Cuticular proteins of Anopheles gambiae

描述(由申请人提供)：将确定冈比亚按蚊角质蛋白(CP)的编码基因，并确定每个基因或一组相关基因表达的时间和空间模式。CP是昆虫中含量最丰富的多基因家族之一。到目前为止，已鉴定的序列中有一半以上都有一个保守的基序，即R&R共识，它赋予了结合几丁质的能力。这个基序有两种形式，一种与软质层的蛋白质有关，另一种与硬质角质层的蛋白质相关。从其他昆虫中已知的CP基序将用于计算机分析按蚊基因组。一旦推测的CP被确定，它们的时间表达模式将用定量RT-PCR来确定。然后在不同发育阶段的动物的组织切片上进行原位杂交，确定具有代表性的mRNAs的空间位置。在形成角质层下的表皮中发现mRNAs将表明候选蛋白质注定要分泌到角质层中。定位也将提供初级蛋白质结构与角质层类型的关联。为了了解缺乏R&R共识的假定CP是否也与几丁质结合，将表达具有代表性的蛋白质的cDNA，纯化它们的蛋白产物并测试其与几丁质柱的结合。通过使用鸟枪式蛋白质组学方法从完整和提取的角质层中鉴定可被蛋白酶释放的多肽，将获得关于角质层中蛋白质的进一步信息，包括那些未被强变性物质溶解的蛋白质。这是对单一物种CP的首次全面分析，将阐明不同类型的CP在角质层结构和功能中所起的作用，特别是因为所确定的序列也将受到二级结构分析和进一步的结构建模的影响。这些信息从冈比亚按蚊的基因组延伸到其角质层的组成和结构，可能会为控制这种强大的媒介提供新的策略。

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.