Study Of Drought Resistance Of C₂H₂ Zinc Finger Protein PuZFP103 Gene In Populus Ussuriensis

Drought is one of the abiotic stresses that seriously affects the growth,development of plants and restricts the development of forest economy.Improving the drought resistance of trees and cultivate new varieties of drought resistance are of great significance to the development of the environment in the future.In the process of signal transduction from sensing stress signal to stress response,the functions of various transcription factors enable plants to adapt to stress.C2H2 zinc finger protein(ZFP,Zinc Finger Proteins)gene is involved in the transcriptional regulation of abiotic stress and developmental regulation.At present,there are few reports on the function of C2H2 zinc finger protein gene in woody plants.In this paper,the expression characteristics and function of PuZFP103 in Populus ussuriensis under stress were analyzed.The results are as follows:(1)We cloned the PuZFP103 gene from P.ussuriensis.Bioinformatics analysis showed that the PuZFP103 protein contained a typical C2H2 zinc finger domain as well as the conserved sequence QALGGH in the zinc finger.There is an EAR motif at its C terminal region.(2)The expression patterns of PuZFP103 gene under NaCl,PEG6000,ABA and NAP treatments at different time points(0h,6h,12h,24h,48h,72h and 96h)in roots and leaves of P.ussuriensis were analyzed.It was found that the expression of PuZFP103 gene changed differently under various stresses,in which the expression of PuZFP103 in leaves was higher under NaCl treatment,but there was little difference in roots.The expression of PuZFP103 gene in roots and leaves increased significantly under PEG6000 and ABA treatments.Especially in the root,the expression level increasedobviously.Additionally,we used ABA inhibitor NAP for two hours,and then treated with PEG6000 and ABA for different periods of time.we found that the expression of PuZFP103 gene decreased significantly in root,indicating that the gene is likely to be related to ABA in root.(3)The promoter element of PuZFP103 gene was analyzed and predicted by PlantCare,and it was found that the promoter region contained stress response element,such as TC-richrepeats and ABA response element ABRE etc.The plant clone vector pGWB3-PuZFP 103pro::GUS,was constructed for transient infection and conducted GUS staining of P.ussuriensis,which treated with PEG6000 and ABA.The result showed that it mainly showed high expression activity in roots.(4)Then we constructed pBI121-PuZFP103-GFP and pUC19-PuZFP103-GFP plant expression vectors to investigate their subcellular localization.The results showed that PuZFP 103 protein was mainly expressed in the nucleus;The transcriptional activation experiments confirmed that PuZFP103 transcription factors had no transcriptional activity.Then we used protoplast transfection to determine the activity of GUS/LUC enzyme to verify that PuZFP103 was a transcriptional inhibitor.(5)In order to further study the biological function of PuZFP103 gene,we obtained PuZFP103 overexpression(35S::PuZFP103)and inhibitory expression(RNAi)transgenic plants.Compared with wild type P.ussuriensis.(WT),the number of adventitious roots increased,and the number of lateral roots decreased in transgenic plant lines.After drought stress,the number and length of adventitious roots of overexpressed plants were significantly increased than those of WT plant.The phenotype of the overexpressed strain was opposite to that of the inhibitions expressed plants lines.Through anaysis of NBT,DAB staining,MDA,proline and soluble sugar content,it was found that under 7%PEG6000 stress,the cell damage degree of overexpression of PuZFP103 gene was significantly lower than that of WT plant.Together,the PuZFP103 overexpressing in P.ussuriensis improve drougt resistance.