Construction, Expression Of PTD-Cry1Ac Vectors And Insecticidal Activity Of The Expressed Fusion Proteins

The environmentally friendly insecticide crystal proteins (ICP) produced by Bacillus thuringiensis (Bt)are of great scientific interest because of their potency and specificity to a wide range of insect pests. Because of their highly specific toxicity, Bt toxins offer tremendous benefits for insect pest management. Moreover, Bt genes are the most important genes transferred to many crops, which plays an incearsing important role in insect pest management. However, due to the widely use of ICP, there is a fatal menace to the application of transgenic crops and Bt formulations, that is insect pest resistance. The rational and effective application of Bt has gained great attention and discussion worldwide. Protein transduction domain (PTD) is a kind of peptide fruitfully containing arginine, which has been proved to cargo exogenous macromolecules or charged compounds into living cells. In recent years, unprecedented progress has been made in the PTD application in iatrology. The transduction characteristic has been suscussfully applied in preclinical therapy. In this study, TAT was used to explore the interaction between PTD and ICP of Bt. The fusion protein of PTD-Cry1Ac was constructed, expresses and purified, and bioassay and immunoassay detection proved that PTD enhanced the insecticidal activity of Cryl Ac. The main results are as follows:1. Four kinds of double-stranded oligomeric nucleotide of TAT protein transduction domain encoding 6His and 11 amino acids were synthesized. The espression vectors of 6His-TAT-CrylAc-6His and 6His-Cry1Ac-6His recombinants were constructed and expressed in E.coli. They are pET-1AC vector including pET-21b-Cry1Ac activity gene, for short P;pET-T1AC vector including pET-21b-TAT-Cry1Ac activity gene, for short T;pET-PQ1AC including pET-21b+Cry1Ac partial activity gene, for short PQ;pET-TQ1AC vector including pET-21b-TAT- Cry1 Ac partial activity gene, for short TQ. The contraction was confirmed by immunoassay detection.2. We got the soluble expressed proteins by expressing the vectors. The soluble proteins were much more than the precipitable proteins by using 20 ℃, 116 rpm, fostered 26 h and the concentration of IPTG was 0.1 mol/ml. It was convenient to purification.3. Bioassay of the espressed fusion protein against P.xylotella of laboratory colony and populations collected from the field was conducted. Results of the insecticidal activity results are as follows: For the population collected from the field, LC₅₀ of pET-T1AC(T)with TAT was 60.20 μg/ml, LC₅₀ of pET-T1AC(P) was 80.82 μg/ml, and the synergism radio was 1.34-fold;LC₅₀ of pET-TlAC(TQ)with TAT was 100.70 μg/ml, LC₅₀of pET-TlAC(PQ) was 116.67 μg/ml, and the synergism radio was 1.16 -fold. For the laboratory colony, LC₅₀ of pET-TlAC(TQ)with TAT was 20.51 μg/ml, LC₅₀ of pET-TlAC(PQ) was 97.60 μg/ml, and the synergism radio was 4.76- fold. These results indicated that PTD-Cry1 Ac significantly increased the insecticidal toxicity of Cry 1 Ac.4. The four highly expressed fusion proteins were purified. The affinity of the purified proteins with BBMV isolated from P.xylotella was analysed by ELISA. Results showed that the enhanced toxicity was correlated with that PTD increased the affinity of purified proteins with BBMV. The synergistic effect of PTD to Bt, as well as its potential to overcome Bt resistance was first identified, which proved a new way to overcome insect resistance by using the carrier ability of PTD in the future.