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1.
为评估转cry1Ab/cry2Aj、cry1Ab/vip3DA玉米对棉铃虫Helicoverpa armigera(Hübner)、甜菜夜蛾Spodoptera exigua(Hübner)和斜纹夜蛾Prodenia litura(Fabricius)的抗虫性,在室内测定了3个转cry1Ab/cry2Aj玉米品系和1个转cry1Ab/vip3DA玉米品系对3种害虫幼虫存活和生长发育的影响,研究了该系列Bt玉米不同组织器官对害虫的杀虫活性和控制效果。结果显示,棉铃虫初孵幼虫取食各品系Bt玉米叶片96h后死亡率为87.50%~90.00%,取食花丝和雌穗的幼虫96h后几乎全部死亡;甜菜夜蛾初孵幼虫取食各品系Bt玉米叶片、花丝和雌穗168h后死亡率为22.50%~68.33%,幼虫的生长发育受到明显抑制,体重抑制率达85.00%~95.00%;斜纹夜蛾初孵幼虫取食各品系Bt玉米叶片、花丝和雌穗96 h后死亡率显著高于非转基因亲本对照,168h后幼虫死亡率达90.00%以上。研究表明,转cry1Ab/cry2Aj和cry1Ab/vip3DA玉米品系对棉铃虫和斜纹夜蛾的初孵幼虫表现出较好的抗性,可以作为转多基因抗虫玉米育种的备选材料。 相似文献
2.
依据对夜蛾科具有高毒力cry基因的序列,设计特异性引物对实验室保存的对鳞翅目害虫高效的菌株资源进行筛选。通过比较质粒图谱,对其多样性进行了研究。进一步测定Bt菌株对甜菜夜蛾的杀虫活性,筛选出高毒力的菌株。从192株菌株中筛选出15株对夜蛾科害虫具有高活性的菌株,包含的基因类型有:cry1A、cry1C、cry2A、cry9和vip3A类基因,晶体形态包含:双锥体形、方形、球形;其中2株菌表达130ku和60ku的蛋白,1株菌表达130ku和70ku的蛋白,其余菌株均表达约130ku的蛋白;这15株菌通过质粒图谱分析分为8种类型;3株菌对甜菜夜蛾幼虫具有高毒力,其中菌株PS3-C2对甜菜夜蛾LC50为1.87μg/mL。本研究成功筛选出对甜菜夜蛾具有高毒力的菌株3株,其中PS3-C2菌株与对照菌株KN11活性相当,为新型杀虫基因分离克隆与制剂的研发奠定了基础。 相似文献
3.
利用生物活性测定方法从本实验室已分离保存的Bt菌株中筛选出了5株对甜菜夜蛾具有较高杀虫毒力的菌株。该5株Bt菌株对甜菜夜蛾初孵幼虫的LC50值为0.556~0.914mg/mL胞晶混合物,其中GS3菌株杀虫活性最高,LC50值为0.556mg/mL胞晶混合物;包含晶体形态主要是球形、大菱形、小菱形等;所含基因类型主要是cry1Aa、cry1Ab、cry1La、cry1Ia、cry1Ib、cry2Ab、cry2Ad、cry2Ah、cry7Ab、cry9Ba等;5株菌株均表达约60kD和130~150kD蛋白,其中2株还表达较弱的80kD蛋白。从GS3菌株中克隆得到一种新的cry2A基因,GenBank登录号为KT692984,由Bt基因国际命名委员会命名为cry2Ah5。该基因开放阅读框为1899bp,编码632个氨基酸,编码蛋白分子量为70.8kD,等电点pI为8.18,与其他Cry2Ah蛋白序列相似性为97%~99%。该基因在大肠杆菌BL21(DE3)中表达约70kD蛋白,与预测分子量相符。本研究筛选得到5株对甜菜夜蛾具有较高杀虫毒力的Bt菌株,克隆表达了一种新的cry2A基因cry2Ah5,为甜菜夜蛾的生物防治提供了新的菌株及基因资源。 相似文献
4.
草地贪夜蛾是一种世界性重大农业害虫，给玉米等多种主要粮食和经济作物造成严重危害。为实现对草地贪夜蛾的高效、绿色、持续防控，亟需筛选高毒力苏云金芽胞杆菌菌株资源。本研究以前期实验室储备的对斜纹夜蛾、小地老虎具有杀虫活性的363株野生菌株库为候选菌株，基于菌株杀虫基因差异及进化关系，去除冗余菌株后获得172株候选菌株。通过培养获取这些菌株胞晶混合物，进行杀虫蛋白定量后，测定杀虫活性，获得27株对草地贪夜蛾初孵幼虫具有较高杀虫活性的菌株，其中菌株B14-D2的杀虫活性最高，LC50为0.155 μg/g。克隆了该菌株中的cry1Ea3基因并在HD73-无晶体突变株中表达，Cry1Ea3蛋白对草地贪夜蛾初孵幼虫LC50为1.789 μg/g。本研究为新的Bt产品和杀虫基因的开发利用提供了丰富资源。 相似文献
5.
为探究甜菜夜蛾Spodoptera exigua中肠碱性磷酸酶(alkaline phosphatase protein 2,ALP2)是否为Cry1Ac杀虫蛋白的受体,采用同源克隆和RACE技术克隆了编码alp2基因的完整c DNA序列,利用荧光定量PCR比较了甜菜夜蛾幼虫中肠不同龄期ALP2表达量的差异,利用Ligand blot方法检测了中肠ALP2与Cry1Ac杀虫蛋白的结合。结果表明,alp2基因序列全长1 629 bp(Gen Bank序列号为KP420013),编码542个氨基酸,预测在氨基酸序列N端包含1个由21个氨基酸组成的信号肽,在C端存在1个GPI修饰的锚定位点,且在整个氨基酸序列中存在多个糖基化修饰位点。在整个甜菜夜蛾幼虫期均有ALP2表达,但不同龄期的表达量差异显著,1龄幼虫期表达量最低,4龄幼虫期最高。Ligand blot方法检测结果表明原核表达的ALP2片段与活化的Cry1Ac杀虫蛋白可以结合。研究表明,甜菜夜蛾中肠的ALP2可能是Cry1Ac的受体之一。 相似文献
6.
筛选对草地贪夜蛾高毒力Bt菌株，对于开发绿色高效防治草地贪夜蛾新产品具有重要意义。本研究从自行筛选的对夜蛾科害虫高毒力的Bt菌株资源中，选取9株菌株进行草地贪夜蛾室内毒力测定，并对高毒力菌株进行杀虫基因分析；选取室内毒力测定活性最高的3株菌株进行田间小区药效试验。结果表明，9株Bt菌株在药后7 d对草地贪夜蛾初孵幼虫均表现出显著的毒杀效果，LC50范围在0.07～3.38 μg/g，其中毒力最高的3个菌株分别是KN50、KN11和KNR8，LC50分别为0.07、0.23和0.43 μg/g，表现非常显著的致死效果；对KN50、KN11和KNR8等6种高毒力的Bt菌株的杀虫基因分析发现，他们都含有vip3Aa、cry2Ab、cry1Ac、cry1Ia杀虫基因。KN50、KN11和KNR8菌株的32000 IU/mg制剂在田间对草地贪夜蛾表现出不同程度防效，其防效高低与室内毒力结果一致，其中KN50药后7 d防效最高，在200和400 g/667 m2下对1～3龄幼虫防效分别可达86.09%和95.22%，对不同龄期幼虫混发的防效为72.61%和86.59%。因此KN50、KN11和KNR8这3个菌株能作为备选高效菌株用于防治草地贪夜蛾新产品开发，为实现草地贪夜蛾的绿色防控、化学农药合理减替提供重要保障。 相似文献
7.
在室内用生测法研究了表达Cry1Ab杀虫蛋白的Bt玉米MON810和Bt11小苗和心叶对粘虫Mythimna separata (Walker)初孵幼虫以及玉米叶片对暴食期4龄和5龄幼虫的杀虫效果.结果表明,2种Bt玉米小苗及心叶对粘虫初孵幼虫都具有很好的控制效果,取食MON810和Bt11小苗的粘虫初孵幼虫第1 天的存活率分别为69.44%和86.11%,到第3 天时,取食Bt11小苗的幼虫全部死亡,此时取食MON810小苗的幼虫存活率仅为4.17%,到第7 天全部死亡,而此时在各自相对应的对照玉米DK647和NX4906小苗上幼虫存活率分别为91.66%和88.89%.取食MON810和Bt11心叶的粘虫初孵幼虫第2 天时幼虫存活率急剧下降,分别为13.89%和19.45%,到第7 天时,取食两种Bt玉米心叶的初孵幼虫全部死亡,而取食对照玉米心叶幼虫存活率分别为94.45%和97.22%,差异极显著.两种Bt玉米对粘虫4龄幼虫有很好的杀虫效果,取食的第11天 4龄幼虫全部死亡,对照幼虫存活率均为100%;在Bt11和MON810叶片上取食的粘虫5龄幼虫也不能存活,分别在取食的第11天和12天全部死亡,对照存活率分别为96.00%和98.00%,差异极显著.表明Bt玉米对粘虫初孵幼虫和暴食期的4龄和5龄粘虫都具有显著的致死作用. 相似文献
8.
菌株FB是1株对小菜蛾Plutella xylostella幼虫具有高毒力的苏云金芽胞杆菌Bacillus thuringiensis （Bt）。本研究通过扫描电子显微镜、大质粒电泳、总蛋白SDS-PAGE及菌株生长特征观察的方式研究了菌株FB特征,克隆得到了基因cry1Ia、cry1Ea、cry1Ab、cry2Ab和vip3Aa全长,依据全基因组测序结果得到了1个cry8基因部分片段,首次在Bt菌株中同时发现基因cry1类和cry8类,这五种基因推导的氨基酸序列与已知基因序列相比,最高相似性分别为99%、98%、99%、100%、99%,而cry8半长基因与已知基因仅为63%。生测结果表明,蛋白Cry1Ia、Cry1Ea和Cry1Ab对小菜蛾幼虫具有较高杀虫活性。 相似文献
9.
《中国生物防治学报》2015,(4)
苏云金芽胞杆菌Bacillus thuringicnsis(Bt)是一种广泛存在于自然界中的生防微生物,其杀虫活性主要来源于杀虫基因编码的内毒素(insecticidal crystal proteins,ICPs)、营养期杀虫蛋白(vegetative insecticidal protein,VIP)和分泌期杀虫蛋白(secreted insecticidal protein,Sip),相比于以上2种杀虫蛋白,Sip的研究相对较少。本研究从实验室中的Bt野生菌株中克隆sip基因,其中编号为DQ89的Bt菌株成功克隆得到1188 bp大小的sip基因,编码395个氨基酸,与已知的Sip1A蛋白的相似性为87%,序列提交到Gen Bank并获得登录号KP114614。将该基因在大肠杆菌中表达的蛋白进行生物活性测定,结果显示其对大猿叶甲Colaphellus bowringi Baly具有一定的杀虫活性,其LC50为1.542μg/m L。 相似文献
10.
转Bt基因抗虫玉米对亚洲玉米螟幼虫取食行为的影响 总被引：1，自引：0，他引：1
室内研究了转cry1Ab杀虫蛋白基因的Bt抗虫玉米M ON810和Bt11对亚洲玉米螟(Ostrinia furnacalis)初孵幼虫和3龄幼虫的取食行为、取食选择性和存活率的影响。在48h的非选择性试验中,玉米螟初孵幼虫在M ON810和Bt11玉米心叶组织上的取食率随时间的增加而下降,在对照玉米上的幼虫取 相似文献
11.
Toxicity of chlorantraniliprole to Cry1Ac-susceptible and resistant strains of Helicoverpa armigera 总被引：1，自引：0，他引：1
Guangchun Cao Lili Zhang Gemei Liang Kris A.G. Wyckhuys 《Pesticide biochemistry and physiology》2010,98(1):99-103
Transgenic Bt cotton expressing Cry1Ac is important in controlling various agricultural pests, including Helicoverpa armigera. Especially for transgenic crops that are cultivated in large expanses, avoiding resistance development is a key for ensuring sustainability of Bt technologies. Integrated pest management, in which transgenic crops are strategically combined with rational pesticide use, may help to prevent H. armigera resistance acquisition in Bt cotton. In this study, we evaluated the toxicity of a novel insecticide (chlorantraniliprole) on Cry1Ac-susceptible and resistant individuals of H. armigera. More specifically, we assessed the effect of chlorantraniliprole on the activity of two enzymes and conducted laboratory bioassays to determine its toxicity on H. armigera larvae. Chlorantraniliprole increased esterase and glutathione-S-transferase activities in Cry1Ac susceptible and resistant populations of H. armigera. Cry1Ac resistant populations XJ-F (Cry1Ac resistance ratio 21.8-fold), XJ-10.0 (95.8-fold) and BTR (3536.5-fold) did not show cross-resistance to chlorantraniliprole, with LC50 values of 0.0733 (μg/mL) in XJ-F, 0.0545 (μg/ml) in XJ-10.0 and 0.0731 (μg/mL) in BTR, which were close to that in the susceptible strain 96S (0.0954 μg/mL). Our work shows that chlorantraniliprole could be considered to be integrated in Bt cotton management schemes to delay the H. armigera resistance development. 相似文献
12.
A number of cadherin mutants conferring resistance to Bt toxin Cry1Ac have been reported in three major lepidopteran pests, including Helicoverpa armigera. Unlike most of the cadherin mutants conferring recessive resistance, an allele (r15) with a 55aa deletion in the intracellular domain of cadherin (HaCad) was previously identified to cause non-recessive resistance to Cry1Ac in H. armigera. In the present study, a DNA-based PCR method was developed to screen the r15 allele from field populations of H. armigera collected from the main cotton planting areas of China in 2011 and 2012. Three heterozygous r15 alleles were detected from 562 moths collected from northern China (with intensive Bt cotton planting), and r15 allele frequency was estimated to be 0.0027. However, no r15 allele was detected from 314 moths collected from Xinjiang (with limited Bt cotton use). Although all the r15 alleles have the same deletion in the cDNA sequence, at least four different indels causing loss of exon 32 have been detected in the genomic DNA sequences flanking exon 32 of HaCad. Multiple origins of the r15 alleles illustrate parallel genotypic adaption of H. armigera to the selection pressure of Bt cotton. 相似文献
13.
为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。 相似文献
14.
室内毒力测定结果表明,溴虫腈EC对甜菜夜蛾卵的孵化有一定的抑制作用,但作用较小,20 mg/L药液浸卵后,卵孵化抑制率为43.11%。溴虫腈EC对甜菜夜蛾1~2龄幼虫的毒力很高,3龄后随着虫龄的增大,甜菜夜蛾对溴虫腈的敏感性显著降低。室内钵栽试验表明,20 mg/L溴虫腈EC对甜菜夜蛾2龄幼虫的减退率在91.43%~100%之间,持效期15 d以上。田间试验表明,溴虫腈对甜菜夜蛾有优异的防治效果,每公顷施用5%溴虫腈EC 1 200 mL,防治效果在95％以上,持效期为20 d。 相似文献
15.
为阐明苏云金杆菌Bacillus thuringiensis(Bt)与白蛾周氏啮小蜂Chouioia cunea Yang的协同作用,分别用103~108孢子/m L的6个Bt浓度处理美国白蛾Hyphantria cunea 2龄幼虫后,通过室内试验测定了Bt对其致死、亚致死效应,及亚致死浓度Bt对寄生蜂子代出蜂的影响。结果表明,浓度106孢子/m L处理组美国白蛾均未能化蛹;而106孢子/m L处理组均能化蛹并羽化,且幼虫发育历期随Bt浓度的增加而延长,平均蛹重在74.92~92.18 mg之间,均高于对照的68.66 mg,但蛹重不呈线性规律变化。当Bt对美国白蛾平均校正死亡率低于17.17%时,幼虫发育历期延迟不明显,但平均蛹重为101.11~124.34 mg,均高于对照;接蜂后能够正常出蜂,出蜂率为33.13%~51.25%,每蛹出蜂量为117.43~143.42头,均显著高于对照。表明用平均校正死亡率低于20%的Bt处理对白蛾周氏啮小蜂的寄生有利,2种生防措施协同作用存在增效潜力。 相似文献
16.
Shudong Luo Guirong Wang Kong Ming Wu Lianyang Bai Yuyuan Guo 《Pesticide biochemistry and physiology》2006,85(2):104-109
Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three 125I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no significant difference in Cry1Ac-binding affinity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (Rt decreased from 5.87 ± 1.40 to 2.23 ± 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance fitted the “mode 1” pattern of Bt resistance described previously. 相似文献
17.
The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC50 and LC99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC50 and LC99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour. 相似文献
18.
国产Bt-Cry1Ab和Bt-(Cry1Ab+Vip3Aa)玉米对草地贪夜蛾的抗性测定 总被引：1，自引：0，他引：1
草地贪夜蛾Spodoptera frugiperda(J. E. Smith)是世界性重大农业害虫。种植转基因Bt作物是主要的防治手段之一。我们利用室内生物测定方法评价了国产Bt-Cry1Ab玉米(转化体C0030.3.5)和Bt-(Cry1Ab+Vip3Aa)玉米(转化体DBN3601和DBN3608)对草地贪夜蛾1～4龄幼虫的毒力。结果显示,供试两种转基因玉米共6个品种皆可高效表达目标杀虫蛋白并对草地贪夜蛾具有很强的毒杀作用,对1龄幼虫的致死率达到59%～100%,存活幼虫的生长发育亦受到显著抑制。表明国内研发的Bt-Cry1Ab玉米和Bt-(Cry1Ab+Vip3Aa)玉米对草地贪夜蛾具有良好的控制效果,其中Bt-(Cry1Ab+Vip3Aa)玉米的防治效果显著优于Bt-Cry1Ab玉米,两种转基因玉米皆具有较好的商业化应用前景。 相似文献
19.
Ashraf O. Abd El-Latif 《Pesticide biochemistry and physiology》2010,96(3):155-159
Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera. 相似文献