RESUMEN
Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Geraniltranstransferasa/genética , Matricaria/enzimología , Matricaria/genética , Oxilipinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Aminoácidos , Biocatálisis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Genes de Plantas , Geraniltranstransferasa/química , Geraniltranstransferasa/aislamiento & purificación , Matricaria/efectos de los fármacos , Matricaria/crecimiento & desarrollo , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/química , Proteínas de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.
Asunto(s)
Genes Fúngicos , Geraniltranstransferasa/genética , Poria/enzimología , Poria/genética , Triterpenos/metabolismo , Acetatos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Biocatálisis/efectos de los fármacos , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Cromatografía de Gases , Clonación Molecular , Ciclopentanos/farmacología , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Geraniltranstransferasa/aislamiento & purificación , Datos de Secuencia Molecular , Oxilipinas/farmacología , Filogenia , Pichia/efectos de los fármacos , Pichia/metabolismo , Poria/efectos de los fármacos , Poria/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.