The Influence Of SiRNA Molecular Interference On The Expression Of Genes Related To The Population Effect Of Lactobacillus Paracasei

Lactobacillus paracasei HD 1.7 was isolated from Chinese sauerkraut fermentation broth.L.paracasei HD 1.7 secreted a kind of bacteriocin Paracinl.7 during fermentation process.The Paracinl.7 could inhibit the growth of some G+bacteria,some G-bacteria and Saccharomyces cerevisiae.We investigated the relationship between the L.paracasei HD1.7 quorum sensing related genes,i.e.prcK and prcR and the Paracinl.7 production.This research could be a potential basis for the exploration of the function of prcK and prcR and the mechanism of L.paracasei HD 1.7 quorum sensing.This research aimed to reveal the relationship between the Paracinl.7 production and the quorum sensing.In this research,gene silence technology was used to explore the function of prcK and prcR of L.paracasei HD 1.7.According to the sequences of prcK and prcR,we designed eight specific siRNAs and one 5'-FAM siRNA.Electrotransformation technique was adopted to transform these siRNAs into L.paracasei HD1.7.The expression level of related genes in prcK and prcR silence mutant strains in the 24-hour culture were detected by real time PCR.The results showed that siRNA-K4,siRNA-K5,siRNA-R4 and siRNA-R6 significantly inhibited genes repression.Antibacterial tests were earried out to observe the antibacterial condition of mutant strrains.The antibacterial activities of mutant strains were lower than the original strain.The titer decrease by 22.48%,20.61%,21.43%and 20.70%,respectively.This result testified that the silence of prcK and prcR had an influence on paracin 1.7 production.Furthermore,gene over expression technology was used to explore the function of prcK and prcR.Specific primers were designed by Primier 5 according to the sequences of prcK and prcR derived from GenBank.Then his-tag was added to the 5' end of the target gene.His-prcK gene and His-prcR gene were amplified and ligated to pMD18-T vector.Via the restriction enzyme digestion analysis and nucleotide sequencing,the positive recombinant plasmids pMD18-T-prcK and pMD18-T-prcR were obtained which contained His-prcK and His-prcR fusion gene,respectively.Then His-prcK and His-prcR were cloned to the expression vector pRSFDuet-1 and the recombinant expression vectors pRK-tet and pRR-tet were obtained.These constructed plasmids were introduced into L.paracasei HD 1.7 by electrotransformation.The mutant strains with over expression of prcK and prcR were selected by teracyline resistance screening.The level of over expression of the two genes in mutant strains in the 24-h culture was detected by real time PCR.The results showed that expression level of related genes in mutant strains were higher than that in the original strain by 23.28%and 31.40%respectively.His-prcK protein and His-prcR protein were successfully detected by SDS-PAGE and Western blotting.Expression levels of related proteins in mutant strains were higher than that in the original strain by 119.37%and 131.04%respectively.Antibacterial tests were done to observe antibacterial condition of mutant strains.The results demonstrated that the antibacterial activities of mutant strains were stronger than the original strain.The titer reached an increase of 17.86%and 20.69%,respectively.This data testified that the over expression of prcK and prcR had an influence on Paracin 1.7 production.By constructing and over expressing the silence mutant strains of prcK and prcR,as well as conducting the antibacterial tests,this study revealed that the antibacterial activities of silence mutant strains decreased and over expression mutant strains increased compared with that in the original strain.It suggested that prcK and prcR had n influence on Paracin 1.7 production.Preliminary reaserch found that prcK and prcR were similar with HPK10 subfamily of HPK and LytR/AlgR family respectively.Taking into account all the results,the work could testify that prcK and prcR was sensor kinase and response regulator.This research could provide foundation for the construction of regulation system and the optimization of bacteriocin production.It provided more broad development prospect,application space and could benefit human.