Study On The Correlation Of Endophytic Fungi And Its Secondary Metabolites In Rabdosia Rubescens

Objective: To study the correlation between endophytic fungi and its secondary metabolites in Rabdosia rubescens. Study on the biological diversity of these endophytic fungi. Found out the active fungi which have the ability to produce oridonin. By using the methods of mutagenesis, try to find a higher yielding strain, and optimize this stable high-yielding mutant strain’s fermentation process to improve its production of oridonin. By the molecular biological identification of M-J-5 to determined its taxonomy position. And inhibition test was used to detect the invitro human breast cancer by these fungi researching for its antitumor activity. Methods: The topic uses the different parts(leaf and stem)of Rabdosia rubescens, The method of tissue separation was used to separating these fungi, Identifition the enpdophytic fungi which separated from Rabdosia rubescens, and study these fungi’s species composition and distribution; Through the Thin-Layer Chromatography method and the High Performance Liquid Chromatography method, try to found out the fungi which have the ability to produce oridonin, and study on its biological characteristics; Through the UV and Na NO2 mutagenesis and UV- Na NO2、Na NO2-UV compound mutation methods by the high-yield mutations fungus and content its oridonin’s production; Optimization of fermentation about the mutant strain by single factor and four factors of three levels orthogonal test; By using molecular biology method to indentify M-J-5; The MTT assay was used to detected the inhibitory effect on multiplication of human breast cancer cells by oridonin. Result 1. The population composition of Rabdosia rubescens endophytic fungi.From the Rabdosia rubescens 256 endophytic fungi had been isolated, these fungi can be identified in 4 orders, 6 families, 9 generals, and there are 77 fungi belong to Penicillium which has accounted for 30.1%. Acorroding to all fungi which have been identified the Penicillium is the major species in Rabdosia rubescens endophytic fungi. 2. Study on the secondary metabolites of Rabdosia rubescens endophytic fungi.Use the methods of Thin-Layer Chromatography, High Performance Liquid Chromatography to detective the endophytic fungi which separated from the leaf and stem of Rabdosia rubescens, there are 2 fungi have found out a fungi which have the capacity for oridonin, and the highest production of oridonin strain is M-J-5. This fungus’ s oridonin production was reached 0.211g/L 3. The high yield mutagenic test of the M-UH-4 which producing oridoninThe methods of UV and Na NO2 single factor mutagenesis and UV- Na NO2、Na NO2-UV composite mutagenesis, finally found a fungi of M-HU-4 which can be identified a high-yielding mutant fungus, this fungus’ s production of oridonin has reached 0.219 g/L, by contrast with M-J-5, the oridonin production had yielded 4.11%. 4. The best fermentation test of the M-UH-4 which producing oridoninBy using the temperature, rotational speed, PH, inoculation, fermentation day single factor method and orthogonal experiment. An optimal fermentation conditions have been found out: choose PDB medium as the basical medium, PH is 7.0, glucose is extract on 10.0g/L, with 2% inoculum yeast had added to 5.0g/L,, temperature constant at 26℃, rotational speed is130 r /min and cultured for 6 days. By contrast with the original fermentation conditions, the oridonin production has reached 0.214 g/L, increased1.47%. Mycelial biomass has reached on 8.13g/L, increased 6.77%,5. The molecular biological identification of the M-J-5 which producing oridoninUsing the method of CTAB try to extract the DNAwhich from M-J-5, then contrast its ITS sequence, choose some high relevance sequence from NCBI database and contrast by BLAST, comprehensive analysis by morphology characteristics identification, M-J-5 can be identified to Penicillium。6. MTT assay was used to detected the inhibitory effect on multiplication of human b-reast cancer cells by the strain M-UH-4 extraction of fermentation brothThe MTT assay was used to detected the inhibiting effect on multiplication of human breast cancer cells by the different concentration extraction of fermentation broth,during 24h、48h、72h the lethality rate wer78.27%、83.62%、83.96% respective by dilute with 10-3. The result showed that there are obvious inhibitory effect of human breast cancer cells by secondary metabolite of the strain M-UH-4. Conclusion: By discussion of the endophytic fungi in Rabdosia rubescens and its secondary metabolites, preliminary identify the population composition of Rabdosia rubescens endophytic fungi, description these fungi have biological diversity; found a active fungus which can produce oridonin and use mutagenesis methods selected a stable high-yielding mutant strain, The optimal fermentation test showed a best condition to cultured M-HU-4.;The strain MJ5 is identified as Penicillium; According to the inhibition test which used to detective the invitro human breast cancer, this fungns had antitumor activity.