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Rhizospheric actinomycetes and halophilic microbial metabolites preliminary study

Author: ChenYi
Tutor: SunChengHang；ZhangYuQin；JinWenZao
School: Peking Union Medical College , China
Course: Microbial and Biochemical Pharmacy
Keywords: Arabidopsis Rhizosphere actinomycetes Feng added neomycin Seven neomycin Root length Moderately halophilic actinomycetes Multi-spore actinomycetes Separate Purification
CLC: S154.3
Type: Master's thesis
Year: 2009
Downloads: 250
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Abstract

The first part of the Arabidopsis thaliana root length inhibition model put in the rhizosphere actinomycetes screening in principle, under the guidance of the the rhizosphere dialogue (or interaction), the model plant Arabidopsis seed germination and root length as an indicator of microbial secondary metabolites - plant root length inhibition \Plant hormones 6 - benzylaminopurine (6-BA) as a positive control, and distilled water as a negative control Arabidopsis root length - agar mixed drug evaluation method found antibiotic isolated by our laboratory abundance plus neomycin (Toyocamycin similar) with 6-BA regulating the activity of Arabidopsis root growth. 6-BA Hefung plus adriamycin inhibition of Arabidopsis root elongation of 50% of the desired concentration (50% inhibitory concentration) were 0.096 mg / L, and 0.200 mg / L, the activity was slightly higher at the same concentration ,6-BA in abundance plus amphotericin. To facilitate the screening, combined with the agar disk diffusion method optimized the Arabidopsis root length inhibition model Arabidopsis root length - agar diffusion screening methods and medicinal plants in Yunnan 628 actinomycetes isolated from soil rhizosphere fermentation products as a screening object to that found with Arabidopsis root growth inhibitory activity of the compounds. The research results show that 628 rhizosphere actinomycetes, 25 bacteria fermentation broth with different degrees of inhibition of Arabidopsis root growth, the ethyl acetate extract of the fermentation broth the I06A-02110 and I06A-02381 proposed Arabidopsis root growth has a strong inhibitory effect. I06A-02381 secondary metabolites chromatography and activity tracking evaluation experiments show that, with a strong inhibition of the activity of the component content in the Arabidopsis model minimal, with weak activity and both anti-Bacillus subtilis and rice blast fungus activity compound content. High content, a low active ingredient to the chromatographic separation to give a code-2381 compound. At 254 nm, the HPLC analysis showed a purity of 100%. Compound 2381 by high resolution mass spectrometry, ultraviolet spectrum, the hydrogen spectrum, 13C NMR, DEPT spectrum, HSQC spectrum HMBC Spectral confirmed compound 2381 seven known antibiotic neomycin A hydroformylation product. ,6-BA in DMSO and distilled water as negative control and positive control, agar mixed drug evaluation method, determination of the inhibitory activity of the compound of 2381 pairs of Arabidopsis root length, the 50% inhibitory concentration of greater than 1 mg / L, and by calculating, its theoretical value of 7.285 mg / L 6-BA for 50% inhibitory concentration of 0.139 mg / L, the Hydroformylation the seven amphotericin A has a very weak inhibition of the Arabidopsis root growth. Due to various reasons, although not separated into the active target product through this model has been found and traced to the positive strains and the active ingredient, the study further found that by using the model Arabidopsis seed germination and root growth inhibitory activity of the microorganisms metabolites laid the foundation. The second part halophilic microbial metabolites preliminary study based on the new species may produce new compounds assumptions Aydin Lake saline soil in Xinjiang isolated a moderately halophilic actinomycetes I06A-01 708 is selected as the research object. The polyphasic taxonomic research that I06A-01708 multi-spore Actinomyces the new the species (Actinopolyspora nov.sp.), the optimal growth salt concentration of 10% (w / v) NaCl is a moderately halophilic actinomycetes. Preliminary activity studies have shown that the secondary metabolites of the bacterium Bacillus subtilis, Staphylococcus aureus (209P) and Escherichia coli (E. coli) was inhibited, which strongest inhibitory activity of Bacillus subtilis, select subtilis identification of strains of Bacillus subtilis as activity tracking. I06A-01708 growth and fermentation period is very long, we optimize the growth conditions, the generation time of the active product to determine the optimum fermentation period, while its secondary metabolites. The fermentation liquid extraction using ethyl acetate, the residue was purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, thin layer chromatography and high performance liquid chromatography and other separation methods, the separation to a molecular weight of the compound of approximately 1110. It has the activity of inhibiting the growth of Bacillus subtilis, because of its low yield, no sufficient compound for further study. Another component without ultraviolet absorption, but has a strong inhibition of the activity of Bacillus subtilis is the main component of the secondary metabolites of the active ingredient. This is the first test of the separation and purification of the full multi-Actinomyces New Species (Actinopolyspora nov.sp.), secondary metabolites, especially for the separation of UV-absorbing compounds were explored initially identified its separation and purification The basic flow, further separation of work underway.

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