The role of mosquito microRNAs in blood digestion.

描述(申请人提供)：雌性蚊子需要血粉才能繁殖，在吸血期间，许多毁灭性的媒介传播疾病在人类身上传播。更深入地了解与蚊子血粉过程、繁殖事件和抗病原体免疫相关的分子机制，对于设计创新的媒介控制策略特别重要。基于强大的初步数据，我们认为microRNAs(MiRNA)在媒介蚊子的血液消化功能中发挥着重要而保守的作用。我们已经开发了用于耗尽埃及伊蚊和冈比亚按蚊miRNAs的反配子及其功能特征。我们已经确定了两个对血液消化有强烈影响的miRNAs。通过利用GAL4-UAS、miRNA坚韧诱饵或海绵功能丧失转基因蚊子来评估这些miRNAs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。将使用多算法计算靶标预测方法来确定miRNA靶标。然后，携带32个候选miRNA靶标的荧光素酶报告构建体将被导入蚊子细胞系，并将在 正在研究的miRNA的异位共转染。在转基因功能丧失的miRNA蚊子中，通过RNA干扰敲除目的基因将实现表型拯救。作为另一种方法，我们将识别与编码血液消化关键酶的基因的32UTRs互补的miRNAs，并研究它们各自的作用。这一应用为人类疾病的两种最重要的蚊媒中miRNAs的功能鉴定提供了一种全面的方法。

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.