Construction Of Expression Vector Of Sirna Specific To ERG9 And Fermentation Production Of Coenzyme Q₁₀ By Schizosaccharomyces

Coenzyme Q₁₀ comprises a benzoquinone ring linked to a polyisoprenyl chain of 10 units. It's main physiological function come from the oxidation-reduction of benzoquinone and the physical speciality of polyisoprenyl chain. Coenzyme Q₁₀ is an essential electron carrier in the mitochondrial respiratory chain, and widely used as an essential component of ATP generation in the oxidative phosphorylation process and as an lipid-soluble antioxidant preventing lipid peroxidation. Its metabolization is relevant to several diseases. Coenzyme Q₁₀ is extensively researched as an important biochemical remedy and hygienical nurture.Restraining and silencing the expression of squalene synthetase, Schizosaccharomyces pombe Linder's Coenzyme Q₁₀ anabolism was researched, Firstly, To construct eukaryotic vector expressing siRNA (small interference RNA) of ERG9. Designing three different siRNA targeting the coding sequence of the ERG9, the mU6 ERG9 siRNA was constructed by inserting the designed siRNA to the eukaryotic expression vector mU6 pro. We got mU6-ERGP-shRNA10,mU6-ERGP-shRNA32,mU6-ERG9-shRNA585 three mutants. Inserting zeo into ERG9 gene, pGEM-ERG9-zeo gene-knocked expression frame was constructed successfully, the components of the annealing buffer were optimized. Keeping NaCl hyper- 150mmol/L, under pH 7.5,the annealing efficiency reach 90%.By the radicalization of UV, 06-2-8,06-3-15,06-5-27,06-6-11 four high yield mutants were found, The optimum dealing time was 90s, Rate of plus mutation reached 8.33%. the components of the C/N were optimized. The optimum C/N was 1.6, The effect of precursors of Coenzyme Q₁₀ on it's production on the study of metabolic regulation of s.pombe were researched. The results showed that adding precursors could enhance the yield of Coenzyme Q₁₀.the yield of Coenzyme Q₁₀ increased about 14% compare with no adding.The results of zeo antibiotics endurance experimentation showed that the optimum concentration of zeo was 150μg/ml. The yield of Coenzyme Q₁₀ of S.pombe ERG9* mutants reduced about 5.62% compared with the original strains. But they had steady transmissibility and high growth biomass. after 72 generations, the periods of anabolism was shortened 6h, Compared with the original strains, the relative differences of yield of Coenzyme Q₁₀ reduced 6.61%. The results showed that there were a continual degradation and a high mutation with S.pombe mu6-siRNA mutants. The fermentation level reduced about 10.7%, The experimentation has achieved elementary research of RNAi in eukaryotic cell, The results of study lay the foundation for further studying on enhancing the metabolic flux of the CoQ₁₀ pathway by the inhibitive effect on synthesis of squalene.The methods of extracting Coenzyme Q₁₀ were investigated in this thesis. The optimum method was that the cells were pretreated with HC1 at hot water, then extracted with acetone. The optimum temperature and time of pretreating with HCl were 80℃and 80min, and that of extracting Coenzyme Q₁₀ with acetone were room temperature and 2.5 hours.