RESUMEN
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
Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 µg/g, when the concentration of MeJA was 300 µM after 72 h induction.