| Pests are one of the main factors affecting the productivity of agriculture and forestry.With the increasing pollution of chemical pesticides to the environment,the application of biological pesticides is becoming more and more common.Bacillus thuringiensis?Bt?can produce different types of insecticidal proteins,such as:Insecticidal crystal proteins?ICPs?,and vegetative insecticidal proteins?Vips?.Due to the differences in insecticidal spectrum and mechanism of action between Vips and ICPs,Vips has always been a rese arch hotspot.Since the discovery of Vip3A protein,although there are a lot of studies on Vip3A protein,its three-dimensional structure has not yet been resolved,the relationship between the structure and function of Vip3A protein is poorly understood,and there are a series of questions such as the improvement of the insecticidal activity.In this study,Vip3Aa39,which has broad-spectrum insecticidal activity against Lepidoptera pests,was selected as the object,and the key functional areas and key amino acid sites of Vip3Aa39 were identified by constructing chimeric proteins and mutants.The inactive Vip3Ad is a new selection in this study,which is used to construct chimeric gene with Vip3Aa39 in order to obtain key amino acid fragments.By comparing the nucleotide sequences of vip3Aa39 and vip3Ad,using homologous recombination technology,the amino acid fragments of Vip3Aa39 and Vip3Ad were exchanged by overlapping extension PCR method,and 10 chimeric genes were constructed to induce the expression of protein and to determine the biological activity of three Lepidoptera insects,Helicoverpa armigera,Plutella xyllostella and Spodoptera exigua,and the three-dimensional structure information of the modified Vip3Aa39 proteins were predicted by homology modeling,and the key amino acid fragments of Vip3Aa39 were obtained by comparative analysis of the biological activity and structural relationship of the chimeric proteins.In order to further study the key amino acid sites of Vip3Aa39,analysis of amino acid homology of Vip3Aa11and Vip3Aa39 was conducted,and it was found that there were only 39 amino acid differences between them.Using bioinformatics method,15 different targets were identified,and the corresponding amino acid in Vip3Aa39 protein was mutated into the corresponding amino acid of Vip3Aa11,and 15 mutants were constructed.After induction and expression,the biological activity of Helicoverpa armigera,Plutella xylostella and Spodoptera exigua were measured.Combined with the predicted three-dimensional structural information of Vip3Aa39 mutant protein,five key amino acids affecting the activity of Vip3Aa39 protein were determined.The main results are as follows:1.Screening the inactive vip3Ad gene of chimeric protein construction material.In order to obtain inactive Vip3A protein and highly active Vip3Aa39 protein to study the key areas of amino acid,we used temperature screening method and PCR method to screen new genes from 4034 soil samples.The vip3Ad gene was obtained from strain BJG810.The coding frame length of vip3Ad gene was 2361 bp,and the nucleic acid sequence similarity to vip3Ad2?CAI43276?reached99.36%.The sequence was submitted to Gen Bank and the accession number was KP346519.After transformation into BL21?DE3?,expression was induced to obtain 88k Da protein.The biological activity assay showed that the protein had no insecticidal activity against the above three Lepidopteran test insects Helicoverpa armigera,Plutella xylostella and Spodoptera exigua.Compared with the Vip3Aa39 sequence,the amino acid homology of the two proteins was 85%.Therefore,the genetic material for chimeric protein construction was obtained.2.Constructed the chimeric proteins and obtained the key amino acid fragment of Vip3Aa39protein.Four Vip3Ad Aa proteins starting from the N-terminus of Vip3Ad protein were successfully constructed.By replacing the N-terminal amino acid fragment of Vip3Aa39 with the corresponding fragment of Vip3Ad protein,4 recombinant proteins were obtained,namely Vip3Ad Aa-1?amino acids 1-60 were replaced by Vip3Ad?,Vip3Ad Aa-2?amino acids 1-116 were replaced by Vip3Ad?,Vip3Ad Aa-3?amino acids 1-344 were replaced by Vip3Ad?,Vip3Ad Aa-4?amino acids 1-455were replaced by Vip3Ad?.Compared with Vip3Aa39,the LC50 of Vip3Ad Aa-1 is lower than that of three insects,and the insecticidal activity is significantly increased.Vip3Ad Aa-1 and Vip3Aa39have only three different amino acids?T6,N45,E60?;The insecticidal activity of Vip3Ad Aa-2against Helicoverpa armigera and Plutella xylostella was not significantly different from that of Vip3Aa39;however,the insecticidal activity against Spodoptera exigua was significantly reduced;compared with Vip3Aa39,the insecticidal activity of Vip3Ad Aa-3 against Helicoverpa armigera was significantly improved and its activity against Spodoptera exigua was reduced.Compared with Vip3Aa39,the insecticidal activity of Vip3Ad Aa-4 against Plutella xylostella was significantly increased.Three proteins of Vip3Aa Ad Aa class were successfully constructed,by replacing intermediate amino acid fragments of Vip3Aa39 to corresponding fragments of Vip3Ad.They are Vip3Aa Ad Aa-1?amino acids 116-210 replaced by Vip3Ad?,Vip3Aa Ad Aa-2?amino acids 210-345replaced by Vip3Ad?,Vip3Aa Ad Aa-3?amino acids 455-632 replaced by Vip3Ad?.The insecticidal activity of Vip3Aa Ad Aa-1 against Spodoptera exigua was significantly reduced,while the insecticidal activity against Helicoverpa armigera and Plutella xylostella were almost lost.And amino acid comparison found that Vip3Aa Ad Aa-1 and Vip3Aa39 only have three different amino acid positions?A134V,I176M,S200P?;the insecticidal activity of Vip3Aa Ad Aa-2 against Helicoverpa armigera significantly improved and the insecticidal activity against Plutella xylostella reduced;the insecticidal activity of Vip3Aa Ad Aa-3 against Plutella xylostella and Spodoptera exigua significantly reduced.Three Vip3Aa Ad proteins ending in the C-terminus of Vip3Ad protein were successfully constructed.They are Vip3Aa Ad-1?amino acids 345-789 are substituted?,Vip3Aa Ad-2?amino acids 727-789 are substituted?,Vip3Aa Ad-3?amino acids 778-789 are substituted?.The insecticidal activity of the protein showed that Vip3Aa Ad-1 and Vip3Aa Ad-2 lost their toxicity to three kinds of insects,while Vip3Aa Ad-3 had insecticidal activity against all three kinds of insects.Compared with Vip3Aa39,the insecticidal activity of Vip3Aa Ad-3 against Helicoverpa armigera increased and the insecticidal activity against Plutella xylostella reduced.Based on the above analysis,61-116 and 116-210 of Vip3Aa39 are the key amino acid fragments affecting the activity against Helicoverpa armigera;1-60,210-345,778-789 amino acid fragments of Vip3Aa39 can be modified to increase insecticidal activity aga inst Helicoverpa armigera.116-210,210-345,455-632,778-789 of Vip3Aa39 are the key amino acid fragments affecting the insecticidal activity against Plutella xylostella;1-60,1-455 of Vip3Aa39 can be modified to enhance the insecticidal activity against Plutella xylostella.61-116,116-210,1-344,455-632 of Vip3Aa39 are the key amino acid fragments affecting the insecticidal activity against Spodoptera exigua.Among them,116-210 of Vip3Aa39 are the key amino acid fragments against these three insects,and 1-60 can be modified to enhance the biological activity of the three test insects.The combination of two important amino acid sites was clarified.After modification of the combination of?A134V,I176M,S200P?the insecticidal activity against three s pecies of insects lost,indicating that it is a key amino acid site combination of Vip3Aa39;After modification of the combination of?T6A,N45D,E60D?,the insecticidal activity against three test insects increased significantly,indicating that the biological activity can be improved by modifying the combination of amino acid sites.3.Obtained 15 mutant proteins and identified 5 key amino acid sites of Vip3Aa39 for insecticidal activity.15 mutants?N9S,A10T,T193S,L194S,S200G,N543S,L544I,G546E,E547D,V681T,I685T,R686S,L780I,K782H,and N784Y?were successfully constructed.Through the determination of insecticidal activity and structural analysis of mutant proteins,the results showed that:compared with Vip3Aa39,the insecticidal activity of t he mutant protein against three insects had changed to varying degrees.The insecticidal activity of L544I,R686S and N784Y mutant proteins against Helicoverpa armigera and Plutella xylostella were significantly improved.The insecticidal activity of N9S,S200G,N543S,L544I,E547D,V681T,I685T and K782H mutant proteins against Spodoptera exigua all significantly increased.Therefore,the five amino acid positions of L194,N543,L544,K782,and N784 may be identified as the key sites for the insecticidal activity of Vip3Aa39 protein.4.The results of tryptic digestion of chimeric protein s and mutant proteins showed that,except Vip3Aa Ad-2 and A10T,the rest were digested into stable 62k Da fragments,confirming that trypsin was involved in the activation of the engineered protein;this result further showed that The reason why Vip3Aa Ad-2,A10T protein does not have insecticidal activity may also be that the protein cannot be digested by trypsin into the core fragment.In summary,based on the differences in the insecticidal protein activity of Vip3Aa39 and Vip3Ad,Vip3Aa39 and Vip3Aa11,and the difference in amino acid sequence,this study modified Vip3Aa39,constructed the chimeric proteins and mutant proteins of Vip3Aa39,and obtained the key active areas related to the insecticidal activity,which are of guiding significance for the in-depth study of the function of Vip3Aa insecticidal gene and the relationship between function and structure.Additionally,this study obtained the highly active Vip3A a insecticidal protein,which provides genetic materials for the construction of efficient engineering bacteria and the cultivation of transgenic plants,delaying insect resistance,and also provides a theoretical basis for the application of such genes in the fields of biological pesticides and genetic modification. |
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