Study On The Mutagenic Effect And Mechanism Of D-ribose Producing Strain By Ion Implantation

D-ribose is a kind of very important pentose. It is the constituent of biological geneticmaterial mRNA, rRNA,5s RNA, et. Various kinds of RNA and many vitamin andcoenzyme. In organisms usually it transports together with the phosphate, have veryimportant physiological, biochemical effect. And in the pharmacy industry,clinical medicine,foodindustry,cosmeticsandfeedindustryandotherrelatedindustrieshavewidelyapplication.IndustrialproductionofD-riboseusuallybyfermentation.At present, in China the fermentation strains for production is Bacillus subtilistransketolase mutant. It has poorer tolerance to the fermentation product, fermentationconversionrateislow,it’s alsomoredifficultextracting highpurityfermentationproductfromthe fermented liquid.Although using a variety of physical and chemical methods of mutationbreeding,inChina,thetolerationtoD-riboseofthestrainhasnoobviouslyimprovement,thenitsfermentationlevelalsohasnoobviouslyimprovement.Asakindofuniquemutagenicmechanismandbiologicaleffectofmutagenicsource,theapplications of ion beam in mutation breeding is very quickly. Ion implantation is anintegrated mutation method,is the characteristics collection of physical mutagenesis inducedand chemical mutagenesis. When used in low dose injection and the cell damage is lighter,itcan strongly influence biological cell physiological, biochemical properties, resulting in thechangementbasic unit ofthe geneticmaterial──bases, inducing thevariation ofchromosomestructure.Thistechnologyhassmallerphysicaldamage,andgetahighermutationrate,a widespectrum of mutation, and the equipment is simple, easy to use, low cost, to the human bodyand environment harmless etc. In the microbial mutation breeding research, the use of ionimplantationonimprovedstrainshasmaderemarkableresults.Thispaperaimstostudythemutageniceffectandthemechanismofactionoflowenergynitrogen ion implantation D-ribose producing strain, and screen out the high yield strain. Thedissertationisdividedthecontentintofourparts,specificasfollows：Thefirstpart：investigatethetransformationperformanceofthebiologicalstrain Investigate the D-ribose transformation performance of the laboratory existing bacteria,theresultsasfollows:Theactivatedstraingrowsfaster,staysingoodcondition,hasstablegeneticperformance,theshakeflask fermentation cultivationof72h,thesugaryield can bestabilized at about65.3g/L,canbeusedinsubsequentimplantationmutation.The second part：the mutagenic effect of low energy nitrogen ion implantation to theD-riboseproducingbacteria.Using ion implantation technology mutation D-ribose producing bacteria, bacillussubtilis ketol enzyme variants, results show that the induced strains survival rate and N+ionimplantation dose relationship present typical saddle type curve, mutation rate with theincrease of the dose increased, after reach a certain level, then increase the dose, the mutationrate down, are more mutations appeared in the low dose, and the more negative changeappeared in high doses. According to the induced strains survival rate and positive mutationrateof10keV, calculate and determine theion implantation energy induced into thebest doseof12x10¹⁴ion/cm². Under optimum mutagenic dose selection for the plant stable high yieldmutantstrain.The third part：study the effect mechanism of the low energy ion to the D-riboseproducingstrainStudied after different dose of low energy nitrogen ion irradiation of D-ribose producingstrain changes in protein expression level, explore the ion implantation of thalli proteinmolecules expression level. After ion implantation mutation of the bacterium total proteinSDS-PAGE electrophoresis experiment, protein electrophoresis, according to the results ofvarious strains of protein under different mutagenic dose effect, in many bands on thepresence of and the change of light and shade, showed that ion implantation damage inbacteria is heritable and multiple loci broad spectrum model, so as to lay the foundations forthefurtherorientationtransformationstrain.Thefourthpart：optimizethefermentationconditionofthehighyieldstrain Investigation the genetic stability of the high yield mutagenesis strain, grope thecultivation and fermentation conditions for the high yield strain, maximize the biologicaltransformationperformanceofthehighyieldstrain,improvetheproductionofD-ribose.According to the results of orthogonal test, optimum fermentation culture mediumcompositionis:Glucose21.0%,Cornstarch2.8%,（NH₄）2SO₄0.9%,MnSO4·4H2O0.005%,CaCO32.0%. Determine the optimum culture conditions for the fermentation:37℃, initialpH7.0, quantity of12%, with fluid amount to25/250ml, table speed230rpm.The optimumfermentation culture medium and the optimum culture conditions, after72h shaking flaskfermentation, Bacillus subtilis W-N-23’s D-ribose producing an average of98.2g/L, up50.38%comparedwiththestartingstrainproductionperformance.Can consider to amplify the strains used in fermentation experiments, further explore thestraincapacity,inordertoappliedintheindustrializationassoon aspossible,obtaineconomicbenefits.