Insecticidal fusion proteins for the control of Coleopteran pests
Insecticidal fusion proteins for the control of Coleopteran pests
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用于控制鞘翅目害虫的杀虫融合蛋白
DOI：
--
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
E. Back
中科院分区：
文献类型：
--
作者：
E. Back
含有与载体结构域融合的毒素的融合蛋白已被证明是有效的口服活性杀虫剂。功能性重组融合蛋白的表达，其中包括雪花莲凝集素（Galanthus nivalis 凝集素；GNA）与印度红蝎子毒素（Mesobuthus tamulus 毒素；ButaIT）和蓝山漏斗网蜘蛛（Hadronyche versuta 毒素；ω-ACTX-Hv1a (ω-ACTX)）的毒素融合； κ-ACTX-Hv1c (κ-ACTX)) 在酵母（毕赤酵母）和植物（拟南芥）表达系统中进行。添加纯化标签、改变融合蛋白的组装设计和毒素序列的点突变都被研究以提高产量并减少表达和纯化过程中的蛋白水解切割。
测定了重组蛋白对作为鞘翅目动物模型的栗色砗磲的口服毒性。融合 ButaIT 和 ω-ACTX 毒素的融合蛋白表现出的毒性范围从喂食 1mg g-1（(his)6-GNA-ω-ACTX 和 ω-ACTX-GNA-(his)6）时的完全死亡率到喂食 2mg g-1（ButaIT-GNA-(his)6）时的 65% 死亡率。尽管单个成分具有功能，但掺入 κ-ACTX 和 GNA 的融合蛋白被证明是无毒的。缺乏毒性是由于昆虫肠道环境中的高蛋白水解裂解。获得的数据支持使用谷盗作为金针虫（Agriotes spp.）的模型，金针虫是英国马铃薯的严重害虫。
选定的融合蛋白在转基因拟南芥中表达。
ButaIT-GNA 作为融合多肽的表达在转化体中很容易检测到，估计表达水平约为叶组织中总可溶性蛋白含量为 0.15%。当表达 ButaIT-GNA 融合蛋白的植物被喂食番茄蛾 (Lacanobia oleracea) 幼虫时，融合蛋白显示出完全的功能，其毒性水平与之前的人工饲料生物测定中所见的毒性水平相当。在拟南芥中表达的基于 ω-ACTX 的构建体在植物中受到高水平的蛋白水解切割，因此没有进行毒性分析。
Fusion proteins containing a toxin fused to a carrier domain which directs transport across the insect gut epithelium have been shown to be effective orally active insecticides. Expression of functional recombinant fusion proteins comprising of snowdrop lectin (Galanthus nivalis agglutinin; GNA) fused to toxins from Indian red scorpion (Mesobuthus tamulus toxin; ButaIT) and Blue Mountains funnel-web spider (Hadronyche versuta toxins; ω-ACTX-Hv1a (ω-ACTX); κ-ACTX-Hv1c (κ-ACTX)) was carried out in both yeast (Pichia pastoris) and plant (Arabidopsis thaliana) expression systems. Addition of purification tags, altering the design of assembly of the fusion protein and point mutation of toxin sequence were all investigated to improve yield and reduce proteolytic cleavage during expression and purification.
Recombinant proteins were assayed for oral toxicity against T. castaneum as a model coleopteran species. Fusion proteins incorporating ButaIT and ω-ACTX toxins showed toxicity ranging from complete mortality when fed at 1mg g-1 ((his)6-GNA-ω-ACTX and ω-ACTX-GNA-(his)6) to 65% mortality when fed at 2mg g-1 (ButaIT-GNA-(his)6). Fusion proteins incorporating κ-ACTX and GNA were shown to be non-toxic despite individual components being functional. Lack of toxicity was due to high proteolytic cleavage in the insect gut environment. Data was obtained to support the use of Tribolium as a model for wireworm (Agriotes spp.), serious pests of potatoes in the UK.
Selected fusion proteins were expressed in transgenic Arabidopsis.
Expression for ButaIT-GNA as a fusion polypeptide was readily detectable in transformants with estimated levels of expression of approx. 0.15% total soluble protein in leaf tissue. When plants expressing ButaIT-GNA fusion protein were fed to larvae of the tomato moth (Lacanobia oleracea) the fusion protein was shown to be fully functional with levels of toxicity comparable to that seen in previous artificial diet bioassays. ω-ACTX based constructs expressed in Arabidopsis were subject to high levels of proteolytic cleavage in planta and so were not assayed for toxicity.
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影响因子：
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作者：
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DOI：
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发表时间：
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期刊：
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影响因子：
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