Cx-Cellulase Binding Peptides Increase The Toxicity Of δ-endotoxin Against Longhorned Beetle

Longhorned beetle is one of the branch-borers that have caused a mass damage to wood. Holes that formed in truck by honghorned beetle lervae in xylem of living trees affect wood development and ultimate wood quality, thus cause a great loss in forestry production and a big damage to the environment. Although there are many strategies in controling longhorned beetles, the results are still unsatisfactory. Chemical pesticides can not control longhorned beetle larvae effectively because their larvae hide in trucks, and refuse to the influence from external environment. Biological control exhibit advantages on safe and strong selectivity compared to traditional methods of pest control. Bacillus thuringiensis is one of the important microorganism that produce insecticidal crystal proteins exhibiting specific toxicity for susceptible insects. Transgenic Bt crops are worldwide planted because of their insecticidal traits. Similarly, genetic engineering should be an effective way to control the longhorned beetle damage.Our laboratory have isolated a Bt886-Cry3 Aa gene that exhibited a high activity against Coleoptera insects. Insect bioassay performed on Anoplophora glabripennis Motsch and Apriona germari Hope showed that the mortality of larvae fed with the product of this gene was over 60%. However, both transgenic poplar with native Cry3 Aa and with modified-Cry3 Aa by using poplar-prefered codons did little effects on longhorned beetles probably due to its low expression level in poplar.Two peptides P1(LPPNPTK) and P2(TPHRSPL) that specificaly bind to cellulase from midgut of longhorned beetle lavae were screened out previously in our laboratory. Cellulase is the primary digestion enzyme in the midgut of larvae. In this study, these peptides were fused to Cry3 Aa protein to creat the binding between Cry3 Aa with cellulase. There are two potential mechanisms leading to the enhanced activity of Cry toxin against longhorned beetles: 1) the remainning time of fused proteins in midgut may be prolonged, which is beneficial for Cry3 Aa to fully play its role I; 2) The inhibit of the cellulase activity affects the development of longhorned beetles.The DNA sequence coding for P1 and P2 peptides were added in the 5’-end of the designed primers. Six fused DNA fragments, of which 4 have one peptide(P1/P2), and 2 have two peptides(P1+P2) were obtained by PCR amplication. Seven prokaryotic expression vectors named p ET30a(+)-Cry3 Aa, p ET-30a(+)-P1-Cry3 Aa, p ET-30a(+)-P2-Cry3 Aa, p ET-30a(+)-Cry3Aa-P1, p ET-30a(+)-Cry3Aa-P2, p ET-30a(+)-P1-Cry3Aa-P2, p ET-30a(+)-P2-Cry3Aa-P1 were constructed by routine molecular cloning technique we produce.The target protein in expression products was idenbtified using SDS-PAGE gel electrophpresis, Western Blot, ELISA, LC-MS/MS assays. The results of insect bioassay performed on A. germari Hope larvae showed that the toxicity of P1-Cry3 Aa, Cry3Aa-P1 and P1-Cry3Aa-P2 was enhanced obviously compared to Cry3 Aa. The corrected mortality rate of P1-Cry3 Aa was almost three times higher than that of Cry3 Aa. The inhibition of the growth of larvae fed with P1-Cry3Aa-P2 measured by weight reached to 62.4%, was three times more than that with Cry3 Aa alone. Therefore, P1 and P2 fused with Cry3 Aa produced fused-Cry3 Aa proteins that exhibit higher toxicity than Cry3 Aa alone. This provides a new strategy to enhance the activity of Cry3 A toxins against longhorned beetles.The retaining time of fused Cry3 Aa in midgut and the effect of fused Cry3 Aa on cellulase activity in midgut of A. germari Hope were used to analyze the mechanism of the toxicity enhancement of the fused Cry3 Aa. Retaining time analysis was performed on excretas that collected at different times. The result showed that the fused Cry3 Aa was centralized at 6 h, whereas Cry3 Aa at 4 h. This indicated that the retaining time of fused Cry3 Aa is longer in midgut compared to Cry3 Aa. Meanwhile, The Cry3 Aa concentration in midgut guice after fed with fused Cry3 Aa was higher than that with Cry3 Aa alone. This result is consistent with the result that the higher Cry3 Aa content was found in in excreta fed with Cry3 Aa alone. Therefore, the remainning time of fused Cry3 Aa is prolonged after binding with cellulase, and the enhanced toxicity of fused Cry3 Aa is due to the prolonged retaining time.We also analyzed the cellulase activity when bond with fused Cry3 Aa or Cry3 Aa alone. The result showed that the fused protein does not affect the activity of cellulase. Therefore, the enhancement of Cry3 Aa avtivity against longhorned beetles in this experiment is no due to the inhibition of cellulase activity, but to the prolonged retaining time. These fused Cry3 Aa proteins could enhance the toxicity against to longhorned beetle larvae thus the new fused Cry3 Aa genes, if its expressed fused Cry3 Aa proteins exhibiting a stable toxicity to longhorned beetles in transgenic poplar, would make its contribution in resistance breeding of trees against loanghorned beetles.