The Effect Of Sporulation Killing Factor Gene Knockout On The Biomass And Amylase Production By Bacillus Subtilis

Bacillus subtilis is widely used in agricultural bio-control and industrial enzyme preparation field.However,in the stationary PHase,cells srarting the expression of genes related to spore formation will also start the expression of sporulation killing factor(Skf)gene,further to kill cells who have not yet started gene related to spore formation and sensitive to Skf expression,and then obtain their nutrition and delay their spore formation,finally results to the decrease of number of living cells and bacteria biomass,this PHenomenon described above is called cannibalism.Bacillus subtilis will synthesize a large number of enzyme protein during the spore formation,including amylase and protease,but it has not been reported whether cannibalism can reduce the amount ofthese enzymes synthesis,In this study,we used Bacillus subtilis WB800 lack of eight kinds of extracellular proteaseadopts as the research object,by the method of homologous recombination,to knockout skf gene,further to achieve strains lacking skf gene,finally to explore skf gene knockout whether can improve the amylase activity when improving WB800 biomass.The main results were as follows:1.According to skf ABCEF gene sequences of bacillus subtilis strain subspecies 168 strain,we designed primers and cloned skf ABCEF genes and their upstream 1140 bp sequences and downstream 591 bp sequence from WB800 strain genome.By comparison,these sequences similarity were high to 100% compared to multiple subspecies of bacillus subtilis skf ABCEF gene sequence published in the Gene Bank,which Showed that skf gene is very conservative in bacillus subtilis.2.Through three overlapping PCR reaction,we fused the three fragments respectively skf ABCEF upstream of homologous sequences(1140 bp),?tetracycline resistance genes expression box(1436 bp)and the skf ABCEF downstream homologous sequences(591 bp).And then,we connected the fusion sequencing with p MD19-T Simple vector,and sequence the fused fragment.The sequence blast result showed that connection order and base sequence of DNA fragments to construct homologous recombination knockout skf ABCEF gene are both correct.3.Using the method of modified chemical Spizien,we transformed the DNA fragments into WB800 to knockout skf ABCEF gene by homologous recombination,and then we conduct the transgenerational genetic stability test and PCR identification,further to get a stable genetic transformant WB800 ?skf ABCEF tetr called strain NO.5.4.Shake flask culture the transforment NO.5 and WB800 in LB medium,and OD value determination results culture and microscopic observation both showed that the number of bacteria and spore of transforment was 108.67% and 108.72% higher than original strain.At the same time,the determination results of showed that the ?-amylase vitality in medium of transformant increased by 30% ~ 65% compared with the original strain.