| Lactic acid bacteria(LAB)is a class of Gram-positive bacteria that can ferment carbohydrates and produce lactic acid.Lactobacillus has many beneficial functions,such as the improvement of the body's antioxidant capacity,the increase of the intestinal bacteria and the reduction of harmful bacteria,the treatment of infectious diseases and gastrointestinal diseases,the stimulation of the body's immune function,the reduction of cholesterol and blood pressure.There are about 30 genera of lactic acid bacteria,including Bifidobacterium,Streptococcus,Lactobacillus,,Pediococcus,Rothia,Leuconoostoc and so on.Lactobacillus and Lactococcus can adhere and survive in the gastrointestinal tract,urinary,reproductive system or mucosal parts and no pathogenicity,therefore,which are being widely concerned.Over the past decade,the construction of Lactobacillus expression system and the expression of foreign protein to be a new vaccine carrier using genetic engineering technology is being widely concerned.However,Lactobacillus are Gram-positive bacteria,with very thick and dense rigid cell wall,resulting in the extraction efficiency of endogenous proteins,non-secreted exogenous proteins,nucleic acids,and other non-secreted metabolites is extremely low and transformation efficiency of exogenous DNA into the recipient cell is also very low,which severely restricted the research of molecular biology of Lactobacillus and the development of genetic engineering technology,and the efficient transformation of Lactobacillus is a necessary prerequisite for heterologous expression of exogenous gene in Lactobacillus.Therefore,improving transformation efficiency of lactic acid bacteria is lactic acid in molecular biology and genetic engineering in the development of key technical problems be solved.Therefore,the key issue is to explore an effective cell wall-breaking method and method of improving transformation efficiency of Lactobacillus is the research of molecular biology and the development of genetic engineering technic.The main research content is divided into the following two parts:1.Research on the cell wall-breaking methods of LactobacillusFour different methods including repeated freezing and thawing,lysozyme digestion,zirconia beads disruption and ultrasonic treatment were used to treat Lactobacillus.plantarum1.557 cells,respectively.And gram staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)were used as evaluation method to analyze the difference of cell wall-breaking effects and time costs.In addition,the activity of protein extracted by zirconia beads method was tested by the ?-glucuronidase(GUS)assays.The quality of RNA extracted by zirconia beads method was tested by agarose gel electrophoresis.Results showed that zirconia beads method was better than other methods,the GUS protein extracted by this method has the activity of protein.the OD260/OD280 ratio of RNA extracted by this method is between 1.8 and 2.1,which is in accordance with the standard ratio,and the purity is high and the degradation degree is low.Therefore,the zirconia beads method is suitable for rapid extraction of lactic acid bacteria bacterial proteins and total RNA.Then,the weight of bacteria,the grinding buffer and time were tested and selected as important aspects to optimize the wall-breaking method,and then,the breaking effect was analyzed and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Under optimized conditions,six different species of strains from lactic acid bacteria were tested for cell wall-breaking treatments with the zirconia beads method,after that,the breaking effects were analyzed and evaluated by SDS-PAGE.Results showed that,on condition that the ratio of bacteria,grinding buffer and zirconia beads(W/V/W)is 0.013 g:300?L:1g,grinding time of 15 min,the protein migration with clear banding,high abundance;RIPA,PBS,PENP,GUS as the grinding buffer,the protein migration pattern with clear banding,high abundance,while 8M urea as the grinding buffer,the protein migration pattern with obscure banding,low abundance;After six strains of lactic acid bacteria were broken through this method,the protein migration pattern with clear banding,high abundance.Therefore,zirconia beads method is a combined rapid and efficient method for breaking lactic acid bacteria cell wall.And the method of extracting lactic acid bacteria protein has laid a foundation for the further study of the effect of lactic acid bacteria restriction-modification system on the transformation efficiency(electroporation with in vitro modified plasmid DNA).2.Research on the effect of Restriction-Modification system ontransformation efficiency of LactobacillusIn this paper,we used the method of electroporation to research on the effect of Restriction-Modification system on transformation efficiency of Lactobacillus in order to improve the electroporation efficiency.Their logarithmic growth phase was determined by measuring the growth curve of five strains of Lactobacillus-Lactobacillus plantarum 1.557,Lactobacillus plantarum WCFS1,Lactobacillus paracasei,Lactobacillus acidophilus,Lactobacillus delbrueckii.Then,five strains of Lactobacillus in the logarithmic growth phase were transferred into medium containing different concentrations of glycine,respectively,and measuring the growth curve to determine the appropriate concentration and time of adding glycine.On this basis,competent cells from five strains of Lactobacillus were prepared.At the same time,five strains of Lactobacillus were cultured in medium containing different concentrations of erythromycin and their growth situation was observed to determine the appropriate concentration of erythromycin used to screen the transformants after electroporation.Results showed that Lactobacillus plantarum 1.557,Lactobacillus plantarum WCFS1,Lactobacillus paracasei,Lactobacillus acidophilus,Lactobacillus delbrueckii reached the end of logarithmic phase at 12 h,16 h,16 h,20 h,20 h,respectively,and for these five strains of Lactobacillus,the appropriate concentration of erythromycin is 0.5?g/mL,10?g/mL,5?g/mL,1?g/mL,1?g/mL,respectively.2%glycine can be used as cell wall weakening agent for these five strains of Lactobacillus,which is conducive to the formation of competent cells.Then,under the condition of successful preparation of competent cells,we researched on the effect of Restriction-Modification system on transformation efficiency of Lactobacillus by three methods:temperature treatment,in vivo DNA modification and in vitro DNA modification.Results showed that the transformation efficiency of L.plantarum 1.557?L.acidophilus 1.1878 and L.delbrueckii subsp.bulgaricus 1.2161 can be significantly improved after incubation at 50?.The transformation efficiency of L.plantarum 1.557?L.acidophilus 1.1878 and L delbrueckii subsp.bulgaricus 1.2161 can be significantly improved after electrotransformation with modified plasmids in vivo of Lactococcus lactis.The research provided foundation for further study of establishing high-efficiency expression system of lactic acid bacteria and the development of genetic engineering technology. |
PDF Full Text Request