The Study On Plasma Mutation Breeding Of Marine Pharmaceutical Fungus

The costs and threshold of pharmaceutical research is increasing day by day and always restricts the development of new drug. Plasma technology, a new means of breeding bacterial species, has brought the hope to solve the pharmaceutical bottleneck problem. There are more and more reports concerning about plasma mutation breeding technology, but its appliction in marine pharmaceutical microbial strains breeding and genetic modification are less reported. It is hardly known as to be used in marine pharmaceutical fungus.In this paper, the experiment was based on original strain, a sporogenous marine fungus named Aspergillus unguis DLEP2008001who had the characteristic of anti-tumor and anti-bacterial, for small scale of plasma mutagenesis. We selected51bacterial colonies in the induced strains randomly and the rate of positive mutation reaches15.7%. The rate of mutant strains whose active were more than20%had reached3.9%. The experiment preliminary confirmed plasma induction can improve the yield of active substance in the original strain.Plasma,5-fluorouracil, diethyl sulfate and Lithium chloride served as inductors which applied to original strain Aspergillus unguis DLEP2008001and obtained the rule curve between the fatality rate and revulsant dose. We made a composite mutation plan which referenced to the above rule curve, getting three groups of composite conditions which was suitable to mutagenesis. Exploring experiment confirmed the8groups of suitable mutagenesis condition, they were plasma inducing by the80s, DES1%6min+plasma inducing9s, LiCl4.5%+plasma inducing30s, LiCl3%+DES1%6min+plasma inducing40s, DES6%12min+plasma inducing80s, LiCl6%,5-FU:320μg/mL, DES6%12min.In this paper, we carried out shaking fermentation of the original strain, and observed the yield and richness of its secondary metabolite under different fermentation days, eventually determined7d as the best fermentation days for original strain.In this paper,we carried out large-scale mutagenesis based on6groups of good conditions which got from the exploring experiment. We determined10groups of mutant strains who had character of high yield through preliminary screening (96-well plates table, activity test of resistance of E. coli AB3027in the way of filter), they are2-1-F-10,6-20-18,3-9-D-2,4-D-2-1,8-5-F-11,1-3-C-6,6-10-C-7,2-4-C-9,10-4-B-4,2-7-A-7. Secondary screening (fermentation extract, activity test of resistance of E. coli AB3027in the way of filter, TLC, bioautography, half the preparation of active track, HPLC-DAD-MS), MS qualitative analysis(Agilent Mass Hunter Qualitative Analysis B.06.00) and database retrieval (Dictionary of Natural Products on DVD2011), it also proved that the plasma mutation breeding technology can stimulate bacterial strains to produce more diversified and new structures、produce leading compounds.To summarize, through the application of plasma induction in marine pharmaceutical fungus Aspergillus unguis DLEP2008001, and studied on secondary metabolites of mutant strains.We carried out that plasma induction had a certain potential in Marine pharmaceutical fungus’ breeding, perhaps plasma induction is a new way to solve pharmaceutical bottleneck problem of marine Pharmaceutical’s research.