Genome-wide analysis of bHLH gene family in Coptis chinensis provides insights into the regulatory role in benzylisoquinoline alkaloid biosynthesis

Coptis chinensis Franch is a perennial species with high medical value. The rhizome of C. chinensis is a traditional Chinese medicine widely used for more than 2000 years in China. Its principal active ingredients are benzylisoquinoline alkaloids (BIAs). The basic helix-loop-helix (bHLH) transcription factors play an important regulatory role in the biosynthesis of plant secondary metabolites. However, the bHLH genes in C. chinensis have not been described, and little is known about their roles in alkaloid biosynthesis. In this study, a total of 143 CcbHLH genes (CcbHLHs) were identified and unevenly distributed on nine chromosomes. Phylogenetic analysis divided the 143 CcbHLH proteins into 26 subfamilies by comparison with Arabidopsis thaliana bHLH proteins. The majority CcbHLHs in each subgroup had similar gene structures and conserved motifs. Furthermore, the physicochemical properties, conserved motif, intron/exon composition, and cis-acting elements of CcbHLHs were analyzed. Transcriptome analysis revealed that 30 CcbHLHs were significantly expressed in the rhizomes of C. chinensis. Co-expression analysis revealed that 11 CcbHLHs were highly positively correlated with contents of various alkaloids of C. chinensis. Moreover, yeast one-hybrid experiments verified that CcbHLH001 and CcbHLH0002 could interact with the promoters of berberine biosynthesis pathway genes CcBBE and CcCAS, suggesting their regulatory roles in BIA biosynthesis. This study provides comprehensive insights into the bHLH gene family in C. chinensis and will support in-depth functional characterization of CcbHLHs involved in the regulation of protoberberine-type alkaloid biosynthesis.