The Toxicity Analysis Of The Fusion Protein And Construction Of Plasmid Vector For Transgene Insect-resistance Cotton

Pests and diseases has caused great influence to crops, not only reducing crop yield and quality, but also significantly affecting the enthusiasm of the farmers. To some extent, the application of chemical pesticides play a role in controlling of pests and diseases; but the usage of it at present, has caused great damage to environmental protection and ecological balance. People pay more attentions to these defects, and then Biological Control came into being.The academician of Japan, Ishiwatari, the first time has isolated Bt(Bacillus thuringiensis) from the infected silkworm. As the materials used in transgenic Bt cotton genetically modified crops, there has been unceasing new discoveries. As the wide range of insect resistant transgenic crop cultivation, insects with a single gene has the effect of insecticide resistance. The resistance is mainly due to the original activition of the insecticidal toxic protein and the intestinal receptor mutation. The main strategy to solve the problem is developping a multivalent different mechanism insecticidal gene.into insect-resistant crop plants.This study according to the bias of the Gossypium hirsutum codon, optimized codons of vip83, cry2A and cry9C, which code Vip83 (Vegetative Insecticidal Protein), Cry2A (Insecticidal Crystal Protein) and Cry9C (Insecticidal Crystal Protein), respectively. The genes were synthesized after optimization, and then ligated the two genes by polylinker, which encodes the enterokinase recognize amino acid sequence sequence (GATGATGATGATAAG) to make the two merge into synthesized a coding frame, constructing the bivalent genes vip83s-cry2As and vip83s-cry9Cs, respectuvely.In order to proof-test the toxicity of the hybrid protein. The five genes cry2As, cry9Cs, vip83s, vip83s-cry2As and vip83s-cry9Cs were constructed to restructure plasmids pGEX-6p-cry2As,pGEX-6p-cry9Cs,pGEX-6p-vip83s, pGEX-6p-vip83s-cry 2As and pGEX-6p-vip83s-cry9Cs, with the prokaryotes expression vector pGEX-6p-1. They all successfully achieved restructuring expression in E.coli BL21 (DE3). With different buffer (PBS buffer pH 7.0 and Gly-NaOH buffer pH 10.0), SDS-PAGE analysis shows that Cry2A、Cry9C、Vip83-Cry2A and Vip83-Cry9C were unsolvable in PBS buffer, Vip83 protein corresponding to the same condition has the solubility of 40%.Whereas,in the Gly-NaOH buffer, Cry2A, Cry9C, Vip83-Cry2A and Vip83-Cry9C have the solubility of 50%. The Vip83 protein even comes up to 65%.The result of Enterokinase cleavage activity in vitro by fused protein Vip83-Cry2A and Vip83-Cry9C indicates that the fusion protein have been separated into two parts correctly. By the sequence of strong promoter in a plasmid,Ωsequence, strong terminator and PolyA sequences of pG4AB, ligating cry2As and vip83s-cry2As with the plant expressed vector pCAMBIA2300, were constructed the vector pC35SΩAN-2A,and pC35SQAN-V-2A,to promote the levels of insecticidal protein expression in plants.Subsequently, bioassay was conducted by using Diamondback moth, which showed the Vip83-Cry2A has more important insecticidal activity than the Vip83 and Cry2A respectively, which laid the foundation of breeding new insect-resistance cotton. Vip83-Cry9C also has more high insecticidal activity than the individual proteins.Added the pG4AB’s strong promoter,Ωsequence, strong terminate and PolyA, construction pC35SQAN-2A and pC35SΩAN-V-2A by pCAMBIA2300 and cry2As vip83s-cry2As genes. Because of the expressed elements,which can enhance the proteins in the cotton, improve the insecticidal activity, extending the spectrum of insecticide, and reducing the production cycle of insect resistance.