The role of mosquito microRNAs in blood digestion.

描述（由申请人提供）：雌蚊需要吸血来繁殖，在吸血过程中，许多毁灭性的病媒传播疾病在人类中传播。更深入地了解与蚊子血食过程、繁殖事件和抗病原体免疫相关的分子机制，对于制定创新的病媒控制策略尤为重要。基于强有力的初步数据，我们认为microRNAs （miRNA）在媒介蚊的血液消化功能中起着重要且保守的作用。我们在埃及伊蚊和冈比亚按蚊中开发了用于mirna耗竭及其功能表征的拮抗剂。我们已经确定了两个对血液消化有强烈影响的mirna。利用GAL4-UAS miRNA Tough Decoy或海绵丧失功能转基因蚊子来评估这些miRNA的时空特异性。了解miRNA功能的一个重要步骤是确定它们的靶标。一种多算法计算靶标预测方法将用于确定miRNA靶标。然后将携带候选miRNA靶点的32个utrs的荧光素酶报告基因构建体转染到蚊子细胞系中，并检测其表达水平

DESCRIPTION (provided by applicant): Female mosquitoes require a blood meal for reproduction, and during blood feeding spread many of the devastating vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes, reproductive events and anti-pathogen immunity is of particular importance for devising innovative vector control strategies. Based on strong preliminary data, we suggest that microRNAs (miRNA) play important and conserved roles in blood digestive functions of vector mosquitoes. We have developed antagomirs for depletion of miRNAs and their functional characterization in Aedes aegypti and Anopheles gambiae. We have identified two miRNAs with strong effects on blood digestion. Evaluation of spatiotemporal specificity of these miRNAs will be achieved by utilizing GAL4-UAS miRNA Tough Decoy or sponge loss-of-function transgenic mosquitoes. An important step in understanding miRNA function is determining their targets. A multi-algorithm computational target-prediction approach will be used for determination of the miRNA targets. Luciferase reporter constructs carrying 3 2UTRs of the candidate miRNA targets will then be transfected into mosquito cell lines, and their expression levels will be tested after ectopic co-transfection of a miRNA that is being studied. A phenotype rescue will be achieved by RNA interference knockdown of a target gene in the transgenic loss-of-function miRNA mosquitoes. As an alternative approach, we will identify miRNAs complementary to 32UTRs of genes encoding key enzymes involved in blood digestion and study their respective roles. This application presents a comprehensive approach for functional characterization of miRNAs in two of the most important mosquito vectors for human diseases.