http://medicalcannabis.weebly.com/uploads/2/7/9/4/2794475/identication_of_candidate_genes_affecting_thc_biosynthesis_in_cannabis_sativa.pdf
RNA isolated from the glands of a
Cannabis glands.
Abstract
RNA isolated from the glands of a
D9-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
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.
