苏云金芽胞杆菌杀虫晶体蛋白研究进展

苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt),属于蜡样芽胞杆菌族,芽胞杆菌属的革兰氏阳性细菌,与其它芽胞杆菌的区别是在形成芽胞的同时,伴随有杀虫晶体蛋白(Cry蛋白)的产生。Cry蛋白对鳞翅目、鞘翅目、膜翅目和双翅目等多种害虫具有特异的杀虫作用,因此,Cry蛋白以微生物杀虫剂或转Bt基因植物的形式而被广泛应用于害虫生物防治中。本文从Bt菌株的发掘、cry基因的转录调控机制、Cry蛋白的结构功能及作用机制等方面进行综述,为Bt杀虫蛋白的推广应用、Bt蛋白的定向改造和构建高效广谱的工程菌提供参考。     

苏云金芽胞杆菌分离过程中无晶体芽胞杆菌的分析

本研究选择了在苏云金芽胞杆菌Bacillus thuringiensis(Bt)分离过程中发现的与Bt芽胞相似、没有伴胞晶体的8株芽胞杆菌,对其种属、质粒等生物学特性和杀虫活性进行了初步研究.对分离菌株所产芽胞进行形态观察及质粒图谱分析,发现其与Bt相似;但肽指纹图谱分析和16S rDNA聚类研究表明所分离菌株与蜡样芽胞杆菌B.cereus(Bc)匹配最高;杀虫活性测定表明这8株菌对猿叶甲Colaphellus bowringi幼虫均无毒杀作用,而15-3、W23-1和W25-3 3个菌株及其培养上清液对小菜蛾Plutella xylostella幼虫有一定的杀虫活性,说明它们能产生活性物质.这些结果说明在Bt菌株的分离过程中要对无晶体菌株加以重视,其含有的活性物质具有潜在的研究和利用价值.     

小菜蛾对苏云金芽胞杆菌抗性研究进展

自１９９０年Ｔａｂａｓｈｎｉｋ首次报道小菜蛾田间种群对苏云金芽胞杆菌产生抗药性以来,国内外学者在小菜蛾对苏云金芽胞杆菌的抗性监测和抗性筛选、交互抗性、抗性遗传以及抗性机制等方面作了大量,深入的研究。本文对这些方面的研究进展作一综述。     

北京植物园苏云金芽胞杆菌菌株的分离鉴定

[目的]从北京植物园采集的土壤样品中分离高毒力的苏云金芽胞杆菌.[方法]采用温度法筛选Bt菌株,比对大质粒DNA图谱,区分不同类型的Bt菌株,PCR-RFLP方法对cry1～cry40类基因型进行鉴定,SDS-PAGE分析杀虫晶体蛋白,测定Bt分离株对大猿叶甲、小菜蛾幼虫的杀虫活性,筛选出高毒力的菌株.[结果]从149份土壤样品中分离出147株Bt,分为12种类型,含有菱形、方形、球形和不规则等晶体类型;6株菌中分别含有fry1Aa、cry1Ab、cry1Ac、cy1Ah、cry1Ba、cry1Be、cry1Ia、cry1La、cry2Ab和cry7Aa等基因类型,其余6株中不含有已知基因.有9株表达约130 ku蛋白,1株表达约70 ku蛋白,2株表达约150 ku蛋白,有3株既表达130 ku蛋白又表达60 ku蛋白;发现一株对大猿叶甲具有较高毒力的菌株ZWY-7,1株对小菜蛾有高毒力的菌株ZWY-9,2株菌都没有检测到已知基因型.[结论]北京植物园中Bt分布广泛,类型多样,其中发现两株高毒力的菌株,有望获得新的杀虫基因.     

对植物寄生线虫具有活性的苏云金芽胞杆菌研究进展

植物寄生线虫是重要的植物病原物之一,给农业生产带来了巨大的经济损失。长期以来,一直缺乏有效防治植物寄生线虫的手段。苏云金芽胞杆菌（Bacillus thuringiensis,Bt）是重要的昆虫病原细菌,广泛应用于鳞翅目、双翅目、鞘翅目害虫等农林及卫生害虫的防治;部分Bt菌株对植物寄生线虫具有很高的活性。本文总结了杀植物寄生线虫Bt菌株筛选的模型与方法的建立、Bt菌株杀植物寄生线虫的作用机理及其相关应用、以及杀植物寄生线虫的伴胞晶体蛋白与相关基因等,可望为杀植物寄生线虫Bt制剂的研究与开发提供借鉴。     

苏云金芽胞杆菌安全性的研究进展

苏云金芽胞杆菌（Bacillus thuringiensis,简称Bt）是目前应用最为广泛的一类微生物农药。本文分析比较了我国及美国Bt产品登记和安全评价等情况,并对Bt制剂的风险评价进行了分析。Bt菌株不是人或其他哺乳动物的已知病原体,对哺乳动物无毒性、感染性和致病性;其种群数量在环境中呈逐渐减少趋势,无残留风险;对非靶标昆虫的生存和繁殖及土壤微生物群落结构无显著影响。     

苏云金芽胞杆菌防治植物寄生线虫的研究进展

植物寄生线虫是一类重要的植物病原生物,给农业生产造成巨大的损失.传统方法防治植物寄生线虫都存在一定的局限性.研究发现苏云金芽胞杆菌对线虫有毒杀活性,为防治植物寄生线虫寻找到了一条新途径.文章介绍了苏云金芽胞杆菌防治植物寄生线虫的研究现状,着重阐述了苏云金芽胞杆菌的杀线虫晶体蛋白及其基因、杀线虫机制等方面的研究进展,并探讨了其中存在的问题及其对策.     

治理昆虫对转苏云金芽胞杆菌杀虫蛋白基因植物的抗性

治理昆虫对转苏云金芽胞杆菌杀虫蛋白基因植物的抗性华中农业大学植保系（４３００７０）李建洪邓望喜华中农业大学生命科学院（４３００７０）喻子牛迄今为止，通过遗传工程的方法，已获得了４０多种表达苏云金芽胞杆菌杀虫晶体蛋白（ＩＣＰＳ）毒素的植物。美国ＥＰＡ于...     

苏云金芽孢杆菌防治鞘翅目害虫的研究进展

本文综述了国内外有关防治鞘翅目害虫的苏云金芽孢杆菌菌株、杀虫晶体蛋白及作用方式、生物测定以及制剂和应用等方面的研究进展。     

苏云金芽胞杆菌晶体毒素的多样性

苏云金芽胞杆菌是生物防治中应用最为广泛的一种杀虫剂,它对多种昆虫和其他一些无脊椎动物具有特异的毒杀活性。苏云金芽胞杆菌的杀虫活性主要来自于菌体在形成芽胞期间生成的伴胞晶体毒素,这些晶体毒素在结构和生物学功能方面表现高度的多样性。本文介绍了苏云金芽胞杆菌晶体毒素的分类、命名方法,深入讨论了晶体毒素的氨基酸序列、三维结构、靶标生物和杀虫机理的多样性。评价了各种分类方法的特点,并展望了晶体毒素作用机制研究和未来晶体毒素新基因的发掘。     

菌株NDBJJ-BFG的鉴定及其对马铃薯甲虫不同虫态的毒力测定

为筛选出对马铃薯甲虫Leptinotarsa decemlineata(Say)具有防治潜力的菌株,本研究从马铃薯甲虫僵虫虫体上分离菌株NDBJJ-BFG,通过形态学特征和分子生物学方法明确其分类地位,采用喷雾法和覆土法测定了不同浓度下该菌株对马铃薯甲虫室内毒力的致死中浓度LC50及半致死时间LT50。结果表明,经形态特征与r DNA ITS序列分析最终确定菌株NDBJJ-BFG为球孢白僵菌Beauveria bassiana(Bals.)Vuill。该菌株对1、2、3和4龄马铃薯甲虫幼虫的LC50分别为0.91×10~6、1.51×10~6、5.09×10~6和6.84×10~6个/m L;采用喷雾法和覆土法处理蛹的LC50分别为1.43×10~7个/m L和8.15×10~6个/m L,成虫的LC50分别为5.08×10~7个/m L和2.97×10~7个/m L。在孢子悬浮液浓度相同时LT50随虫龄的增大而延长,其中成虫的LT50最长,其次是蛹;在相同龄期下LT50随着孢子悬浮液浓度的增大而缩短。表明菌株NDBJJ-BFG对马铃薯甲虫1龄和2龄幼虫具有高毒力,其在马铃薯甲虫的生物防治中将具有较大的应用潜力。     

印楝素对马铃薯甲虫的控制作用

为探讨植物源杀虫剂印楝素对马铃薯甲虫的防治作用,采用叶碟法测定印楝素乳油对其成虫的拒食作用,并通过药效试验测定印楝素乳油对马铃薯甲虫的控制效果。结果显示,印楝素对马铃薯甲虫成虫有较强的非选择性拒食作用,且拒食率随取食时间的增加而升高。在取食36 h后,浓度为6、7.5、10、15和30 mg/L印楝素的拒食率分别为55.5%、59.1%、53.4%、76.1%和63.3%,拒食率与浓度无显著相关性;但印楝素对马铃薯甲虫的选择性拒食率较低,各浓度处理间均无显著差异,取食36 h时,选择拒食率约20%;在田间10 mg/L印楝素溶液施药1 d和3 d后,虫口减退率分别为26.52%和50.76%,显著高于对照组,施药9 d后,马铃薯甲虫的校正防治效果为48.86%。表明印楝素对马铃薯甲虫有一定的控制效果。     

Evaluation of the resistance of transgenic potato plants expressing various levels of Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the laboratory and field

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, is a destructive pest. The CPB is a quarantine pest in China, but has now invaded the Xinjiang Uygur Autonomous Region and is continuing to spread eastwards. To control the damage and overspreading, transgenic potato plants expressing Cry3A toxin were developed, and their resistance to CPB was evaluated by bioassays in the laboratory and field in 2009, 2010 and 2011. RESULTS: The insect resistance of the high‐dose (HD) transgenic lines was significantly greater than the middle‐dose (MD) and low‐dose (LD) transgenic lines regarding leaf consumption, biomass accumulation and mortality. The HD and MD transgenic lines showed 100% mortality when inoculated with first‐ and second‐instar larvae; however, the LD transgenic lines showed about 50% mortality. The HD transgenic lines exhibited a significantly higher yield than the MD and LD transgenic lines owing to their high CPB resistance. CONCLUSION: Commercially available transgenic potato plants with above 0.1% Cry3A of total soluble protein and NT control refugia could control damage, delay adaptation and halt dispersion eastwards. The two HD transgenic lines developed in this study, PAH1 and PAH2, are ideal for use as cultivars or germplasm to breed new cultivars. Copyright © 2012 Society of Chemical Industry     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.     

Density—yield relationships for Colorado potato beetle adults on potatoes

A model composed of two straight lines describes the relation between overwintering spring adults of Colorado potato beetle [Leptinotarsa decemlineata (Say)] and potato yield. Potato vines (Solarium tuberosum (L.), cv. ‘Superior’) had a damage boundary of around 5.8 spring adults per plant. However, under conditions favorable for reproduction, overwintering adults of 0.07 beetles per plant would produce a large population of first-generation larvae that would reduce considerably the potato yield if not controlled. There is no appreciable seasonal overlapping between adults and young larvae in the field. Monitoring activities should therefore be directed at the beginning of the season against overwintering adults and later against first-generation larvae. For summer adults, an action threshold of 10 per plant is proposed.     

新疆北疆马铃薯甲虫成虫对新烟碱类杀虫剂的敏感性变化

采用点滴法于2009和2010年监测了新疆维吾尔自治区北疆马铃薯甲虫Leptinotarsa decemlineata 9个田间种群成虫对新烟碱类杀虫剂吡虫啉、啶虫脒、噻虫嗪和噻虫啉的敏感性变化,发现其对吡虫啉和噻虫嗪的敏感性逐年降低。2009年监测的6个种群中有3个对啶虫脒和噻虫嗪低抗(抗性倍数5.0~10.0);2010年监测的6个种群全部对噻虫嗪产生了抗性,其中中抗(抗性倍数10.1~40.0)和低抗种群各3个。噻虫嗪与高效氯氟氰菊酯可能存在交互抗性。     

Target site insensitivity and mutational analysis of acetylcholinesterase from a carbofuran-resistant population of Colorado potato beetle, Leptinotarsa decemlineata (Say)

The BERTS strain of Colorado potato beetle (CPB) was found to be highly resistant to N-methyl carbofuran but relatively susceptible to azinphosmethyl. N-Methyl carbofuran resistance was found to correlate well with acetylcholinesterase (AChE) insensitivity. In becoming resistant to N-methyl carbofuran, the AChE of the BERTS strain became more sensitive to N-propyl carbofuran inhibition. This negative cross-insensitivity correlated well to the increased relative toxicity of the BERTS strain to N-propyl carbofuran compared to the susceptible SS strain. BERTS beetles were sorted into BERTS-R and BERTS-S substrains using their AChE activity profiles. Sequence comparisons of AChE cDNAs from the two substrains revealed the presence of the point mutation that results in the S291G substitution previously found in the AChE of the azinphosmethyl-resistant AZ-R strain of CPB. A novel mutation present only in BERTS-S CPB, however, resulted in an additional I392T substitution in the AChE and apparently reverses the resistance conferring properties of the S291G substitution.     

利用新鲜蓝藻藻泥发酵制备苏云金芽胞杆菌杀虫剂

富营养化湖泊中蓝藻爆发已成突出的环境问题。以云南昆明滇池蓝藻新鲜藻泥为培养基,通过接种Bt菌种,测定发酵过程中Bt的活菌数及芽胞数,观察其生长过程中形态变化,同时测定杀虫晶体蛋白产量及杀虫毒力效价,并与常规LB培养基比较,探究了利用蓝藻藻泥培养基接种苏云金芽胞杆菌(Bt GIM1.32)制备生物源杀虫剂的可行性。结果表明:Bt GIM1.32菌种在新鲜蓝藻培养基中能正常生长并完成芽胞和伴胞晶体的合成;当蓝藻含固率为3%、pH 7、接种活化10 h处于对数生长期的菌种、接种量为2%、培养温度为30℃时,灭菌后直接接种Bt GIM1.32菌株,发酵48 h左右其活菌数可达到相对稳定值(6.79×108CFU/mL),54 h左右芽胞数达到相对稳定值(7.01×108CFU/mL);其130 ku晶体蛋白的质量浓度为4.81 mg/mL,对3龄小菜蛾的致死中浓度(LC50)为0.180μL/mL,毒力效价为1 600.00 IU/μL;发酵性能较常规LB培养基优良。研究表明,蓝藻作为发酵生产生物源农药Bt的培养基具有很好的开发潜力和应用前景。     

四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

Neural actions of imidacloprid and their involvement in resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say)

BACKGROUND: The development of resistance to imidacloprid in eastern US populations of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), threatens this critical use for neonicotinoid insecticides. Previous pharmacokinetic studies with resistant adult CPBs provided no explanation for the high resistance level (over 200-fold) to topically applied imidacloprid. The authors assessed the neural activity of imidacloprid by recording spontaneous activity from a motor nerve leaving the isolated central nervous system to compare the sensitivity of the latter to imidacloprid between susceptible and resistant CPBs. RESULTS: On the isolated central nervous system, imidacloprid was initially neuroexcitatory, and neuroinhibitory at higher concentrations. The neuroexcitatory action of imidacloprid was blocked by coapplication of a specific nAChR antagonist, methyllycaconitine, indicating that it is a result of action on nAChRs. The sensitivity to the neuroexcitatory and inhibitory activities of imidacloprid varied independently among individuals in each population. The sensitivity of the central nervous system of resistant CPBs to excitation by imidacloprid did not differ from that of susceptible insects, but the sensitivity to inhibition by imidacloprid was reduced 52- to 58-fold, indicating a possible change in the sensitivity of at least one subgroup of nAChRs. CONCLUSION: This study provides evidence that reduced nerve sensitivity to the blocking action of imidacloprid is associated with imidacloprid resistance in the CPB.