Characterization Of Zinc-Finger Protein MdZFP2 In Response To Drought And Freezing Stress In Apple

China is the largest apple producing and consuming country worldwide,but frequently occurred drought and freezing stress have limited the development of apple industry in the Loess Plateau of China.It is of great significance to improve the stress tolerance of apple by its own genetic resources.In this study,we cloned a zinc-finger gene from the cultivar'Golden delicious' and named it as MdZFP2.The sequence characteristics,subcellular localization and expression pattern were then investigated.By yeast two-hybrid screening,we identified two interacting proteins of MdZFP2,which were then verified by bimolecular fluorescence complementation.In addition,by using apple transgenic plants,we conducted long-term drought experiments as well as ion-leakage experiments to explore the biological function of MdZFP2 in response to drought and low temperature stress.The results are as follows:1.We cloned MdZFP2 gene that is located on chromosome 15 with an open coding reading frame of 912 bp.Encoding 303 amino acids,its molecular weight is 33.93 kD.Sequence analysis indicated that MdZFP2 has a conserved DNA binding domain of zinc finger protein,DOF.Subcellular localization experiments indicated that MdZFP2 is localized in the nucleus,similar to typical transcription factors.Two cis elements,responsive to MeJA and ABA signaling on MdZFP2 promoter were identified.2.Tissue-specific expression results showed that MdZFP2 was expressed in roots,stems and leaves of apples,with the highest expression in roots.MdZFP2 was induced by PEG treatment and natural drought treatment but repressed by cold stress.The expression of MdZFP2 with PEG treatment for 6 hours was 6 times higher than that before treatment.During the natural drought treatment,the expression level of MdZFP2 increased with the time prolonged,while the expression level after 4 hours cold treatment was only 0.17 times of control.In addition,MdZFP2 was also responsive to jasmonic acid(JA)and salicylic acid(SA)hormone treatments,and the expression level decreases with the hormone treatment time prolonged.3.The MdZFP2-pGBKT7 vector was constructed to investigate the expression activity of MdZFP2 protein in yeast.It was found that MdZFP2 protein had self-activating activity,while the truncated fragment MdZFP2-510aa-BD abolished this self-activation.We used this fragment as a bait to screen interacting proteins of MdZFP2,and identified twocandidate proteins,MdMIEL1 and MdSNF1?2.The former encodes a RING-type E3 ligase,and the latter is one of the subunits that make up the SnRK1 phosphorylation complex.The protein interactions were further verified by yeast two-hybrid and bimolecular fluorescence complementation.Besides,MdZFP2 downstream target genes were predicted by chromatin immunoprecipitation experiments.4.The MdZFP2 RNAi and overexpression vector were constructed and transformed into tissue-cultured GL-3 apple to obtain transgenic apple plants.Long-term drought treatment showed that the plant height,stem diameter,photosynthesis parameters,water conductivity and biomass of the aboveground and underground parts of MdZFP2 RNAi plants under drought were significantly lower than those of GL-3,indicating MdZFP2 RNAi plants were more sensitive to long-term drought treatment.Subsequently,the leaf natural dehydration experiment was carried out and we found that the RNAi plants lost more leaf water than GL-3.In contrary,the overexpression plants showed the opposite trend.It is concluded that MdZFP2 is involved in the response of apple to drought stress and positively regulates the drought resistance of apple.In addition,the cold tolerance of RNAi transgenic apple plants was investigated by ion leakage experiment.It was found that the relative ion-leakage of RNAi plants was significantly higher than that of GL-3 at-4oC and-6 oC,indicating that MdZFP2 RNAi plants are more tolerant to low temperature stress.