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The physiological functions of ABC transporters in Tribolium castaneum and their role in protecting insects from pesticides and their metabolites

赤拟谷盗ABC转运蛋白的生理功能及其在保护昆虫免受农药及其代谢物侵害中的作用

基本信息

批准号：
272721159
负责人：
Professor Dr. Hans Merzendorfer
金额：
--
依托单位：
Abteilung Molekularbiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/272721159?language=en
关键词：
physiological functions ABC transporters Tribolium

项目摘要

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across cellular membranes. While ABC transporters from vertebrates have been extensively studied, there is only little insight into their physiological functions in insects. Therefore, we conducted a comprehensive RNAi study and silenced the expression of all 73 ABC genes identified in the genome of the red flour beetle, Tribolium castaneum. We monitored development to screen for evident phenotypes that could link the respective gene with a specific function. Using this approach, we identified ten genes that lead to abnormal phenotypes during development when their expression was knocked-down. Two of these genes are associated with eye pigmentation (TcABCG-9A/B), two are related to protein biosynthesis (TcABCE-3A and TcABCF-2A), two appear to have functions in lipid transport affecting waterproofing of the epicuticle (TcABCG-4C and TcABCH-9C), one seems to be involved in an ecdysterone-related processes (TcABCG-8A), and the other three may have other developmental functions (TcABCA-9A/B and TcABCB-5A). In the proposed research, we will pursue several aspects related to the functions of selected ABC transporters in T. castaneum. 1) We aim to clarify the physiological function of TcABCG-8A and address the question how this ABC transporter modulates ecdysterone responses. This will involve the analysis of transport properties using vesicles from baculovirus-infected insect cells that express different mutated versions of TcABCG-8A. 2) We will examine the roles of TcABCG-4C and TcABCH-9C in the transport of cuticular lipids. Next to GC/MS determination of the lipid composition in the cuticle of control and dsRNA-injected larvae, we will identify the transport substrates using vesicle preparations of insect cells expressing these TcABC genes. 3) We will further analyze the sulfonylurea receptor homolog TcABCC-9A, which has been suggested to mediate the insecticidal effects of benzoylurea compounds known to inhibit chitin synthesis. Utilizing a newly developed membrane preparation from T. castaneum pupal elytrae, we will perform in vitro analysis to verify or falsify this hypothesis. 4) As T. castaneum is also an excellent model for investigating insecticidal effects, we will perform an RNAi screen to identify TcABC genes that are involved in the elimination of insecticides and/or their metabolites. This study will be complemented by fluid secretion assays using large Malpighian tubules isolated from the superworm, the larvae of the tenebrionid beetle, Zophobas artratus, which is related to T. castaneum and equally amenable to RNAi. 4) Finally, we will systematically address the question whether inhibitors of ABC transporters are suited to increase susceptibility to insecticides of tolerant Tribolium strains.

ATP 结合盒 (ABC) 转运蛋白属于一个大的蛋白质超家族，在所有生物体中具有重要的生理功能。大多数是整合膜蛋白，可跨细胞膜运输多种底物。虽然来自脊椎动物的 ABC 转运蛋白已被广泛研究，但对其在昆虫中的生理功能了解甚少。因此，我们进行了全面的 RNAi 研究，沉默了赤拟谷盗基因组中发现的所有 73 个 ABC 基因的表达。我们监测发育，以筛选可以将相应基因与特定功能联系起来的明显表型。使用这种方法，我们鉴定了十个基因，当它们的表达被敲低时，这些基因会在发育过程中导致异常表型。其中两个基因与眼睛色素沉着相关（TcABCG-9A/B），两个与蛋白质生物合成相关（TcABCE-3A 和 TcABCF-2A），两个似乎具有影响表皮防水的脂质运输功能（TcABCG-4C 和 TcABCH-9C），一个似乎参与蜕皮甾酮相关过程（TcABCG-8A），其他三个可能具有其他发育功能（TcABCA-9A/B 和 TcABCB-5A）。在拟议的研究中，我们将探讨与赤霉中选定的 ABC 转运蛋白功能相关的几个方面。 1）我们的目的是阐明TcABCG-8A的生理功能，并解决该ABC转运蛋白如何调节蜕皮甾酮反应的问题。这将涉及使用表达 TcABCG-8A 不同突变版本的杆状病毒感染昆虫细胞的囊泡来分析运输特性。 2）我们将研究TcABCG-4C和TcABCH-9C在角质层脂质运输中的作用。接下来用 GC/MS 测定对照和注射 dsRNA 幼虫角质层中的脂质成分，我们将使用表达这些 TcABC 基因的昆虫细胞的囊泡制剂来鉴定运输底物。 3）我们将进一步分析磺酰脲受体同源物TcABCC-9A，它被认为介导已知抑制几丁质合成的苯甲酰脲化合物的杀虫作用。利用新开发的来自赤霉蛹鞘翅的膜制剂，我们将进行体外分析以验证或证伪这一假设。 4) 由于赤霉也是研究杀虫效果的优秀模型，我们将进行 RNAi 筛选，以鉴定参与消除杀虫剂和/或其代谢物的 TcABC 基因。这项研究将通过使用从超级蠕虫、拟步甲虫 Zophobas arttratus 的幼虫中分离出的大马氏小管进行的液体分泌测定来补充，该超级蠕虫与 T.castaneum 有亲缘关系，同样适合 RNAi。 4）最后，我们将系统地解决ABC转运蛋白抑制剂是否适合增加耐受性谷盗菌株对杀虫剂的敏感性的问题。