Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. However, there are potential risks of the evolved resistance of insects to Cry toxin owing to decreased binding of toxins to target sites in the brush border membranes of the larva midgut. The Cry toxins with different binding sites in the larval midgut have been considered to be a good combination to deploy in delaying resistance evolution. Bioassay results demonstrated that the toxicity of different Cry toxins ranked differently for each species. The toxicity ranking was Cry1Ac>Cry1Ab>Cry2Ab for Helicoverpa armigera, Cry1B>Cry1C>Cry2Ab for Spodoptera exigua, and Cry2Ab>Cry1B> Cry1C for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with 125I-Cry1Ab, ^(125)I-Cry1Ac, 125I-Cry1B, 125I-Cry1C, 125I-Cry2Ab and the brush border membranes vesicles (BBMV) from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of Cry1Ab and Cry1Ac was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV (K_d =(45.00±2.01) nmol L-1 and (12.80±0.18) nmol L-1, respectively; B_(max) =(54.95±1.79) ng and (55.44±0.91) ng, separately). The binding of Cry1B was shown to be saturable by incubating with increasing concentrations of S. exigua BBMV (K_d =(23.26±1.66) nmol L-1; B_(max) =(65.37±1.87) ng). The binding of ^(125)I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, Cry1B and Cry1C showed some combination with the BBMV of S. litura, and a certain amount of Cry2Ab could bind to the BBMV of A. ipsilon. These observations suggest that a future strategy could be devised for the focused combination of specific cry genes in transgenic crops to control target pests, widen the spectrum of insecticide effectiveness and postpone insect resistance evolution.
LU QiongCAO Guang-chunZHANG Li-liLIANG Ge-meiGAO Xi-wuZHANG Yong-junGUO Yu-yuan
Cry toxins produced by Bacillus thuringiensis(Bt) are effective biological insecticides against certain insect species.In this study,bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua.We also identified a cadherin-like gene in S.exigua that could enhance the toxicity of Cry1B and Cry1C.The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE).The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD.BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects.Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages.The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.
LU QiongZHANG YongojunCAO Guang-chunZHANG Li-liLIANG Ge-meiLU Yan-huiWU Kong-mingGAO Xi-wuGUO Yu-yuan
Credible evidence shows that odorant binding proteins(OBPs)are required for insect olfaction perception and play a key role in transporting hydrophobic odorants across the sensillum lymph to the olfactory receptors(ORs).In the present study,a novel OBP(AlinOBP3)gene from the lucerne plant bug,Adelphocoris lineolatus,was cloned and expressed.The expression pattern of AlinOBP3 was evaluated by qPCR,which indicated that AlinOBP3 was dominantly expressed in antennae.The binding properties of AlinOBP3 with 9 cotton volatiles and 5 sex pheromone analogs were measured by fluorescence competitive binding assays with the fluorescence probe 1-NPN.The results revealed that of 9 cotton volatiles,Myrcene,β-Ocimene and α-Phellandrene can bind with AlinOBP3.α-Phellandrene especially bound to AlinOBP3 with a high binding affinity,with a dissociation constant of 56.68 μmol/L.Of the 5 sex pheromone analogs,Hexyl butyrate had the strongest binding affinity with AlinOBP3,with a dissociation constant as 59.53 μmol/L.Butyl butyrate,trans-2-Hexenyl butyrate and Ethyl butyrate had medium binding affinities with AlinOBP3,with dissociation constants of 227.39,108.77 and 143.47 μmol/L,respectively.The results suggest that AlinOBP3 might be a pheromone binding protein(PBP)with a dual-function for the perception of sex pheromones and plant volatiles.
The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera (Hübner).The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including (E)-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional (3D) model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.
