Function Analysis Of PuZFP2 Gene For Drought Tolerance In Populus Ussuriensis

C2H2-type zinc finger protein transcription factors can regulate a variety of physiological and biochemical reactions in plants,and then play a regulatory role in plant stress tolerance.At the same time,C2H2-type zinc finger protein is also related to the growth and development of plants.In recent years,there is relatively little research on C2H2-type zinc finger protein in woody plants.In this paper,the PuZFP2 gene of C2H2 zinc finger protein C1-2i subclass of Populus ussuriensis was selected to study the drought tolerance function and the molecular mechanism of drought stress response.The main results are as follows:(1)The expression pattern of PuZFP2 under 6%PEG6000,abscisic acid(ABA),methyl jasmonate and salicylic acid was analyzed by qRT-PCR,the results indicated that PuZFP2 could be significantly induced by 6%PEG6000 in roots and leaves of P.ussuriensis and could be significantly induced by ABA in roots.The PuZFP2 gene promoter was cloned from P.ussuriensis,and proPuZFP2::GUS transgenic plants of P.ussuriensis were obtained by Agrobacterium tumefaciens-mediated leaf disc method.GUS staining analysis of transgenic plants in tissue culture showed that the staining in roots and leaves was shallow under normal culture conditions,while the staining in roots and leaves deepened after 6%PEG6000 stress.The promoter element of PuZFP2 gene was predicted by PlantCare analysis,and it was found that the promoter region contained drought stress response element MBS and ABA stress response element ABRE,etc.(2)The subcellular localization experiment was carried out by using poplar protoplast transient expression method,and it was found that PuZFP2 transcription factor was located in the nucleus.Transcriptional activity of PuZFP2 was analyzed by transient expression method of poplar protoplast,the vector containing four GAL4 DNA binding sites and GUS reporter gene and the vector containing luciferase(LUC)reporter gene were transfected into poplar protoplasts simultaneously with four effector vectors.and the relative GUS activity(GUS/LUC)was calculated with LUC as an internal reference.The results showed that compared with BD,BD-PuZFP2 significantly inhibited the expression of GUS reporter gene,which indicated that PuZFP2 had transcriptional inhibitory activity.(3)The overexpression vector of PuZFP2(PuZFP2-OE)driven by the promoter 35S was constructed.and the genetic transformation of P.ussuriensis was carried out by A.tumefaciens-mediated leaf disc method,and 14 strains of PuZFP2-OE transgenic plants were obtained.The drought tolerance of WT and PuZFP2-OE strains under 6%PEG6000 stress was compared.Under 6%PEG6000 stress,compared with WT.the dry weight of roots and stems and leaves of PuZFP2-OE strain increased significantly.the relative conductivity.malondialdehyde and hydrogen peroxide content in roots and leaves decreased significantly,and the activities of superoxide dismutase and peroxidase and proline content increased significantly.The above results indicate that PuZFP2,as a transcription inhibitor located on the nucleus,is related to the drought tolerance of P.ussuriensis,and the overexpression of PuZFP2 gene can improve the drought tolerance of P.ussuriensis by enhancing the ability of scavenging reactive oxygen species and accumulating more proline.