Comparative transcriptome analysis reveals putative transcription factors components of the regulatory network for cannabidiol biosynthesis in Cannabis sativa L.

BACKGROUND: Cannabidiol (CBD), a unique secondary metabolite of Cannabis sativa L. (cannabis), is widely used in medicine, food, cosmetics, and forage. Elucidation of the transcriptional regulation mechanism can provide a theoretical basis for molecular breeding with higher CBD content. However, the regulatory network for cannabidiol biosynthesis in cannabis is largely unknown. METHODS: Transcriptome analysis of four cannabis tissues was conducted to screen differentially expressed genes. Short time-series expression miner (STEM) analysis combined with strain-specific expression correlation analysis was performed to filter core transcription factors (TFs) associated with CBD biosynthetic enzyme genes. Yeast one-hybrid and yeast two-hybrid assays were applied to verify the binding and protein interaction relationships of key TFs, and subcellular localization assay was performed in tobacco leaf. RESULTS: A total of 1314 genes specifically upregulated in female flowers were identified by transcriptome analysis of four different tissues with distinct CBD contents and clustered into 19 profiles using short time-series expression miner (STEM) analysis. Profile 8, including 16 TFs and 6 enzyme genes related to the CBD biosynthesis pathway, demonstrated similar trends in the CBD contents of the four tissues. Among the 16 TFs, 7 exhibited similar strain-specific expression profiles and their expression levels positively correlated with those of the six enzyme genes. Moreover, a yeast one-hybrid assay found that CsMYB6 and CsMYB29 bind to the promoters of four and two CBD biosynthetic enzyme genes, respectively. Meanwhile, a yeast two-hybrid assay revealed that CsMYB6 interacts with two regulators of CBD biosynthesis, CsMYB1 and CsWRKY1, while CsMYB29 interacts with CsWRKY1. In addition, CsMYB29 and CsMYB6 were localized in the nuclei. CONCLUSIONS: In summary, the two TFs, CsMYB6 and CsMYB29, identified in this study provide valuable insight into the complex regulatory mechanisms of CBD production in cannabis and offer a genetic resource for the genetic improvement of CBD content.