Breeding Of Highly Virulent New Strains And Effect Of MmsB And CysC Genes Related On Crystal Protein Formation In Bacillus Thuringiensis

Bacillus thuringiensis（Bt）is an important insect pathogenic bacterium that produces insecticidal crystal proteins（ICPs）while forming spores.The Bt preparation has the characteristics of strong target specificity and environmental friendliness,and has developed into the world’s most widely used microbial pesticide in the fields of agriculture and forestry.However,Bt preparation has some shortcomings,such as slow insecticidal effect,limited virulence,and insects are prone to resistance.Therefore,it is necessary to continuously breed new strains or carry out genetic modification on the existing strains to obtain Bt strains with high toxicity and improved performance,which has important theoretical significance and practical value.Bt D18 isolated from soil in our laboratory was selected as the starting strain.Atmospheric and room temperature plasma（ARTP）combined with nitrosoguanidine（NTG）compound mutagenesis was used to select mutant strains of Bt that are highly toxic to Plutella xylostella.It was determined that the optimal range of processing time for ARTP was 30 s～60 s.And 50 s was selected as the processing time for subsequent mutagenesis.Using light microscopy for initial screening of the slant cultures,197 mutants were screened for virulence rescreening experiments from the 956 mutants isolated from four rounds of ARTP iterative mutagenesis and one round of ARTP-NTG compound mutagenesis.It was found that the average mutation rate of each round of mutagenesis can reach more than 20%,and the average round-by-round correction of mortality increased by about 20%.Finally,two positive mutants,AN-L5-1 and AN-L5-7,with stable traits were verified by ICPs yield examination and virulence LC₅₀.Compared with the starting strain D18,AN-L5-1 and AN-L5-7 increased the yield of ICPs by80.47%and 88.31%,respectively,and their insecticidal virulence against Plutella xylostella significantly increased.Moreover,the high-virulence Bt 4.0718 was selected as the starting strain,and the biological functions of mmsB gene and cysC gene were studied by gene knockout technology.The mmsB gene encodes3-hydroxyisobutyrate dehydrogenase（Hibadh）,an essential enzyme in the metabolism of valine.A homologous recombination knockout plasmid,constructed on the basis of the temperature-sensitive vector p Rec-Mob-Ts,was introduced into the recipient bacterium Bt 4.0718 by conjugation.And the mmsB gene knockout strain Bt-ΔmmsB was obtained.The complementation mutant of mmsB gene was also constructed.Growth curves showed that the Bt-ΔmmsB entered the declining period much earlier,and the stationary phase was obviously shortened,as compared with that of Bt 4.0718.Consistently,spore and crystal formation and mother cell lysis also occurred much earlier in Bt-ΔmmsB as evidenced by phase-contrast microscopy.The yield of crystal protein and insecticidal toxicity against Helicoverpa armigera were comparable to that of Bt 4.0718.The phenotype of complemental strain has recovered.Furthermore,Hibadh protein was expressed in E.coli in order to find the target of the protein,and further reveal the key role of mmsB gene in spore and crystal protein formation process.The cysC gene encodes adenosine 5’-phosphosulfate kinase（APSK）,which is a key enzyme in the cysteine biosynthesis pathway.The same knockout system was used to obtain the cysC gene knockout strain Bt-ΔcysC.And the complementation mutant of cysC gene was also constructed.Results showed that,compared with Bt 4.0718,Bt-ΔcysC could form spores normally,but could not form crystals.The yield of crystal protein and insecticidal virulence to Helicoverpa armigera were obviously reduced.The phenotype of complemental strain has recovered.At the same time,APSK protein was expressed in E.coli.It provides a new theoretical basis for finding the key regulators of ICPs formation.Taken together,by using ARTP technology for breeding of Bt high-virulence strains,two positive mutant strains with significantly enhanced insecticidal activity against Plutella xylostella were obtained.Moreover,by knocking out of the two functional genes mmsB and cysC in Bt 4.0718,respectively,two engineering strains were obtained.Bt-ΔmmsB had the comparable insecticidal virulence with Bt 4.0718,but the initiation of crystal formation and mother cell lysis were much earlier,which shortened the fermentation cycle.In view of energy consumption and production costs,this phenotype is expected to be developed and utilized in production.The crystal protein production of Bt-ΔcysC and its insecticidal virulence against Helicoverpa armigera were reduced,indicating that cysC is a key functional gene that affects the formation of ICPs.It lays a theoretical foundation for studying the metabolic regulation of Bt ICPs biosynthesis and highly toxic strains constructing.