abiotic and biotic stress in Arabidopsis,tobacco and rice.In this study,we researched the expression pattern of MhCPKl in Malus hupehensis,and isolated MdCPK1a ORF sequence which shares a high degree of homology with MhCPK1,sub-cellular localization of MdCPK1a protein and stress tolerance of MdCPK1a-overexpressing nicotiana benthamiana plants under biotic and abiotic stresses were studied.These researches provide reference for the breeding of new varieties of apple.The results are as follows:1.Based on the published sequence of(MDP0000153100)in the apple genome database,a pair of specific primers(MhCPK1)were designed to isolate MhCPK1 ORF sequence in Malus hupehensis.The deduced full-length sequence of MhCPK1 cDNA consisted of 1701 nucleotides which encoded a 566-amino acid polypeptide with a calculated molecular mass of 62.92 kDa and an isoelectric point of 5.24.Because this putative Malus hupehensis CDPK shares a high degree of homology with MDP0000153100 from apple,we named the gene MhCPK1.Expression analysis indicated that MhCPK1 was expressed in fruits at different developmental and ripening stages,as well as in several tissues such as roots,stems,leaves,flowers,and moreover,expression levels of MhCPK1 were higher in fruits than in other vegetative tissues.MhCPK1 was found to be upregulated upon Alternaria alternata,cold and high temperature treatments,and downregulated upon abscisic acid,NaCl and drought treatment.2.In order to investigate the sub-cellular localization of MdCPK1a,the MdCPK1a gene encoding frame lack of termination codon was connected with N terminal of green fluorescent protein(GFP),we generated a 35S:MdCPK1a-GFP-pCAMBIA1300 fusion construct and expressed the contruct in Nicotiana benthamiana leaves by agroinfiltration.The subcellular localization of MdCPK1a-GFP and GFP control were visualized with laser scanning confocal microscopy.The results revealed that MdCPK1a-GFP was localized in both the plasma membrane and the nucleus,whereas the GFP control was localized in multiple subcellular compartments including the cytoplasm and the nucleus.3.The effects of over expressing the MdCPK1a gene in transgenic plants of tobacco on their responses to abiotic stresses were investigated.There was no difference between WT and transgenic plants from the phenotype under drought and high temperature(42℃).To elucidate the role of MdCPK1a in cold stress tolerance,3-week-old T2 plants of WT,T1,T2,and T3 genotypes were exposed to 4 ℃ for 10 days,then the plants were returned to normal growing conditions for two week.Results indicated that the WT plants were more affected by the cold treatment than the A4,A3 6,and A2 lines overexpressing the MdCPK1a gene.60-96%of the MdCPAK1a-overexpressing A4,A3 6,and A2 lines survived,while only 17%of WT plants survived.After 25d suffering from salt stress and two week recovery,the WT plants grew weaker and the leaves were more etiolated than transgenic plants.The average dry weights of shoots and roots of the MdCPK1a-overexpressing were heavier than those of WT plants,Moreover,the transgenic tobacco promotes stress tolerance against tobacco Brown Spot disease.These result suggest that transgenic tobacco over expressing the MdCPK1a gene showed tolerance to cold,salt and disease stress,and the cold resistance is more apparent.4.In view of the remarkably improved cold tolerance in the transgenic lines,several physiological,molecular and biochemical analysis were performed in order to identify the mechanism underling the improved cold tolerance.In this study,after cold stress,a lower EL and MDA level were detected in the transgenic lines,WT exhibited clearly more intense DAB and NBT staining in comparison with the transgenic lines,which implied that the accumulation of ROS in transgenic plants were less than that in WT plants.And the activities of CAT,POD,and SOD were higher in the transgenic lines than in WT weather under normal conditions or cold stress.To further elucidate the molecular mechanisms of MhCPK1 in cold stress signal perception,the expression levels of stress-responsive and ROS-related genes were analyzed.The expression levels of ROS-related genes(NtSOD,NtGPX and NtCAT)and stress-responsive gene NtSPS,NtDREB10C,NtLEA5,NtDREB3 were higher in transgenic plants than in WT under cold stress.However,the expression levels of NADPH oxidase gene NtrbohD was lower in transgenic plants than in WT.These results suggest that plants showed enhanced tolerance to cold stress by enhancing ROS-scavenging system,repressing ROS accumulation and regulating the expression of stress-responsive gene. |
