Study On The Mechanism Of PagbHLH35 Gene In Response To Salt Stress In Populus Alba×P.Glandulosa

The bHLH transcription factor family is involved in plant growth and stress response,regulating growth and developmental processes such as embryonic growth,seed germination,cell division,element enrichment and response to stress through signal transduction pathways.In this study,we used Populus alba×Populus glandulosa as experimental material,cloned PagbHLH35 gene and explored its resistance mechanism by spatiotemporal expression analysis,and laid the foundation for the study of the molecular mechanism of resistance of bHLH transcription factor family in poplar,with the following results:The 735 bp PagbHLH35 gene CDS,which encodes 244 amino acids,was cloned from Populus alba×Populus glandulosa.Predictions revealed that the protein is an unstable hydrophobic protein that does not contain a transmembrane helix or signal peptide;it shares a conserved structural domain with other bHLH family transcription factors.Transcriptional activation activity assays and subcellular localization demonstrated that the protein has no self-activating activity and is localized in the nucleus.The expression level of PagbHLH35 gene was significantly increased under salt stress.Analysis of the promoter of the PagbHLH35 gene revealed that the promoter contains many hormone response elements and several transcription-related elements.Many light response elements,copper response elements,drought response elements and stress response elements were also identified.Transgenic tobacco and Populus alba×Populus glandulosa were grown by constructing PagbHLH35 gene overexpression vector and using Agrobacterium-mediated leaf disc transformation method.The transgenic strains showed significant advantages over the wild type under salt and drought stress conditions.The POD activity,SOD activity,proline content,and chlorophyll content of the transgenic strain were significantly higher than those of the wild type,while the MDA content,H₂O₂ content,and relative conductivity were significantly lower than those of the wild type.This indicates that the transgenic plants can reduce the damage caused by salt stress by improving the scavenging ability of reactive oxygen species and proline accumulation level,thus enhancing the stress resistance of the transgenic plants.It was demonstrated by yeast single hybridization experiments that PagbHLH35 protein can interact with elements such as G-box and E-box.It is shown that PagbHLH35 regulates its expression by binding to elements such as G-box and E-box in the promoters of downstream genes.In summary,the PagbHLH35 gene may be involved in the abiotic stress response process and enhances the stress tolerance of transgenic poplar.