Abstract:

Objective: The MYB-related transcription factor family plays an important role in plant growth and development and stress response, but functional studies on members of this family in poplar have rarely been reported. In this study, a MYB-related gene PagMYBR96 that responds to salt stress was identified in Populus alba × P. glandulosa '84K' based on transcriptome sequencing data. Further, the gene was cloned and its characteristics and the involved biological processes were analyzed in order to provide a reference for subsequent gene function and regulatory mechanism studies and forest genetic engineering breeding. Method: The PagMYBR96 gene was amplified from 84K poplar cDNA using PCR, and the physicochemical properties of the encoded protein sequence were analyzed. The conservative domain, evolutionary relationship, hydrophilicity, hydrophobicity, transmembrane region, signal peptide, phosphorylation site, and secondary structure of the protein were analyzed using bioinformatics analysis. Agrobacterium-mediated transient transformation of tobacco leaves was used to analyze the subcellular localization of the protein. The activity of transcriptional activation of the protein was analyzed by yeast transformation screening assay. The temporal and spatial expression patterns of the gene were analyzed by RT-qPCR. The biological processes and metabolic pathways involved in the gene were analyzed using gene co-expression and functional annotation and enrichment analysis. Result: The 882 bp PagMYBR96 gene was cloned from 84K poplar, and could encode protein with 293 amino acids, with a molecular weight of 33 306.33 Da, and a theoretical isoelectric point of 6.32. The protein contains a conservative MYB domain and its characteristic amino acids. The protein is closely related to the homologous protein of Salix dunnii, and has no transmembrane region and signal peptide. It may be a hydrophilic protein. PagMYBR96 is a nuclear localization protein and has no activity for transcriptional activation in yeast. The gene spatio-temporal expression pattern showed that PagMYBR 96 was able to respond to salt stress in both roots and leaves of 84K poplar, and its expression was up-regulated at the early stage of stress, and then gradually returned to normal after reaching the peak. PagMYBR96 and 518 co-expressed genes were related to cellular processes, metabolic processes, biological regulation, response to stimulation, developmental processes, and were significantly enriched in GO terms such as ubiquitin-like protein conjugating enzyme activity, ubiquitin-like protein transferase activity, ubiquitin-protein transferase activity, ubiquitin conjugating enzyme activity. In addition, these genes were also significantly enriched in the pathways of aminoacyl-tRNA biosynthesis, ubiquitin mediated proteolysis, and pyruvate metabolism. Conclusion: The PagMYBR96 gene of 84K poplar can respond to salt stress in different tissues, and participate in plant growth and development and stress response through different biological processes. It is worth noting that the functions of PagMYBR96 and its co-expressed genes are related to processes such as protein degradation mediated by the ubiquitin proteasome system and pyruvate metabolism, which is also the direction that needs to be explored in future research.

Key words: Populus alba × P. glandulosa '84K', salt stress, MYB-related transcription factor, expression analysis