Detection Of The Vegetative Insecticidal Protein (Vip3A) Gene In Bacillus Thuringiensis Strains And Cloning Of Vip3A Gene From Bt Strain WY-197

Chapter 1: A new rapid method to detect the vegetative insecticidal protein gene(vip3A) in Bacillus thuringiensis strains has been developed. It is based on PCR screening in first-tier, SDS-PAGE analyzing with strain culture-supernatant proteins secondly, and at last bioassay with strains' culture-supernatant proteins to target insect. Vip3A, detected in the culture supernatant during the vegetative growth in some B. thuringiensis strains, has been described as a novel toxin with a wide spectrum against lepidopteran insects, and has a molecular weight of about 88kDa. Based on the known vip3A genes, we designed a new pair of primer for PCR. And the target band of vip3A gene was detected in 10 out of 15 Bt strains by PCR. So, this pair of primer can be considered as a special primer for vip3A gene's detection. All 15 strain's supernatant proteins were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). A band, about 88kDa, was observed in almost all the PCR positive strains except for one strain (L16). It has proved the validity of PCR. In bioassay, the culture supernatant of all the positive strains has high toxicity to Spodoptera exigua and Helicoverpa armigera, while the negative strains have no toxicity to either of them. Otherwise, the boiled supernatants of the positive strains have no toxicity to S. exigua and H. armigera. It indicate that the insecticidal protein in the supernatant is thermal instable, which corresponding with the property of Vip3A protein. Chapter 2 :The full-length vip3A gene,about 2,370bp,coding 790aa, from a wild Bacillus thuringiensis WY-197, has been cloned by the full-length PCR and sequenced. Comparing those reported vip3A genes with the amino acid sequence, it has the similarity about 99% and above to them. It has only three different amino residues from the Vip3A(a) (K303→Q, A466→T, E762→K), and has only a few difference with other vip3A genes too. When vip3A was expressed in prokaryo, we couldn't detect the deduced band of vip3A gene by SDS-PAGE and the insecticidal activity against Spodoptera exigua and Helicoverpa armigera in bioassay, but could observe the inhibiting effect to the larva of two pests. It was presumed to be associated with the inductive conditions.