Construction And The Toxicity Analysis Of Bacillus Thuringiensis Chimeric Insecticidal Protein

As an important insecticidal microorganism, the Bacillus thuringiensis (Bt.) has been widely used for biological control and genetic modification field. A problem that has been widely concerned is the growing resistance of agricultural pests. Therefore, extending novel insecticidal gene from Bacillus thuringiensis and finding the way to slow insect resistance become the current issues in biological control and genetically modified studies.Vegetative insecticidal proteins (VIPs) are regarded as a second-generation insecticidal proteins. Different from the traditional widely used insecticidal crystal proteins (ICPs), VIPs have a distinct insecticidal mechanism and a broad insecticidal spectrum. Recently genetically modified crops containing VIPs have been successfully bred. However, the study of the synergistic between VIPs and ICPs was rarely reported around the world. This study used genes, vip3Aa7and several crys optimized for expression in Gossypium hirsutum maintained in this laboratory to construct several new fusing genes which expressed chimeric insecticidal proteins and then studied their feature of toxic. The main results are as follows:1. The construction of Bt. chimeric insecticidal proteins with plant expression vector and pCPANEK genie plant expression vector.Using muticopy enhancer and CaMV35S promoter, TmvΩ and Kazak translation enhancing sequences, PolyA sequence, GSP sequence and the Nos terminator sequence,we construnted the expression box. The pCAMBIA2300plant expression vector was used as an original plasmid. We constructed the pCPANC1C, the pCPANC2A, pCPANC9C, pCPANV3AC1C, pCPANV3AC2A and pCPANV3AC9C plant expression vectors. These plant expression vectors were delivered to the Chinese Academy of Agricultural Sciences Cotton Research Institute for breeding a new transgenic Bt cotton.We used the expression box and pCAMBIA2300vector construct a pCPANEK current plant expression vector. A enterokinase site (EK site) was added between5’ and3’ expression box, to directly built the bivalent chimeric protein in a simple way. Using the pCPANEK vector could reduce the complicated cloning operation.2. Fusing the vegetative insecticidal protein Vip3Aa7and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvaeIn this thesis, we constructed several chimeric proteins using Vip3Aa7and Cry1Ca Cry2Aa, Cry9Ca genes. In chimeric proteins, V3AC9C and vip3Aa7are at5’region preceded by a GST-tag and removed the stop codon. N-terminus of the Cry proteins was at3’region, with the start codons linked to the3’region of vip3Aa7. GST-tag, vip3Aa7and N-terminus of cry proteins were in the same expression cassette. All the expression plasmids were transformed into E. coli BL21cells. The results are shown that:Most proteins were correctly expressed in BL21. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the V3AC9C chimeric proteins containing Vip3Aa7and N-terminus Cry9Ca was markedly higher than either of the single toxins. The LC50value for chimera was0.322μg/mL, whereas the LC50for the single N-terminus of Cry9Ca was0.915μg/mL, and the LC50for the Vip3Aa7alone was4.9μg/mL. The value for chimera was15.3-fold higher than the single Vip3Aa7,2.8-fold higher than the single Cry9Ca. The total expression of the chimeric protein was61.3%of total expression of single non-chimeric N-terminus of Cry9Ca.Treatment of the V3AC9C chimeric protein, Vip3Aa7and N-terminus of Cry9Ca with trypsin, the products were separated by SDS-PAGE.The results indicated that the chimeric protein could be cleaved in two single toxins in the midgut.Vip3Aa7and Cry9Ca were assumed to have a mass ratio of1:1in the V3AC9C chimeric protein. Hence, we mixed the single Vip3Aa7and the single N-terminus of Cry9Ca (mass ratio of1:1) as a control. As we designed, the chimeric protein was processed into two single toxin when it entered the midgut of the insect, Vip3Aa7and Cry9Ca,to separately exert their toxic effects, the chimeric protein should have the same toxicity with the mixture of Vip3Aa7and Cry9Ca toxins (mass ratio of1:1). the toxicity of the mixture of the Vip3Aa7and Cry9Ca toxins (mass ratio of1:1) was only1.056μg/mL. Calculating the SF for Vip3Aa7and Cry9Ca in chimeric protein V3AC9C was4.79. Meanwhile the SF in the mixture of the Vip3Aa7and Cry9Ca toxins (mass ratio of1:1) is only1.46. Solubility analysis and microscopic examination showed that the chimeric protein Vip3Aa7can help Cry9Ca dissolve in alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly attributed to the increase in solubility.These results lay a foundation on the development of a new generation of bio-insecticides and multi-gene transgenic plants.3. Bacillus thuringiensis vegetative insecticidal protein Vip3Aa7cysteine mutant.Previous studies have shown the toxicity of Bt. insecticidal proteins was related to its solubility. In our research, Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7protein by site-directed mutagenesis. The SDS-PAGE analysis indicated the solubility of three mutants was increased. However, bioassays tested on Plutella xylostella showed the toxicity of C401S and C507S were completely abolished.In additional, We found the mutant C507S was sensitive to treatment of trypsin. The Vip3Aa7wild-type, C292S and C401S mutants were processed into62kDa core-fragment. But C507S mutant was degraded rapidly and hardly formed62kDa core-fragment. So it was speculated conserved amino acid sequence resulted in preserved spatial structure, which ensured that Vip3A-type proteins were not completely degraded by trypsin. The change of Cys507→Ser507altered the structure and reduced the quality of the62kDa core-fragment thereby resulting in loss of toxicity. The result suggested a possible association between insecticidal activity and trypsin sensitivity of Vip3A. This study serves a guideline for the study of Vip3A protein structure and active mechanism. Although the attempt which increasing toxicity of Vip3Aa7by mutation of cysteine residues was failed. We still believe that this idea may be applied on the rest of the Bt. toxin.