Cuticular proteins of Anopheles gambiae

冈比亚按蚊的角质层蛋白

基本信息

批准号：
8490666
负责人：
JUDITH WILLIS
金额：
$ 31.09万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
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/8490666
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

项目摘要

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.

描述（由申请人提供）：疟疾的主要载体Anopheles Gambiae使用了约2％的蛋白质编码基因来实现结构性表皮蛋白。这些基因已被注释，确定它们的表达模式，并确定了许多表达的动物区域。其他实验室牵涉到杀虫剂和耐药性，伴侣识别以及响应血液粉的合成中的表皮蛋白。我们建议通过验证血液粉后其成分蛋白的转录水平的增加以及表皮蛋白基因的表达是否会因响应对干燥而变化，来确定角质层是否是动态结构。定量的实时RT-PCR将验证是否已涉及对血液粉的反应的基因上调。简单的生理实验将证明肛门是否适应了干燥挑战。如果这样做，对转录本的全基因组分析将表明是否涉及表皮蛋白基因。作为更好地理解角质层组件和结构的第一步，选定蛋白质的角质层内的位置将通过可视化用胶体黄金标记的二级抗体来确定，这些抗体与EM截面上的原代抗体结合。下一步迈向更好地理解组件和结构的一步将是确定所有表皮蛋白对几壳的亲和力，以及在自然环境中彼此之间的亲和力，首先先用缓冲液提取粉碎的整个动物，以去除可溶性蛋白质，然后以增加与几丁质结合的浓度的浓度。各种馏分中表皮蛋白的补体将由胰蛋白酶级分的定量质谱法确定。如果代表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Catechol的交联的肽。从上面描述的研究中，将选择表皮蛋白的基因，RNAi将用于降低其转录水平，以确定这种治疗方法是否损害了蚊子的形式和功能，可能揭示了可以在控制疟疾中利用的特征。