ANNOUNCEMENTS
MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs that play a central role in plants by regulating gene expression post-transcriptionally. This study focuses on miR167, a key regulator of auxin signalling pathways and root development. In the allotetraploid crop Brassica juncea, 15 homologs have been identified due to whole-genome duplication and hybridisation events evenly dispersed on the chromosomes from progenitor genomes. The study aimed to characterise the sequence and regulatory variation between MIR167 promoters from Brassica juncea. In silico analysis of ~2 kb upstream regions showed sequence divergence among the 15 MIR167 promoter homologs, along with differentially present transcription factor binding site (TFBS) profiles. Some promoters, such as Bju-MIR167m and Bju-MIR167g, exhibited high TFBS density and unique clustering in phylogenetic analyses, suggesting potential neofunctionalisation. The enrichment of stress-responsive TF families such as WRKY, NAC, and MYB further implied a role in environmental adaptation. Under the control of the CaMV 35S promoter, the expression of the RUBY vector was evaluated in Nicotiana benthamiana (transient) and Arabidopsis thaliana (stable) transformation systems. Visual red pigmentation confirmed successful expression and established RUBY as a non-invasive tool for promoter strength assays. To functionally evaluate promoter activity of MIR167 homologs, a promoterless RUBY reporter vector was designed. Select MIR167 promoters (Bju-MIR167a, Bju-MIR167d, and Bju-MIR167n) were cloned into intermediate vectors and sequenced, enabling future functional studies in planta.
Keywords: MicroRNA, Brassica, Polyploidy Promoters analysis, TFBS, RUBY Reporter.
