本项目以淡水水域常见种蚤状溞为研究对象，拟通过转录组测序构建蚤状溞miRNA表达谱和蚤状溞不同世代的基因表达谱；利用生物信息学手段对miRNA表达谱和基因表达谱进行比较分析，筛选出在序列和表达模式上相互关联的miRNA和靶基因；克隆差异表达基因获得其cDNA全长；对相互关联的miRNA和靶基因，利用Real-time PCR、Western Blotting和整体原位杂交技术、整体原位免疫组化方法进一步定量和定位分析；采用RNA干扰技术，对蚤状溞体内的miRNA进行沉默，检测蚤状溞体内与所沉默miRNA相关联基因的表达水平，验证miRNA对相应基因的调控作用，并结合与衰老相关的形态学表型指标变化，深入分析miRNA在蚤状溞衰老过程中对相应信号通路的调控作用。本研究旨在探索枝角类生殖模式的变化规律和分子基础，搭建枝角类miRNA功能解析技术平台，为实现枝角类生殖转化的人为调控奠定基础。

Combining the basis of previous research, and synthesizing transcriptome, cell biology, molecular biology and immunohistochemical methods and techniques, the miRNA expression profiling and gene expression profiles of different generations in Daphnia pulex, live in fresh waters, are constructed by transcriptional genome sequencing; The miRNA expression profiling and gene expression profiles are analyzed comparatively by use of bioinformatics, and miRNA and target genes interrelated in sequence and expression pattern are screened; The cDNA of differential expression genes are cloned by the RT-PCR and rapid amplification of cDNA ends (RACE); Expression and location of miRNA and target genes are analyzed by Real-time PCR、Western Blotting, whole-mount in situ hybridization and whole-mount in situ immunohistochemistry; Then using RNA interference technique to suppress miRNA expression and detecting changes of target genes at spatial and temporal expression, and verify the regulating effect of miRNA to target genes. Combined with morphological phenotypic indicators of aging-related ulteriorly, analyze regulation of miRNA in the corresponding signal pathway of D. pulex in the aging process. We aim is to explore the variation and molecular basis of cladocerans reproductive patterns, build technology platform of miRNA function analysis (identification and functional identification of target genes), provides a theoretical basis for molecular mechanisms of regulating switch reproductive transformation of cladocerans.