Studies On Expression Of Bt Toxin CryIAb/Ac Genes In Pichia Pastoris GS115

Bacillus thuringiensis is an important and widely used microorganism as microbial insecticides in agriculture production. Because there are several disadvantages during the application and the development of insecticide resistance, people have tried to transfer Bt toxic protein genes to plants or others microorganisms through biotechnological means to expand the range of insecticides and decrease the pest-resistance to toxic protein. In this study, cryLAb/Ac was cloned and transferred into Pichia pastoris GS115 and a genetic modified strain with cryLAb/Ac was constructed successfully.Primers with restriction enzyme cutting site recognized by Not I and EcoR I were designed with the help of software named Primer5, cryLAb/Ac gene about 1.8kb encoding three structure domain of toxic protein was amplified by PCR and the two gene fragments were subcloned to construct the vector named pMD-cryLAb/Ac. The vector was sequenced and there are 2 bases has changed in cryLAb gene which cause the change of coding to one amino acid but not affect the coding of active center of toxic protein, cryLAc have the same sequence as the original one. Choose pPIC9K as expression vector, digested the pMD-cryLAb/Ac and expression vector pPIC9K with EcoR I and Not I, and rejoin with T4 ligase to construct a secretory recombinant vector pPIC9K-cryLAb/Ac. Transfered to Pichia pastoris GS115 by electrotransformation, the positive transformants were selected. PCR result proved the cryLAb/Ac gene has been transferred into Pichia pastoris GS115 successfully.With the induction of methanol, CryLAb/Ac toxic protein has been effectively secreted and expressed. By SDS-PAGE gel electrophoresis analysis, all the molecular weight of recombinant protein expressed by cryLAb/Ac gene are around 70kDa. The induced protein expression is at the highest level in fermentation flask for 72 hours at pH 6.6. Bioassay results showed that the toxic protein coded by cryLAb/Ac has a strong toxic effect to both Spodopteru exigue and Chilo suppressalis. LC₅0 of CrylAc toxic protein on the Spodopteru exigue is 0.28μg/ml. CryLAb toxic protein on the Spodopteru exigue is 0.76/μg/ml. the LC₅0 of CryLAb toxic protein on the Chilo suppressalis is 0.40μg/ml, the LC₅0 of CrylAc toxic protein on Chilo suppressalis is 0.28μg/ml.