BASIC RESEARCH ON INSECTICIDAL PROTEINS PRODUCED BY SYMBIONTS OF NEUROPTERA INSECTS

神经翅目昆虫共生体产生的杀虫蛋白的基础研究

• 批准号: 13660113
• 负责人: MATSUDA Kazuhiko
• 金额: $ 2.3万
• 依托单位: KINKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660113/
• 关键词: Neurootera; antlion; symbionts; Enterobacter aerogenes; Bacillus cereus; insecticidal protein; bacterial disease; symbiont; insecticidal protein; Bacillus cereus; zinc methalloprotease inhA2; sphingomyelinase C; GroEL; chaperonin

The aim of this study is to isolate symbionts producing insecticidal proteins from Neuroptera insects, test for their toxicities against insects and elucidate their mode of actions. Enterobacter aerogenes and Bacillus cereus have been isolated from larvae of Myrmeleon bore. E. aerogenes was found to produce GroEL-like protein as one of active substances. On the other hand, B. cereus is known to secret phospholipase C as one of insecticidal toxins. GroEL like protein and phospholipase C were immunologically detected from the regurgitant of the antlion. Active principles, Toxins 1 and 2 were isolated as insecticidal proteins other than phospholipase C from the culture broth of B. cereus by testing their toxicity by injection. Amino acid sequencing of these proteins suggested that Toxins 1 and 2 are homologous to zinc methalloprotease inh2A and sphingomyelinase C, respectively. The genes for these toxins have been cloned and sequenced to deduce their primary structures.Since the insectici … More dal proteins produced by the symbionts exerted their toxicity rapidly, these proteins could attack nervous systems of insects. To prepare an assay system for the proteins, structural properties and sensitivity to neonicotinoid insecticides of nicotinic acetylcholine receptors (nAChRs) were also investigated by deploying homomer-forming chicken α7 nAChR subunit and two-electrode voltage-clamp electrophysiology as well as site-directed mutagenesis. It has been shown that G189 in loop F of the α7 nAChR is located close to the nitro group of imidacloprid a neonicotinoid in the binding site. On the basis of the crystal structure of acetylcholine binding protein from a snail Lymnaea stagnate that is highly homologous to the N-terminal agonist binding domain of the α7 subunit, Q79 in loop D of the α7 nAChR was found to be located in the proximity of G189 in loop F. Thus Q79 was replaced by either acidic or basic residues and the mutant receptors were functionally expressed in Xenopus oocytes to evaluate role of the residue in its interactions with neonicotinoids in the nAChR. The neonicotinoid sensitivity of the α7 nAChR was markedly reduced by the Q79E mutation, whereas it was enhanced by the Q79K and Q79R mutations, suggesting the data were the result of electronic interactions of the nitro group of neonicotinoids with the added residues. These neonicotinoid-resistant and sensitive nAChRs can also be used for testing the insecticidal proteins and comparing their mode of actions with that of neonicotinoids. Less

本研究的目的是从脉翅目昆虫中分离出产生杀虫蛋白的共生菌，测试它们对昆虫的毒力，并阐明它们的作用方式。从桃金娘幼虫中分离到产气肠杆菌和蜡状芽孢杆菌。产气假单胞菌可产生类GroEL蛋白作为活性物质之一。另一方面，蜡样芽孢杆菌作为一种杀虫毒素，能分泌磷脂酶C。免疫组织化学检测到反流液中有GroEL样蛋白和磷脂酶C。通过注射毒性试验，从蜡样芽孢杆菌发酵液中分离到除磷脂酶C以外的杀虫蛋白毒素1和毒素2。氨基酸序列分析表明，毒素1和毒素2分别与锌甲醇蛋白水解酶inh2A和鞘磷脂酶C同源。这些毒素的基因已经被克隆和测序，以推测它们的一级结构。由于昆虫…这些共生菌产生的更多的DAL蛋白迅速发挥其毒性，这些蛋白可以攻击昆虫的神经系统。为建立烟碱型乙酰胆碱受体(α-7nAChR)的蛋白质检测体系，采用同源形成鸡AChR亚基、双电极电压钳电生理和定点突变的方法，研究了烟碱型乙酰胆碱受体(NAChRs)的结构特征和对新烟碱类杀虫剂的敏感性。研究表明，α7nAChR环F中的G189位于新烟碱类化合物吡虫啉的硝基附近。根据与α7亚基N末端激动剂结合域高度同源的钉螺α7nAChR的乙酰胆碱结合蛋白的晶体结构，发现该蛋白D环的Q79位于F环的G189附近。因此，Q79被酸性残基或碱性残基取代，突变的受体在非洲爪哇卵母细胞中得到功能性表达，以评估残基在其与nAChR中新烟碱类化合物相互作用中的作用。α7nAChR的新烟碱敏感性因Q79E突变而显著降低，而因Q79K和Q79R突变而增强，提示该数据是新烟碱硝基与添加残基的电子相互作用的结果。这些新烟碱抗性和敏感的nAChRs也可以用来检测杀虫蛋白，并将它们的作用方式与新烟碱的作用方式进行比较。较少

项目成果

N.Yoshida, K.Oeda, T.Mikami, Y.Fukita, K.Nishumura, K.Komai, K.Matsuda: "Chaperonin turned insect toxin"Nature. 411. 44 (2001)

N.Yoshida、K.Oeda、T.Mikami、Y.Fukita、K.Nishumura、K.Komai、K.Matsuda：“伴侣蛋白转变为昆虫毒素”性质。

M.Ihara, K.Matsuda et al.: "Diverse actions of neonicotinoids on chicken α7,α4β2 and Drosophila -chicken SADβ2 and ALSβ2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes"Neuropharmacology. 45. 133-144 (2003)

M.Ihara、K.Matsuda 等人：“新烟碱类杀虫剂对非洲爪蟾卵母细胞中表达的鸡 α7、α4β2 和果蝇鸡 SADβ2 和 ALSβ2 杂合烟碱乙酰胆碱受体的不同作用”45. 133-144 (2003)。

M.Shimomura, H.Okuda, K.Matsuda et al.: "Effects of mutations of a glutamine residue in loop D of the α7 nicotinic acetylcholin receptor on agonist profiles for neonicotinoid insecticides and related ligands"British Journal of Pharmacology. 137. 162-169 (

M.Shimomura、H.Okuda、K.Matsuda 等人：“α7 烟碱乙酰胆碱受体环 D 中谷氨酰胺残基的突变对新烟碱类杀虫剂和相关配体的激动剂谱的影响”《英国药理学杂志》137. 162。 -169（

M.Ihara, K.Matsuda et al.: "Diverse actions of neonicotinoids on chicken α7, α4β2 and Drosophila chicken SADβ2 and ALSβ2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes"Neuropharmacology. 45. 133-144 (2003)

M.Ihara、K.Matsuda 等人：“新烟碱类杀虫剂对非洲爪蟾卵母细胞中表达的鸡 α7、α4β2 和果蝇鸡 SADβ2 和 ALSβ2 杂合烟碱乙酰胆碱受体的多种作用”45. 133-144 (2003)。

K.Matsuda, S.D.Buckingham, D.Kleier, J.J.Rauh, et al.: "Neonicotinoids : insecticides acting on insect nicotinic acetylcholine receptors"Trends in Pharmacological Sciences. 22. 573-580 (2001)

K.Matsuda、S.D.Buckingham、D.Kleier、J.J.Rauh 等人：“新烟碱类杀虫剂：作用于昆虫烟碱乙酰胆碱受体的杀虫剂”药理学科学趋势。