Identification of candidate genes affecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa

Abstract

RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

Fig. 1.

Isolation of glandular trichomes from mature female bracts. (A) Female inflorescence 8 weeks after germination. (B) Leaves associated with the female inflorescence (note arrow). (C) Glands on the bracts of pistillate florets, each bearing a pale bifid style (note arrow indicating glands. (D) Scanning electron micrograph of capitate glands. (E) Micrograph of bract after removal of glands. (F) Micrograph of isolated glands. Bar in (D) and (F)=80 μm.

Fig. 2.

Biochemical pathways leading from primary metabolites to THCA showing candidate Cannabis unigenes encoding enzymes in the pathway. (A) Production of THCA. (B) De novo fatty acid pathway leading to the formation of hexanol. (C) Breakdown of fatty acids leading to the formation of hexanol. (D) MEP pathway leading to geranyl pyrophosphate. Candidate Cannabis unigenes are shown, as well as the relative gland over leaf expression ratios for a subset of the candidates (see Table 2).

Fig. 3.

Comparison of PKS-related unigenes from Cannabis sativa glandular trichomes. Accession numbers: Arabidopsis thaliana CHS, NP_196897; hop (Humulus lupulus) VPS, BAB121202; hop CHS, CAK19319; Cannabis CHS, AAL92879. The alignment was generated as described in the Materials and methods. The distance relationships are described in the main text.

Fig. 4.

Analysis of PKS activity for two candidate gene products expressed in Cannabis glands. (A) SDS-PAGE analysis of PKS proteins encoded by CAN1069 and CAN24 expressed in E. coli harbouring the corresponding gene expression plasmids. (B) HPLC separation of the reactants malonyl-CoA (mal), coumaroyl-CoA (cou), and hexanol-CoA (hex), and the potential product olivetol. Separation by HPLC of reaction products obtained using CAN1069 protein (C) or CAN24 protein (D) with either hex or cou as substrates. Numbered peaks represent products not seen in control reactions containing boiled protein. (E) Absorption spectrum of product 2 (solid line) compared to naringenin (dashed line). The molecular mass of the most abundant ion species is shown in the insert. (F) Absorption spectrum of product 3 (solid line) compared to olivetol (dashed line). The molecular mass of the most abundant ion species is shown in the insert. Product 1 yielded the same mass and spectrum as product 3 shown in (F).

Fig. 5.

Comparison of potential products produced by two or three decarboxylative condensation reactions. (A) Prediction of pyrone production from two decarboxylative condensations. (B) Expected product from three decarboxylative condensations.