Construction of an rahD Gene Konckout Mutant of Cholesterol-lowering Lactobacillus plantarum Lp10
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Abstract:
Based on the analysis of transcriptome data, it was speculated that rhaD gene in Lactobacillus plantarum was involved in metabolism of rhamnose metabolism responsible for the biosynthesis of exopolysaccharide (EPS). As a result, the cholesterol-lowering effect of L. plantarum is realized by the adsorption of EPS whose biosynthesis was regulated by the expression of rhaD gene. In order to verify the function of rhaD gene, rhaD gene knockout vector of L. plantarum Lp10 was constructed by homologous recombination technology, the knockout vector pNZ5319-rhaD was transformed into L. plantarum Lp10 competent cells by the method of electroporation. Resistance plate was used to screen the transformants followed by the performances of PCR identification and DNA sequencing using the genome DNA as template. Results showed that the knockout vector pNZ5319-rhaD was constructed successfully, and the DNA sequencing results indicated that rhaD gene was successfully knocked out, and the cholesterol lowering ability of knockout strain increased slightly. This research not only provides technical support for the construction of L. plantarum gene knockout vectors, but also lays the theoretical foundation for the subsequent functional verification of genes related to the cholesterol-lowering effects of L. plantarum.
