Knockout Of Uridine Diphosphate Glucose Dehydrogenase Gene And Studies On Mutants Genetic Characteristics

An extracellular polysaccharide produced by Xanthomonas campestris is called xanthan gum.The synthesis of xanthan gum is a complicated and delicate process that requires the participation of various enzymes and other factors such as transferase I-V,ketotransferase,acetylase,uridine diphosphate glucose dehydrogenase,polymerase and so on.The molecular genetics research of Xanthomonas campestris is also paid more and more attention.Through the elucidation of its biosynthetic pathway and the establishment of Xcc genetic research system,some progresses have been made in the engineering of directional transformation and construction of recombinant strains by genetic engineering.This project selected Xanthomonas campestris uridine diphosphate glucose dehydrogenase gene as the research object to explore its effectim to genetic characteristics of Xanthomonas campestris.Genomic DNA was extracted from Xanthomonas campestris as the original strain;the uridine diphosphate glucose dehydrogenase gene was amplified;the uridine diphosphate glucose dehydrogenase gene was ligated into the pMD~?19-T vector and transformed into E.coli Competent cells;identified uridine diphosphate glucose dehydrogenase gene pK18mobSacB plasmid linked to competent cells into E.coli,identified and then transferred to X.campestris competent cells;through the gene recombinant Xanthomonas campestris mutants were obtained.Comparison of growth curve between mutant and original strain,plate culture analysis of mutant and original strain,comparison of xanthan gum yield and xanthan gum viscosity,Comparison of production among protease,amylase and cellulase between mutant and original strain,Indicating that the genetic characteristics of the mutant may be affected by mutations in the uridine diphosphate glucose dehydrogenase gene.To further verify that the mutation of uridine diphosphate glucose dehydrogenase gene is a factor affecting the genetic characteristics of X.campestris,the overexpression of the target gene was performed.The target gene was linked to the pHM1 plasmid,first transferred into E.coli competent cells,and then transformed into X.campestris mutant competent cells to obtain mutant overexpression strains.Comparison of the growth curves of mutants,original strains and overexpressing strains mutants,comparison of plate culture analysis,Xanthan gum yield and xanthan gum viscosity,comparison of productionamong protease,amylase and cellulose.Furthermore,the genetic characteristics of the bacteria are regulated by the uridine diphosphate glucose dehydrogenase gene.