Construction Of CDNA Library By Suppresion Subtractive Hybridization In VBNC Salmonella Typhimurum And Clone And Analysis Of Differentially Expressed Genes

Salmonella is a kind of intestinal pathogenic bacteria, it has infection for most kind foranimals and people and most of that have strong pathogenicity. VBNC (viable butnon-culturable): living non-culturable state, that is, when the bacteria in the adverse conditions,the bacteria morphology will be transformative, the bacteria can not grow in ordinary culture,but using a special method still demonstrate that their activity. It is a serious disease, severecases can lead to death of people and animals.When in VBNC the ‘missing check’ of bacteria inVBNC will be potential hazard in food security, so the problem of VBNC bacteria receivedmore and more attention. Currently, there was no conclusion in the study of the molecularmechanisms of bacteria in VBNC. But when in VBNC, some related genes expressions werehigher than that of normal state. Isolating differentially expressed genes directly in bacteria inVBNC can announce abjectively in the molecular mechanisms of bacteria in VBNC.In this research, Salmonella and Salmonella mutant were induced by differentconcentrations of acetic acid and4℃to set up a quick inducing method to get stableSalmonellaand Salmonella mutant in VBNC strains. Then we can get full amount strains using asexperimental materials in a short time.In this study, using SSH technique to construct VBNC Salmonella-Salmonella, VBNCSalmonella-VBNC Salmonella mutant cDNA library and obtained differential gene fragments,after T vector and cloned into competent E. coli. We obtained E. coli strains carrying differentialfragments, by PCR screening verification and digoxin hybrid screening. Finally we obtained28and23strains carrying different fragments of e.coli strains after sequencing. Afterbioinformatics software analysis, according to the sequencing results showed that there were5groups besides19unknowed gene fragements including cell structure gene, metabolic-relatedgene, protein expression-related gene, signal transduction gene and immune gene. The SSHlibrary was constructed, and the molecular mechanisms of bacteria in VBNC related genes wereobtained to provide basic data to deeply study these genes, such as glutathione s-transferases,peptidase PmbA, ClpXP protease specificity-enhancing factor, glutaredoxin2.