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

atp结合盒（ABC）转运蛋白属于一个大的蛋白质超家族，在所有生物体中都具有重要的生理功能。大多数是完整的膜蛋白，运输广泛的底物穿过细胞膜。虽然脊椎动物的ABC转运蛋白已经被广泛研究，但对它们在昆虫中的生理功能却知之甚少。因此，我们进行了一项全面的RNAi研究，并沉默了红粉甲虫Tribolium castaneum基因组中所有73个ABC基因的表达。我们监测发育以筛选可能将各自基因与特定功能联系起来的明显表型。使用这种方法，我们确定了10个基因，当它们的表达被敲低时，在发育过程中导致异常表型。其中两个基因与眼睛色素沉积有关（TcABCG-9A/B），两个与蛋白质生物合成有关（TcABCE-3A和TcABCF-2A），两个似乎在影响表皮防水的脂质运输中起作用（TcABCG-4C和TcABCH-9C），一个似乎参与了一个激素相关过程（TcABCG-8A），另外三个可能具有其他发育功能（TcABCA-9A/B和TcABCB-5A）。在本研究中，我们将从几个方面探讨与甘蔗中选定的ABC转运体的功能有关的问题。1)我们的目的是阐明TcABCG-8A的生理功能，并解决这个ABC转运体如何调节蜕皮酮反应的问题。这将涉及使用杆状病毒感染的昆虫细胞中表达不同突变版本的TcABCG-8A的囊泡来分析运输特性。2)我们将研究TcABCG-4C和TcABCH-9C在表皮脂质运输中的作用。除了GC/MS测定对照和注射dsrna的幼虫角质层的脂质组成外，我们将利用表达这些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，该同源物被认为介导了抑制几丁质合成的苯甲酰脲类化合物的杀虫作用。我们将利用新开发的一种从castaneum蛹elytrae制备的膜，进行体外分析来验证或证伪这一假设。4)由于castaneum也是研究杀虫效果的一个很好的模型，我们将进行RNAi筛选，以确定参与消除杀虫剂和/或其代谢物的TcABC基因。这项研究将辅以液体分泌分析，使用从超级蠕虫中分离出来的大马尔比氏小管，这是拟黄甲虫的幼虫，Zophobas artratus，它与T. castaneum有关，同样适用于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Tribolium菌株对杀虫剂的敏感性。

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.