Response of Cytochrome P450 Expression to Maize Volatiles in Helicoverpa armigera (Hner)

The 7-ethoxycoumarin O-deethylase (ECOD) activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera (Hner). The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.     

Characterization and functional analysis ofβ-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

Carbohydrate chains are the principal antigens by which Bacillus thuringiensis(Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length c DNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques(GAL-Harm, Gen Bank accession no.: GQ904195.1) with two potential N-glycosylation sites(157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1 Ac enhanced the toxicity against the si RNA-treated larvae compared with non-si RNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1 Ac in H. armigera.     

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera (Hübner)

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.     

Response of Cytochrome P450 Expression to Maize Volatiles in Helicoverpa armigera (Hübner)

The 7-ethoxycoumarin O-deethylase (ECOD) activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera (Hübner). The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.     

Polycalin is involved in the action mechanism of Cry2Aa toxin in Helicoverpa armigera(Hübner)

Receptor proteins on the brush border membrane of the insect midgut epithelium are involved in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis(Bt). Polycalin has been identified as a binding protein of the Bt Cry1 Ac toxin in several Lepidoptera including Helicoverpa armigera, but its role in the action mechanism of Cry2 Aa is still unclear. In this study, we investigated the binding characteristics of polycalin from the midgut of H. armigera with Cry2 Aa and its role in the toxicity of Cry2 Aa. The results demonstrated that heterologously expressed H. armigera polycalin peptide could bind with Cry2 Aa with high affinity(K_d=32 nmol L~(–1)). The toxicity of Cry2 Aa decreased by 27% after H. armigera larvae ingested polycalin antisera. These results suggested that polycalin could be a potential functional receptor for Cry2 Aa, and it plays an important role in the susceptibility of H. armigera to Cry2 Aa.     

Inhibition of the transcription of P450 monooxygenase gene CYP9A14 in Helicoverpa armigera (Hübner) by baculovirus-mediated RNAi

采用实时荧光定量PCR,测定棉铃虫Helicoverpa armigera (Hübner)细胞色素P450 CYP9A14基因在氯氰菊酯抗性棉铃虫中肠里的表达量,结果表明抗性棉铃虫是敏感棉铃虫中肠表达量的17倍.将棉铃虫中肠P450 CYP9A14基因的一个490bp反向重复片段以双链RNA干扰(double-stranded RNA interference,dsRNAD的方法重组到棉铃虫核型多角体病毒(helicoverpa nuclear polyhedrosis virus,HaNPV)中,结果表明以该重组病毒注射氯氰菊酯抗性棉铃虫体内后,幼虫中肠CYP9A14基因的转录水平显著下降.     

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera(Hübner)

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.