Transcription factors involved THCAS promoter in cannabinoid biosynthesis pathway of Cannabis sativa L.

Abstract

Cannabinoids are important secondary metabolites found in Cannabis sativa L. that play a crucial medical role in pain relief and mood adjustment. The main cannabinoid substances in cannabis are delta-9-tetrahydrocannabinolic acid (THCA), and cannabidiolic acid (CBDA). THCA biosynthesis is regulated by the THCA synthase (THCAS) gene which is regulated by the THCAS promoter. However, a comprehensive understanding regarding the regulation of gene expression by transcription factors (TFs) in cannabis is lacking. This study identified the TFs involved in regulating the THCAS promoter in Cannabis sativa L. ‘Hang Kra Rog’ by first amplifying the nucleotide sequences in the promoter of the THCAS gene from cannabis plants using the PCR technique. Subsequently, the THCAS promoter was cloned into the pENTR-5’-TOPO vector for sequencing purposes and then transferred into the R4L1pDEST_HISi vector by the LR reaction for yeast one-hybrid (Y1H) screening to study the interaction between TF proteins from the transcription factor cDNA library and the THCAS promoter within yeast cells. The TF proteins involved in the THCAS promoter were identified into seven groups: ARID, B3, bZIP, CPP, GeBP, HD, and NAC with further verification of their functions in cannabis plant cells using transient effector-reporter analysis.