| Lactobacillus plantarum is a species of lactic acid bacteria during malolactic fermentation(MLF),which possesses the potential to improve the aroma profile of wine,while most of them can not cope with acid environment in wine.Exploring the possible acidresistance gene in L.plantarum and evaluating their biological function play a crucial role in explaining the evolution of microbial population during MLF and offering guidance for industrial practice and application.The SA-680 acid-resistance marker gene in L.plantarum XJ25,which was separated from fermented must,was disrupted by gene editing to study its physiological role.The main results are as follows:(1)Plasmid methylation is the major barrier in the electrotransformation of L.plantarum XJ25;collecting time of competent cells is another obstacle for electrotransformation,transformation efficiency of competent cells of OD600=0.2 reached the peak of 1.83×105 CFU/?g DNA;with the increasing electrical field strength,transformation efficiency of L.plantarum XJ25 climbed to 2.08×105 CFU/?g DNA when 7.5 k V/cm was given and then dropped;transformation efficiency of L.plantarum XJ25 was negatively correlated with plasmid concentration and had no clear relationship with pulse times.(2)Plasmids with different sg RNAs,p LCNICK-?mdt-sg RNA1 and p LCNICK-?mdtsg RNA2,were constructed through overlap extension-PCR and multiple fragments one step cloning strategy.No mutation was introduced by sequencing analysis.After electrotransformation,transformants could be obtained using p LCNICK-?mdt-sg RNA1,while p LCNICK-?mdtsg RNA2 could not do that.Off-target effect might be occurred using p LCNICK-?mdt-sg RNA2;positive mutant L.plantarum XJ25-?mdt was harvested through streaking 3 times on nonantibiotic MRS plates.(3)After losing the SA-680 acid-resistance marker gene,the maximum biomass of L.plantarum XJ25-?mdt had no significant difference compared with wild-type L.plantarum XJ25(p<0.05). |
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