Analysis Of Drought Tolerance Function Of PdabHLH1 Gene In Populus Davidiana × Populus Alba Var.pyramidalis

The growth and development process of plants often faces multiple abiotic stresses.Drought,as one of several common abiotic stresses,has various effects on plant growth and development,such as affecting plant growth phenotype,physiological and biochemical indicators,and regulation of gene expression,and so on.The bHLH transcription factor family can participate in the regulation of plant resistance to stress（drought,high salt,low temperature,iron deficiency,etc.）and growth and development.To identify the drought tolerance function of PdabHLH1 gene in Populus davidiana×Populus alba var.pyramidalis,bioinformatics,expression pattern,transgene function and regulatory analysis were performed in this study:The full length of PdabHLH1 gene was cloned,and the open reading frame of PdabHLH1gene was 678 bp long,encoding 225 amino acids.PdabHLH1 was identified as a bHLH family gene by sequence alignment.Subcellular localization using protoplast transformation of P.davidiana×P.alba var.pyramidalis showed that the PdabHLH1 protein is localized in the cytoplasm.Analysis of transcriptional activation activity showed that the full-length PdabHLH1 protein did not exhibit transcriptional activation activity.The expression pattern of PdabHLH1 gene was analysed using qRT-PCR and the results of tissue expression pattern showed that the expression of PdabHLH1 gene in the leaves of P.davidiana×P.alba var.pyramidalis was higher than that in the roots and stems,which was 2.09times that of the roots.The expression level in the roots and stems was similar;The expression pattern of abiotic stress showed that the PdabHLH1 gene could be induced to express under both PEG₆₀₀₀and Na Cl treatments.Especially under PEG₆₀₀₀simulated drought stress,the expression level of PdabHLH1 gene in roots,stems,and leaves was significantly induced to be upregulated,indicating that PdabHLH1 gene may participate in the drought response process of P.davidiana×P.alba var.pyramidalis.The overexpression vector pBI121-PdabHLH1-GFP was constructed and ten transgenic lines were obtained by stable transformation of P.davidiana×P.alba var.pyramidalis using the Agrobacterium-mediated leaf disc method.Natural drought treatment of wild type（WT）and transgenic plants（OE）observed that a higher degree of wilting of WT than OE terminal buds,and after rehydration,WT did not regain uprightness,OE3 regained all uprightness and OE5partially regained uprightness.Both chemical staining analysis and physiological measurements indicated that WT had more severe cell damage and accumulated more reactive oxygen species（ROS）in their bodies than OE.It is therefore hypothesized that overexpression of PdabHLH1 in stably transformed plants could improve drought tolerance by enhancing the activity of SOD and POD,eliminating reactive oxygen species from the body and reducing the accumulation of O₂^-and H₂O₂.To verify whether PdabZIP1 regulates the PdabHLH1 gene,the PdabZIP1 gene with drought tolerance function was cloned and two PdabZIP1 overexpression stable transformed lines were obtained.PdabZIP1 was identified to recognize the ABRE cis-acting element using yeast single hybridization（Y1H）,and PdabZIP1 was able to recognize and bind the PdabHLH1 promoter region containing the ABRE element.Chromatin immunoprecipitation（Ch IP）further verified that PdabZIP1 could regulate PdabHLH1 expression by recognizing and binding to the ABRE element in the PdabHLH1 promoter region.In summary,this study identified the drought-tolerant PdabHLH1 gene and its upstream regulatory gene PdabZIP1,providing reference data for in-depth research on the drought tolerance mechanism of the PdabHLH1 gene of P.davidiana×P.alba var.pyramidalis,and providing materials for drought tolerant breeding research of P.davidiana×P.alba var.pyramidalis.