Cloning and Expression Analysis of miR408 Precursor Sequences from Salvia miltiorrhiza

MiR408 is a highly conserved miRNA in plants and responds to copper availability by targeting genes encoding copper-containing proteins. Its expression is significantly affected by plant growth and development and environmental conditions. MiR408 is critical for the HY5-SPL7 gene network in Arabidopsis, and mediates a coordinated response to light and copper, illustrating its central role in the reaction of plants to the environment. Based on the genomic survey database of Salvia miltiorrhiza established in our lab, a 366 bp miR408 precursor sequence with stable stem loop structure was cloned and named Sm-MIR408. The 723 bp promoter region upstream of Sm-MIR408 was obtained by PCR. The GenBank accession numbers of Sm-MIR408 and the promoter sequence are KU360384 and KU360385, respectively. Bioinformatic analysis showed that the promoter region of Sm-MIR408 shared the same cis-acting elements as those of Ath-MIR408 in Arabidopsis thaliana and Osa-MIR408 in Oryza sativa, and included G-box, CGTCA-motif, TGACG-motif and GTAC-motif, whereas HSE and CATT-motif elements existed only in the Sm-MIR408 promoter region. Mature miR408 is highly conserved among different species. A phylogenetic tree of miR408 from different species showed that miR408 precursor sequences from monocotyledons were clustered on one branch and those from dicotyledons were clustered on another. Real-time quantitative PCR showed that the expression of Sm-MIR408 in the roots, stems, leaves and flowers of S. miltiorrhiza was the highest in the flowers and the lowest in the roots. Expression of Sm-MIR408 could be suppressed by methyl jasmonate, wounding, and continuous light or darkness. The cloning and expression analysis of Sm-MIR408 presented in this research has laid a foundation for studying the function of miR408 in S. miltiorrhiza in the future.