Functional Analysis Of PsnHB63 Gene From Populus Simonii×P.nigra In Salt Stress Tolerance

HD-Zip transcription factor is unique to higher plants,and participates in the process of plant growth and development and abiotic stress.In this study,a 717 bp cDNA fragment of the coding region of the PsnHB63 gene was cloned from Populus simonii×P.nigra,which encodes 238 amino acid residues.It was an unstable hydrophilic protein with a conserved domain of the Homeobox/HALZ family.qRT-PCR analysis showed that the expression of PsnHB63 gene is tissue-specific and induced by salt stress and drought stress.The subcellular localization results showed that PsnHB63 protein was localized in the nucleus.The 1456 bp DNA fragmentof upstream of PsnHB63 gene was cloned from Populus simonii×P.nigra,and the prediction results indicate that the PsnHB63 gene promoter sequence contains cis-acting elements related to transcription,plant response to stress,and growth and development.The results of GUS staining of transgenic Arabidopsis thaliana showed that the PsnHB63 gene promoter drives GUS gene expression in roots,stems and leaves,which is consistent with the tissue-specific expression characteristics of PsnHB63 gene.Using yeast one-hybrid technology to identify proteins that interact with the promoter region of PsnHB63 gene include plant defense and stress response related proteins,oxidoreductase activity related proteins,plant growth and development related proteins and transcription related proteins.Construct PsnHB63 gene overexpression vector and genetically transform Populus simonii×P.nigra nigra to obtain PsnHB63 gene overexpression plant.Under 200 mM NaCl stress,the growth of overexpressing Populus simonii×P.nigra nigra was better than the wild type;the SOD,POD activity and chlorophyll content of the overexpressing Populus simonii×P.nigra nigra were higher than that of the wild-type,but the MDA content,relative conductivity and water loss rate were lower than wild-type.Under stress and non-stress conditions,the PsnHB63 gene overexpressing Populus simonii×P.nigra nigra had more developed root systems than wild-type plants,chlorophyll content is more than wild type.The pGBKT7-PsnHB63 vector was constructed and transferred into yeast cells.It was found that the PsnHB63 Transcription factor has transcriptional self-activation activity,and its transcription activation domain is within 56aa of the C-terminal of the protein;and using yeast two-hybrid technology to find 2 proteins that interact with the PsnHB63 transcription factor from the Populus simonii×P.nigra nigra library:proteins interacted with transcription factors were enriched in proline proteins that did not indicate characteristics and heavy metal-related prenylated plant protein 5 subtype X8.The effector vector pGADT7-Rec-PsnHB63 and the reporter vector pAbAi-HBS1,pAbAi-HBS2,pAbAi-DRE,and pAbAi-MYB-P were subjected to yeast one-hybrid experiments.The results showed that the PsnHB63 transcription factor recognizes HBS 1,HBS2,DRE,MYB-P stress resistance elements..