植物乳杆菌降胆固醇Lp10菌株的rhaD基因敲除株的构建

Construction of an rahD Gene Konckout Mutant of Cholesterol-lowering Lactobacillus plantarum Lp10

摘要:

以转录组数据为依据推测植物乳杆菌（Lactobacillus plantarum）中rhaD基因参与胞外多糖（exopolysaccharide，EPS）合成代谢中的鼠李糖代谢过程，从而通过调节胞外多糖合成并利用其吸附作用达到降胆固醇效果。为了进一步验证rhaD基因的功能，本论文研究利用同源重组技术，构建rhaD基因敲除载体，采用电击转化方法将构建的敲除载体导入植物乳杆菌Lp10菌株感受态细胞，并在抗性平板上筛选转化子，以转化子的基因组DNA为模板进行PCR及测序鉴定，再利用邻苯二甲醛法测定突变株与初始菌株的降胆固醇能力。结果表明，敲除载体pNZ5319-rhaD被成功构建，测序结果显示rhaD基因被成功敲除；敲除菌株的降胆固醇能力略有上升。本论文研究结果不仅为植物乳杆菌基因敲除载体的构建提供了实验技术支持，同时为植物乳杆菌降胆固醇相关功能基因的验证分析奠定了理论依据。

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.