Proteomics Analysis Of Salt-tolerant Lactobacillus Plantarum FS5-5 Based On The ITRAQ Technology

Dajiang is a popular traditional fermented food in Northeast of China. As a main condiment, salt is added in the fermentation process, where salt tolerant bacteria group contain lactic acid bacteria and yeast. Lactobacillus plantarum, as a putative probiotic, is often isolated from the dajiang samples. Proteomic study of salt-stress Lactobacillus plantarum could provide a theoretical basis to study the mechanism salt-stress of lactic acid bacteria.This study act Lactobacillus plantarum FS5-5 as the research object, which was from traditional fermented food in Northeast China, drew the growth curve under different salt concentrations, compared the cell morphology under different salt concentrations, and analyzed the expression of protein under different salt concentrations using iTRAQ technology, verified the genes of the key proteins by real-time quantitative PCR technology.1. By measuring the OD value in 600nm, a series of growth curves of Lactobacillus plantarum FS5-5 were drew, which NaCl concentration were 0.0%,1.5%,3.0%,4.0%,5.0%, 6.0%,7.0% and 8.0%(wt/vol), respectively. From these growth curves, the time point reached the mid of logarithmic growth phase were 5h,5h,6h,7h,8h, 10h,12h and 12h, respectively.2. Bacterial samples, from the mid of logarithmic growth phase, were carried out the transmission electron microscopy experiment, which NaCl concentration were 0.0%,1.5%, 3.0%,4.0%,5.0%,6.0%,7.0% and 8.0%(wt/vol), respectively. From the transmission electron microscopy pictures, bacterial cells were in the shape of short rod, reproductive mode was binary fission. With the increasing of NaCl concentration, the osmotic pressure between inside and outside of bacterial cell was increased, and the cell morphology of Lactobacillus plantarum FS5-5 also had some deformation, some cells appeared the separation of the cytoplasmic wall, even appeared the obvious leakage of the internal dissolution.3. Bacterial samples, from the mid of logarithmic growth phase, were carried out the isobaric tag for relative and absolute quantitation experiment, which NaCl concentration were 0.0%,1.5%,3.0%,4.0%,5.0%,6.0%,7.0% and 8.0%(wt/vol), respectively. The test results showed that all spectra identified number was 343524, the spectra matched number was 85516, the peptides identified number was 11215, and the proteins matched number was 2056 in 1578_UNI_Lactobac database.The test results regarded fold change more than 1.2 and p-value less than 0.05 as choosing condition, and compared with NaCl concentration 0.0%, when the NaCl concentration was 1.5%,3.0%,4.0%,5.0%,6.0%,7.0% and 8.0%(wt/vol), differently expression proteins in total were 1944.The function and metabolic pathways of differently expression proteins were noted through go database and kegg database. Under salt stress environment, differently expression proteins mainly participated in stress proteins, ribosome, DNA repair and recombination proteins, and carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, peptidoglycan biosynthesis, energy metabolism, and so on.4. The gene expression levels of the key proteins in iTRAQ were verified by qRT-PCR experiment. The results showed that the level of gene expression was consistent with the level of protein expression, and the accuracy of iTRAQ results was proved in mRNA expression level.