Screen And Primary Identification Of Key Genes Resisting Phage Infection In Bacillus Thuringiensis

As the widespread abuse of antibiotics,bacteria antibiotic resistance is aggravating.People are searching substitutes for antibiotics,phage therapy is one of these substitutes.However,the models of the interaction between phage and host are mainly focused on model strains,which restricts people’s awareness of the interaction between phage and host.Recently,as underground pests,including root-knot nematodes,cyst nematode,grubs,cutworms,flea beatles and root maggot are aggravating,they are hazarding farming industry enormously.Based on the fact that Bacillus thuringiensis(Bt)has made great progress in preventing underground pests and series of products have been developed,but,in our application,these Bt formulation have poor adaptability and unstable controlling effect in soil ecosystem.Our research group proved that the persistence and survival of Bt can be reduced notablely by soil lytic bacteriophage.To provide new perspective for the interaction between phage and host and provide scientific proof for genetic modification and directional selection to enhance the adaptability of Bt products,We utilize Bt as the material to identify the key genes resisting phage infection in Bt,harnessing different methods to investigate the interaction between phage and Bt from whole genome Main results are as follows:1.The research on key factors and mechanisms Bt fighting against phage infection rapidly by comparative genomics.We find that YBT-1518 can mutant in a few hours after phage BMBsp1 infection.Then we proved that this phenonenom is not limited to YBT-1518.Based on this,we take YBT-1518 as a model to investigate the mechanism Bt resisting phage infection quickly.we coculture YBT-1518 and 5 different lytic phages and have screened 31 phage resistant strains under 5 different phage pressure.And we have analyzed 28 mutant strains the mutation site and the mutation pattern finding that 71 mutant genes in the chromosome,58 of them are in prophage zone,60 SNP in the intergenic region,all of them are in the prophage zone and 46 mutant genes in the plasmid,88 SNP in the intergenic region.It is the first time to find Bt can mutate quickly after phage infection and that this fast resistance mechanism is related to prophage.Later we will use CRISPRi to knock out candidate genes and investigate the function and mechanisms of key genes.2.The research on key factors and mechanisms Bt fighting against phage infection by proteomics.Our group use proteomics to identify the different expression proteins between phage infection strain and no phage infection strain.We find that 28 up-regulation genes after phage infection.We find that ABC transporter relevant proteins upregulate specifically after phage infection,we take three ABC transporter genes prior to others to knock out.We find that there is no difference between C15 and ABC transporter knocking-out strain.Afterwards we will use CRISPRi to knock out other candidate genes and check the phenotype of phage resistance.3.The research on key factors and mechanisms Bt fighting against phage infection by mutant libraryOur laboratory constructs the transposon insertion mutant library by using transposon mini-T10 and identifies 14 mutant strains the insertion site.We check the phage resistance phenotype of these 14 strains and identify 4 key genes in Bt resisting phage infection,two IS element transposase、two hypothetical protein.Besides we identify two transcriptional regulator nprr and plc R by checking the phage resistance phenotype of the constructed Bt single gene mutant strain.