Affects Of Site-directed Mutagenesis Of Domain Ⅲ Of Bacillus Thuringiensis Cry1Aa Toxin On Structure And Function

Several studies confirm that proteolytic activation ofprotoxins in the gut of susceptible insects is one of the mostimportant steps in the Cry toxin insecticidal mechanism andresistance to Bacillus thuringiensis toxins. The eliminationof internal chymotrypsin and trypsin sites has proved to be anefficient approach to improve the insecticidal action of crytoxins. A modified SDS-alkaline method was used to extractplasmids of Bt 4.0718 strain, by analyzing the consensus regionof different kinds of cry1Aa gene with the National Center forBiotechnology information's BLAST WWW server, primers weredesigned to amplified the full-length cry1Aa gene from plasmids,the PCR products were treated with SalI and BamHI, then theproducts were inserted into the pUC19 vector, and weretransformed into E. coli DH5α, to get the strain UAaH19, thentransited the 4.2 kb fragment from plasmid pUAaH19 into shuttlevector pHT315, and expressed in crystal minus strain cry~-B, theprotoxins were tested by SDS-PAGE, and a 130kDa protein wasoverexpressed.In this study, on potential chymotrypsin and trypsin sitesin DomainⅢof Cry1Aa insecticidal crystal protein, mutagenicprimers were designed, using plasmid pUAaH19 as template,according to the instruction of QuikChange Site-DirectedMutagenesis Kit and by PCR method, the residues Phe and Arg onexpected sites were changed individually to alanine,altogether ten mutants: F536A,R543A,F550A,F565A,R566A,F570A,F574A,F576A,F583A and F590A were obtained, then the4.2 kb fragment containing the mutant sites were inserted into shuttle vector pHT315, and expressed in crystal minus straincry~-B, and the protoxins were tested by SDS-PAGE, except forF574A, the 130kDa protein were highly expressed in othermutants.In bioassays, F536A,R566A and F590A showed up to 20％,40%,40% increase in toxicity against Spodoptera exigua Hüner larvewhen compared to the wild type, and F536A,F565A showed 6%,10% increase against Heliothis armigera Hubner than the wildtype. Toxicities of some mutants were altered greatly, and thesame mutants were shown to have different toxicities againstthose two insects. The changes of mutants' structure wereforecasted by DeepView/Swiss-PdbViewer, and it supposed thatthe changes of side-chain affect the folding of protein or thesensitivity of protease, then affect the toxicity. Theseresults suggest that those mutated sites play important rolesin the larvicidal activity of Cry1Aa.In this study, some residues of potential chymotrypsin andtrypsin sites Phe and Arg on DomainⅢof Bacillusthuringiensisδ-endotoxin Cry1Aa were changed individuallyto Ala, then the expression of proteins were tested, theformation of insecticidal proteins were observed and itschanges of structure were forcated, in order to explore thefunction of single site in insecticidal proceas, and therelation of structure and function. It has important theoreticand practical sense for exploring deeply the function of DomainⅢ, and elucidating the Cry toxin insecticidal mechanism andresistance to Bacillus thuringiensis toxins, and selectingstains with higher efficient, broader spectrum, and shortermortal time.