Conserved Sequences in Cannabaceae

Doty, J.A., Horrocks, J., and Lackeyram-Owen, S.

University of Maine at FarmingtonDepartment of Natural Sciences

Results

Methods

Conclusions

Cannabis has been shown to successfully treat a wide variety of chronic health conditions (1-2). Treatment success comes from the phytocannabinoid compounds present in Cannabis, which are known to function through the human endocannabinoid system (3). Phytocannabinoids bind to CB1 receptors present in CNS and PNS tissues, or CB2 receptors present in immune and hematopoietic systems (4), where they elicit various responses (Figure 1).

Table 1. THC:CBD ratios in the three Cannabis chemotypes (6).

The goal of this project is to identify conserved sequences in THCA and CBDA synthetase genes, and use these sequences to develop DNA barcodes for chemotype identification. We predict these barcodes will more accurately identify Cannabis chemotype.

Introduction

Conserved sequences were identified through published studies and by comparing published THCA and CBDA synthetase sequences using Jalview sequence alignment software.

Hemp seed (C. ruderalis) DNA was isolated and these conserved sequences were targeted for amplification and sequencing. Sequence data was analyzed and validated using published sequences in GenBank.

Phytocannabinoid chemotaxonomic studies have identified three distinct phytocannabinoid profiles in Cannabis (6). These chemotypes are characterized by their THC:CBD ratios (Table 1).

Chemotype inheritance is governed by a mono-allelic system involving the genes that produce either CBD or THC from CBG. These synthetase enzymes, THC-acid (THCA) synthetase and CBD-acid (CBDA) synthetase, are simply isoforms of the same enzyme encoded at the same locus (B) (7). BT codes for the THCA synthetase isoform and BD codes for the CBDA synthetase isoform. A BT/BT genotype codes for Chemotype I, BT/BD genotype codes for Chemotype II, and BD/BD codes for Chemotype III (7). Genotype tests have been developed for chemotype identification, but these tests fail to capture phytocannabinoid profile variation within chemotypes (8).

Figure 1. Endocannabinoid system model showing phytocannabinoid binding to endocannabinoid receptor CB1 (5).

These confirmed consensus sequences in C. ruderalis provide a basis for DNA barcoding. Once our DEA Schedule 1 permit is secured, we will target these same conserved sequences in C. sativa and C. indica to confirm their utility for this purpose. Recent studies indicate THCA and CBDA synthetase genes vary not only in copy number, but also in expression levels (8). This may necessitate using quantitative PCR to fully characterize Cannabis chemotype.

1. Johnson , J., et al. 2010. Multi-center, double-blind, randomized, placebo-controlled, parallel-group study of the efficacy, safety and tolerability of THC:CBD and THC extracts in patients with intractable cancer-related pain. Journal of Pain and Symptom Management. 39(2):167-179.

2. Bucatello ,E., et al. 2011. Acute and chronic cannabinoid extracts administration affects motor function in a CREAE model of Multiple Sclerosis. Journal of Ethnopharmacology. 133(3):1033-1038.

3. Pacher, P., S. Bataki, and G. Kunos. 2006. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol. Rev. 58:389-462.

4. Pertwee, Roger. 1997. Pharmacology of CB1 and CB2 Receptors. Pharmacology and Therapeutics. 74(2):129-180.5. Guzman, Manuel. 2003. The endogenous cannabinoid system. Nature Reviews Cancer. 3:745-755.6. deMeijer, E.P.M., et al. 2003. The inheritance of chemical phenotype in Cannabis sativa L. Genetics. 163:335-346..

References7. deMeijer E.P.M. and K.M. Hammond. 2005. The inheritance of chemical phenotype in Cannabis sativa L. Euphytica. 145:189-198.8. Allen, L.N., et al. 2016. Complex variability within the THCA and CBDA synthetase genes in Cannabis species. J Forensic Investigation.

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Figure 3. Section of conserved regions within the CBDA (top) and THCA (bottom) gene sequences generated via Jalview sequence alignment software. Sequences were obtained through GenBank (CBDA Accession number: AB035796.1, THCA Accession number: KT876047.1).

This research was supported by Maine IDeA Network of Biomedical Research Excellence (INBRE) funding from the National Institutes of GeneralMedical Sciences of the National Institutes of Health under grant number P20-GM103423.

Figure 2. THC and CBD biosynthetic pathway, showing enzymatic and non-enzymatic conversion.

Figure 4. Percent match between amplified THCA and CBDA synthetase fragments and published sequence data in GenBank for two different primer sets. THCA-1 and CBDA-1 target a 1,600 bp region while THCA-2 and CBDA-2 target a 500 bp region.

Figure 5. Section of a CBDA sequence for a Chemotype III sample.