转Bt基因抗虫作物对鳞翅目非靶标昆虫生态影响的研究进展

任何转基因作物在进入商业化应用之前都必须经过严格的环境风险评价。评价转基因作物特别是抗虫作物对农田重要非靶标节肢动物的生态影响是其中一项重要内容。当前,全球种植的转基因抗虫作物大多表达对鳞翅目害虫具有活性的Cry1或Cry2类杀虫蛋白。由于非靶标鳞翅目昆虫如斑蝶、家蚕等与靶标害虫具有较近的亲缘关系,其幼虫可能同样对这类杀虫蛋白敏感。因此,这类转基因抗虫作物对非靶标鳞翅目害虫的潜在影响引起了研究者的广泛关注。在总结国内外相关研究数据的基础上,系统分析了转基因抗虫作物对非靶标蝶类和蚕类昆虫的潜在影响,获得以下结论：虽然蚕类和蝶类昆虫对Cry1或Cry2类杀虫蛋白敏感,但在自然条件下这类非靶标昆虫暴露于Cry杀虫蛋白的水平很低,抗鳞翅目害虫转基因作物的种植不可能显著影响田间蝶类昆虫的种群密度,也不会给我国的蚕丝产业带来负面影响。     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

棉铃虫中肠氨肽酶APN4与Cry1Ac、Cry2Aa结合能力的比较

【目的】Bt杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶N(Aminopeptidase N)是一类重要的Bt受体蛋白。因此,分析该受体与Bt杀虫蛋白的结合能力,可为进一步明确不同Bt的分子作用机制、Bt的抗性治理以及新Bt的开发应用等提供借鉴。【方法】本文利用Ligand blot和Elisa方法比较了棉铃虫Helicoverpa armigera中肠APN4(Aminopeptidase N4,APN4)与Cry1Ac、Cry2Aa的结合能力。【结果】原核表达的APN4片段与活化的Cry1Ac、Cry2Aa都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】APN4片段与Cry1Ac、Cry2Aa的结合能力在数量级上不存在显著性差异。     

抗虫转基因植物对非靶标节肢动物生态影响的研究进展

重组DNA技术的发展为培育高效的抗虫作物提供了前所未有的便利条件。通过转基因技术,全世界已培育出众多转基因抗虫植物品系。其中,表达苏云金芽孢杆菌(Bt)基因的作物品系如Bt棉花和Bt玉米已在很多国家大规模种植,在害虫控制方面发挥了重要的作用。转基因抗虫作物可能带来的生态风险问题,如对农田非靶标节肢动物的潜在影响,一直受到相关研究者及民众的广泛关注。至今,已有大量研究论文发表。本文在总结、归纳前人研究的基础上,阐述了从实验室到田间多层次评价转基因抗虫作物对非靶标生物影响的一般研究程序和方法,并简要综述了Bt玉米和Bt棉花2种已商业化种植的转基因抗虫作物对农田非靶标节肢动物生态影响的研究进展。现有研究表明:当前种植的Bt作物所表达的Cry蛋白杀虫专一性非常强,对农田非靶标节肢动物没有毒性;且Bt作物的利用降低了广谱化学杀虫剂的施用量,从而提高了非专一性害虫天敌的种群密度,加强了对害虫的控制,并有效地保护了生态环境和农民健康。因此,Bt作物可以作为害虫综合防治(IPM)的一个策略,结合其他防治措施可加强对害虫的有效控制。     

Bt杀虫基因研究现状与趋势

Bt(Bacillus thuringiensis)是一种革兰氏阳性昆虫病原菌,对哺乳动物等非靶标生物安全无害,cry基因、cyt基因、vip基因编码的杀虫蛋白是其主要活性物质.目前,Bt除了用作生物杀虫剂外,表达Bt杀虫蛋白的转基因抗虫植物也已经被广泛用于害虫的防治,Bt在应用方面取得的巨大成功,极大地促进了该领域的研究发展.本文对Bt杀虫基因克隆技术、Cry蛋白结构多样性、杀虫基因命名与专利保护、研究论文发表等现状与趋势进行分析,发现我国的Bt基础研究和应用基础研究已经与国际接轨,但是产业化明显滞后,企业投入偏少.从发展趋势来看,我国在该领域仍然有较大的发展空间,参考美国转基因作物产业化与企业创新产出的时序关系,随着我国转基因作物向产业化推进,企业的研究与创新将成为一个新的增长点.     

Bt Cry毒素抗虫模拟物靶向创新设计

Bt Cry毒素是当前研究最深入、应用最广的生物抗虫蛋白，对农业害虫的绿色防治发挥了重大作用。然而，随着其制剂和转基因抗虫作物的广泛应用，由此驱动诱发的靶标害虫抗药性及潜在生态安全风险等问题日益凸显。探寻具备模拟Bt Cry毒素杀虫功能的新型抗虫蛋白材料，不仅可为农作物持续健康生产保驾护航，也能在一定程度上缓解靶标害虫对Bt Cry毒素的抗药性压力。近年来，笔者团队以抗体“免疫网络学说(immune network theory)”中Ab2β类型抗独特型抗体(anti-idiotype antibody, Anti-Id)具备模拟抗原结构和功能的特性为理论依据，借助噬菌体展示抗体库及特异性抗体高通量筛选与鉴定技术，设计Bt Cry毒素抗体为包被靶点抗原，从噬菌体抗体库中靶向筛选到了一系列具备模拟Bt Cry毒素抗虫功能的Ab2β类型抗独特型抗体(即Bt Cry毒素抗虫模拟物)，其中活性最强的Bt Cry毒素抗虫模拟物对靶标害虫的致死率接近相应原Bt Cry毒素的80%，初步实现了Bt Cry毒素抗虫模拟物的靶向设计。本文从理论依据、技术条件、研究现状等方面进行系统概述，并就相关技术发展...     

鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系

随着Bt Cry作物在我国的广泛应用和推广,靶标害虫对其抗性风险已成为Bt Cry作物生态安全研究的重要内容.氨肽酶N(Aminopeptidase N,APN)是位于昆虫中肠刷状缘膜囊泡(Brush Border Membrane Vesicles,BBMV)上Bt Cry毒素重要的受体蛋白之一,它与Bt Cry毒素...     

Cry毒素自然进化与作用机制研究进展

苏云金芽胞杆菌(简称Bt)产生的杀虫Cry毒素被广泛的运用于转基因作物害虫的有效防治。在Cry毒素家族中,Cry毒素的三维结构域引导它们自然进化,并产生大量结构和行为方式相似但在不同的昆虫中表现出不同特异性的Cry蛋白,同时Cry毒素家族也是对昆虫特异性分子研究的基础。简述了Bt本身及其发展历程,还详细阐述了Cry毒素在自然中完成昆虫特异性的进化及其作用机制。最后,对于Cry毒素杀虫活性的进化提出了一般性的策略。     

Bt水稻田重要非靶标节肢动物暴露于Cry2Aa蛋白的程度分析

根据风险=危险×暴露的原理,在实验室条件下评价转基因作物对非靶标节肢动物影响时,所选择的代表性非靶标生物通常是在农田系统中较高地暴露于转基因外源杀虫蛋白的节肢动物种.为了弄清Bt稻田主要节肢动物暴露于Cry蛋白的程度,选择合适的非靶标节肢动物,用于转基因抗虫水稻的风险评价,本文采用酶联免疫技术检测了水稻不同生长期从转cry2Aa基因水稻田采集的不同节肢动物体内Cry2Aa蛋白的含量.结果表明: 不同节肢动物种体内的Cry蛋白含量差异显著.一些节肢动物体内不含Cry蛋白,而一些节肢动物体内含有较高的Cry蛋白;相对于花期后采集的节肢动物,在Bt水稻花期采集的节肢动物,特别是捕食性节肢动物体内的Cry蛋白含量较高;寄生性节肢动物体内未检测到Cry蛋白.这为在实验室条件下评价转基因水稻对农田非靶标节肢动物的影响奠定了基础.     

Bt水稻田重要非靶标节肢动物权露于Cry2Aa蛋白的程度分析

根据风险=危险×机露的原理,在实验室条件下评价转基因作物对非靶标节肢动物影响时,所选择的代表性非靶标生物通常是在农田系统中较高地关露于转基因外源杀虫蛋白的节肢动物种.为了弄清Bt稻田主要节肢动物暴露于Cry蛋白的程度,选择合适的非靶标节肢动物,用于转基因抗虫水稻的风险评价,本文采用酶联免疫技术检测了水稻不同生长期从转cry2Aa基因水稻田采集的不同节肢动物体内Cry2Aa蛋白的含量.结果表明:不同节肢动物种体内的Cry蛋白含量差异显著.一些节肢动物体内不合Cry蛋白,而一些节肢动物体内含有较高的Cry蛋白;相对于花期后采集的节肢动物,在Bt水稻花期采集的节肢动物,特别是捕食性节肢动物体内的Cry蛋白含量较高;寄生性节肢动物体内未检测到Cry蛋白.这为在实验室条件下评价转基因水稻对农田非靶标节肢动物的影响奠定了基础.     

Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata

Environmental risk assessment for genetically modified crops producing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) includes the evaluation of adverse effects on non-target organisms. Although ELISA concentration measurements indicate the presence of Cry proteins, sensitive insect bioassays determine whether there is biological activity. The insecticidal activity of the coleopteran-active Cry3Bb1 expressed in different tissues of Bt maize, contained in maize-fed herbivores, and in spiked soil was measured in sensitive insect bioassays using larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Biological activity was confirmed of Cry3Bb1 contained in pulverized Bt maize pollen, roots, leaves, silk, and Bt maize-fed spider mites and western corn rootworm adults. When test substances were incorporated into artificial diet at the same concentrations of Cry3Bb1 (measured by ELISA), maize pollen and leaf litter exhibited lower toxicity than fresh plant material and maize-fed arthropods. This suggests that nutritional quality of food and degradation of Cry proteins may influence toxicity to insects. When soil was spiked with Cry3Bb1, the Bt protein was highly adsorbed and retained its full biological activity. Because toxicity of Cry proteins contained in different matrices cannot always be determined from ELISA values alone, sensitive insect bioassays can improve hazard and exposure assessments in environmental risk assessment of Bt crops.     

Structure of Cry2Aa suggests an unexpected receptor binding epitope

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.     

昆虫类钙粘蛋白与Bt Cry1A蛋白之间的相互作用

类钙粘蛋白(cadherin-likeprotein)位于昆虫中肠刷状缘膜囊泡(brushbordermembranevesicles,BBMV)上,是苏云金芽孢杆菌(Bacillusthuringiensis,Bt)产生的杀虫晶体蛋白(BtCry蛋白)的主要受体之一。它能够与BtCry蛋白结合,引起细胞膜的渗透性发生改变,促进BtCry蛋白对敏感昆虫的毒杀作用。类钙粘蛋白基因的突变还能导致敏感昆虫对BtCry蛋白产生抗性。因此,研究昆虫类钙粘蛋白与BtCry蛋白之间的相互作用,将有助于揭示BtCry蛋白杀虫作用机理。文章对昆虫类钙粘蛋白种类、结构特征、在昆虫体内的分布、及其与BtCry蛋白之间的相互作用等方面的研究现状进行详细论述。     

Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.     

The distinct properties of natural and GM cry insecticidal proteins

The Cry toxins are a family of crystal-forming proteins produced by the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of juvenile insects. These pores allow pathogen entry into the hemocoel, thereby killing the insect. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance insect resistance in genetically modified (GM) crops. In most countries of the world, such GM crops are regulated and must be assessed for human and environmental safety. However, such risk assessments often do not test the GM crop or its tissues directly. Instead, assessments rely primarily on historical information from naturally occurring Cry proteins and on data collected on Cry proteins (called ‘surrogates’) purified from laboratory strains of bacteria engineered to express Cry protein. However, neither surrogates nor naturally occurring Cry proteins are identical to the proteins to which humans or other nontarget organisms are exposed by the production and consumption of GM plants. To-date there has been no systematic survey of these differences. This review fills this knowledge gap with respect to the most commonly grown GM Cry-containing crops approved for international use. Having described the specific differences between natural, surrogate and GM Cry proteins this review assesses these differences for their potential to undermine the reliability of risk assessments. Lastly, we make specific recommendations for improving risk assessments.     

DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions

Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.     

A critical assessment of the effects of Bt transgenic plants on parasitoids

The ecological safety of transgenic insecticidal plants expressing crystal proteins (Cry toxins) from the bacterium Bacillus thuringiensis (Bt) continues to be debated. Much of the debate has focused on nontarget organisms, especially predators and parasitoids that help control populations of pest insects in many crops. Although many studies have been conducted on predators, few reports have examined parasitoids but some of them have reported negative impacts. None of the previous reports were able to clearly characterize the cause of the negative impact. In order to provide a critical assessment, we used a novel paradigm consisting of a strain of the insect pest, Plutella xylostella (herbivore), resistant to Cry1C and allowed it to feed on Bt plants and then become parasitized by Diadegma insulare, an important endoparasitoid of P. xylostella. Our results indicated that the parasitoid was exposed to a biologically active form of the Cy1C protein while in the host but was not harmed by such exposure. Parallel studies conducted with several commonly used insecticides indicated they significantly reduced parasitism rates on strains of P. xylostella resistant to these insecticides. These results provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides, and describe a test to rigorously evaluate the risks Bt plants pose to predators and parasitoids.     

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.