Study On The Regulation Of Histone Acetylation On Ganoderic Acids And Polysaccharide Biosynthesis In Ganoderma Lucidum

Ganoderma lucidum,a precious treasures in the traditional Chinese medicine,was also well known as one of the world's large medicinal fungi.Ganoderic acids(GA)and polysaccharides were two kinds of secondary metabolite produced by G.lucidum.A lot of researches revealed that they had many important pharmacological activities,such as anti-cancer and anti-HIV,and so on.The regulation of epigenetic played an important role in many filamentous fungal secondary metabolites biosynthesis.Histone modification was considered to be one of main epigenetic modifications.Among the various modifications,histone acetylation was the most widely studied modification method.At present,there is no information about epigenetic mechanism for regulation of GA and polysaccharides biosynthesis in liquid static culture of G.lucidum.In this paper,Vorinostat(SAHA),an inhibitor of histone deacetylases,was applied to control histone acetylation level of G.lucidum cells.The focus of this work was on understanding the possible regulation mechanism of histone acetylation on growth,spore formation,GA and polysaccharides synthesis and morphology in G.lucidum.Liquid submerged cultivation and two-stage liquid static cultivation were two main cultivation methods for G.lucidum.Previous research suggested that two-stage cultivation was an efficient way to enhance total GA biosynthesis.In this study found that liquid submerged cultivation was benefit to polysaccharides production by G.lucidum.The maximum yield of extracellular polysaccharide(EPS)and intracellular polysaccharide(IPS)production were 10-fold and 1.32-fold that of two-stage liquid static culture.Western blot(WB} analysis revealed that the level of histone H3,H4 acetylation(H3ac,H4ac)were significant higher in the liquid static culture and the relative level were 1.5-,3.0-fold respectively that of liquid submerged culture.Cell was treated with different concentration of SAHA(0,0.6,60,120,180 ?M)during the two-stage liquid static cultivation of G.lucidum.The results showed that the mycelia growth,sporulation and pigment synthesis in the G.lucidum were significantly inhibited compared to control when SAHA were added in liquid static culture.The mycelia morphology were changed when 120 and 180 ?M SAHA were used.The sporulation rate and cell growth were inhibited by SAHA.When the concentration of SAHA were 60 and 180 ?M,the EPS production reached 0.67±0.005 g/L and 0.63±0.04 g/L,respectively,which were 1.20-and 1.11-folds higher than those in the control without SAHA.The maximum contents of IPS in the G.lucidum cell treated with 60 and 180 ?M SAHA were 32.87±0.39 and 34.19±0.56 mg/100 mg dry cell weight(DW),which were 2.47 and 3.72 mg/100mgDW higher than those obtained in the control group.The biosynthesis of GA was significantly inhibited by SAHA,the content of GA was lower by about 15.5%to 36.2%than those of the control.The transcription levels of GA biosynthesis genes(e.g.hmg,sqs,se and Is)were examined by real-time PCR.The results showed that the transcription levels were up-regulated in experiment grops on 6th day and on 9th day.To further investigate the influence of SAHA on biosynthesis pathway of GAs,the transcrption of global regulator laeA and velvet gene were detected.Their expression levels were decreased drastically,and were 5.3-folds and 15.5-folds down-regulated compared to control.The transcrption levels of polysaccharides biosynthesis genes(e.g.ugp,gls,pgm)were also examined,Treatment with 60 ?M SAHA,the expression level of polysaccharides biosynthesis gene were reached a high level,which were 2.6-folds,3.5-folds and 2.5-folds that of the control.The novel rpd3(HDAC)and elp3(HAT)genes from G.lucidum were cloned in silico based on the corresponding G.lucidum EST sequence in NCBI database.Some characters of the proteins were analyzed by information tools.The rpd3 gene from G.lucidum was 1593 bp and it encoded 530 amino acids localized in the cytoskeleton,there was not trans-membrane helical regions,was not a secreted protein,and the protein relative molecular mass was 58.60079 KDa,isoelectric point value was 5.20.The secondary structure were mainly composed of random coil.It contains a typical super family structure of histone deacetylases.The amino acid encoded by rpd3 gene in G.lucidum was highly homologous with those encoded by rpd3 genein Dichomitus squalens and Laetiporus sulphureus.The elp3 gene from G.lucidum was 1665 bp and it encoded 554 amino acids localized in the cytoplasm,there was not trans-membrane helical regions,was not a secreted protein,and the protein relative molecular mass was 62.59957 KDa,isoelectric point value was 8.38.The secondary structure were mainly composed of a-helix.It contains a typical super family structure of histone deacetylases.The amino acid encoded by elp3 gene in G.lucidum was highly homologous with those encoded by elp3 gene in Dichomitus squalens and Trametes versicolor.In this paper,the effect of histone acetylation on GA and polysaccharides biosynthesis were studied,epigenetic regulation mechanism of histone modification on cell growth and secondary metabolism was also discussed.Moreover,this research would be helpful to study on the epigenetic regulation mechanism on other higher medicinal fungi.