Abstract
Cannabis sativa L. produces unique phytocannabinoids, which are used for their pharmaceutical benefits. To date, there are no reports of in vivo engineering targeting the cannabinoid biosynthesis genes to greater elucidate the role each of these genes play in synthesis of these medically important compounds. Reported here is the first modulation of cannabinoid biosynthesis genes using RNAi via agroinfiltration. Vacuum infiltrated leaf segments of the Cannbio-2 C. sativa strain, transfected with different RNAi constructs corresponding to THCAS, CBDAS, and CBCAS gene sequences, showed significant downregulation of all cannabinoid biosynthesis genes using real-time quantitative PCR. Using RNAi, significant off-targeting occurs resulting in the downregulation of highly homologous transcripts. Significant (p < 0.05) downregulation was observed for THCAS (92%), CBDAS (97%), and CBCAS (70%) using pRNAi-GG-CBDAS-UNIVERSAL. Significant (p < 0.05) upregulation of CBCAS (76%) and non-significant upregulation of THCAS (13%) were observed when transfected with pRNAi-GG-CBCAS, suggesting the related gene’s ability to synthesize multiple cannabinoids. Using this approach, increased understanding of the relationship between cannabinoid biosynthesis genes can be further elucidated. This RNAi approach enables functional genomics screens for further reverse genetic studies as well as the development of designer cannabis strains with over-expression and/or downregulation of targeted cannabinoid biosynthesis genes. Functional genomics screens, such as these, will further provide insights into gene regulation of cannabinoid biosynthesis in Cannabis.
Highlights
Cannabis sativa L. is one of the earliest domesticated and cultivated plants with records of its use in central Asia dating back more than 6,000 years (Li, 1973)
To establish RNAi in C. sativa, THCA synthase (THCAS), CBDA synthase (CBDAS), and CBCAS gene sequences were determined by BLAST querying the Cannbio-2 genome sequence assembly with publicly available sequences (Table 1)
Building upon the approach developed by Deguchi et al (2020), vacuum infiltration was achieved in leaf segments of Cannbio-2, a cultivar with a ratio 1:1.8 their biologically active forms Δ9tetrahydrocannabinol (THC) to CBD, to significantly reduce the relative expression of cannabinoid biosynthesis genes THCAS, CBDAS, and CBCAS
Summary
Cannabis sativa L. is one of the earliest domesticated and cultivated plants with records of its use in central Asia dating back more than 6,000 years (Li, 1973). Manipulation of Cannabinoids by RNAi pathway which produces biologically inactive compounds, such as Tetrahydrocannabinolic acid (THCA) and Cannabidiolic acid (CBDA; Matchett-Oates et al, 2021a) which when decarboxylated are converted to their biologically active forms Δ9tetrahydrocannabinol (THC) and Cannabidiol (CBD) displaying psychoactive and non-psychoactive properties, respectively (Kogel et al, 2018). THC has been the primary cannabinoid studied in cannabis research since its discovery (Gaoni and Mechoulam, 1964), but considerable interest exists in understanding the activity of the other major cannabinoids and their possible therapeutic properties. The common precursor of all cannabinoids is CBG, which is enzymatically synthesized into the unique phytocannabinoids, giving cannabis its therapeutic potential (Borrelli et al, 2013)
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