Application Of Mcherry Red Fluorescencent Protein Labelled Lactobacillus Plantarum WCFS1

Lactic acid bacteria(LAB)is a common flora of the human gastrointestinal tract.Most lactic acid bacteria,especially lactobacilli,are known for their applications in fermented foods and probiotics.Therefore,the research on the physiological characteristics,functional proteins and other molecular biology of lactobacilli has become the frontier of lactic acid bacteria research.In addition to the study of the physiological characteristics of Lactobacillus,some recognized probiotic strains have been considered to play an active role in the treatment of human gastrointestinal diseases.There are reports about the immune system of LAB and host,but the mechanism of LAB plays a beneficial role in the gastrointestinal tract is not clear.It is true that the effective molecular markers of lactic acid bacteria will make it possible to study the functional factors and mechanisms of LAB.At the same time,the development and use of labeling technique will also effectively monitor the activity process of LAB in the gastrointestinal tract.The mechanism of Lactobacillus survival in gastrointestinal tract,interaction between LAB and pathogens is more intuitive.The method of labeling lactic acid bacteria with plasmid type and integrated mcherry gene was established in this thesis,which lay the foundation for further research on the molecular biology of LAB.In this paper,Lactobacillus plantarum WCFS1 was selected as the research object.The genome of this strain had been sequenced.It was a Lactobacillus strain with early sequencing,more complete biological information and more molecular markers,such as protein gene.In addition,many literatures have reported the successful expression of foreign protein in WCFS1.Therefore,the fluorescence labeling of Lactobacillus plantarum WCFS1 is successful,and it also provides a theoretical basis for more studies on molecular markers of LAB.First of all,two kinds of fluorescent protein markers,GFP and mCherry,were selected to mark Lactobacillus plantarum WCFS1.GFP and mCherry encoding genes were inserted into the expression plasmid pSIP of Lactobacillus,respectively,and the expression of different fluorescent labeling genes in Lactobacillus plantarum WCFS1 was investigated.The experimental results showed that although the recombinant expression of GFP and mcherry genes could be successfully recombinant(Western Bloting detected the recombinant protein band),the fluorescence background of the control group was high under the condition of GFP green fluorescent protein detection,and the green fluorescence of the experimental group was not obvious in the experimental group.On the contrary,under the condition of mCherry red fluorescent protein detection,the fluorescence background of the control group was very low,and the red fluorescence of the experimental group YMW460 was brighter.After the strain was induced to express 16h,the fluorescence activity reached the highest value of 32934RFU.The results confirmed that the mcherry gene was used to label Lactobacillus plantarum WCFS1 in this study.Secondly,in order to realize the continuous expression of mcherry gene in Lactobacillus plantarum WCFS1,in this thesis,three kinds of continuous expression promoters,namely P1dhL,PslpA and P32,were applied to study mcherry gene expression.The results showed that all kinds of promoters could achieve continuous expression of mcherry gene.Among them,under the promoter PldhL operation,the fluorescence activity of mcherry gene was the best,and the fluorescence value of recombinant YMPldhLW460H run up to 34569RFU.The results provide favorable conditions for the integration expression of mcherry gene on the WCFS1 chromosome of Lactobacillus plantarum.Finally,the homologous recombination strategy was used to integrate the mcherry gene under the PldhL promoter to the WCFS1 chromosome of Lactobacillus plantarum.Then,Cre-lox-Based system was used to remove the resistance screening markers in the homologous recombination process,and the mcherry integrated lactobacillus strain WCFS1 mutant strain YMW5328,which was constructed,was successfully constructed.After 18h culture,the fluorescence value reached22346RFU.The maximum fluorescence intensity of the strain was lower than that of the plasmid type fluorescent label strain YMPldhLW460H,but the fluorescence gene was integrated on the chromosome,the fluorescence activity of the strain was stable and the generation was not easy to lose.In conclusion,this study systematically studied the method of mcherry gene fluorescence labeling of Lactobacillus plantarum WCFS1.The best scheme of plasmid type and integrated mCherry fluorescent protein to label Lactobacillus plantarum WCFS1 has been established successively.It provides an effective means for further research on the physiological characteristics of LAB and the molecular mechanism of enteric probiotics.