[Regulation of alkaloid biosynthesis by transcription factor CcWRKY40 in Coptis chinensis]

The biosynthetic pathway of alkaloids in Coptis chinensis has been preliminarily elucidated, while the regulatory mechanism of alkaloid biosynthesis remains to be thoroughly investigated. This study screened a transcription factor CcWRKY40 with high expression under methyl jasmonate induction from the previous transcriptome data of C.chinensis. To investigate the regulatory mechanism of CcWRKY40 on alkaloid biosynthesis, this study cloned CcWRKY40 and conducted bioinformatic analysis of this gene. The tobacco transformation experiment was conducted to predict the subcellular localization of CcWRKY40 and real-time fluorescence quantitative PCR was performed to analyze the tissue-specific expression of CcWRKY40. The yeast one-hybrid assay was employed to examine the interactions between CcWRKY40 and the promoters of five main functional genes. The dual luciferase assay was carried out to validate the transcriptional activation function of CcWRKY40. Molecular docking was performed to predict the possible binding sites of CcWRKY40 to promoters. The results indicated that the open reading frame(ORF) of CcWRKY40 was 951 bp, encoding 316 amino acid residues. CcWRKY40 was located in the nucleus. The RT-PCR results demonstrated that CcWRKY40 was most highly expressed in the fine roots in 1-to 5-year-old C. chinensis. The results form yeast one-hybrid and dual-luciferase assays indicated that CcWRKY40 was able to bind to the promoters of CcCNMT and Cc4'OMT and activate their transcription. The binding sites were predicted at the core domain(RKYGQK) of CcWRKY40. In summary, CcWRKY40 can bind to the promoters of functional genes CcCNMT and Cc4'OMT, thereby activating their transcription. The present study lays a foundation for elucidating the regulatory mechanism of CcWRKY40 on alkaloid biosynthesis in C. chinensis.