Breeding High-yield Nucleic Acid Saccharomyces Cerevisiae With Atmospheric And Room Temperature Plasma Mutation Technology And Its Characteristic Analysis

Ribonucleic acid(RNA)not only plays a crucial role in gene expression and protein biosynthesis,but also participates in many physiological functions of cells.RNA is widely used in food industry,medicine and health care as well as animal husbandry.The content of RNA in yeast is generally higher than in other microorganisms,meanwhile the content of RNA in yeast is much higher than that of DNA in yeast.It is easy to extract RNA with excellent quality.Yeast can be quickly cultured in a large scale and the demand for the medium is very low.ARTP mutation breeding technology has successfully carried out mutagenesis on nearly 60 kinds of microorganisms including bacteria,actinomycete,fungi,yeast,microalgae and so on.ARTP has superior performance,which can lead to varieties of DNA damage.Finally,a large capacity mutation library is obtained.High-yield nucleic acid Saccharomyces cerevisiae was obtained by ARTP mutation breeding technology.We could quickly obtain mutants with high cell biomass,high RNA content,excellent performance and stable genetic.It would be able to solve the two key problems: low cell biomass and RNA production of high nucleic acid yeast in China.As a result,it could reduce the production costs and increase the economic efficiency,even provide the foundation for the development of food industry,medical health care and animal husbandry.In this study,Y17 screened from the environment was used as the original strain to isolate the haploid strain.Three consecutive ARTP mutagenesis were carried out.The mutant strain Y17aM3-12 was screened by the sensitivity to potassium chloride and obtained a significant increasing in RNA content.Then the 26 S rDNA identification,physiological and biochemical characteristics and passage stability analysis of Y17aM3-12 were carried out and the cultivation was optimized.The results of the study are as follows:When Y17 was induced to produce spores in the MacConkey medium,sporulation rate was 32% at the seventh days.We obtained 4 type a haploid strains and 3 type alpha haploid strains.Determining the content of RNA,a type a haploid strain had the highest content of RNA with 90 mg-RNA/g-DCW.It was used as the original strain and repeatedly screened of with high RNA content strain.Finally,a mutant strain Y17aM3-12 with the RNA content of 39% higher than that of original strain Y17 was obtained.26S rDNA of Y17aM3-12 was identified and the results showed that the 26 S rDNA sequence had 100% homology with Saccharomyces cerevisiae NL9-9.The morphological characteristics,sugar fermentation,carbon source assimilation and nitrogen assimilation ability of Y17aM3-12 were similar to those of Y17.The result is consistent with the manual of Saccharomyces cerevisiae identification.Y17aM3-12 glucose tolerance concentration was 10%,KCl tolerance concentration was 160 g/L,and the best growth was at pH5.5 acidic condition.The culture medium and conditions of Y17aM3-12 were optimized.Through the experiment of single factor,we finally established the optimum inoculum concentration was 10%,the optimum pH was 5.5,the optimum temperature was 26 ℃,the best carbon source was molasses,the best nitrogen source was peptone,and the best phosphorus source was phosphoric acid.Through orthogonal test,we established the best combination of culture medium compositions and culture conditions.The inoculum concentration was 5%,the culture temperature was 30 ℃,the initial pH was 5.5,Molasses concentration was 10%,peptone concentration was 2%,and phosphoric acid concentration was 1.0 m L/L.In this case,the OD value of growth of Y17aM3-12 increased from 14 to 23 and increased by 64%;and the content of RNA increased from 112 mg-RNA/g-DCW to 142 mg-RNA/g-DCW increased by 26%.