Genome-wide characterisation and analysis of bHLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza

Abstract

Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.

Figure 1. An un-rooted phylogenetic tree of the bHLH gene family in S. miltiorrhiza.

The amino acid sequences were aligned using Clustal W, and the phylogenetic tree was constructed using neighbour-joining criteria. The letters (A-Y) represent the main subfamilies. The putative bHLH genes involved in the regulation of tanshinone biosynthesis are SmbHLH37 (subfamily R), SmbHLH51 (subfamily R), SmbHLH53 (subfamily R), SmbHLH60 (subfamily W), SmbHLH74 (subfamily H), SmbHLH92 (subfamily P), and SmbHLH103 (subfamily P).

Figure 2. The structural features of each bHLH gene in subfamily A.

The exons are represented by green round-cornered rectangles. The black lines connecting two exons represent introns. The numbers above the line represent the intron phase.

Figure 3. The distribution of conserved motifs in each bHLH gene.

The relative positions of each conserved motif within the bHLH protein are shown in colour.

Figure 4. Heatmaps representing the expression profiles of S. miltiorrhiza bHLH genes in the flower, leaf, root and stem.

The numbers to the right of the figure indicate the bHLH gene name. The colour scale is shown at the top. Higher expression levels are shown in green. The genes with RPKM values of 0 in the four tested organs are not included in the figure.

Figure 5. Expression patterns of 7 putative bHLH genes and the DXS2 gene.

Fold changes in gene expression levels in S. miltiorrhiza R (root), S (stem), L (leaf), F (flower), R1 (periderm), R2 (phloem), R3 (xylem), T0 (treatment with carrier solution for 12 h), and T12 (treatment with MeJA for 12 h) are shown. The transcript levels in the leaf after treatment with the carrier solution for 12 h were normalised to 1.