| Bacillus thuringiensis (Bt), an insect pathogen can produces insecticidal protein toxins (ICPs)during sporulation within a crystalline inclusion in the mother cell, additionally it can secrete vegetativeinsecticidal proteins (VIPs) into the extracellular medium. This insecticidal activity against manyimportant insect pests has allowed Bt to be developed as a biological control agent and an alternative tosynthetic chemicals. The toxin genes have also been expressed in crops to control agricultural pests.Many laboratories have an interest in collecting Bt strains and screening them for new toxin genes inorder to expand target spectrum and of existing toxins and virulent novel genes. Various approacheshave been developed to identify toxin genes from Bt strains. Whilst these approaches were notpracticable for large strain collections with disadvantages of challenge and time-consuming screeningprograms. There was a need then to be able to solve these problems. We have demonstrated that it wasan efficient way to mine novel genes from a pooled DNA sample from a very large culture collection,moreover, it provided theory foundation for the promotion and application of searching for genes.In this work we cloned15vip3A genes. We assessed the practicability of mining a mixture ofgenomic DNA from a two thousand strain collection for vip3A genes successfully. Based on thealignment of vip3A-type genes, one forward primer and two alternative reverse primers were designedthat together should amplify any vip3A gene containing these conserved regions. Using PCR-RFLPmethod, a total of500recombinant clones were randomly selected from which it was found that288contained a vip3A gene in the correct orientation for expression. The PCR-RFLP analysis of thefragments revealed six distinctly different profiles. Based on sequenceing and blast, fifteen novel vip3Agenes were registered in GenBank and given names by the nomenclature committee, includingvip3Aa52, vip3Aa53, vip3Ad3, vip3Ad4, vip3Ag7, vip3Ag8, vip3Ag9, vip3Ag10, vip3Ag11, vip3Ag12,vip3Ag13, vip3Ag14, vip3Ag15, vip3Aj1as well as vip3Aj2and vip3Aj1gene belonging to the thirdholo-type novel genes.We found Vip3Ag8protein showed activity against Pseudaletia separate. In order to assesswhether there were any differences in toxicity between the various Vip3A toxins, the correspondinggenes were cloned and expressed in E. coli Rosetta (DE3). SDS-PAGE analysis showed that a circa88kDa band was present for all clones. None of the Vip3A toxins showed any toxicity towards Ostriniafurnacalis, Helicoverpa armigera, Plutella xylostella, Agrotis ipsilon, Pseudaletia separata orSpodoptera exigua, except Vip3Ag8showed activity against Pseudaletia separate with the mortality of54.68%. A multiple sequence alignment of those Vip3Ag protiens reveals that155Vand176MofVip3Ag8protein are particularly different from all the other variants, which may affect the activity.Moreover, the beginning of trypsin-activated of Vip3Aa may forcus on the position of No.39aminoacid. To determine the region of activited Vip3Aprotein was unclear, we optimize the conditions ofaffinity purification and digestion. Activation of the Vip3A proteins, either in crude extracts or after affinity purification, was carried out with trypsin. These four proteins, including Vip3A production,purification and activation were tested against S. frugiperda. Bioassays showed that protoxin from crudeextract was significantly more active than activated toxins from crude extract. Metal-chelate affinitychromatography purification resulted in a significant loss of activity of Vip3Aa protoxin. Again, Vip3Aatoxicity was reduced after digestion, while they all stay growth inhibition. The results of N-terminalamino acid sequencing and peptide mass fingerprint analysis showed that N-terminal amino acid oftrypsin-activated of Vip3Aa may begin from the position of No.39amino acid. The study on sequenceanalysis of activated Vip3Aa protein will promote the interaction mechanism between Vip3Aa andinsects. |
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