| Ganoderma lucidum is a traditional and precious Chinese medicine in our country.Its active ingredients are mainly Ganoderma lucidum polysaccharide and Ganoderma lucidum triterpenes.At present,there are a large number of glossy ganoderma health products on the market,cosmetics and skin care products added with glossy ganoderma components.With the aggravation of social pressure and population aging,people of all ages pay more and more attention to health.With more and more thorough research on the pharmacological effects of Ganoderma lucidum and people's wider acceptance of the concept of homology of medicine and food,it can be predicted that the market demand for Ganoderma lucidum products will also be more vigorous in the future.Therefore,both scientific research and industry urgently need high-quality raw materials for product research and development.However,due to the lack of innovation in Ganoderma lucidum breeding technology in recent years,the special germplasm resources with high polysaccharide content required by the market are very scarce.Therefore,this thesis intends to use the high polysaccharide strain as the starting strain and use UV and ARTP mutagenesis techniques to mutagenize the protoplasts of the starting strain to obtain the mutant strain with high polysaccharide.The main biological research results obtained in this paper are as follows:(1)Optimizing the screening of excellent starting strains and protoplast preparation conditions.Protoplast optimization and protoplast viability identification were carried out for the starting strain.The optimal preparation conditions of protoplast were obtained as follows:mycelium on the 8th day,enzymolysis time for 3h,enzymolysis temperature for31?,centrifugal force of collected protoplast for 1811g g.Under these conditions,the concentration of protoplast prepared reached 1.3×10~8,and the viability was confirmed by staining identification.The regeneration rate of protoplast on common regeneration plate could reach 4‰.(2)Obtaining optimized mutagenesis treatment conditions of ultraviolet and ARTP.The protoplasts prepared from G157 were optimized by UV irradiation and ARTP mutagenesis respectively.It was found that the lethal time for UV irradiation protoplasts to reach 70%was 4 s.The response method was used to optimize the mutagenesis conditions of ARTP.It was found that when the treatment conditions were aeration rate=9.8slm,treatment time=12.88s and protoplast sample volume=30?L,the lethal rate of ARTP to protoplasts reached 90%.Through the somatic incompatibility experiment of ARTP mutant strain,it was found that the aeration rate was in direct proportion to the treatment duration and mutation rate.When the treatment duration exceeded 35s,the effect of aeration rate on mutation rate began to decrease.(3)18 mutant strains with high polysaccharide content were obtained.104 mutant strains were shake-flask fermented and mutant strains with higher polysaccharide content than the original strain were screened.A total of 19 strains have polysaccharide content higher than that of the starting strain G157,of which 12 strains have polysaccharide content higher than 5%of that of the starting strain,of which the polysaccharide content of the three strains A-434,A-194 and A-197 have increased by20%compared with the starting strain,of which A-434 has the highest polysaccharide content,increased by 46%.After comparing the biomass of mutant strains with high polysaccharide content,it was found that the proportion of biomass increase reached 58%,of which 11 strains,A-434,A-316,A-147,A-141,A-189,A-227,A-148,A-194,A-197,A-246,A-51,all exceeded the growth rate of the starting strain,of which the biomass of A-246 exceeded the starting strain 219%.The polysaccharide yield of 104 mutant strains was analyzed.A total of 18 strains were found to have higher polysaccharide yield than the original strain,of which the yield of A-246 was 268%higher than the original strain.The shake flask biomass of strain A-351 was greatly increased by improving the liquid culture medium,which was 15.4 times higher than the original biomass.(4)Confirming the genetic stability and diversity of mutant strains with high polysaccharide content.By comparing the growth rates of the first generation and the tenth generation of high polysaccharide strains and comparing their ITS sequences,it is found that the growth rates of the first generation and the tenth generation have not changed significantly,and their ITS genes have not changed,indicating that the mutagenized strains obtained are genetically stable.RAPD polymorphism analysis of the high polysaccharide mutant strain showed that the genetic similarity of the high polysaccharide mutant strain was between 0.5 and 0.97,and through the observation of the mycelium morphology of the mutant strain,it was found that the high polysaccharide strain obtained in this paper had rich genetic diversity.(5)It is found that the mutant strain with high polysaccharide has biological activity of resisting oxidation and stimulating macrophages to release NO.Through the DPPH radical scavenging experiments of high polysaccharide strains,it was found that the antioxidant capacity of mutant strain A-482 was significantly higher than that of the original strain and other high polysaccharide strains.By measuring the effect of water extracts of different strains on stimulating macrophages to release NO,it was found that the water extract of strain A-316 could induce macrophages to release more NO,which indicated that the ability of the strain to regulate immunity was better than that of the original strain and other high polysaccharide strains. |
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