Research On Vegetative Insecticidal Protein From Bacillus Thuringiensis 4.0718

Vegetative insecticidal protein of Bacillus thuringiensis wasdiscovered by Estruch in 1996 for the first time. Which express andexcrete into the medium Culture in vegetative period.It has broadspectrum and high insecticidal activity on lepidopter.The nucleotidesequence is different from the known nucleotide sequence of insecticidalcrystal protein.The mechanism is also different from all the crystalprotein,so that Vip3A is a new insecticidal pesticidal protein.Study on theVip3A will help to extending the pesticidal spectrum and retarding theresistance of insect.A series of experiments was conducted on the vegetative insecticidalproteins,the result as Follows:1. The gene of vip3A was identified in different B.thuringiensissubspecies of my laboratory by PCR .And the vip3A gene from Bt 4.0718was cloned ,expressed and the bioassay was conducted with Spodoptraexigua and Helioths vitesces.A pair of primers was designed based on the conserved regions ofvip3A genes, the genome of Bt subsp, tenebrionis 4AA1, Bt subsp.Wuhanensis 140, Bt subsp, kurstaki HD-73 ,Bt subsp.israelensis 4Q5 andBt 4.0718 was used as the template of PCR.only the Bt 4.0718 subspeciescan amplify the special band using the method of PCR. The 2.4-kb PCRproduct was cloned into the pMD18-T vector using standardprocedures.Then the PCR product was sequenced, proved that the Bt4.0718 subspecies contain vip3A gene. The sequence of the vip3-Bt4 hasbeen deposited in the GenBank database under accession numberDQ497637.According the result of bioassay with the supernatant of Bt4.0718 culture, We found it have high virulence to the Spodoptra exigua. And according to the result of SDS-PAGE, we can speculate thepesticidal activity of culture supernatant of Bt 4.0718 is based on theVip3A.The 2.4-kb PCR product ofvip3-Bt4 from Bt 4.0718 was cloned intothe pET28a vector using standard procedures named pEVB.pEVB wastransform into the E.coli BL21,induce the culture using the IPTG with theconcentration 1 mol/L after 1 hour, the expression of Vip3-Bt4 could bedetected by SDS-PAGE.Using the Akta purifier His-tag affinitychromatography pillar to purify the Vip3A. Then Vip3A antibody wasprepared by inject rabbit with pure Vip3A protein. Bioassay wasconducted with first-instar larvae of the Spodoptra exigua and Heliothsvitesces. The LC₅₀ on Spodoptra exigua is 0.9851mg/ml.And the LC₅₀ onHelioths vitesces is 1.5 mg/ml. The mortality determined after 96h was94.8% and 69.44% respectively.2. Using EGFP to detect vegetative insecticidal protein, and study on therelation between vegetative insecticidal protein and spores effect.We can obtain the vector named pHTVG via fusion of the geneegfp and vip3A. The gene of vip3A and egfp were placed under the controlof the promoter of vip3A, insertion into the vector pHT315, namedpHTVG.Transform pHTVG into the B. thuringiensis acrystalliferousstrain CryB.A expressed 110kDa fusion protein was find by SDS-PAGEand Western-blotting.Using fluorescent microscope to detect theemergence of fluorescence,it appeared after 15h and along with the timego on, the emission of fluorescence increased gradually. Distill the sporeafter 72 hours for incubation, the emission of fluorescence can not bedetected. It prove Vip3A doesn't make up of the spore. So we speculateVip3A is concerned with insecticidal activity of spore via devouting byinsect,when spore enter into midst intestines,it can grow into sperm, andexpress the Vip3A, get along with the crystal protein,destruct midstintestines of insect,result in the death of insect finally.