miRNA对核基因表达的调控是生物界普遍存在的表观遗传调控途径。然而，miRNA对细胞器基因的调控目前仅在高等动物线粒体中发现，在单细胞真核生物中相关研究还未见报道。申请者前期工作中利用单细胞真核模式生物——莱茵衣藻作为研究对象，针对衣藻叶绿体基因组编码的psbA设计人工miRNA，结果显示psbA的转录表达受到显著抑制，初步实现miRNA对衣藻叶绿体编码基因的转录后调控，预示莱茵衣藻叶绿体中可能存在miRNA的基因调控机制。本项目拟通过探索RISC核心组分（AGO蛋白等）在衣藻叶绿体的定位及生物学功能，利用CLIP-seq探索AGO蛋白结合的衣藻叶绿体mRNA，并通过RNA-seq测序技术分析衣藻细胞核与叶绿体基因组中潜在编码叶绿体miRNA的序列及其靶基因，以期揭示在真核微藻中miRNA对叶绿体基因的调控机制，进一步拓宽对单细胞真核微藻miRNA表观调控机制的理解。

The regulation of nuclear gene expression by microRNAs is a common epigenetic pathway in biology. However, the regulation of organelle genes by microRNAs is only found in the mitochondria of higher animals, and the related studies in single-cell eukaryotes have not been reported. In our previous work, a significant improvement in photobio-H2 production was observed in the eukaryotic model organism Chlamydomonas reinhardtii, which employed a newly designed strategy based on a heat-inducible artificial miRNA expression system targeting a chloroplast genome coding gene, psbA. The post-transcriptional regulation of chloroplast coding genes by microRNAs was preliminarily realized, which suggested that there might be a gene regulation mechanism of microRNAs in the chloroplast of C. reinhardtii. In this study, we aim to explore the location and biological functions of RISC core components (AGO proteins, etc.) in Chlamydomonas chloroplasts. We will also identify the AGO-binding mRNAs in Chlamydomonas chloroplasts by CLIP-seq technique. Furthermore, we will analyze the coding sequence and target genes of potential microRNAs in the genome of Chlamydomonas chloroplast by RNA-seq technology. Moreover, we plan to verify the relationship between microRNAs identified previously from Chlamydomonas nuclear genome, and genes encoded by Chlamydomonas chloroplast genome. We hope the results obtained from this study will reveal for the first time the regulation mechanism of microRNAs on chloroplast genes in eukaryotic microalgae, and this mechanism will further broaden our understanding on the mechanism of epigenetic regulation of microRNAs.