Preliminary Study On Mechanism Of Triterpenoids Accumulation Induced By Hydrogen Sulfide In Phellinus Igniarius Mycelium

In this thesis, the one which had the highest content of betulin was chosed as the subsequent material from four Phellinus igniarius mycelium strains. Then the influence of H2S and CaCl2on the content of betulin, the effects of H2S treatment on different forms calcium content, and activity of calcium key enzymes were explored. In the meantime, transcriptome of Phellinus ignirius mycelium was studied. The results were as follows:1.0.1mM,0.5mM,1mM,2mM,5mM,10mM,15mM,20mM,50mM of NaHS were added into Phellinus igniarius mycelium culture, the results showed that low concentration of NaHS increased the content of betulin, and high concentration of NaHS reduced it,2mM NaHS was the optimum concentration, and the content of betulin improved90.95%compared with control; Based on the result of before, NaHS was added into Phellinus igniarius mycelium culture for12h,24h,48h,72h,96h at the fouth, eighth, twelfth days, the results showed that the content of betulin improved except4d48h and8d12h, and8d48h of NaHS treatment was the best effect which increased by112.43%compared with control on the content of betulin.1mM,5mM,10mM,15mM,20mM,50mM CaCl2were added into Phellinus igniarius mycelium culture, the results showed that the content of betulin increased gradually with the increase of the concentration of CaCl2. When the concentration was10mM, the effect was the best, and the content of betulin improved102.86%compared with control. Along with the increase in the concentration, the content of betulin had a downward trend. CaCl2was added into Phellinus igniarius mycelium culture for12h,24h,48h,72h,96h at the fouth, eighth, twelfth days, the results showed that the content of betulin improved and8d96h of CaCl2treatment was the best effect which increased by99.83%compared with control on the content of betulin.1mM,5mM,10mM,15mM,20mM,50mM of CaCl2were added with2mM NaHS, the results indicated that the content of betulin presented the downward trend after rising first, and lOmM CaCl2treatment was optimum and the content of betulin increased by143.37%compared with control. Calcium channel blockers were added with2mM NaHS treatment, then the content of betulin decreased. So the results showed that H2S and CaCl2interactioned and then improved the content of betulin.2.0.5mM,2mM,10mM of NaHS were added into Phellinus igniarius mycelium culture for3h,6h,9h,12h,18h,24h,48h, and the results showed that the content of calcium in different forms and activity of calcium key enzymes had different degrees of change. The overall trend was2mM>0.5mM>10mM, and the treatment of (2mM,48h) was the optimum one. Calcium channel blockers were added with the2mM NaHS, then the content of calcium in different forms and activity of calcium key enzymes decreased. As a word, calcium mediated the role of H2S in promoting.3.0.5mM,2mM,10mM NaHS were added into Phellinus igniarius mycelium culture for3h,6h,9h,12h,18h,24h,48h, and the results indicated that the changes situation of souble protein content was the same with acticity of calcium key enzymes, and (2mM,48h) was the optimum treatment. However, the changes of superoxide anion generation rate and total protease activity were contrary. The results implied that (2mM,48h) of NaHS treatment made mycelium activity the highest. Calcium channel blockers were added with the2mM NaHS treatment and then souble protein content decreased and another two increased, the results showed that calcium mediated the role of H2S in promoting to mycelium activity.As a word, interactions of H2S and CaCl2improved the content of betulin, and calcium mediated the role of H2S in promting the synthesis of betulin.4. Using the Illumina high-throughput sequencing technologies to sequence transcriptome and de novo assembling, the results showed that25811unigenes were got, in which19266unigenes were annotated to the NR database,86.2%of the genes had high homology with Fomitiporia mediterranea,7831unigenes were annotated to the COG database,6845unigenes were annotated to the GO database, and11088unigenes were annotated to108metabolic pathways. Digital gene expression profile analysis was used between four samples which were control、treatment of H2S-3h、treatment of H2S-12h and treatment of H2S-24h. The results illustrated that4836unigenes expressed differentially in control vs H2S-3h, and4017unigenes expressed differentially in control vs H2S-12h and4222unigenes expressed differentially in control vs H2S-24h. GO classification results were consistent with the whole transcriptome, which were positioned on the cell structure, executing catalytic activity and participating in the metabolic process. KEGG annotation was used to analyze metabolic pathways of terpenoid, the results showed that a large number of unigenes which presented thiolase, HMGS, HMGR, MVD, IDI, FPPS in metabolic pathways of terpenoid expressed up-regulatly.