Study On Local Transcriptional Regulation Mechanism Of Of Fructooligosaccharides In Lactobacillus Plantarum

In Lactobacillus plantarum,fructooligosaccharides(FOS)metabolism is controlled by both global and local regulatory mechanisms.Although catabolite control protein A has been identified as a global regulator of FOS metabolism,the functions of local regulators remain unclear.In order to explore the local transcriptional regulation mechanism of FOS metabolism by L.plantarum,this subject took SacR1 and SacR2 proteins as the research objects,and the specific combined of the transcription factor binding sites(TFBS)of these two proteins and the promoter region of the two gene clusters was verified through in vivo and in vitro experiments.The Ch IP-seq experiment was used to explore the regulatory genes of SacR1 and SacR2 proteins,and analyzed together with the genes regulated by Ccp A to construct a regulatory network for the metabolism of FOS by L.plantarum.The main conclusions of the paper are as follows:(1)In order to analyze the role of SacR1 and SacR2 in the metabolic FOS process,two gene knockout strains ?sac R1 and ?sac R2 were constructed and compared with the wild type growth and metabolism differences.Comparing the same carbon source,the growth curve and growth rate of the three strains were not significantly different.Considering that the global regulation may be more significant due to the presence of glucose,ribose was selected as the carbon source for further research.Compared with ribose,when wild-type strains use FOS as the carbon source,the expression levels of the three key genes are significantly increased;on the contrary,after the sac R1 and sac R2 genes are inactivated,the expression of these key genes does not change significantly,Indicating that SacR1 and SacR2 may be involved in the metabolism of FOS as repressor proteins,FOS can play a role in inducing or derepressing.Through prediction,a total of 4 potential TFBS were found in the three promoter regions in the two gene clusters of sac PTS1 and sac PTS26.(2)In order to verify the binding of SacR1 and SacR2 proteins to TFBS,two protein E.coli exogenous expression vectors were constructed respectively.The recombinant protein was verified by EMSA experiment,which confirmed the specific binding of SacR1 and SacR2 to 4 TFBS in 3 promoter regions.In order to further verify the in vivo binding of SacR1 and SacR2 proteins to TFBS,two overexpression vectors were constructed respectively.Ch IP-q PCR analysis showed that transcription factors are highly enriched in the DNA fragments of the promoter region,again proving SacR1 and SacR2 Interaction with TFBS.(3)In order to search for genes regulated by SacR1 and SacR2 proteins,a genome-wide scan of TFBS combined with these two local transcriptional regulators was performed.Through the Ch IP-seq experiment,it was found that 409-Flag-sac R1 captured a total of 100 peaks,a total of 121 can be matched to CDSs,and 10 can be matched to a gene interval;409-Flag-sac R2 captured a total of 108 peaks,a total of 111 Can be matched to CDSs,and 24 can be matched to gene intervals.Most potential TFBS are located upstream of operons encoding specific carbohydrate catabolism-related functions,suggesting that SacR1 and SacR2 may be involved in related regulation.Finally,the mechanism of L.plantarum metabolism of FOS is comprehensively analyzed.The metabolism of FOS in L.plantarum is an extremely complex regulatory network.In this network,the combined action of global and local regulatory factors coordinate the transcription of various units,enabling L.plantarum to better utilize the carbon source.