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1.
用转座穿梭系统构建了携带绿色荧光蛋白基因(gfp)的重组棉铃虫核型多角体病毒rHa-FGP,以其多角体添食感染棉铃虫3龄幼虫,室内饲养3代,各代均可见自然光下发绿色荧光的棉铃虫幼虫,其中子代不再重复感染。F0、F1、F2代发绿色荧光的棉铃虫幼虫所占百分比分别为34%、20%、8%。提取虫体内的病毒多角体DNA,以PCR和斑点杂交鉴定表明,gfp不仅在亲代棉铃虫体内正常表达,而且在子代幼虫中表达,HaNPV通过卵实现了垂直传播。  相似文献   

2.
粉纹夜蛾颗粒体病毒重组增效蛋白的增效作用   总被引:11,自引:2,他引:9  
采用时间 剂量 死亡率模型 ,分析了粉纹夜蛾 (Trichoplusiani)颗粒体病毒重组增效蛋白P96对棉铃虫 (Helicoverpaarmigera)核型多角体病毒 (HaNPV)感染棉铃虫幼虫的增效作用。结果显示 :感染后 11d ,HaNPV P96组的LC50 值为 3.4 7× 10 3 多角体 /mL ,比HaNPV组 ( 3.89× 10 4 多角体 /mL)降低了 91.0 8% ;在 1.6× 10 4 ~ 1.6× 10 6多角体 /mL浓度范围内 ,HaNPV P96组的LT50 值较HaNPV组缩短 0 .3~ 1.8d。P96显著提高了HaNPV对棉铃虫幼虫的毒力  相似文献   

3.
以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组的DNA为模板设计引物,PCR扩增病毒增效蛋白(Enhanicn)基因,然后经BamH Ⅰ/Pst Ⅰ双酶切消化,得到近乎全长的约2.6kb的增效蛋白基因片段,再与pQE32质粒连接,构建了重组表达载体pQE32/En,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为102kD的融合蛋白并命名为P102,纯化的P102包涵体显示了明显的增效活性,在感染后168h时统计可提高HaNPV对棉铃虫幼虫的感染死亡率6.25%-27.09%,缩短LT5012.3h以上;在感染后72h时统计可提高Bt对棉铃虫幼虫的感染死亡率28.18%,缩短LT5012.33h。  相似文献   

4.
棉铃虫颗粒体病毒增效蛋白基因2.6kb片段的表达   总被引:7,自引:2,他引:5  
以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组的DNA为模板设计引物,PCR扩增病毒增效蛋白(Enhanicn)基因,然后经BamHI/Pst I双酶切消化,得到近乎全长的约2.6kb的增效蛋白基因片段,再与pQE32质粒连接,构建了重组表达载体pQE2/En,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为102kD的融合蛋白并命名为P102,纯化的P102包涵体显示了明显的增效活性,在感染后168h时统计可提高HaNPV对棉铃虫幼虫的感染死亡率6.25%~27.09%,缩短LT5012.3h以上;在感染后72h时统计可提高Bt对棉铃虫幼虫的感染死亡率28.18%,缩短LT5o12.33h.  相似文献   

5.
陈法军  吴刚  戈峰 《昆虫学报》2004,47(6):774-779
通过室内饲养实验研究了在高CO2浓度(738.8±25.7μL/L)中生长的小麦对棉铃虫 Helicoverpa armigera (Hübner)生长发育,繁殖以及营养效应的影响。结果表明: (1)取食高CO2浓度大气中生长的麦粒的棉铃虫对食料的取食量和粪便排泄量增加,与对照相比,取食量和粪便排泄量分别增加46.3%(P<0.05)和37.8%;(2)大气CO2浓度增加影响了麦粒中的营养成分的含量,其中,可溶性蛋白、游离氨基酸、葡萄糖和总糖的含量及碳氮比(C∶N)都显著增加,果糖和粗蛋白的含量都显著降低;(3)大气 CO2浓度升高所导致的麦粒营养成分的变化影响了棉铃虫幼虫的食物利用效率,与对照组相比,棉铃虫幼虫对食物的毛转化率和净转化率分别降低27.2%和25.4%,对食物的相对取食率则显著提高58.8%(P < 00.1)。据此推测,未来高CO2浓度的大气环境会降低春小麦的营养价值,从而影响棉铃虫的生长发育,加重其对小麦的危害。  相似文献   

6.
将带有棉铃虫颗粒体病毒增效蛋白(HaGV enhancin)基因的重组表达载体pET-30a-En分别用Bal Ⅰ和Dra Ⅰ酶切后连接,构建重组表达载体pET-30a-Ben和pET-30a-Den,其外源基因的开放阅读框分别为HaGV增效蛋白基因5'端的1.7kb和2.2kb.IPTG诱导表达产生66.7kD和85.1kD的多肽并命名为Ben和Den.初步纯化的Ben和Den显示了对棉铃虫核多角体病毒(HaNPV)及Bt的增效活性.Ben可对棉铃虫核多角体病毒增效10.5%~26.5%,LT50缩短0.9d,Den增效10.2%~33.0%,LT50缩短1.2d~1.9d.重组增效蛋白可对Bt(12500稀释)增效20.7%~35.4%,Den增效16.7%~31.5%,Ben增效¨.7%~27.4%.这为进一步研制抗虫工程菌打下了良好的基础.  相似文献   

7.
以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组DNA为模板,设计引物PCR扩增病毒增效蛋白(Enhancin)基因,然后经SacI/PstI双酶切消化,得到5′端截短的约2.1kb增效蛋白基因片段,再与pQE30质粒连接,构建了重组表达载体pQE/EnC,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为78×103 D的融合蛋白并命名为P78,纯化的P78包涵体显示了明显的增效活性,可提高AcMNPV对小菜蛾幼虫的感染率27.88%~32.92%。  相似文献   

8.
昆虫病原线虫共生细菌是寄生在昆虫病原线虫肠道的一种细菌,二者互惠共生。实验采用6个不同种的菌株为筛选材料。共生细菌菌株的培养液经85%饱和度的(NH4)2SO4盐析,浓缩冻干得到杀虫粗提物。以粗提物注射大蜡螟Galleria mellonella、饲喂玉米螟Ostrinia furnacalis和棉铃虫Helicoverpa armigera,发现Xenorhabdus nematophilus D43、X.bovienii A54、Photorhabdus luminescens HZL和CB-8等4个菌株发酵液的粗提物对昆虫有高的血腔毒性,菌株A54对昆虫又有高的胃毒效果。由此确立A54为高毒力的菌株,其杀虫活性表现为:注射大蜡螟48 h的死亡率为80%,96 h为93.3%;粗提物饲喂玉米螟,72 h死亡率为53.3%,120 h死亡率为100%;饲喂棉铃虫,72 h死亡率为80.1%,120 h死亡率为90%。杀虫粗提物经DEAE-52柱层析分离,得到一个穿透峰和三个盐的梯度洗脱峰,其中穿透峰对昆虫有很好的胃毒效果,但没有血腔毒性;三个盐峰均有很高的血腔毒性,但没有胃毒作用。穿透峰样品饲喂2龄、3龄棉铃虫也有很好的杀虫活性,96 h 2龄棉铃虫的死亡率为65%,3龄棉铃虫的死亡率为30%;处理96 h的棉铃虫同处理前相比体重下降,未死棉铃虫体重明显低于对照。  相似文献   

9.
将带有棉铃虫颗粒体病毒增效蛋白 (HaGVenhancin)基因的重组表达载体 pET 30a En分别用BalⅠ和DraⅠ酶切后连接 ,构建重组表达载体pET 30a Ben和 pET 30a Den ,其外源基因的开放阅读框分别为HaGV增效蛋白基因 5′端的 1 7kb和 2 2kb。IPTG诱导表达产生 66 7kD和 85 1kD的多肽并命名为Ben和Den。初步纯化的Ben和Den显示了对棉铃虫核多角体病毒 (HaNPV)及Bt的增效活性。Ben可对棉铃虫核多角体病毒增效 1 0 5%~2 6 5% ,LT50 缩短 0 9d ,Den增效 1 0 2 %~ 33 0 % ,LT50 缩短 1 2d~ 1 9d。重组增效蛋白可对Bt(1∶2 50 0稀释 )增效 2 0 7%~ 35 4% ,Den增效 1 6 7%~ 31 5% ,Ben增效 1 1 7%~2 7 4%。这为进一步研制抗虫工程菌打下了良好的基础  相似文献   

10.
乳糖不仅可以诱导棉铃虫颗粒体病毒增效蛋白表达,而且表达量与IPTG(异丙基-β-D-硫代半乳糖苷)作为诱导剂时相比,并不低于后者,这样大规模生产棉龄虫颗粒体病毒增效蛋白的成本就可能被降低。乳糖的最适浓度经优化为2.2%~2.5%(W/V)。对乳糖的不同除菌处理表明:0.70×10~5Pa 15min蒸汽灭菌的乳糖可以诱导此增效蛋白表达,这种处理比滤膜过滤除菌更为方便和经济,因此就为棉铃虫颗粒体病毒增效蛋白的生产提供了经济实用的前景。  相似文献   

11.
棉铃虫核型多角体病毒(HaNPV)分别与三氟氯氰菊酯、溴氰菊酯、氰戊菊酯、灭净菊酯、灭多威、辛硫磷、甲基对硫磷和乙酰甲胺磷等化学杀虫剂混合饲喂棉铃虫幼虫,统计致死中浓度LC50,计算增效比,测定虫体内与抗性有关的三种重要酶:多功能氧化酶(MFO)、羧酸酯酶(CarE)、乙酰胆碱酯酶(AChE)的活性。研究大豆卵磷脂对HaNPV致病性的影响。结果表明:HaNPV与化学杀虫剂混合饲喂抗性棉铃虫,生测统计增效比均大于1.0,特别是病毒与甲基对硫磷混用,增效比更是达到3.53,表现出良好的增效作用。混剂感染抗性棉铃虫,虫体内MFO的活性比化学杀虫剂单用时降低3~12倍,CarE和AChE的活性也比化学杀虫剂单用时低,HaNPV明显抑制了化学杀虫剂对MFO和CarE的诱导作用。HaNPV与大豆卵磷脂混用,提高了HaNPV对棉铃虫的感染致死率,缩短了致死中时间(LT50)。  相似文献   

12.
粘虫颗粒体病毒的增效因子提高杆状病毒的感染   总被引:13,自引:4,他引:9  
丁翠  邓塔 《昆虫学报》1995,38(4):407-413
一株美洲粘虫GV(增效品系)在东方粘虫幼虫上进行增殖,所获粘虫GV(PuGv-Ps>对东方粘虫NPV(PsNPV)、棉铃虫NPV(HaNPV)和黄地老虎NPV(AsNPV)进行增效试验。 实验结果证明PuGV-Ps对三种NPV都有明显的增效作用, 其中以对PsNPV为最强,增效率达55%一85%;对HaNPV为30%-80%;而对AsNPV只有15%-35%。用凝胶过滤技术从PuGV-Ps中分离增效因子PusF-Ps,其对PsNPV和A,NPV的增效作用亦同样明显,250μg的PuSF-Ps可以提高PsNPV的感染能力达80%。经ScPhadcf-150柱(1.6X 90)和使用四种标准蛋白(细胞色素c、胃蛋白酶、牛血清清蛋白和乳酸脱氢酶)测得PuSF-Ps的分子量为160 000左右。  相似文献   

13.
王燕  丁翠 《昆虫学报》1997,40(1):7-14
AsNPV+HasNPV、AsNPV+HaNPV、AsNPV+PsNPV分别感染烟青虫、棉铃虫和粘虫幼虫,对分离到的核型多角体病毒(AsNPV+HasNPV)-Helicoverpa assulta、(AsNPV+HaNPV)- H.Armigera和(AsNPV+PsNPV)-Pseudaletia separata,经电镜观察,多角体蛋白及病毒粒子蛋白SDS-PAGE电泳,病毒核酸的限制性内切酶酶解分析等研究,证明各病毒的多角体形态不规则,大小差异极大,病毒粒子为杆状,(AsNPV+HasNPV)-H.Assulta 和(AsNPV+HaNPV)-H.Armigcra病毒粒子有单粒和多粒包埋类型, (AsNPV+PsNPV)-P.Separata为多粒包埋型。各病毒的多角体蛋白基本上只有一种多肽,分子量为25 000道尔顿左右。(AsNPV+HasNPV)-H.Assulta、(AsNPV+HaNPV)-H.armigera和(AsNPV+PsNPV)-P.Separata的病毒粒子分别有10、14、5条多肽,分子量大小在13 500~98 000,13 000~88 000,18 500~52 000道尔顿之间。病毒核酸经EcoRI,HindIII,HindIII+BamHI酶解,其DNA的酶切位点,大小及DNA的总分子量与AsNPV和原寄主的Has-NPV,HaNPV和PsNPVDNA的酶切图谱存在一定差异,混合病毒侵染昆虫后新复制的病毒核酸发生一定的变化,从而导致病毒蛋白和病毒形态的变化。混合感染后AsNPV对Has-NPV、HaNPV和PsNPV的侵染有明显的增效作用,其机理有待深入研究。  相似文献   

14.
Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is serious pests of cotton and several other crops. Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) can be important alternative to synthetic insecticides for the management of H. armigera. However, the efficacy of HaNPV can vary in horizontal and vertical transmission. In the current study, we evaluated the efficacy of HaNPV of a virulent strain (vertically transmitted up to six generations) and wild strains (used after isolation from the field infected larvae). Both strains were applied to the 2nd instar larvae of H. armigera @ 1 × 109 polyhedral inclusion bodies (PIB)/ml. There were six replications of each strain (strains). The results indicated higher mortalities in larvae exposed to virulent strains (68.33 ± 6.07%) as compared to wild strain (45 ± 2.24%). Virulent strains killed the larvae quite faster than wild strain. The lethal time (LT50) to kill 50% of the larvae by virulent strain was 7.15 days and for wild strain it was 19.47 days. The results showed that multiple passage of HaNPV through several generations enhances its efficacy to kill H. armigera larvae faster. The results of this study will be helpful to manage H. armigera and other related lepidopoterous pests.  相似文献   

15.
Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus.  相似文献   

16.
The chickpea pod borer, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), is an important pest of many legume crops, including chickpea, Cicer arietinum L. (Fabaceae). We field-tested the efficacy of two pathogens, Helicoverpa armigera nucleopolyhedrosis virus (HaNPV) and Bacillus thuringiensis Berliner subsp. kurstaki (Btk), both alone and in combination with the parasitoid Habrobracon hebetor Say (Hh) (Hymenoptera: Braconidae), for control of H. armigera larvae in chickpea fields. A randomized complete block design was used at two sites, so that wasp release treatments at one site would not contaminate pathogen-only treatments at the other. Pathogens were applied when most pest larvae were in the second instar, and wasp releases were made 2 days later. Number of H. armigera larvae per plant, number of damaged pods, yield (kg ha−1), treatment cost (US$ ha−1), and benefit:cost ratio were all determined. Treatments separated significantly at 14 days after treatment, with the HaNPV+Hh and Btk+Hh treatments providing the best control, suggesting a synergistic impact of the wasp and the pathogens when applied together. These treatments also produced the fewest damaged pods, the highest yields, and the greatest yield increments in comparison to untreated controls. However, costs were also highest for these combined treatments, which reduced their net benefit in comparison to use of Hh or Btk alone, although more efficient mass production of the pathogens might make combined applications feasible. We conclude that both HaNPV and Btk are suitable for integration with releases of H. hebetor against H. armigera in chickpea fields, provided parasitoid releases are delayed until 2 days after pathogen application.  相似文献   

17.
Nucleopolyhedrosis viruses can be utilized for effective management of agriculture pests. Their efficacy can be increased if they are mixed with certain insecticides. In the current study, HaNPV was mixed with two insecticides: spinetoram and emamectin benzoate in various combinations and applied to larvae of H. armigera in laboratory conditions. There were a total of 15 combinations of HaNPV with each of the two insecticides in addition to five doses of HaNPV and three doses of insecticides alone. The synergistic and antagonistic effects of combinations were explored. The results revealed that there was synergistic effect of HaNPV @ 0.5 × 109 PIB/ml × Spinetoram @ 40, 20, 10 ml/100 L of water. In case of emamectin benzoate, synergistic effects were recorded at 1 × 109 PIB/ml HaNPV × emamectin benzoate @ 100 ml/100 L of water. However, 0.5 × 109 PIB/ml HaNPV has synergistic effects with all three doses of emamectin benzoate. The results suggested that HaNPV can be used in combination with spinetoram and emamectin benzoate for the management of resistant population of H. armigera.  相似文献   

18.
棉铃虫对Bt生物农药早期抗性及与转Bt基因棉抗虫性的关系   总被引:19,自引:0,他引:19  
用饲料感染法建立了棉铃虫Helicoverpa rmigera(Hubmer)敏感品系(SUS1)对Bt生物农药的敏感毒力基线和区分剂量,1995年测定了五省六县棉铃虫初孵幼虫对Bt生物农药的敏感性,结果表明:山东阳谷、河北邯郸、河南新乡、安徽萧县及江苏丰县棉铃虫已产生早期抗性,抗性个体百分率为5%~10%,与敏感品系相比,LC50值稍有增加,但斜率b值明显变小;而江苏东台棉铃虫仍属敏感。这是国内外首次诊测到棉铃虫对Bt生物农药抗性。用棉叶喂饲法测定比较了转Bt基因棉花品系对不同种群棉铃虫的抗虫性效果,结果表明:用早期抗性的阳谷和新乡棉铃虫初孵幼虫接虫5d后平均死亡率较敏感品系下降16%~29%,说明棉铃虫对Bt农药与转Bt生物基因棉花品系间存在交互抗性。还讨论了Bt农药的抗性治理对策。  相似文献   

19.
以棉铃虫Helicoverpa armigera (Hübner)室内敏感品系和田间品系为寄主,研究了亚致死浓度的Bt杀虫蛋白对中红侧沟茧蜂Microplitis mediator (Haliday)生长发育的影响。结果表明: 当寄主一直取食,或者在被寄生前12小时开始取食含Bt杀虫蛋白浓度为0,0.5,1.0, 2.0,4.0,8.0 μg/g的饲料时,与对照相比,中红侧沟茧蜂的卵-幼虫历期延长,茧重和成虫体重降低,成虫寿命缩短,但对茧期没有明显影响。Bt杀虫蛋白能有效抑制两个棉铃虫品系幼虫的生长,显著降低棉铃虫蛹重;当Bt蛋白浓度为4.0 μg/g时,显著降低棉铃虫化蛹率。用转双基因抗虫棉SGK321(表达Cry1A+CpTI蛋白)饲喂两个棉铃虫品系初孵幼虫,室内品系的第2、3、4和5天校正死亡率分别为48.5%、87.8%、96.6%和 95.8%,显著高于田间品系(30.9%、59.6%、80.9%及86.1%)。本研究表明,不论是田间品系还是室内品系,棉铃虫取食含Bt杀虫蛋白的饲料后,对中红侧沟茧蜂的生长发育都具有显著的负面作用。  相似文献   

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