Functional Study Of Extracellular Polysaccharide-related Gene CpsA Of Lactobacillus Plantarum JM113

Probiotics can promote animal growth,inhibit pathogenic bacteria,and can be used as feed additives to replace antibiotics as growth promotors.Exopolysaccharides(EPS)is an important metabolite of bacteria,which has an effect on the physiological and biochemical characteristics of bacteria.Extracellular polysaccharides of probiotics have recently attracted wide attention due to their various biological activities.However,the research on the regulation of extracellular polysaccharide synthesis is just beginning.Lactobacillus is a common probiotic bacterium,which is widely used in food,medicine and animal husbandry industries.In this study,a probiotic Lactobacillus plantarum JM113 was used for our research.Firstly,a traceless knockout system was constructed for this strain.Then,the effect of cpsA on the phenotype of Lactobacillus plantarum was explored by knocking out the target gene,and the transcriptional regulation of related gene clusters by cpsA was explored by qRT-PCR.The main results are as follows:1.Based on the principle of homologous recombination,a vector pLPK?cpsA was constructed for traceless knockout of cpsA in Lactobacillus plantarum JM113.The target gene knockout strain JM113-?cpsA was successfully obtained.In addition,the complement plasmid pAR-NcpsA was successfully constructed to obtain the complement strain JM113-?cpsA-cpsA.2.Phenotypic analyses showed that cpsA affected the growth curve of the strain at 37?.After knocking out cpsA,production of exopolysaccharides,the agglutination ability and the biofilm formation ability all decreased.The production of exopolysaccharides decreased 22.3%,and the biofilm formation ability decreased 66.3%.3.By analyzing the qRT-PCR data,it was found that the transcriptional expression levels of cps3,cps4 and cpsWC gene clusters related to exopolysaccharide synthesis increased significantly in the complement bacteria.In summary,in this study,we successfully constructed a vector pLPK-?cpsA to knockout the target gene in a traceless manner and studied the effect of this gene on the phenotype of Lactobacillus plantarum and expression of the downstream genes.provided a molecular theoretical basis for studying the regulation mechanism of exopolysaccharides synthesis in Lactobacillus.