Preparation, Regeneration And Genetic Transformation Of Bacillus Licheniformis Protoplasts

Bacillus licheniformis is one of the most important strains for industrial biotechnological processes, and widely used in fields such as medicine, plant diseases, food, feed, and environment. The performance of B. licheniformis wide type strain generally can not meet the production need, therefore, genetic modification is needed. The transfer of alien DNA into B. licheniformis is the premise and foundation for molecular operation and genetic study to host, while protoplast transformation is one of the major means of genetic transformation in Bacillus. For there is great difference in protoplast formation,regeneration and transformation of B. licheniformis strains, it's need to carry on the optimized analysis to its influential factors. B. licheniformis 303 is an excellent host strain of Bacillus licheniformis. In this paper, B. licheniformis 303 was served as a host strain, systematic investigation was carried out on factors affecting formation and regeneration of protoplast, prelimiariarily optimized protoplast transformation of B. licheniformis 303, and laid the foundations for further molecular operation.Firstly, some parameters involved in preparation and regeneration including growth phase, lysozyme concentration, cell treatment, osmotic stabilizer and medium composition were optimized. The results showed that the optimal yield of B. licheniformis protoplast was obtained with 8×109/mL when the late logarithmic-phase cultures of 8 h were digested with 100 mg/mL lysozyme at 37℃with gentle shaking (100 r/min) for 40 min, and SMMP (adjusting pH to 6.5) was used as osmotic pressure stabilizer. For the regeneration of protoplast, the DM3 medium containing 1 mol/L sodium succinate was more efficient. The peak of regeneration frequency is 17%.Based on these results, main factors influencing protoplast transformation were studied, including protoplast concentration, plasmid concentration, Mg2+ concentration and post-cultured ways. The results showed that the optimal transformation conditions was obtained under conditions of adding amount of methylated pHY-P43-secQ 2μg, taking 40% PEG6000+20 mmol/L Mg2+ for fluxing agent, concentration of protoplast 3×109/mL, treatment time 5 min, and incubated at 30℃with gentle shaking (100 r/min) for 150 min, the average transformation rate was 15 CFU/μg DNA.