Characterization and functional analysis of β-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.     

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.     

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.     

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera (Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli. The real time quantitative PCR (RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isoborneol, nerolidol, 2-nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33–38 (α1), 40–48 (α2), 62–72 (α3), 80–96 (α4), 98–108 (α5), and 116–119 (α6), two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trp101, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.     

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.     

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.     

Response dynamics of three defense related enzymes in cotton leaves to the interactive stress of Helicoverpa armigera (Hübner) herbivory and omethoate application

In order to explore the response dynamics of the activities of defense related enzymes in cotton leaves towards the interactive stress of Helicoverpa armigera herbivory and omethoate application, the activities of phenylalanine ammonia-lyase(PAL), lipoxygenase(LOX), and polyphenol oxidase(PPO) were examined from 6 to 126 h after cotton leaves were treated 12 h of H. armigera herbivory, and then sprayed with 800 mg L–1 omethoate. The results showed that the changes in the activities of PAL, LOX and PPO that occured under the interactive stress of H. armigera herbivory and omethoate application reflected the interactive effects of the two stresses on cotton defense. The similarity between the response dynamics of PAL, LOX, and PPO activities in cotton leaves under the interactive stress and that under H. armigera herbivory treatment alone showed that the induction of H. armigera herbivory on the activities of PAL, LOX and PPO in cotton leaves played a leading role in the interactive effects, and the effect of omethoate application played only a minor role. A joint factor analysis was performed according to a method which has been used to analyze the joint toxicity of pesticides; this analysis sought to clarify if there was a synergistic, antagonistic, or additive effect on PAL, LOX, and PPO activity in cotton leaves resulting from the interactive H. armigera herbivory and omethoate treatment. In the interactive effect on the response of PAL activity in cotton leaves, antagonistic effects of the omethoate application towards H. armigera herbivory were observed at 6 and 12 h. Synergistic effects were then observed at 18 and 30 h. Antagonistic effects were observed from 54 to 78 h and synergistic effects were finally observed at 126 h. The correlation between H. armigera herbivory and omethoate application in the interactive effect on cotton defense responses of LOX activity also fluctuated from synergism to antagonism during the time course. In the interactive effect on PPO activity, only antagonism was observed between H. armigera herbivory and omethoate application. In the interactive stress of H. armigera herbivory and omethoate application on cotton defense responses, omethoate affected the defense responses of cotton to H. armigera herbivory by producing antagonistic and synergistic effects. These results will be useful to understand the relationship between host plant and herbivorous pest.     

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

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)-ß-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.     

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.     

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera （Hvbner）

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera(Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli.The real time quantitative PCR(RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings.Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles.The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde,whereas methyl phenylacetate,2-decanone,1-pentanol,carvenol,isoborneol,nerolidol,2nonanone and ethyl heptanoate have relatively weak binding affinity.Moreover,the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38(α1),40-48(α2),62-72(α3),80-96(α4),98-108(α5),and 116-119(α6),two pairs of disulfide bridges Cys49-Cys55,Cys75-Cys78.The two amino acid residues,Ile94 and Trp101,may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.     

Effects of Tebufenozide on the Biological Characteristics of Beet Armyworm (Spodoptera exigua Hübner) and Its Resistance Selection

In this article, the selection of tebufenozide to beet armyworm (Spodoptera exigua Hubner) was studied by the treatments to alternative generations' 3rd-instar larvae with LC50 dose and to continuous generations' larvae with LC10 dose; the effects of tebufenozide on the biological characteristics of current and subsequent generations were examined by the treatments to 3rd-instar larvae and egg pods in different concentrations. After treatments with LC50 dose till F11, the toxicity of tebufenozide to beet armyworm had no significant change, whereas the pupation rate, pupal weight, and fecundity were reduced markedly. After treatments with LC10 dose till F19, the beet armyworm only developed 3.52-fold resistance, and the main biological characteristics were nearly accordant in each generation. The livability was reduced 72 h later after treatments to 3rd-instar larvae, respectively in 2.5-40 (ig mL-', and larval duration, pupation rate, and pupal weight changed considerably with the increase in concentrations. The fecundity, larval livability, larval weight and pupal weight of subsequent generations were reduced as the dose increased over 10 ug mL-1. The hatching rate of egg pods did not differ with that of the controls obviously after treatment in 10-300 ug mL-1. But the larval livability, larval weight and pupal weight were reduced when eggs were exposed to 50 ug mL-1 dose or more. The results indicated that tebufenozide had low resistance risk to the current and subsequent generations of beet armyworm even if tebufenozide had significant effects on the biological characteristics of this insect.     

棉铃虫(Helicoverpa amigera Hübner)滞育蛹和非滞育蛹血淋巴某些生化特性的比较研究

试验研究了棉铃虫滞育蛹与非滞育蛹血淋巴在甘油、氨基酸、脂肪酸以及蜕皮激素含量上的不同。结果表明：1）滞育蛹血淋巴17种游离氨基酸的总量比非滞育蛹增加,其中脯氨酸、天冬氨酸、精氨酸、赖氨酸的含量明显高于非滞育蛹,已解除滞育的蛹血淋巴中氨基酸的组成与非滞育蛹仍有所不同,其中丝氨酸、苯丙氨酸、组氨酸的比例明显增加。2）滞育蛹和非滞育蛹血淋巴总脂中的饱和脂肪酸均以棕榈酸为高。不饱和脂肪酸中均以亚油酸含量为高,且滞育蛹的饱和脂肪酸比例高于非滞育蛹。3）血淋巴中甘油的含量两者无显著差异。4）滞育蛹在滞育期血淋巴中20－羟基脱皮酮滴度一直维持在极低水平。当滞育开始解除时,20－羟基脱皮酮的滴度明显上升。     

玉米螟(Pyrausta nubilalis Hübner)及其防治的初步研究

1.本試驗于1954—1955年在杭州華家池浙江農学院農場進行。2.玉米螟一般形态作了簡單的記述。幼虫各分期形态的記載可供作区別令期的資料。3.玉米螟在杭州地区一年發生四代,以老熟幼虫在玉米稈越冬,越冬死亡率在5月中旬为最高达27.27％,成虫都在晚上活动,產卵在玉米叶背面主脈附近,离叶尖20—30厘米处最多,幼虫孵化在上午9—11时左右。4.春玉米可用1％可湿性DDT乳剂和0.125％r可湿性六六六噴射。第一次每畝用100市斤,第二次每畝用150市斤。在螟蛾盛發期施用效果顯著。5.玉米播种試驗說明在7月中旬播种的螟災比較輕,主要原因是由于7中旬以后玉米螟卵塊赤眼蜂的寄生率提高至97％以上。     

A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua(Hübner)

Odorant binding proteins(OBPs) in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors(ORs) located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP c DNA, namely Sexi OBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR(q PCR) measurement indicated that Sexi OBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of Sexi OBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, Sexi OBP13 displayed a high binding affinity(Ki=3.82 μmol L–1) to Z9,E12–14:Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles(Ki27 μmol L–1). The behavioral tests further confirmed that Z9,E12–14:Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that Sexi OBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed.     

拟除虫菊酯的应用研究 Ⅳ.拟除虫菊脂一杀虫脒合剂棉虫防治应用领域的研究—(2)合剂对棉铃虫(Heliothis armigera Hübner)的田间增效试验初报

从害虫综合防治体系对药剂使用的基本原则要求,以及从更好地提高拟除虫菊酯类药剂的经济使用效益和节约能源等角度考虑,我们认为这类高效、低残毒的广谱系药剂应用中需要解决的重要问题是较大幅度降低施药剂量和减少施药次数。为解决这些问题,我们从二代棉铃     

pH influences the profiles of midgut extracts in Cnaphalocrocis medinalis(Guenée) and its degradation of activated Cry toxins

Midgut extracts play crucial roles in food digestion and detoxification. We evaluated the effect of pH on the profiles of the midgut extracts in rice leaffolder, Cnaphalocrocis medinalis and the degradation of activated Bt-toxins by the midgut extracts under different pH conditions. Total protease activity increased slightly with the increase with the simulated pH in the midgut extracts and the maximal protease activity was observed at pH 10.5, while an upward trend was observed as the pH of reaction buffer increased. Activity of chymotrypsin-like enzymes increased with pH, both in the buffer and midgut extracts, while the activity of trypsin-like enzyme was unaffected. Degradation of the activated Cry2 A by the midgut extracts enhanced as the pH increased. Cry2 A was fully degraded into smaller segments at pH 9.0–10.5. Activated Cry1 C protein at pH 9.0–10.5 was partially degraded by the midgut extracts. Activated Cry1 Aa and Cry1 Ac were partially degraded into fragments by the midgut extracts at high pH. These results will facilitate our further understanding of the interactions between C. medinalis and the Cry toxin.     

Expression of Aminopeptidase N1 （APN1）, the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.