Study On The Differential Expression Of Proteins In Different Lactobacillus Plantarum Of Cholesterol Reducing Ability

With the improvement of people’s living standard, the diet structure has changed a lot and the highcholesterol content of dietary has increased too. High levels of cholesterol is one of the most important reasonthat leading to the death in the word. It has been recognized by scientists that Lactobacillus plantarum couldlower the cholesterol. In recent years, many domestic and foreign scholars are able to find the Lactobacillusplantarum could lower cholesterol but fail to clarify the mechanism. The experiment aims to analysis thereason why the cholesterol lowering ability difference in two Lactobacillus plantarum from gene level toprotein level.This experiment has pre screened two strains with different cholesterol reducing ability. One is the ordinaryLactobacillus plantarum Lp and the other is Lactobacillus plantarum Lp-UVs29which is treated by mutagenictreatment to plantarum Lp. The cholesterol lowering ability is in great difference. The cholesterol loweringability of Lactobacillus plantarum–UVs29has increased by97.97%than the original strain capacity. And thenwe screen different expressed protein by means of two dimensional gel electrophoresis and go to verificationby means of mass-spectrometric technique of two Lactobacillus plantarum. Five of those proteins which hasrelated to cholesterol metabolism are selected to proceeding experiment.Design the primers according to the published gene sequence of NCBI and send it to Shanghai SangonBiological Co.Led after cloned. The result show that the five genes exist in two Lactobacillus plantarum. Thesequencing result showed that Enolase, NADH peroxidase reductase，Enoyl-ACP reductase，Bile salthydrolase，3-hydroxy-3-methyl glutaryl coenzyme A reductase gene in homology could reach above99%withthe published Lactobacillus plantarum.Real-Time PCR technology is able to quantify the expression of mRNA in the sample. We have achievedfive target genes by cloning and sequencing. Using cDNA as template in two strains of Lactobacillusplantarum fluore scence quantitative PCR to the five genes.The experimental results show that the five gene amplification efficiency is very well. The expressionof enolase gene and NADH peroxide gene in Lactobacillus plantarum Lp was higher than that of Lactobacillusplantarum Lp-UVs29, expression of Enoyl-ACP reductase gene was lower than that of Lactobacillusplantarum Lp-UVs29, bile salt hydrolase gene and3-hydroxy-3-methyl glutaryl coenzyme A reductasegene expression are higher than that of Lactobacillus plantarum Lp-UVs29, and the expression of BSH genein Lactobacillus plantarum Lp-UVs29amount is4.5times the amount of the expression in Lactobacillus plantarum Lp.We successful detected5target protein and9target peptide in two Lactobacillus plantarum through theestablishment of mass spectrometry and MRM method. The result showed that the mass peak area of peptidesis higher in Lactobacillus plantarum Lp-UVs29and indicating the higher expression. The expression of this5protein in Lactobacillus plantarum Lp-UVs29is at least3times higher than in Lactobacillus plantarum Lp.