Establishment Of Genetic Transformation And Recombination System And Exploration Of Gene Knock-out Methods In Bacillus Subtilis 210

With high resistance and antifungal activity,Bacillus subtilis has been widely applied in controlling plant diseases.In previous work,our laboratory has screened out a B.subtilis strain?named B.subtilis 210?with high antifungal activity and broad application prospect.In this thesis,the genetic transformation and recombination systems were established in B.subtilis 210.I have also attempted to establishthe gene knock-out system in B.subtilis 210.My work would lay the foundation for the application of B.subtilis 210 in controlling plant fungi disease.I studied the effects of cell age,electric field intensity,trehalose concentrations and recovery times on transformation efficiency of B.subtilis 210 with the plasmid pWB980 as the donor.The optimal electroporation parameters were as following:OD_600nm=6.44,electric field is 2.0 KV/mm,rehalose concentrations is 0.5 M and revive 3 hours after electroporation.The results showed that cell age and electric field intensity have the strongest effect on the transformation efficiency of B.subtilis 210 and trehalose which may protect cells after electric shock can moderately improve transformation,the highest transformation efficiency of B.subtilis 210 was 9.32×10³ cfu/?g DNA.Although B.subtilis 210was transformable by electroporation,its transformation efficiency is much lower than B.subtilis WB800.Homologous recombination was examinedin B.subtilis 210 by the Spizizen's two-step transformation procedure and the following results were obtained:B.subtilis 210 and WB800 recombinants were obtained with genomic DNA of B.subtilis SCK6 which carries the erythrocin resistance gene?erm^r?;PCR examination results showed that the erm^r gene was integrated to the genomes of B.subtilis WB800 and 210.Although homologous recombination occurred in B.subtilis 210,the recombination activity in B.subtilis 210 was much lower than that in B.subtilis WB800.To improve transformational recombination of 210,210K was constructed,which contained the xylose-inducible comK from SCK6.In our laboratory conditions,the highest competence of SCK6 and210K appeared 2 hours after the cessation of exponential growth?G₂?.Compared with the original strain 210,transformational recombination efficiency of 210K was improved more than 50 folds.Based on the establishment of genetic transformation and recombination system in B.subtilis 210,the gene knock-out system was attempted to be established in this strain and the following results were obtained:?1?temperatrue sensitive plasmid based gene knock-out method is time consming and host dependent with high false positive rate;?2?with the PCR product containing a resistance gene flanked by the homologue arms as the donor DNA,the initial attempt to obtain the desired mutant was not successful in the recipient strain WB800K?constructed in Chapter 3?,which carries the xylose inducible comK.In this thesis,the transformation and recombination systems were established in B.subtilis 210,providing conditions for developing and untilizing of this strain in biocontrol.It was also found that transformation and recombination in B.subtilis 210 were much lower than those in B.subtilis BW800 and thus genetic manipulation is relatively more difficult in B.subtilis 210.Finally,the xylose inducible competence factor encoding gene comK was introduced to B.subtilis 210,providing a foundation for exploring gene knock-out method in the wild strain of B.subtilis.