Cloning, subcellular localization, and expression analysis of MtbHLH25 gene in Medicago truncatula

Abstract

Abstract [Objective] bHLH transcription factors are numerous and widely participate in plant growth, development, and stress responses. The experiment used Medicago truncatula R108 as the material to clone the MtbHLH25 gene and preliminarily explored its function, which will help to further study the function of M . truncatula bHLH transcription factors. [Methods] MtbHLH25 gene and its promoter were cloned from M . truncatula using PCR technology. Yeast expression vector was constructed and transferred to Y2H Gold yeast strain using LiAc transformation method. Vectors for subcellular localization assays were constructed and transferred into Agrobacterium EHA105 through freeze-thaw method. The Agrobacterium was injected into tobacco epidermal cells and gene expression was observed using SP8 laser confocal microscopy. The spatiotemporal expression of MtbHLH25 in M . truncatula was observed using fluorescence quantitative PCR technology. [Results] The MtbHLH25 gene and its promoter were cloned from M . truncatula with 882 bp, encoding 293 amino acids. Promoter analysis revealed that it contains ABA response elements, MeJA response elements, GA response elements, and SA response elements. (2) The evolutionary tree showed that the MtbHLH25 protein was highly homologous with the bHLH proteins in Vicia faba and V. villosa Roth. (3) The MtbHLH25 protein was localized in the nucleus. (4) The yeast self-activation test showed that the MtbHLH25 protein has self-activation activity. (5) MtbHLH25 was expressed in roots, stems, leaves, flowers, and fruits of M . truncatula, with the highest expression in roots. Exogenous SA, MeJA, ABA, GA, and salt stress decreased the expression of MtbHLH25, indicating that SA, MeJA, ABA, GA, and salt stress had a negative regulatory effect on the expression of MtbHLH25. Drought stress increased the expression of MtbHLH25, indicating that this transcription factor might play a positive role in drought stress. [Conclusion] The MtbHLH25 gene may be sensitive to salt stress and play a positive regulatory role in drought stress. The MtbHLH25 protein has self-activating activity and may have an activating effect on the downstream reporter genes

Keywords

• mtbhlh25
• medicago truncatula
• hormonal regulation
• transcription of autoactivation