Objective Clone the MtbZIP29 gene from Medicago truncatula to study the self-activation, subcellular localization, and expression characteristics of the bZIP29 transcription factor, providing a theoretical research foundation for elucidating the involvement of the MtbZIP29 gene in the growth and development of M. truncatula and endogenous hormone signaling transduction. Method The MtbZIP29 gene was cloned from the wild-type M. truncatula 'R108', and expression vectors were constructed for experimental purposes. Subcellular localization was observed using a transient fusion protein assay, and using yeast to analyze its self-activation activity. Bioinformatics analyses were conducted on the MtbZIP29 gene, including protein physicochemical property analysis, prediction of promoter cis-acting elements, and prediction of secondary and tertiary protein structures. Additionally, RT-qPCR was employed to analyze the expression patterns of MtbZIP29 across different tissues and under treatments with various hormones (ABA, SA, 6-BA, IAA, and MeJA). Transgenic tobacco plants were obtained using Agrobacterium-mediated method, and the function of MtbZIP29 was analyzed. Result The MtbZIP29 gene was successfully cloned, with a coding sequence (CDS) length of 1,518 bp encoding 506 amino acids. The encoded protein had molecular weight of 55.830 kD, a theoretical isoelectric point (pI) of 6.82, an instability index of 63.60, and is classified as an unstable hydrophilic protein. Secondary structure prediction revealed that α-helixes accounted for 25.89%, extended strands for 0.59%, and the remaining 73.52% as random coils. Subcellular localization indicated the protein was localized to the nucleus. Yeast assays demonstrated the encoded protein has transcriptional self-activation activity. Expression profiling showed that MtbZIP29 transcripts were the most abundant in the leaves, significantly lower in the stems and pods compared to other tissues and markedly influenced by different hormone treatments. Analysis of MtbZIP29-overexpressing plants showed that the rhizomes of transgenic tobacco were significantly enlarged compared with wild-type tobacco. Conclusion The MtbZIP29 gene is involved in root growth and development, responding to different hormones, and may participate in the regulatory processes of root morphology construction and stress adaptability by integrating hormone signals.
