Cuticular proteins of Anopheles gambiae

描述（由申请人提供）：冈比亚按蚊是疟疾的主要媒介，其蛋白质编码基因的约2%用于结构表皮蛋白。这些基因已经被注释，它们的表达模式已经确定，并且已经确定了许多基因表达的动物区域。其他的实验室已经将按蚊的表皮蛋白与杀虫剂和干燥抗性、配偶识别以及对血餐的反应中的合成联系起来。我们建议，以确定是否角质层是一个动态的结构，通过验证其组成蛋白质的转录水平的增加后，血餐和角质层蛋白基因的表达是否可以改变响应干燥。定量实时RT-PCR将验证已经参与对血餐反应的基因被上调。简单的生理实验将证明按蚊是否适应干燥的挑战;如果是这样，对转录本的全基因组分析将显示表皮蛋白基因是否参与其中。作为更好地理解角质层组装和结构的第一步，所选蛋白质在角质层内的位置将通过观察与EM切片上的一抗结合的胶体金标记的二抗来确定。为了更好地理解组装和结构，下一步将是确定所有表皮蛋白质对甲壳素的亲和力，以及在自然环境中相互之间的亲和力，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，然后用增加浓度的试剂干扰甲壳素结合。将通过定量质谱法对胰蛋白酶化组分测定各组分中表皮蛋白的补体。如果充分代表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。从上述研究中，将选择表皮蛋白的基因，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式和功能，可能揭示可用于控制疟疾的特征。

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.