RESUMO
Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue-expressed proteins. These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Proteins extracted from glands, flowers, and leaves were separated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimensional gels from leaves and flowers. The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching. Flower and gland proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified: F1D9.26-unknown prot. (Arabidopsis thaliana), phospholipase D beta 1 isoform 1a (Gossypium hirsutum), and PG1 (Hordeum vulgare). Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.
Assuntos
Cannabis/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/análise , Western Blotting , Canabinoides/metabolismo , Cannabis/enzimologia , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Flores/metabolismo , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Ponto Isoelétrico , Peso Molecular , Mapeamento de Peptídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Distribuição TecidualRESUMO
A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.
Assuntos
Cannabis/enzimologia , DNA Complementar/genética , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Acil Coenzima A/metabolismo , Aciltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Canabinoides/biossíntese , Cannabis/genética , Cannabis/metabolismo , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Malonil Coenzima A/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
Various methods for the analysis of cannabinoids in biological materials, including plant and human body materials, are reviewed. Chromatographic methods, such as TLC, GC and HPLC, and non-chromatographic methods, mainly immunoassays, are discussed and compared. Chromatography is most commonly used in the analysis of plant material, with GC apparently offering the most advantages. Immunoassays, such as radioimmunoassay and fluorescence polarisation immunoassay, and enzyme immunoassay methods, such as enzyme multiplied immunoassay technique and enzyme-linked immunosorbent assay, can be used for human body materials; however, GC-MS is still necessary for confirmation and accurate quantification. Preferred methods are suggested for various specific purposes.
