Identification And Function Of Novel SRNA In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is a gram positive bacteria and has strong toxin to a variety of insects.As a result of this,Bacillus thuringiensis has been developed as a biological pesticide that can compete with chemical pesticide.Small RNA(sRNA)molecules are non-coding RNA that have been implicated in regulation of various cellular processes,allowing bacteria to adapt to changing environmental conditions.Till date,sRNAs have not been reported in Bacillus thuringiensis,not even in Bacillus cereus group.When invading the host,Bt needs to quickly adapt to environment and kill the host.We believe that sRNA is likely to play a regulatory role in the environmental adaptation and virulence in Bt.In order to search for the sRNA in Bt and to study its function,this study was carried out in two parts.1.Analyzed the predicted sRNAs in Bacillus thuringiensis BMB171 and identified their physiological function.The lab has predicted nearly a hundred sRNAs in BMB171,from which this work chose 20 sRNAs to do further study which only exist in Bacillus cereus group,and we carried out the following work.(1)The conservation of candidate sRNAs was analyzed,and then all of these sRNAs unique conserved in Bacillus cereus group,including two sRNAs specific existing Bacillus thuringiensis and Bacillus anthracis.(2)The targets of candidate sRNAs were predicted and COG analysis found that these sRNAs involves in a variety of metabolic pathways.(3)We validated the candidate sRNAs and 8 of the candidate sRNAs were confirmed by reverse transcription PCR.(4)Identified the physiological functions of candidate sRNAs.Construct the sRNA over expression strain,observed phenotypic changes in growth,quorum sensing and stress response.We found that 15 sRNAs over expressed would result in phenotypic changes and 12 sRNAs over expressed affected multiple phenotypic changes.2.BMBsr1 mediated Cry5Ba toxin silencing to help Bacillus thuringiensis breakthrough nematode host feeding cessation defense.BMBsr1,which found in the study of the mechanism of cry5Ba silencing in YBT-1518,is a negative regulator of Cry5Ba toxin.In this study,the following work has been carried out in the center of the regulatory mechanism of BMBsr1.(1)BMBsr1 was been confirmed by reverse transcription PCR and real-time PCR.(2)BMBsr1 was proved to negatively regulate cry5Ba through binding to the RBS region of cry5B mRNA.(3)Combining the expression of Cry5Ba protein and the cry5Ba mRNA,it showed that cry5Ba was regulated at the post transcriptional level by BMBsrl.(4)BMBsrl increase nematode feeding by inhibiting cry5Ba.Bacillus thuringiensis,like most bacteria,can express sRNAs,and these sRNAs involved in the regulation of environmental adaptability and virulence.This study filled in the blank of the sRNA in Bacillus cereus group,and provide the basis on sRNA diversity analysis in Bacillus cereus group.Through the study of sRNAs related to environmental adaptability and virulence,it will be helpful to construct Bt engineering strain with strong environmental adaptability and virulence for us.And through this study,it will be helpful to find the hidden insecticidal crystal protein for us.