| Euglena gracilis,belonging to the Euglenozoa(Euglenophyceae,Euglena),is a unicellular eukaryotic organism,without cell wall.the National Health and Family Planning Commission listed it as a new resource food,the β-1,3 glucan produced by E.gracilis has anti-tumor,immune regulation,uric acid-lowering,and other functions.Therefore,it is of great significance to study its industrial production,metabolic mechanism and genetic transformation.In this study,the fermentation conditions of albinol mutant E.gracilis under heterotrophic conditions were optimized,so that the biomass and β-1,3 glucan content of albinol mutant E.gracilis were greatly increased,and the intracellular small molecule metabolites of wild type E.gracilis and albinol mutant E.gracilis were studied in different periods of fermentation.Finally establish a method for nuclear gene transformation of wild type E.gracilis,the main results are as follows:In order to increase albinol mutant E.gracilis cell number,cell biomass(dry weight)and β-1,3 glucan content,the single factor test,PB test and response surface test were carried out.The results of single factor test showed that when 10 g/L glucose,20 g/L fructose,10 g/L corn steep liquor,5 g/L peptone,and 2 g/L yeast powder were the only carbon and nitrogen sources,cell growth was higher,and sodium nitrate showed inhibition of albinol mutant E.gracilis at different concentrations.The results of PB test showed that peptone>glucose>yeast powder>fructose>corn steep liquor.The peptone,glucose,yeast powder and fructose has a significant effect on albinol mutant E.gracilis growth.The response surface experiment was designed according to the four nutrients with significant effects,the simulation equation was established,and the optimal ratio of the complex medium was determined.The results showed that the concentrations of glucose,fructose,peptone and yeast powder were respectively 8 g/L,20 g/L,5 g/L,3.5 g/L,the cell number was the highest when cultured for 4 days,the concentration of cells about 1×10~7 cells/mL,and the yield of dry weight about 10 g/L.Feeding to the material concentration to the initial medium in second day.the 5 L fermentation tank has achieved a higher cell number about 1.5×10~7 cells/mL,the yield of dry weight about 15 g/L and paramylon 10 g/L.GC-MS was used to detect the thermal stable and volatile intracellular small molecule metabolites of wild type and albinol mutant E.gracilis algal strains in different fermentation stages(lag,exponential,stationary),and 2100 components were detected and with 122 metabolite were identified with 70 match factor in wild type,2191 components were detected and with 100 metabolite were identified with 70 match factor in albinol mutant.According to the PCA,PLS and loading analysis.It was found that the small molecule metabolites of wild type and albinol mutant had changed during the three growth periods.The main changes in the two substances were alcohols,esters,Carbohydrates,amino acids and organic acids,such as D-sorbitol,Myristate myristate,Lactose,L-alanine,Myristate,etc.Different metabolites were obtained by pairwise comparison of metabolites in each growth stage of wild type and albinol mutant algal strains by VIP map,and t-test was performed to obtain metabolites with large differences in different stages.The pCambia1300 plasmid was successfully introduced into wild type E.gracilis genome.But the transformant cells did not exhibit EGFP fluorescence.The constructed ClP1-1300-KsdD plasmid also was introduced into wild type E.gracilis.However,whether KsdD gene is expressed is still need to be validated. |
