| Spodoptera exigua is one of the main serious world-wide agriculture pests with fast ascending resistance to chemical pesticides. Crystal protein Cry1C, produced in sporulation of Bacillus thuringiensis, triggering channel activity in Spodoptera grugiperda and S. exigua cell lines, has specific activity against S. exigua, but its activity is still too low to be used for field application, even little or no toxicity against 3rd or 4th insect larvae. In contrast, Cry1Aa was proved to have high toxicity to the lepidopteran insects, but little toxicity to noctuidae insects, such as S. exigua. As the Cry1Ca domain III can function as a specificity determinant for S. exigua, we hope to swap the domain III between Cry1Ca and Cry1Aa to construct hybrid proteins with high toxicity against the target. In the meantime, to investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future improvement of toxicity against S. exigua, the synergism between the proteins has been evaluated using synergistic factor. For the cloning of cry1Aa and cry1C genes, plasmid pMPCV1 and pMPCV2, which contains cry1Aa and cry1C respectively, were digested with SphI and SmaI. The corresponding fragments of 4.7 kb and 4.2 kb were purified and ligated respectively to SphI-SmaI digested vector pBU4 to get the Eschechia coli recombinants E- pCX1A and E- pCX1C. B. thuringiensis recombinants Bt- pCX1A and Bt- pCX1C were obtained by transforming pCX1A and pCX1C to Bt 4Q7 strain respectively by electroporation. In LB media the two Bt recombinants grow and develop normally, and they could produce crystal bodies during the sporulation. SDS-PAGE showed that the two recombinants could express a 135kD protein and a 130kD protein respectively.The bioassay results showed that when Cry1C and Cry1Aa were mixed with a ratio of 1:1, 1:5 and 5:1, all different mixtures were more toxic than observed activities for Cry1Aa or Cry1C alone, having LC50 values ranging from 0.537 to 1.709 mg/ml. In the combination of 1:1 ratio, the LC50 value was 0.537 mg/ml, and a similar high activity was observed at the LC90 level. It is indicated that all of the mixtures were synergistic, with synergistic facter (SF) values ranging from 1.8 to 4.0 and from 1.1 to 4.4 at the LC50 and LC90 level against S. exigua. The 1:1 ratio of Cry1Aa and Cry1C was the most active combination tested. The SF values of the combinations against H. armigera were less than that of the combinations against S. exigua, the combinations of Cry1C and Cry1Aa were more toxic than Cry1C alone, with the LC50 values of 0.204, 0.673, 0.329 mg/ml at the ratios of 1:1, 5:1, and 1:5 respectively. However, SF values from the data of H. armigera were affected by the difference in toxin concentration, ranging from 1.2 to 2.7 at the LC50 value and from 1.1 to 1.9 at the LC90 value respectively, and the highest toxicity was observed at the combination of 1:1 of Cry1C and Cry1Aa.For the domain swapping, two unique restriction endonucleases XhoI sites were created with site-mutagenesis kit respectively between Domain I and Domain II of Cry1Aa and Cry1C and two Escherichia coli mutants, E- pCR1AM and E- pCR1CM, were obtained. Homonogous recombination cry genes, which contain Domain I from cry1Aa and Domain II, III from cry1C, Domain I from cry1C and Domain II, III from cry1Aa respectively, were constructed successfully. However, the properties and the expression of the two genes are still needed to be investigated. The obtained data may lay the foundation for further research on domain swapping and studies on the structure and function of different domains of Cry proteins.
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