Identification And Functional Analysis Of SRNA Regulating The Virulence Of Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is a kind of Bacillus that can synthesize a variety of crystal proteins with insecticidal activity against invertebrates such as insects,mites and nematodes,as well as some small-molecule insecticidal active substances.It has become one of the main sources for the development of microbial preparations.At present,there have been many reports on the insecticidal mechanism of its insecticidal crystal protein,but the way that Bt,as an insect pathogenic bacteria,interacts with the host in the insecticidal process is not very clear.The research team discovered some pathogenic factors and their modes of action,such as Bel,Bmp1,Nep1,etc.,and also found that the sRNA in Bt participates in the regulation of its virulence to the host.This research studies systematically the effect of sRNA on the pathogenicity of Bt and its regulation from the perspective of sRNA.Through sRNA target prediction,construction of sRNA deletion mutants,phenotype detection and q PCR detection,the influence of sRNA on the pathogenicity of Bt was analyzed,and the following results were obtained.1.Identify 14 virulence-related sRNAs from 34 potentially virulence-related sRNAsThrough target prediction of 34 sRNAs confirmed to be transcribed in strain YBT-1518 that may be related to virulence,29 sRNAs were found to have targets related to Bt pathogenicity,and 11 sRNA deletion mutants were constructed using CRISPR-Cas9 technology.The virulence of the 11 sRNA deletion mutants and the 11 sRNA deletion mutants constructed in the early stage of the laboratory were identified,and 14 sRNAs affecting the pathogenicity of YBT-1518 were found,of which 6 sRNA deletion mutants had increased virulence.8 sRNA deletion mutants have decreased virulence.2.Virulence-related sRNA affects the growth,cry gene expression and spore germination of strain YBT-1518The sRNA deletion mutant that affects the virulence of YBT-1518 was subjected to phenotypic detection of the Bt pathogenic process,and it was found that one of the sRNA(sRNA20)deletion mutants with enhanced virulence increased their growth ability,and one sRNA with reduced virulence(sRNA6)mutants have reduced growth.Two sRNA(sRNA6,sRNA7)deletion mutants with reduced virulence have reduced spore germination rate in vivo,and one sRNA(sRNA20)deletion mutant with enhanced virulence increased the germination rate of spores in vivo.3.Virulence-related sRNA4 weakens the virulence of strain YBT-1518 by inhibiting the expression of potential target gene YBT-1518-26670The deletion of sRNA4 can lead to a significant increase in the virulence of YBT-1518,and the expression of YBT-1518-26670,the target of sRNA4,is up-regulated inΔsRNA4/YBT-1518.The GFP tag confirms that sRNA4 can negatively regulate the expression of YBT-1518-26670.At the same time,the overexpression of YBT-1518-26670 will cause the virulence of YBT-1518 to increase significantly.4.Virulence-related sRNA13 can enhance the virulence of strain YBT-1518 by inhibiting the expression of potential target gene xreThe deletion of sRNA13 will lead to a significant increase in the virulence of YBT-1518,and the expression of its target gene xre will be significantly up-regulated during the spore germination induced by inosine and nematode extracts.At the same time,the loss of xre will lead to the virulence of YBT-1518 to increase significantly.5.Virulence-related sRNA20 may inhibit spore germination through potential target gene ger KA,thereby affecting Bt virulenceThe deletion of sRNA20 will lead to a significant increase in the virulence of YBT-1518,and at the same time the expression of its target gene ger KA will be up-regulated.The spore germination rate of ΔsRNA20/YBT-1518 increased significantly during the germination process induced by inosine.6.Virulence-related sRNA20 inhibits the expression of potential target gene spoⅤAD and negatively regulates the spore formation of strain YBT-1518This study found that there are target genes related to spore formation in all virulencerelated sRNAs.Through the determination of sRNA deletion mutant spore formation rate and the detection of related target genes,it is found that sRNA20 can negatively regulate the expression of spo VAD,and ΔRNA20/YBT-1518 The spore formation rate was also significantly higher than that of YBT-1518,and the overexpression of spo VAD would cause the spore formation of YBT-1518 to be significantly up-regulated.The virulence-related sRNAs identified in this study enriched the virulence regulators of Bt and deepened people’s understanding of Bt as a pathogen.The explanation of sRNA on the regulation mechanism of Bt pathogenicity improves the regulation mechanism of sRNA in Bt on virulence,provides a certain theoretical basis for the construction of high virulence Bt strains,and is expected to promote the application of Bt in the prevention and control of agricultural and forestry pests.The regulation of virulence-related sRNA on the formation of Bt spores provides certain ideas for optimizing the phenotype of Bt spore formation and improving the industrial production of Bt preparations.