Discovery And Application Of Several Regulatory RNAs In Bacillus Thuringiensis Serovar.Chinensis CT-43

One kind of non-coding RNAs which termed regulatory RNA with genetical function like protein regulator,constitutes a significant part of 'central dogma' in bacteria.Discoveries of novel regulatory RNAs and researches on their regulation mechanisms will contribute to our understanding of the diversity of genetic regulation,broaden our eyes in front of the RNA world,even uncover clues making us possible to trace the evolution of life system.Additionally,regulatory RNAs enjoy a sound prospect as tools in bacterial molecular genetic experiments.In this study,we explored several regulatory RNA from Bacillus thuringiensis serovar.chinensis CT-43,by utilising both 'dry lab' and 'wet lab9 skills.In addition,a biosensor was constructed.Integrating the sequencing data of transcriptomes,we collected all transcribed unannotated IGRs from the chromosomal genome of strain CT-43.Following bioinformatical analysis was taken to scan transcriptional signals,20 orphan promoters and 20 orphan terminators were predicted finally,invovled 22 hypothetical sRNA.12 of them were identified as genuine transcripts by white-blue screening with reconstructed promoter trap plasmids.The predicted secondary structures of these sRNA transcripts were rich in steem-loops which strengthened their stabilities.Some of these seemed to be temporal transcribed,thus,they might be clues for our investigation about endo-sporulation and ICPs production.Annotated by Rfam database,a tandem triplication architecture consists of three riboswitches(c-di-GMP-I)was picked out from thouthands of IGRs.They seem to be paralogous elements owing to the conserved sequence and secondary structure.Based on the thermodynamic analysis of terminational conformation and antiterminational conformation,we can presume that each of the three c-di-GMP-I was an inducible transcriptional activation elements.The tandem triplication architecture of c-di-GMP-I were predicted to be different from a single one,manifesting in unique dynamical features:more sensitive to the modification of c-di-GMP level,tighter control of downstream transcription.Gene lacZ was transcriptional fused following the riboswitch encoding sequence as a reporter to investigate the mechanism of genetical regulation,results of both white-blue screening and ?-galactosidase assay supported our presumption.A biosensor-like dual-flourescin reporter system was constructed using tandem triplication architecture of c-di-GMP-I.It is able to monitor the concentration of c-di-GMP,responsing by shift the relative level of Turbo RFP agaist AmCyan.It was confirmed by laser-scanning confocal microscopy imaging and fluorescence spectrophotometry,respectively.This biosenor simulates traffic lights:'red' indicates adherance of cells induced by high level of c-di-GMP,'green' mean the cells are willing to move with a low level of c-di-GMP.