| Bacillus thuringiensis(Bt)is the most widely used insecticidal microorganism,its insecticidal activity mainly comes from the crystal protein produced in the process of spore formation.The research on the molecular structure and function of insecticidal crystal protein has been paid more and more attention.Here,site-directed mutagenesis and bioinformatics techniques have been used to clarify the relationship between structure and function of the parasporal crystal protein.Firstly,bioinformatics technology has been used to analyze the Cry1Ac5 protein domain III,which confirmed that the ?20-?21 loop is a unique functional loop in Cry1Ac5 protein domain III.The relationship between the structure and function of ?20-?21 loop was then studied by using site-directed mutagenesis.Several mutants with increased virulence or stability have been identified.We compared the structure of the Cry1 A protein at various levels and further analyzed the phylogenetic tree of the Cry1 protein domain III and the domain III of the Cry1Ac5 protein was identified as the study object,then analyzed the characteristics of Cry1Ac5 domain III and its ?20-?21 loop and found that in regions of H518FPST522,N543WGNSSI549,F578TSSLGN584 and the like different from other Cry1 A proteins.The ?20-?21 loop of Cry1Ac5 contains F578,S581 and G583 which are highly conserved phenylalanine,serine and glycine residues,so it is inferred that F578,S581 and G583 are among the most important sites and Cry1Ac5 protein ?20-?21 loop structure stability and protein conformation changes may be related to this.Therefore,we determined to ?20-?21 loop as the key research object,through site-directed mutagenesis study of its structure and function.Using the site-directed mutagenesis technique,the 10 residues of Cry1Ac5 protein structure ?20-?21 loop were alanine scanning mutation.All the mutants,including N576 A,F578A,T579 A,S580A,S581 A,L582A,G583 A,N584A,I585 A,V586A,were able to express 130 kDa protein and produce diamond crystals.These results showed that the mutants I585 A and S581 A increased the virulence of cotton bollworm by1.86 and 1.45 times,respectively.However,the mutant G583 A decreased the virulence of Helicoverpa armigera,and the toxicity of other mutants also decreased.Dimensional structure analysis of the Cry1Ac5 protein molecule revealed that the side chain of the 579,580,582,585 position amino acid of the wild-type Cry1 Ac protein extends outwardly toward the surface of the Cry1Ac5 protein molecule,which means Cry1Ac5 protein and its receptor interaction is likely with T579 A,S580A,L582 A,I585A function-related.While the remaining side chains extend inwards towards the inside of the protein molecule,which possibly in relation to the structural stability of the maintenance protein.I585 was further mutated and the I585 E,I585F,I585 T,I585V mutants were constructed and expressed,then we determined its toxicity.The results dhows that I585 F,I585E,I585 T significantly decreased the toxicity,while the I585 V only slightly decreased the virulence.The protease stability and storage stability of I585 A mutant results showed that the protease and storage stability of I585 E,I585T and I585 F decreased,while the protease and storage stability of I585 A were significantly improved.I585 side of the side chain of Cry1Ac5 protein has a negative impact on stability,and the wild-type Cry1Ac5,I585 A crystal morphology and insecticidal activity of the results found I585 A insecticidal activity than wild Cry1Ac1.7 times.For the first time,we found that Cry1Ac5 is unique in the ?20-?21loop structure on domain III,and the function of ?20-?21 loop structure in Cry1Ac5 protein domain III in the function of Cry1Ac5 protein was reported for the first time too.In addition,it explains how Cry1Ac5 protein domain III recognizes and binds to its receptor molecule and how to maintain the structural stability of the protein. |
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