Curticular proteins of Anopheles gambiae

冈比亚按蚊的膜蛋白

• 批准号: 6888899
• 负责人: JUDITH WILLIS
• 金额: $ 29.44万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888899
• 关键词: Anopheles; arthropod genetics; biotechnology; cell growth regulation; chitin; computer assisted sequence analysis; functional /structural genomics; gene expression; gene expression profiling; genome; in situ hybridization; invertebrate cuticle; messenger RNA; polymerase chain reaction; protein binding; protein structure; proteomics

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（由申请方提供）：将鉴定冈比亚按蚊表皮蛋白（CP）的编码基因，并确定每个基因或相关基因组的时空表达模式。CP是昆虫中最丰富的多基因家族之一。迄今为止鉴定的超过一半的序列具有保守的基序，R&R共有序列，其赋予结合几丁质的能力。该基序以两种形式存在，一种与来自软组织的蛋白质相关，另一种与来自硬组织的蛋白质相关。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。一旦确定了推定的CP，将用定量RT-PCR确定其表达的时间模式。然后，代表性的mRNA的空间位置将确定与原位杂交的组织切片上的不同发育阶段的动物。在形成的角质层下面的表皮中发现mRNA将被认为表明候选蛋白质注定要分泌到角质层中。定位还将提供一级蛋白质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CP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在几丁质柱上测试结合。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，将通过使用鸟枪蛋白质组学方法来鉴定可以由蛋白酶从完整和提取的角质层中释放的肽来获得。这是对单一物种CP的第一次全面分析，将阐明不同类型的CP在角质层结构和功能中所起的作用，特别是鉴定的序列也将受到二级结构分析和进一步结构建模的影响。从冈比亚按蚊的基因组延伸到其表皮的组成和结构的这些信息可能为控制这种可怕的载体的新策略指明方向。