Establishing potential for the protection of crops against damage by slugs and aphids using transgenic plants expressing orally toxic fusion proteins.

利用表达口服毒性融合蛋白的转基因植物，建立保护作物免受蛞蝓和蚜虫损害的潜力。

• 批准号: 1784824
• 金额: --
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1784824
• 关键词: Establishing; potential; protection; crops; against

Slugs are the most damaging pest of cereal and oil seed rape (OSR) crops in the UK, and are an important pest of potatoes. Current practice for slug control now relies on just two active ingredients, metaldehyde and ferric phosphate, delivered as pelleted baits in the field. Metaldehyde may in the near future be subject to restricted usage as it has been found in concentrations above the permitted EU drinking water standards. Aphids are also a serious pest of cereals, OSR and potatoes, causing direct damage by feeding, and indirect damage via the transmission of plant viruses (eg. Turnip Yellows Virus). Pest control has become increasingly problematic due to the withdrawal of many chemical pesticides from the marketplace and a shortage of new actives concomitant with the widespread development of resistance to the few products currently permitted for use. The applicants have developed and patented an innovative approach that converts naturally derived peptides that have no oral toxicity, such as toxins produced by spiders to capture prey, into orally effective pesticides. Linkage of such toxins to a "carrier" protein able to cross the gut wall enables transport of the toxin to its site of action, typically the central nervous system of the target pest. Fusion proteins produced recombinantly using yeast as an expression host have been shown to be effective against a range of crop pests, including slugs and aphids, but have no deleterious effects on bees [1]. Proof of concept for this approach in transgenic plants has recently been demonstrated by significant mortality of aphids fed on Arabidopsis expressing the insecticidal fusion protein Hv1a/GNA [2]. The aim of the project is to produce transgenic plants expressing candidate pesticidal fusion proteins and to evaluate progeny for protection against damage by the grey field slug (Deroceras reticulatum) and aphids (peach potato Myzus persicae, cereal Sitobion avenae). Exploitation of fusion protein technology through exogenous applications (eg. bait and sprays) for the control of crop pests is currently being developed in collaboration with industry. A more elegant and targeted approach would be to deliver fusion proteins in the field via transgenic plants. If successful this would offer a highly novel method for the future protection of key UK crops against attack by major pests. With potential for the generation of a variety of fusion proteins using different toxins to target different sites of action in crop pests this research may also offer a platform from which to develop a pyramid approach to facilitate effective pest control and combat resistance development. Candidate fusion proteins incorporating animal or plant derived pesticidal toxins and plant derived carrier proteins will be used to generate transgenic plants. Candidate toxins include the atracotoxin Hv1a spider venom peptide that targets insect voltage-gated calcium channels and pea albumin 1b (Pa1b) a peptide from pea seeds that inhibits V-ATPase. Snowdrop lectin Galanthus nivalis agglutinin (GNA) is well established as a carrier component of pesticidal fusion proteins and a recently identified alternative, mannose-binding lectin from ginger Zingiber officinale (ZoA) would also be utilised. Hv1a/GNA has been selected as it has been shown to have equivalent oral toxicity towards slugs to metaldehyde and toxicity to pyrethroid resistant aphids [3]. We have recently demonstrated that recombinant fusion proteins incorporating Pa1b linked to GNA or ZoA have significantly deleterious effects on aphid survival and fecundity in artificial diet bioassays. Proof of concept will be conducted using the well-established floral dipping technique to create transgenic Arabidopsis expressing fusion proteins under the control of constitutive and/or phloem specific promoters. Homozygous lines would be assayed for protection against attack by juvenile D. reticulatum and M. persicae in whole plant and detached leaf assays. A m

蚯蚓是英国谷物和油菜(OSR)作物中危害最大的害虫，也是土豆的重要害虫。目前控制弹状物的做法现在只依赖于两种有效成分，三乙醛和磷酸铁，在现场以丸状诱饵的形式投放。在不久的将来，三聚氰醛可能会受到限制使用，因为它的浓度超过了欧盟允许的饮用水标准。蚜虫也是谷物、OSR和土豆的严重害虫，通过取食造成直接危害，通过传播植物病毒(例如，萝卜黄化病毒)。由于许多化学杀虫剂从市场上撤出，以及伴随着对目前允许使用的少数产品普遍产生抗药性而出现的新活性物质的短缺，虫害防治变得越来越成问题。申请者开发了一种创新的方法并申请了专利，该方法将没有口服毒性的天然多肽，如蜘蛛为捕获猎物而产生的毒素，转化为口服有效的杀虫剂。这种毒素与一种能够穿过肠壁的“载体”蛋白相联系，使毒素能够运输到其作用部位，通常是目标害虫的中枢神经系统。以酵母为表达宿主的重组融合蛋白已被证明对一系列作物害虫有效，包括虫子和蚜虫，但对蜜蜂没有有害影响[1]。这一方法在转基因植物中的概念验证最近被证明是由以表达杀虫融合蛋白Hv1a/GNA的拟南芥为食的蚜虫的显著死亡[2]。该项目的目的是生产表达候选杀虫融合蛋白的转基因植物，并评估后代对灰田蚯蚓(德罗塞拉斯网纹夜蛾)和蚜虫(桃属土豆Myzus persicae、谷类麦长管蚜)的保护作用。通过外源应用开发融合蛋白技术(例如：目前正在与工业界合作开发防治作物病虫害的杀虫剂(诱饵和喷雾剂)。一种更优雅、更有针对性的方法是通过转基因植物在田间传递融合蛋白。如果成功，这将为未来保护英国主要农作物免受主要害虫的攻击提供一种非常新颖的方法。这项研究有可能利用不同的毒素产生多种融合蛋白来针对作物害虫的不同作用部位，因此也可能提供一个平台，从这个平台开发一种金字塔方法，以促进有效的害虫控制和抗药性发展。含有动物或植物来源的杀虫毒素和植物来源的载体蛋白的候选融合蛋白将用于产生转基因植物。候选毒素包括针对昆虫电压门控钙通道的阿特拉毒素Hv1a蜘蛛毒肽和豌豆白蛋白1b(Pa1b)，豌豆白蛋白1b是一种来自豌豆种子的抑制V-ATPase的多肽。雪花莲凝集素(GNA)是杀虫融合蛋白的载体成分，最近发现的一种可与甘露糖结合的生姜凝集素(ZOA)也将被利用。Hv1a/GNA之所以被选中，是因为已经证明它对蚯蚓的口服毒性与三乙醛相当，对抗拟除虫菊酯的蚜虫具有同等的毒性[3]。我们最近在人工饲料生物测定中证明了含有与GNA或ZOA相连的Pa1b的重组融合蛋白对蚜虫的存活和繁殖力有显著的有害影响。将使用成熟的花浸渍技术进行概念验证，以创建在构成和/或韧皮部特定启动子控制下表达融合蛋白的转基因拟南芥。纯合子品系将在全株和离体叶测定中用于防御网纹石斑菌和烟粉虱幼虫的攻击。上午1：00