Molecular Modification Of Cyt2Aa Toxin From Bacillus Thuringiensis

Bt toxin is the most mature biological insecticide applied worldwide,but it also has problems such as narrow insecticidal spectrum,limited insecticidal effect and resistance.Therefore,it is urgent to study new insecticidal materials.Cry toxins produced by Bt during sporulation are commonly used to control lepidopteran pests,while Cytolysins(Cyt)and Vegetative insecticidal proteins(VIPs)are rarely used in lepidopteran pest control.VIPs have insecticidal activity against Coleoptera,Lepidoptera and Hemiptera,Cytolysin has insecticidal activity against Diptera,but not Lepidoptera.Previously,it has been reported that cytolysin was modified by molecular modification technology and its insecticidal spectrum was successfully expanded.In summary,this paper carried out the design and construction of regional saturated mutant library and gene fusion based on Cyt2Aa,and explored the feasibility of expanding the insecticidal spectrum of Cyt2Aa by some column molecular modification methods.The specific research contents are as follows :1.Construction and evaluation of saturation mutation library of Cyt2AaAccording to the literature review,the loop4 structure in Cyt2Aa toxin is the key region that determines its insecticidal spectrum.In this study,site-saturation mutagenesis was used to perform saturation mutagenesis on the six amino acid sites in the loop4.Degenerate bases were introduced to the selected sites by overlapping extension PCR(SOE-PCR)and the full-length genes containing the mutation sites were amplified.Then,the full-length genes were cloned into the bacteriophage vector and transferred to competent cells of E.coli TG1 to construct the mutant library.The application of the mutant library was evaluated by random colony PCR and sequencing.The results showed that the insertion rate of mutant gene in Cyt2Aa-loop4 mutation library reached 80 %,and the diversity abundance of six selected amino acid sites was also high,which laid a solid material foundation for subsequent screening and identification.2.Screening and identification of Cyt2Aa mutant library combined with aminopeptidase N in midgut of Plutella xylostellaAfter three rounds of enrichment and screening of APN1 protein from Plutella xylostella using Cyt2Aa-loop4 mutant library,the monoclonal colonies were randomly selected from the last round to identify their binding activity with APN1 in Plutella xylostella by ELISA.When the ratio of OD450 value of monoclonal and control hole Cyt2Aa was greater than 2.1,the clones were positive,and six positive clones were tested.The positive cloned amino acids with the highest binding ability were selected for homology modeling and molecular docking with APN receptor.The results showed that the free energy required for binding to APN in Plutella xylostella was significantly reduced after serine at 141 and 143 sites of Cyt2Aaloop4 was mutated to lysine and leucine(Wt141/ Mt141: 0.2/-2.37 kcol/mol;Wt143/ Mt143:-0.95/-2.38 kcol/mol),the synthesis of external binding and molecular simulation studies,from which it can be inferred that the serine at 141 and 143 positions in loop4 region may be the key amino acid for Cyt2Aa specific receptor recognition,which can provide theoretical and technical guidance for its insecticidal spectrum through molecular modification of Cyt2Aa toxin.3.Fusion expression and virulence determination of Cyt2Aa and RTBRTB has a broad-spectrum binding to eukaryotic cells and can endocytosis to enter the cells.In this paper,Cyt2Aa gene and RTB gene were connected by a flexible linker and cloned into prokaryotic expression vector for expression.The binding ability and cytotoxicity of Cyt2Aa-RTB fusion protein were verified.It was proved that Cyt2Aa-RTB fusion protein not only had the ability to bind to Sf9 cells,but also played a cytotoxic role,which could be another effective idea and technical supplement for the modification of Cyt2Aa toxin.