Increased Bacillus thuringiensis δ-endotoxin Cry3Aa toxicity against longhorned beetle by fusing to peptide specifically binding to beetle Cx-cellulase
© Zhao et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Bacillus thuringiensis (Bt) Cry toxins have specific toxicity to susceptible insects. They are being used in transgenic plants or spray to control insect pests in agriculture [1, 2, 3]. Cry3A toxins are used extensively for biological control of coleopteran larvae [4, 5]. A Bt886-Cry3Aa gene that exhibited a high activity against Coleoptera insects isolated Our laboratory. 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 Cry3Aa and withmodified-Cry3Aa by using poplar-prefered codons did little effects on longhorned beetles probably due to its low expression level in poplar.A peptide (LPPNPTK) named PCx that specifically bind to cellulase from midgut of longhorned beetle larvae was screened out from a phage display library previously in our laboratory.
Materials and methods
Fused Cry3Aa genes with PCx coding sequence at 5’ or 3’-end were amplified using pET-30a(+)-Cry3Aa  as a template and designed primers, and. used to construct three recombinant plasmids (pET-30(+)-PCx-Cry3Aa, pET-30(+)-Cry3Aa-PCx and pET-30(+)-Cry3Aa). Target proteins were characterized by Western Blot, ELISA and LC-MS/MS methods. To analyze the activity of PCx-Cry3Aa, Cry3Aa-PCx and Cry3Aa proteins against longhorned beetle larvae, bioassays were performed on A. germariHope larvae by artifical feed with toxins. The retaining time of target proteins in midgut and the cellulase activity of longhorned beetle larvae were measured in order to elucidate the potential mechanism of the fused toxins of PCx and Cry3Aa against longhorned beetle larvae.
These data demonstrate that the cellulase-binding peptide could enhance the toxicity of Bacillus thuringiensis Cry3Aa against the longhorned beetle.We also confirmed that the increased lethality in larvae fed with PCx-Cry3Aa or Cry3Aa-PCx was attributable to the ability of the toxin to bind Cx-cellulase, thereby increasing toxin retention in the midgut. These uniquely modified Cry3Aa proteins have potential use for pest control. The significantly enhanced activity of Cry3Aa fused with the Cx-cellulase-binding peptide provides a new strategy for increasing δ-endotoxin efficacy against the longhorned beetle.
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