Identification of candidate genes affecting THC biosynthesis in Cannabis sativa

Loki7 · Sep 25, 2010

Ever wonder what genes are active in those frosty trichomes making that stony goodness?

Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa

RNA isolated from the glands of a Δ9-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.
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-Loki

Ugyatag · Sep 25, 2010

Very interesting stuff. Does this give us any clues as to the role of THC for the plant? The article mentions similarities to a substance produced by glands in female hop plant that is thought to have some antibiotic properties.

This could be the basis of some real insights into THC, which has been seriously lacking for a substance of such wide use and possible medicinal use.

Loki7 · Sep 26, 2010

Ugyatag said:

Very interesting stuff. Does this give us any clues as to the role of THC for the plant? The article mentions similarities to a substance produced by glands in female hop plant that is thought to have some antibiotic properties.

This could be the basis of some real insights into THC, which has been seriously lacking for a substance of such wide use and possible medicinal use.
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To me, I see two very useful things... really they are the same thing. With the mRNA sequenced (or rather the cDNA that comes from the mRNA) we know the genes that are active, so we can eliminate all the genes that aren't active. This creates a much smaller pool of DNA to fish in. That in itself is huge. But more importantly, they've identified the missing link, even though they themselves didn't realize it.

CBG... mysterious CBG... a prenyltransferase, but more than that...

THC (TETRAHYDROCANNABINOL) ACCUMULATION IN GLANDS OF CANNABIS (CANNABACEAE)

1. THC accumulated in abundance in the secretory cavity where it was associated with the: a) cell walls, b) surface feature of secretory vesicles, c) fibrillar material released from disc cell wall, and d) cuticle. It was not associated with the content of the secretory vesicles. The association of THC with structural components, particularly the wall, fibrillar matrix and surface feature of vesicles, suggests that it may be chemically bound to them rather than being free in the cavity. If THC and other cannabinoids are bound to components in the cavity, their presence and movement may require a source of energy in the cavity. Additional studies are necessary to determine their bound or free status.

2. Little or no THC was detected in the cytoplasm of the disc cells. This suggests that the terpene and phenol precursors, which must occur in the disc cells at some interval, may form the cannabinoids at the surface of the plasma membrane, or in the cell wall facing the secretory cavity.

3. Some THC was present in the cell walls of other cells. Genes for cannabinoid synthesis are present in all cells of the plant, but tissues other than glands produce low levels of these compounds.

4. Reduction or elimination of glands by mutation procedures will reduce significantly the quantity of THC in the plant. However, the pathway for cannabinoid synthesis is controlled genetically: glands are specialized to synthesize high levels of cannabinoids. Thus, a glandless plant can be expected to synthesize very low levels of cannabinoids. We do not know the roles of cannabinoids in the glands. They may be involved in some way with "protection", or other role. The absence of glands may or may not alter the functional role. Since other cells also synthesize these compounds, at very low levels, the quantity may be sufficient to perform the functional role. Therefore, a glandless mutant(s) would serve our purpose to reduce the THC concentration for utilization of such strains in the hemp industry.
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It's a membrane bound prenyltransferase, a CBG pump, if you will... It completes the THC biosynthesis pathway and opens the doors to cannibinoids in other plants.

-Loki

bobba loo · Sep 26, 2010

thc tomatos xD

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Identification of candidate genes affecting THC biosynthesis in Cannabis sativa

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Identification of candidate genes affecting THC biosynthesis in Cannabis sativa

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