Molecular Design, Construction And Expression Of A Fused Insecticidal Gene

Pests seriouly threaten agricultural production. The genetic engineering of insect-resistant plants has been proved very effective in the Integrated Pest Management Program. On the other hand, pests will develop resistance to insect- resistant transgenic plants with the wide planting of these plants. The fused insecticidal protein may delay the process of insects?developing resistance. After investigation of the secondary structure and action mode of Bt insecticidal protein (CryIA) and Cowpea trypsin proteinase inhibitor (CpTI), as well as the action mechanism of insect enterokinase, we devised the fusion gene on the base of maintaining their functions respectively. The method follows: CpTI gene is linked to 3?-end of GFM crylA gene by a nucleotide sequence encoding cleavage site of insect enterokinase. The 282bp of CpTJ gene obtained by PCR amplification was cloned into the plasmid pUC 19 and then the adaptor, which contains a nucleotide sequence encoding cleavage site of insect enterokinase, was linked to 5?-end of CpTJ gene. The CpTI gene with the adaptor was cloned into pG4AB at the 3?-end of the encoding region of GFM cry]A gene and therefore we obtained the plasmid pGF4ABC harboring the fused insecticidal gene CryCI. The CryCI gene and essential regulation elements cut from the plasmid pGF4ABC were inserted into yeast expression vector pPIC9K and plant expression vector pBI 121.1, so we got the secretive yeast expression plasmid pPIC9KBC and plant high-efficiency expression plasmid pGBIF4ABC, Fifty-two His+Muts transformants were obtained after the expression vector pPIC9KBC was introduced into Pichia pasoris, KM7I strain, by electroporation. Five yeast clones were verified to be recombinant clones carrying the fused gene CryCI by PCR amplification with the primers of lit Cry IA gene. The recombinant 96 rate was 9.6 percent. The induction expression of the recombinant yeast clones was carried out by the medium which had the methanol as the only carbon resource and the fused protein of 75kD was expressed. The fused protein from pPIC9KBC/KM71 yeast strain had the insecticidal activity and could be digested by enterokinase in vifro. The tobacco leave discs were transformed with Agrobacterium tumefaciens carrying the plant expression plasmid pGBIF4ABC. We got thiity-four transgenic tobacco plants containing the fused gene CryCI after kanamycin screening, PCR detection and Southern blot analysis. The expression of 75kD fused protein in transgenic plants was proved by western blot. ELISA analysis with the antibody of Bt crystal protein as the first antibody and the inhibition activity analysis of the trypsin proteinase demonstrated that both of the two constitutive parts (Cry 1 A and CpTI) of the fused protein could be expressed effectively in transgenic tobacco plants with the fused gene cryCi Leaf disc bioassay of the total 34 transgenic plants resistant to cotton bollworm (H Armigera) larvae showed that three of them (8.8 percent) had high resistance and 14 of them(4 1.2 percent) had moderate resistance. The result proves that the fused protein has the insecticidal activity. Leaf disc bioassay of the transgenic plants resistant to cotton bollworm (H Armigera) larvae which had developed resistance to monovalent Bt transgenic cotton plants...