Cuticular proteins of Anopheles gambiae

冈比亚按蚊的角质层蛋白

• 批准号: 7884828
• 负责人: JUDITH WILLIS
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7884828
• 关键词: Accounting; Adult; Affinity; Animals; Anopheles Genus; Anopheles gambiae; Antibodies; Binding; Blood; Buffers; Catechols; Chitin; Code; Complement; Consensus; Courtship; Culicidae; Data; Desiccation; Development; Environment; Female; Frequencies; Gene Family; Gene Proteins; Genes; Gold Colloid; Growth; Hour; In Situ Hybridization; Individual; Insecta; Insecticide Resistance; Insecticides; Isoptera; Knowledge; Label; Laboratories; Learning; Life; Limb structure; Location; Malaria; Mass Spectrum Analysis; Membrane; Messenger RNA; Molting; Nymph; Organ; Partner in relationship; Pattern; Peptides; Physiological; Preparation; Property; Proteins; RNA Interference; Recovery; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Rhodnius; Role; Skeleton; Skin; Structural Genes; Structure; Technology; Ticks; Time; Transcript; Wing; base; crosslink; genome-wide; hatching; insight; prevent; public health relevance; research study; resilin; response; species difference; success; vector

DESCRIPTION (provided by applicant): Anopheles gambiae, the major vector of malaria, uses ~2% of its protein-coding genes for structural cuticular proteins. These genes have been annotated, their expression patterns determined, and the regions of the animal where many are expressed have been established. Other laboratories have implicated the cuticular proteins of Anopheles in insecticide and desiccation resistance, in mate recognition, and in being synthesized in response to a blood meal. We propose to determine whether cuticle is a dynamic structure by verifying the increased levels of transcripts of its component proteins after a blood meal and whether expression of cuticular protein genes can change in response to desiccation. Quantitative real-time RT-PCR will verify that genes already implicated in responding to a blood meal are upregulated. Simple physiological experiments will demonstrate whether Anopheles adapts to a desiccation challenge; if it does, genome-wide analyses of transcripts will show whether cuticular protein genes are involved. As a first step toward better understanding of cuticle assembly and structure, the location within the cuticle of selected proteins will be determined by visualizing secondary antibodies labeled with colloidal gold that have bound to primary antibodies on EM sections. The next step toward better understanding assembly and structure will be to determine the affinity of all cuticular proteins for chitin and for each other in their natural environment by extracting pulverized whole animals first with buffer to remove soluble proteins and then with increasing concentrations of agents that interfere with chitin binding. The complement of cuticular proteins in the various fractions will be determined by quantitative mass spectrometry on trypsinized fractions. If well represented proteins have peptides that are not detected, these missing peptides may be those that are participating in catechol-based crosslinking. From the studies described above, genes for cuticular proteins will be selected and RNAi will be used to reduce their transcript levels to establish whether this treatment compromises form and function of the mosquito, possibly revealing features that could be exploited in controlling malaria. PUBLIC HEALTH RELEVANCE: The Anopheles mosquito is the carrier of malaria. As with all insects the exterior of the mosquito - the cuticle - is both its skeleton and its skin and contains many proteins, most of which have been identified in our previous study and constitute about 2% of its protein diversity. We now propose to extend our studies into determining the specific role of these proteins in the life of the mosquito, hoping that this knowledge will contribute to the control of this deadly insect.

描述（由申请人提供）：冈比亚按蚊是疟疾的主要传播媒介，使用约 2% 的蛋白质编码基因来构建角质层结构蛋白。这些基因已经被注释，它们的表达模式被确定，并且许多表达的动物区域已经被确定。其他实验室已表明按蚊的角质层蛋白与杀虫剂和干燥抗性、配偶识别以及响应血粉而合成有关。我们建议通过验证血粉后其成分蛋白转录物水平的增加以及角质层蛋白基因的表达是否会因干燥而改变来确定角质层是否是动态结构。定量实时 RT-PCR 将验证与血粉反应有关的基因是否上调。简单的生理实验将证明按蚊是否适应干燥挑战；如果确实如此，对转录本的全基因组分析将显示角质层蛋白基因是否参与其中。作为更好地了解角质层组装和结构的第一步，将通过观察已与 EM 切片上的一抗结合的胶体金标记的二抗来确定所选蛋白质在角质层内的位置。更好地了解组装和结构的下一步将是确定所有角质层蛋白与几丁质以及在其自然环境中彼此之间的亲和力，方法是首先用缓冲液提取粉碎的整个动物以去除可溶性蛋白质，然后用​​浓度增加的干扰几丁质结合的试剂。各个级分中的角质蛋白的补充将通过胰蛋白酶化级分的定量质谱法来确定。如果表现良好的蛋白质具有未检测到的肽，则这些缺失的肽可能是参与基于儿茶酚的交联的肽。根据上述研究，将选择角质层蛋白的基因，并使用 RNAi 来降低其转录水平，以确定这种治疗是否会损害蚊子的形态和功能，从而可能揭示可用于控制疟疾的特征。 公共卫生相关性：按蚊是疟疾的携带者。与所有昆虫一样，蚊子的外部（角质层）既是其骨骼又是皮肤，并且含有许多蛋白质，其中大部分已在我们之前的研究中鉴定出来，约占其蛋白质多样性的 2%。我们现在建议将我们的研究扩展到确定这些蛋白质在蚊子生命中的具体作用，希望这些知识将有助于控制这种致命的昆虫。