The Study Of The Interaction Between The GlnR Regulatory Protein And Nir From Lactobacillus PlantarumWU14

Nitrite is widely found in vegetable pickled products and meat products.Long-term ingestion of nitrite or one single intake of excess nitrite will be harmful to People's health.Using nitrite reductase produced by microbial metabolism to degrade nitrite can not only effectively remove nitrite in foods,but also microbial metabolites such as bacteriocin and lactic acid,etc can inhibit the growth of pathogenic bacteria and extend the shelf life of fermented products.Nitrite reductase can be divided into four categories according to the different prosthetic groups: CuNiR,nasB,ccNiRs,cd1 NiR.GlnR is a global regulator which can regulate genes associated with nitrogen metabolism.It is divided into two types,the MerR and OmpR families.It can recognize a combination of unique sequences,change the structure of the promoter region,and influence transcription.Based on the rich nitrogen source content,GlnR can inhibit or activate genes involved nitrogen metabolism.In this study,the nitrite reductase gene NiR and GlnR gene were successfully cloned from Lactobacillus plantarum WU14.The amino acid sequence of this NiR was 99% similar to the reported NiR from other sources through the NCBI online comparison.The GlnR amino acid sequence belongs to the MerR family protein after analyzed by software.It was 96% similarity to the GlnR of L.pentosus MP-10.The GlnR was successfully expressed by induction and analysed with mass spectrometry.In order to verify interrelation between the GlnR and NiR,a gel retardation experiment was performed.The results showed that GlnR could bind to the nitrite reductase promoter Pnir,resulting a decrease in the migration rate of the GlnR-Pnir complex on the active gel.To further confirm the interaction of GlnR protein with NiR,a yeast two-hybrid experiment was carried out.The results showed that the GlnR interacted with NiR,activated GAL4 transcription factor,and GAL4 regulated downstream galactosidase gene expression.The results of qRT-PCR experiments showed that under low concentration of nitrite stress,the GlnR transcription increased and NiR gene transcription increased also.Gradually increasing nitrite concentrations obviously decreased the transcription of GlnR and NiR genes.The transcripts of GlnR and NiR were both increased and decreased with the same step in high nitrous acid concentrations.These results indicated that GlnR of L.plantarum WU14 can not only interact with the NiR gene,but also,with the increase of nitrogen sources,GlnR can be activated,which regulated the transcription of NiR gene and increased the expression of NiR.Through gene knock-out experiments,the degradation mechanism of target gene NiR on nitrous acid was further studied,and the positive regulation relationship with GlnR regulatory protein would be revealed.The experimental results showed that the upstream gene of nitrite reductase NiR was knocked out.This would be helpful for the construct of mutant strains and complement mutants to study the growth status of nitrite and nitrite degradation,it was clarified that the operon was importantly regulated by GlnR regulation and nitrite-induced expression.In this thesis,the gel retardation,yeast two-hybrid qRT-PCR,and gene knockout were used to study the interaction of the GlnR on NiR of L.plantarum WU14.The results showed that the GlnR can play a role of positive regulator of NiR,which could lay a foundation for future research on the regulation mechanism of NiR degradation of nitrite.It would be import to elucidate the microbial degradation of nitrite.