Sequence and functional analysis of cis-elements associated with MIR159 loci from Brassica juncea reveal functional diversification and complex transcriptional regulation

摘要

Identification and functional analysis of promoters is critical towards gaining insights into transcriptional regulation, and helps in identifying interacting trans-factors. Analysis of MIRNA promoter can differentiate the function of paralogs and homeologs where identifying the role of members of MIRNA family is difficult based on small RNA blots due to near-identical mature sequences. The role of miRNA as regulators of development and adaptation in polyploid species-Brassica, and the impact of polyploidy on divergence among paralogs and homeologs especially with reference to transcriptional regulation remains unexplored. We identified homologs of MIR159 from 137 species across plants based on their homology to pre-MIR159A, MIR159B, and MIR159C from Arabidopsis and retrieved their promoter sequences. Promoter sequence analysis revealed high level of divergence; albeit clades of closely related taxonomic units were obtained. We detected biased, but no lineage-specific distribution of transcription factor binding motifs (TFBS) in families. Phylogenetic reconstruction in Brassicaceae revealed genome- and sub-genome specific clades. We isolated three homeologs of MIR159A, and one of MIR159C from the A-genome of B. juncea. Functional characterization involved transgenic lines in A. thaliana Col-0 with promoter::reporter transcriptional fusions (p-MIR159::GUS::ter) and monitoring of reporter activity during development, abiotic stresses, and after administering hormones. Comparison of reporter activity of full-length promoters across homeologs and paralogs reveal extensive functional diversification. Comparative analysis of reporter activity of nested deletions, and with distribution of TFBS did not yield a clear correlation. In summary, analysis of GUS during development, hormone treatment and abiotic stresses reveal complex transcriptional regulation of MIR159 expression.