The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa a...The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible（96S） and Cry1Ac-resistant H. armigera strain（Cry1Ac-R）. The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance（SPR） showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A＋Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.展开更多
Bacillus thuringiensis(Bt)insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells o...Bacillus thuringiensis(Bt)insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action.Previous studies suggested aminopeptidase N1(APN1)as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches.In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout.Three APN genes(HaAPN1,HaAPN2 and HaAPN5)were individually knocked out in a susceptible strain(SCD)of H.armigera to establish three homozygous knockout strains.Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout.Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain.This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H.armigera.展开更多
Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low to...Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.展开更多
Evolution of resistance to Cry proteins in multiple pest insects has been threatening the sustainable use of Bacillus thuringiensis(Bt)-transgenic crops.Better understanding about the mechanism of resistance to Cry pr...Evolution of resistance to Cry proteins in multiple pest insects has been threatening the sustainable use of Bacillus thuringiensis(Bt)-transgenic crops.Better understanding about the mechanism of resistance to Cry proteins in insects is needed.Our preliminary study reported that the transcription of HaABCC3 was significantly decreased in a near-isogenic line(LFC2)of a Cry1Ac-resistant strain(LF60)of the global pest Helicoverpa armigera.However,the causality between HaABCC3 downregulation and resistance to Cry1Ac remains to be verified,and the regulatory mechanism underlying the HaABCC3 downregulation is still unclear.In this study,our data showed that both HaABCC3 and HaABCC3 downregulation were genetically linked to resistance to Cry1Ac in LF60.However,no InDels were observed in the coding sequence of HaABCC3 from LF60.Furthermore,F_(1) offspring from the cross of LF60 and a HaABCC2/3-knockout mutant exhibited moderate resistance to Cry1Ac toxin;this indicated that the high resistance to CrylAc toxin in LF60 may have resulted from multiple genetic factors,including HaABCC2 missplicing and HaABCC3 downregulation.Results from luciferase reporter assays showed that promoter activity of HaABCC3 in LF60 was significantly lower than that in the susceptible strain,which indicated that HaABCC3 downregulation was likely mediated by promoter variation.Consistently,multiple variations of the GATA-or FoxA-binding sites in the promoter region of HaABCC3 were identified.Collectively,all results in this study suggested that the downregulation of HaABCC3 observed in the H.armigera LF60 strain,which is resistant to CrylAc,may be mediated by a cis-regulatory mechanism.展开更多
选用棉铃虫幼虫 ,用饲料混毒法测定了B t 与敌百虫和氰戊菊酯混用的增效作用 ,结果表明 :( 1 )对 2龄幼虫 ,敌百虫与B t 以 1∶3相混时显著增效 ;极少量B t 也明显增加敌百虫的毒力 ,同样 ,敌百虫与 B t 2 .5∶1与 5∶1混合时对 5龄幼...选用棉铃虫幼虫 ,用饲料混毒法测定了B t 与敌百虫和氰戊菊酯混用的增效作用 ,结果表明 :( 1 )对 2龄幼虫 ,敌百虫与B t 以 1∶3相混时显著增效 ;极少量B t 也明显增加敌百虫的毒力 ,同样 ,敌百虫与 B t 2 .5∶1与 5∶1混合时对 5龄幼虫也明显增效 ;( 2 )当氰戊菊酯与 B t 以 1∶6.8相混时拮抗 ;而B t 中加入少量氰戊菊酯或氰戊菊酯中加入少量B t 则增效明显 ;( 3)敌百虫和氰戊菊酯与 B t 相混 ,明显缩短了后者对棉铃虫 2龄幼虫的作用时间。展开更多
基金supported by the Key Project for Breeding Genetically Modified Organisms,China (2014ZX08011-002)the National Natural Science Foundation of China (30971921, 31321004)
文摘The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible（96S） and Cry1Ac-resistant H. armigera strain（Cry1Ac-R）. The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance（SPR） showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A＋Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.
基金This work was funded by a grant to YD Wu(No.31530060)from National Natural Science Foundation of China.
文摘Bacillus thuringiensis(Bt)insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action.Previous studies suggested aminopeptidase N1(APN1)as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches.In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout.Three APN genes(HaAPN1,HaAPN2 and HaAPN5)were individually knocked out in a susceptible strain(SCD)of H.armigera to establish three homozygous knockout strains.Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout.Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain.This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H.armigera.
基金supported by the National Key R&D Program of China(2017YFD0200400)the National Natural Science Foundation of China(31272115)
文摘Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.
基金supported by Shenzhen Natural Science Foundation(grant numberJCYJ20200109150629266)Shenzhen Science and Technology Program(grant number KQTD20180411143628272).
文摘Evolution of resistance to Cry proteins in multiple pest insects has been threatening the sustainable use of Bacillus thuringiensis(Bt)-transgenic crops.Better understanding about the mechanism of resistance to Cry proteins in insects is needed.Our preliminary study reported that the transcription of HaABCC3 was significantly decreased in a near-isogenic line(LFC2)of a Cry1Ac-resistant strain(LF60)of the global pest Helicoverpa armigera.However,the causality between HaABCC3 downregulation and resistance to Cry1Ac remains to be verified,and the regulatory mechanism underlying the HaABCC3 downregulation is still unclear.In this study,our data showed that both HaABCC3 and HaABCC3 downregulation were genetically linked to resistance to Cry1Ac in LF60.However,no InDels were observed in the coding sequence of HaABCC3 from LF60.Furthermore,F_(1) offspring from the cross of LF60 and a HaABCC2/3-knockout mutant exhibited moderate resistance to Cry1Ac toxin;this indicated that the high resistance to CrylAc toxin in LF60 may have resulted from multiple genetic factors,including HaABCC2 missplicing and HaABCC3 downregulation.Results from luciferase reporter assays showed that promoter activity of HaABCC3 in LF60 was significantly lower than that in the susceptible strain,which indicated that HaABCC3 downregulation was likely mediated by promoter variation.Consistently,multiple variations of the GATA-or FoxA-binding sites in the promoter region of HaABCC3 were identified.Collectively,all results in this study suggested that the downregulation of HaABCC3 observed in the H.armigera LF60 strain,which is resistant to CrylAc,may be mediated by a cis-regulatory mechanism.
文摘选用棉铃虫幼虫 ,用饲料混毒法测定了B t 与敌百虫和氰戊菊酯混用的增效作用 ,结果表明 :( 1 )对 2龄幼虫 ,敌百虫与B t 以 1∶3相混时显著增效 ;极少量B t 也明显增加敌百虫的毒力 ,同样 ,敌百虫与 B t 2 .5∶1与 5∶1混合时对 5龄幼虫也明显增效 ;( 2 )当氰戊菊酯与 B t 以 1∶6.8相混时拮抗 ;而B t 中加入少量氰戊菊酯或氰戊菊酯中加入少量B t 则增效明显 ;( 3)敌百虫和氰戊菊酯与 B t 相混 ,明显缩短了后者对棉铃虫 2龄幼虫的作用时间。
