Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance a...Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance and fieldevolved practical dual resistance of H.zea to these two toxins have been widely reported.Whether the widespread Cry1Ac/Cy2Ab dual resistance of H.zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear.Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species.To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab,we cloned H.zea cadherin(HzCadherin)cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain-and lossof-function analyses.Heterologous expression of HzCadherin in H.zea midgut,H.zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab,whereas silencing HzCadherin of H.zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab.Likewise,suppressing HzCadherin with siRNA made H.zea larvae resistant to Cry1Ac.These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab,and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab.展开更多
While CrylAc has been known to bind with larval midgut proteins cad- herin, APN （amino peptidase N）, ALP （alkaline phosphatase） and ABCC2 （adenosine triphosphate-binding cassette transporter subfamily C2）, littl...While CrylAc has been known to bind with larval midgut proteins cad- herin, APN （amino peptidase N）, ALP （alkaline phosphatase） and ABCC2 （adenosine triphosphate-binding cassette transporter subfamily C2）, little is known about the recep- tors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry 1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line ofSpodopterafrugiperda （SFg）. As expected, the descending order of cytotoxicity of CrylAc against the three cell lines in terms of 50% lethal concetration （LC50） was midgut （31.0μg/mL） 〉 fat body （59.0μg/mL） and SF9 cell （99.6μg/mL）. By contrast, the fat body cell line （LC50 = 7.55μg/mL） was about twice more susceptible to Cry2Ab than the midgut cell line （16.0/xg/mL）, the susceptibility of which was not significantly greater than that of SF9 cells （27.0μg/mL）. Further, ligand blot showed the binding differences between CrylAc and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of CrylAc, which were enriched in midgut cells.展开更多
MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been succe...MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells.These reporter systems,however,are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase(usually Renilla)used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells.In this study,we replaced the SV40 promoter in the psiCHECK-2 reporter vector,which is widely used with mammalian cell lines,with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors,designated psiCHECK-2-TK and psiCHECK-2-AC5.1,respectively.Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target(7?enz7/a)/reference(firefly)luciferase activity ratios in mammalian(HeLa and HEK293)and insect(Sf9,S2,Helicoverpa zea fat body and ovary)cell lines,while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line.Moreover,psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target,the 3'-untranslated region of heat shock protein 90,in both mammalian and H.zea cell lines,but psiCHECK-2 failed to do so in IT.zea cell lines.Furthermore,psiCHECK-2-TK with the target sequence,HzMasc(H.zea Masculinizer),accurately differentiated between H.zea cell lines with or without the negative regulation factor(miRNA or piRNA)of HzMasc.These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.展开更多
Insects utilize xenobiotic compounds to up-and downregulate cytochrome P450 monooxygenases(P450s)involved in detoxification of toxic xenobiotics including phytochemicals and pesticides.G-quadruplexes(G4)-forming DNA m...Insects utilize xenobiotic compounds to up-and downregulate cytochrome P450 monooxygenases(P450s)involved in detoxification of toxic xenobiotics including phytochemicals and pesticides.G-quadruplexes(G4)-forming DNA motifs are enriched in the promoter regions of transcription factors and function as cis-acting elements to regulate these genes.Whether and how P450s gain and keep G4 DNA motifs to regulate their expression still remain unexplored.Here,we show that CYP321A1,a xenobiotic-metabolizing P450 from Helicoverpa zea,a polyphagous insect of economic importance,has acquired and preserved a G4 DNA motif by selectively retaining a transposon known as HzIS1-3 that carries this G4 DNA motif in its promoter region.The HzIS1-3 G4 DNA motif acts as a silencer to suppress the constitutive and induced expression of CYP321A1 by plant allelochemicals flavone and xanthotoxin through folding into an intramolecular parallel or hybrid-1 conformation in the absence or presence of K^(+).The G4 ligand N-methylmesoporphyrin IX(NMM)strengthens the silencing effect of HzIS1-3 G4 DNA motif by switching its structure from hybrid-1 to hybrid-2.The enrichment of transposons in P450s and other environment-adaptation genes implies that selective retention of G4 DNA motif-carrying transposons may be the main evolutionary route for these genes to obtain G4 DNA motifs.展开更多
基金funded by the USDA National Institute of Food and Agriculture (Hatch Grant ARZT-1360890-H31-164 and multi-state grant ARZ-T1370680-R31-172 (NC246))the National Natural Science Foundation of China (NSFC)–Henan Joint Major Grant (U2004206)+2 种基金the State Key Laboratory of Cotton Biology Open Fund, Zhengzhou University, China (CB2020A06)the Henan Agriculture Research System, China (HARS22-09-G3)the earmarked fund for China Agriculture Research System (CARS-27)
文摘Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance and fieldevolved practical dual resistance of H.zea to these two toxins have been widely reported.Whether the widespread Cry1Ac/Cy2Ab dual resistance of H.zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear.Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species.To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab,we cloned H.zea cadherin(HzCadherin)cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain-and lossof-function analyses.Heterologous expression of HzCadherin in H.zea midgut,H.zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab,whereas silencing HzCadherin of H.zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab.Likewise,suppressing HzCadherin with siRNA made H.zea larvae resistant to Cry1Ac.These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab,and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab.
文摘While CrylAc has been known to bind with larval midgut proteins cad- herin, APN （amino peptidase N）, ALP （alkaline phosphatase） and ABCC2 （adenosine triphosphate-binding cassette transporter subfamily C2）, little is known about the recep- tors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry 1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line ofSpodopterafrugiperda （SFg）. As expected, the descending order of cytotoxicity of CrylAc against the three cell lines in terms of 50% lethal concetration （LC50） was midgut （31.0μg/mL） 〉 fat body （59.0μg/mL） and SF9 cell （99.6μg/mL）. By contrast, the fat body cell line （LC50 = 7.55μg/mL） was about twice more susceptible to Cry2Ab than the midgut cell line （16.0/xg/mL）, the susceptibility of which was not significantly greater than that of SF9 cells （27.0μg/mL）. Further, ligand blot showed the binding differences between CrylAc and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of CrylAc, which were enriched in midgut cells.
基金This work was supported by the National Science Foundation of China(NSFC)-Henan Joint major grant(U2004206)the State Key Laboratory of Cotton Biology(CB2020A06).
文摘MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells.These reporter systems,however,are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase(usually Renilla)used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells.In this study,we replaced the SV40 promoter in the psiCHECK-2 reporter vector,which is widely used with mammalian cell lines,with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors,designated psiCHECK-2-TK and psiCHECK-2-AC5.1,respectively.Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target(7?enz7/a)/reference(firefly)luciferase activity ratios in mammalian(HeLa and HEK293)and insect(Sf9,S2,Helicoverpa zea fat body and ovary)cell lines,while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line.Moreover,psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target,the 3'-untranslated region of heat shock protein 90,in both mammalian and H.zea cell lines,but psiCHECK-2 failed to do so in IT.zea cell lines.Furthermore,psiCHECK-2-TK with the target sequence,HzMasc(H.zea Masculinizer),accurately differentiated between H.zea cell lines with or without the negative regulation factor(miRNA or piRNA)of HzMasc.These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.
基金supported by the National Natural Science Foundation of China(No.31701791)National Science Foundation of China(NSFC)-Henan Joint major grant(No.U2004206)+1 种基金the State Key Laboratory of Cotton Biology(No.CB2020A06)the USDA National Institute of Food and Agriculture Hatch Project(No.ARZT-1370680-R31-R31-172).
文摘Insects utilize xenobiotic compounds to up-and downregulate cytochrome P450 monooxygenases(P450s)involved in detoxification of toxic xenobiotics including phytochemicals and pesticides.G-quadruplexes(G4)-forming DNA motifs are enriched in the promoter regions of transcription factors and function as cis-acting elements to regulate these genes.Whether and how P450s gain and keep G4 DNA motifs to regulate their expression still remain unexplored.Here,we show that CYP321A1,a xenobiotic-metabolizing P450 from Helicoverpa zea,a polyphagous insect of economic importance,has acquired and preserved a G4 DNA motif by selectively retaining a transposon known as HzIS1-3 that carries this G4 DNA motif in its promoter region.The HzIS1-3 G4 DNA motif acts as a silencer to suppress the constitutive and induced expression of CYP321A1 by plant allelochemicals flavone and xanthotoxin through folding into an intramolecular parallel or hybrid-1 conformation in the absence or presence of K^(+).The G4 ligand N-methylmesoporphyrin IX(NMM)strengthens the silencing effect of HzIS1-3 G4 DNA motif by switching its structure from hybrid-1 to hybrid-2.The enrichment of transposons in P450s and other environment-adaptation genes implies that selective retention of G4 DNA motif-carrying transposons may be the main evolutionary route for these genes to obtain G4 DNA motifs.
