Dataset Information

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase.

ABSTRACT: Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

SUBMITTER: Fulvio F

PROVIDER: S-EPMC8466818 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase.

Fulvio Flavia F Paris Roberta R Montanari Massimo M Citti Cinzia C Cilento Vincenzo V Bassolino Laura L Moschella Anna A Alberti Ilaria I Pecchioni Nicola N Cannazza Giuseppe G Mandolino Giuseppe G

Plants (Basel, Switzerland) 20210908 9

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses hav ...[more]

PMID: 34579390

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Project description:Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30-37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3.

Project description:The discovery of phytocannabinoid synthesizing enzymes, tetrahydrocannabinolic acid synthase (THCAs) and cannabidiolic acid synthase (CBDAs) was a breakthrough in Cannabis research. However, their evolutionary aspects and distribution across organisms has not been adequately studied. We searched for THCAs and CBDAs genes in organisms other than Cannabis plants using the database available in NCBI. Both cannabinoid synthases seem to be widely distributed in the plant kingdom. Of several complete or partial sequences of cannabinoid synthases-likes, CBDAs-like from Morus notabilis matched closely to CsCBDAs and CsTHCAs. When amino acid sequences of CsTHCAs, CsCBDAs and MnCBDAs-like were compared to each other, and to the motif database stored in Expasy, all three proteins contained the FAD_PCMH (PCMH-type FAD-binding) domain indicating the conservation of this domain in cannabinoid synthases. Apart from FAD binding, Berberine Bridge Enzyme (BBE-likes), which catalyzes the synthesis of isoquinoline alkaloids in many plants such as mulberry, poplas and citrus, were the other most closely related enzymes to CsTHCAs and CsCBDAs. We also searched for THCAs and CBDAs in fungal and bacterial kingdom but could not find any notably similar sequence. However, partial mRNA from FAD binding enzyme from Trametes versicolor and 6-hydroxy D nicotine oxidase from Aspergillus saccharolyticus matched the CsTHCA sequence and a partial mRNA from a hypothetical protein in Pneumocystis carinii was the most closely matched fungal enzyme to the CsCBDA. Our database search showed that Morus notabilis from mulberry family could be the candidate plant for further studies. Comparative transcriptomic and metabolomic studies for mulberry and Cannabis plants could provide a much clear concepts on the co-evolution of these syanthases. Moreover, the understanding of cannabinoid synthesis pathway is still evolving, in-depth bioinformatics and functional analysis of the enzymes involved are required for pharmaceutical research and industrial advancement.

Dataset Information

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase.

Publications

Analysis of Sequence Variability and Transcriptional Profile of <i>Cannabinoid synthase</i> Genes in <i>Cannabis sativa</i> L. Chemotypes with a Focus on <i>Cannabichromenic acid synthase</i>.

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