| Along with the planting of transgenic crops carrying Bacillus thuringiensis cry genes,the original target insect pests of Cry toxins such as Lepidopteran,Coleopteran and Dipteran will be efficiently controlled.However,hemipteran pest with sap sucking mouth-part,including planthopper,leafhopper,aphid and stink bugs etc,which are not susceptible to Bt toxins will become more harmful in agriculture production.So far,the Cry toxin with obvious toxicity to hemipteran pest is rarely reported.According to the understanding of mechanism of Cry toxins in the midgut of target insects activation of protoxin and binding of activated toxin with appropriate receptor are two essential steps in triggering insecticidal activity of Cry toxins.In this research,we prepared CrylAc,Cry3Aa and Cry4Ba protoxins,which are respectively effective against lepidopteran,coleopteran and mosquitos.as well as a modified CrylAc toxin with deletion of Helix α1.To test the toxicities of these Cry toxins to N.lugens,protoxin and preactivated toxin of each Cry protein were fed to 3-instar nymphs of N.lugens through membrane feeding strategy.Bioassay results indicated that N.lugens nymphs were not susceptible to all tested Cry toxins.Insecticidal activity of Cry1Ac against N.lugens is relatively higher than other toxins.The LC50 of CrylAc protoxin and activated Cry1Ac toxin reached 198.92 μg/mL and 450.18 μg/mL respectively,and the modified CrylAc-α toxin is slightly toxic than protoxin(LC50=166.92 μg/mL).Cry3Aa toxin shows extremely weak toxicity against N.lugens,with LC50=1810.50 μg/mL for Cry3Aa protoxin and LC50=2895.55 μg/mL for preactivated Cry3Aa toxin.The toxicity of Cry4Ba to N.lugens nymphs is not obvious,with LC50=760.83 μg/mL for Cry4Ba protoxin and LC50=523.16 μg/mL for preactivated toxin.These results verified that low toxicity of tested Cry toxins to N.lugens.To further understand the reason of low toxicity of Cry toxins to N.lugens,proteolytically processing of each Cry toxin by gut proteases of N.lugesn were then conducted.Results showed that whether the cysteine protease in the gut proteases was activated or not,Cry1Ac protoxin could be proteolytically processed into a stable～65 kDa fragment by either soluble gut proteases and membrane-bound gut proteases,which is a little larger than the～60 kDa fragment resulted from the processing of Cry1Ac protoxin directly by trypsin.On the other hand,Cry3Aa protoxin could not be processed into stable fragment under the process of gut proteases of N.lugens,while Cry4Ba protoxin could be partially processed into an appropriate fragment which is the same as trypsin activated Cry4Ba toxin.To analysis the binding specificity of Cry toxins with midgut of N.lugens,in vitro binding assay and competitive binding assay were then conducted by binding of each preactivated Cry toxin with gut brush boarder membrane vesicle(BBMV)of N.lugens.Results showed that preactivated CrylAc,Cry3Aa and Cry4Ba toxins could specifically bind with gut BBMV derive from either N.lugens or the susceptible insects.However,CrylAc toxin could not be competed by GalNAc as what shows in the competitive binding of Cry1Ac with H.armigera BBMV.Binding of Cry3Aa with BBMV of N.lugens or T.molitor,cound not be competed by neither GalNAc nor GlcNAc.In contrast,Cry4Ba toxin could not only bind with specific sites in N.lugesn gut but also be competed by GlcNAc.These results give a brief view on the binding of different Cry toxins against N.midgut.In conclusion,this research verified the insusceptable of N.lugens to Cry toxins and briefly revealed the interaction of Cry toxins with the gut of N.lugens.The conclusion may prelimnary explain why Cry toxin shows low toxicity to N.lugens.The results obtained in this research will provide evidences for analysis of action mechanism of Cry toxin in non-target insects,and will be helpful for further research of the development of new Cry toxin effective to such hemipteran insects by molecular modification. |
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