Curticular proteins of Anopheles gambiae

冈比亚按蚊的膜蛋白

• 批准号: 7221912
• 负责人: JUDITH WILLIS
• 金额: $ 27.91万
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7221912
• 关键词: Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Animals; Anopheles Genus; Anopheles gambiae; Arthropods; Binding; Chitin; Class; Code; Computers; Consensus; Cornea; Development; Diagnostic; Drosophila genus; Endopeptidases; Epidermis; Expressed Sequence Tags; Facility Construction Funding Category; Gene Proteins; Genes; Genome; Genomics; Head; In Situ Hybridization; Individual; Insecta; Integumentary system; Internet; Learning; Location; Methods; Molting; Multigene Family; Pattern; Peptide Hydrolases; Peptides; Play; Proteins; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; Role; Role playing therapy; Shotguns; Staging; Structural Models; Structure; Testing; Trachea; Ursidae Family; Variant; Work; capsule; design; research study; vector

DESCRIPTION (provided by the applicant): Genes coding for the cuticular proteins (CPs) of Anopheles gambiae will be identified and temporal and spatial patterns of expression of each gene, or group of related genes, will be determined. CPs are among the most abundant multigene families in insects. Over half of the sequences identified to date have a conserved motif, the R&R consensus, which confers the ability to bind chitin. This motif is found in two forms, one associated with proteins from soft, the other with proteins from hard cuticles. CP motifs known from other insects will be used in computer analyses of the Anopheles genome. Once putative CPs are identified, their temporal patterns of expression will be determined with quantitative RT-PCR. Then the spatial location of representative mRNAs will be determined with in situ hybridization on histological sections of animals of different developmental stages. Finding mRNAs in the epidermis underlying a forming cuticle will be taken to indicate that the candidate proteins are destined for secretion into the cuticle. Localization will also provide a correlation of primary protein structure with cuticle type. To learn if putative CPs that lack the R&R consensus also bind chitin, cDNAs for representative proteins will be expressed, their protein products purified and tested for binding on chitin columns. Further information on proteins in cuticle, including those that are not solubilized by strong denaturing agents, will be obtained by using shotgun proteomic methods to identify peptides that can be released by proteases from intact and extracted cuticles. This first comprehensive analysis of the CPs of a single species will clarify the roles that diverse types of CPs play in the structure and function of cuticle, especially as the sequences identified will also be subject to analysis of secondary structure and further structural modeling. Such information that extends from the genome of Anopheles gambiae to the constituents and construction of its cuticle may point the way to new strategies to control this formidable vector.

描述（由申请人提供）：冈比亚按蚊表皮蛋白（CPs）的基因编码将被识别，每个基因或相关基因组的时空表达模式将被确定。CPs是昆虫中数量最多的多基因家族之一。迄今为止鉴定出的超过一半的序列具有保守的基序，R&R共识，这赋予了结合几丁质的能力。该基序有两种形式，一种与来自软角质层的蛋白质有关，另一种与来自硬角质层的蛋白质有关。从其他昆虫中已知的CP基序将用于按蚊基因组的计算机分析。一旦假定的CPs被确定，它们的时间表达模式将被定量RT-PCR确定。然后通过原位杂交在不同发育阶段动物的组织学切片上确定代表性mrna的空间位置。在形成角质层的表皮中发现mrna将表明候选蛋白注定要分泌到角质层中。定位还将提供初级蛋白结构与角质层类型的相关性。为了了解缺乏R&R共识的推定CPs是否也结合几丁质，将表达代表性蛋白质的cdna，纯化其蛋白质产物并测试其与几丁质柱的结合。关于角质层中蛋白质的进一步信息，包括那些不被强变性剂溶解的蛋白质，将通过使用散弹枪蛋白质组学方法来识别可以由完整的和提取的角质层中的蛋白酶释放的肽。这是对单一物种CPs的首次综合分析，将阐明不同类型CPs在角质层结构和功能中的作用，特别是所鉴定的序列还将进行二级结构分析和进一步的结构建模。这些信息从冈比亚按蚊的基因组延伸到其角质层的成分和结构，可能为控制这种可怕的载体指明了新的策略。