Research On Salt Tolerance Mechanism Of Lactobacillus Plantarum Based On RT-PCR Technique

Lactic acid bacteria is one of the most important industrial fermentation strains. After the fermentation of lactic acid bacteria, proteins are hydrolyzed to amino acids and peptides, which are more easily absorbed by the body. Thereby fermentation process greatly enhances the nutritional value of dairy products. Meanwhile, lactic acid bacteria has many physiological functions including improving gastrointestinal function, anti-tumor, enhancing immunity, lowering cholesterol, maintaining balance of microflora in urinary tract, etc. Because of many advantages of lactic acid bacteria, fermentation technology of lactic acid bacteria has been widely used in food, medicine, biology and other fields. Therefore, the studying of lactic acid bacteria in molecular level has become a hot topics for scholars all over the world. Lactic acid bacteria was faced all kinds of environmental stress during production, processing, storage, transportation and sale, and these stress may cause destruction in some degree. These environmental stress includes osmotic stress caused by high salinity, heat stress caused by high-temperature sterilization, salt stress caused by freezing, acid stress caused by reducing acidity, oxidative stress caused by exposing to the air, starvation stress caused by nutritional deficiencies, etc.The salt-tolerance lactic acid bacteria strain in this experiment was isolated from traditional fermented miso.This experience studied salt-tolerance molecular mechanism of lactic acid bacteria at the transcriptional level by quantitative PCR.This paper is mainly to study the resisting role of lactic acid bacteria to salt stress from several aspects:compatible solutes regulating system, key enzymes of glycolysis regulating system, heat shock protein regulating system,energy synthesis regulating system and proteolysis regulating system. Compatible solutes regulating system genes include Quaternary Ammonium Compound Transporter (qacT), Osmoprotectant Transport System ATP-binding Protein (opuA), Osmoprotectant Transport System Permease Protein (opuB), Osmoprotectant Transport System Substrate-binding Protein (opuQ.Key enzymes of glycolysis regulating system genes include6-Phosphofructokinase(D/fk), Fructose-bisphosphate aldolase (fba), Phosphoglycerate kinase (pgk), Glyceraldehyde3-phosphate dehydrogenase (gapB), L-lactate dehydrogenase (ldh).Heat shock protein regulating system genes include Molecular Chaperon(groEL,groES,dnaK,dnaJ), Heat Shock Protein (hspl,hsp2,hsp3), Universal Stress Protein (usp).Energy synthesis regulating system genes include energy synthesis regulating system gene Proton-transporting ATP Synthase(fifo-ATPase),Proteolysis regulating system genes include X-Pro dipeptidyl-peptidase(PepX). Quantitative PCR results showed that in logarithmic growth phase and stable phase: Compatible solutes regulating system genes qacT, opuA, opuB, opuC were all induced by NaCl in MRS medium and their expressions were upregulated. The higher NaCl concentration was, the more significant up-regulation was. Key enzymes of glycolysis regulating system genes pfk,fba,pgk, ldh were all induced by NaCl in MRS medium and their expressions were upregulated. The higher NaCl concentration was, the more significant up-regulation was Another key enzymes of glycolysis regulating system gene gapB was almost not influenced by NaCl in MRS medium. Heat shock protein regulating system genes groEL, groES, dnaK, dnaJ, hspl, hsp2,usp were all induced by NaCl in MRS medium and their expressions were upregulated. The higher NaCl concentration was, the more significant up-regulation was. Another heat shock protein regulating system gene hsp3was induced by NaCl in MRS medium and its expression was upregulated, but its significant degree of induced up-regulation was not positive correlation with NaCl concentration. The expression of energy synthesis regulating system gene fIfo-ATPase wasn’t influenced by NaCl in MRS medium. Proteolysis regulating system gene pepX was induced by NaCl in MRS medium and its expression was upregulated. The higher NaCl concentration was, the more significant up-regulation was.Comparison of expression of genes induced by NaCl between logarithmic growth phase and stable phase:At the same condition of NaCl concentration in MRS medium, expressions up-regulation of genes opuA、opuC、fQfi-ATPase、pFk、fba、gapB、groEL、groES、dnaJ、 hsp1、hsp2、hsp3、usp induced by NaCl of logarithmic growth phase and stable phase were almost same. Expressions up-regulation of genes qacT、opuB、ldh、pgk、dnaK、pepX induced by NaCl of logarithmic growth phase and stable phase presenced differences in some degree.