Study On Functional Genome Of Novel Bacillus Thuringiensis Strain 4.0718 And Construction Of Engineering Strains With High Efficiency

A novel Bacillus thuringiensis strain 4.0718 could produce typical bipyramidal and cuboidal crystals,and showed high toxicity to insects,such as Helicoverpa armigera.In order to fully demonstrate the insecticidal background and take more advantage of the strain to apply for pests control,the functional genomic of Bt strain 4.0718,including the plasmid profile,protein profile, insecticidal gene content,and protoxin composition were analyzed.The results showed that Bt 4.0718 was a unique strain considering its significant difference in plasmid profile and protein profile.PCR detection revealed that five cry genes (cry1Aa,cry1Ac,cry2Aa,cry2Ab and cry1Ia),vip3A,and chitinase gene were present in the strain 4.0718.Then the proteins in the parasporal crystals from Bt 4.0718 were analyzed by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry(LC-MS/MS),and Cry1Aa and Cry1Ac protoxins were identified in the 130 kDa protein band and Cry2Aa protoxin was identified in the 65 kDa protein band by MS/MS spectra.In order to quickly selecte Bt strains and insecticidal crystal proteins with new insecticidal specificity,a new straightford method was established through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis of in-gelgenerated peptides for protoxin identification.B.thuringiensis subsp,kurstaki HD1,which has clear background in cry gene and protoxin type,was used to model strain to detect the efficiency of this method.Our model study revealed that four protoxins(Cry1Aa,Cry1Ab,Cry1Ac and Cry2Aa)could be rapidly identified from kurstaki HD1.At the same time,the crystal proteins from israelensis 4Q2-72 and aizawa HD133,which has clear cry gene content,were studied by the gel block coupled to LC-MS/MS,and six protoxins(Cry4Aa, Cry4Ba,Cry10Aa,Cry11Aa,Cyt1Aa,and Cyt2Ba)were identified from 4Q2-72, and 6 protoxins(Cry1Ab,Cry1Cb,Cry1Da,Cry1Ad,Cry1Ka,and Cry1Ba) were identified from aizawa HD133,and the Cry1Ad,Cry1Ka and Cry1Ba protoxins have not been reported previously in HD133.The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B.thuringiensis strains.Given its technical simplicity and sensitivity,our method might facilitate the proteomic analysis for B. thuringiensis strains and other biological organization.It is required to obtain novel B.thuringiensis strains with higher toxicity and large-spectrum to increase the application of Bt insecticides.Therefore,a proteinase inhibitor hwtx-11 gene from spider toxin was fused into cry1Ac and several recombinant strains were obtained,and the hwtx-11 gene was successfully expressed in Escherichia coli and B.thuringiensis acrystalliferous strain.The bioassayes of lysis of recombinant strains indicated that Hwtx-11 protein is toxic to Spodoptera exigua and could significantly retard the growth of Spodoptera exigua,and fused Cry1Ac-Hwtx-11 protein showed more toxic to Helicoverpa armigera and Spodoptera exigua larvae than Cry1Ac protein.The study provides an important foundation for constructing fusion protein with different insecticide activity and recombinant strains with higher toxicity.