小立碗藓PpAGO6和PpAGO9在原丝体向茎叶体转化过程中的调控机制研究

The Physcomitrella patens is an irreplaceable model plant due to its high rate of homologous recombination, availability of whole genome sequence and its importance in plant evolution, etc. Argonaute (AGO) family proteins are effectors of gene expression regulation in eukaryotes. Few studies have been reported on the function of bryophyte AGO proteins. Previous work showed that PpAGO6 and PpAGO9 were involved in regulating protonema-to-gametophyte transition of P. patens. We have successfully obtained most transgenic lines of PpAGO6 and PpAGO9 genes in P. patens using homologous recombination technique. They are Ppago6, Ppago9, Ppago6Ppago9, PpAGO6::GFP, PpAGO9::GFP, PpAGO6pro::GUS and PpAGO9pro::GUS transgenic lines. To collect the RNA interacting with PpAGO6 and PpAGO9 proteins，co-immunoprecipitation will be carried out using PpAGO6::GFP and PpAGO9::GFP transgenic lines. The RNA will be analyzed through high-throughput sequencing and bioinformatics. Then, the miRNA interacting with PpAGO6 and PpAGO9 proteins will be selected. The gene knockout lines of selected miRNA will be obtained using homologous recombination technique. We have obtained miRNA knockout lines and their phenotype is similar to that of Ppago6Ppago9 lines. The target gene of the miRNA is the transcription factor in auxin signaling pathway.The biological function of PpAGO6, PpAGO9, miRNA and its target gene will be studied by genetics, molecular and cellular biological technology. This study will elucidate the molecular regulation mechanism of PpAGO6 and PpAGO9 during development, and will provide a new theoretical basis for the mechanism of plant evolution and development.

小立碗藓具有基因同源重组频率高、高质量全基因组序列、在植物进化中重要等特点，是不可替代的模式植物。AGO蛋白与基因表达调控机制密切相关，但苔藓植物AGO蛋白功能尚不清楚。前期工作发现PpAGO6和PpAGO9与原丝体向茎叶体转化相关。我们用同源重组技术获得目的基因敲除、目的基因融合GFP、目的基因启动子融合GUS的转基因株系；以PpAGO6::GFP和PpAGO9::GFP株系为材料，RIP-seq结合生物信息学法分析与目的蛋白相结合的RNA，筛选与目的蛋白互作的miRNA；已获得了与目的蛋白互作、且与目的蛋白有相似表型的miRNA敲除株系，其靶基因是生长素信号传导途径中的转录因子；我们将用遗传、分子和细胞生物学等技术分析目的蛋白、互作的miRNA及靶基因在原丝体向茎叶体转化中的生物学功能,阐明PpAGO6和PpAGO9在发育过程中的分子调控通路，为植物进化和发育机制提供新的理论依据。

小立碗藓具有基因同源重组频率高、高质量全基因组序列、细胞分化能力强等特点，是不可替代的模式植物。AGO蛋白与基因沉默机制密切相关，不同的AGO蛋白选择性地与小RNA结合，形成沉默复合体，在不同水平上以不同方式进行基因表达调控，进而调控植物的生长发育过程和对环境的适应性。但苔藓植物AGO蛋白功能尚不清楚。前期工作发现小立碗藓PpAGO6和PpAGO9与原丝体向茎叶体转化相关，本项目进一步研究其调控机制。本项目用RIP-seq方法获得了与PpAGO蛋白相结合的小RNA，分析了其结合小RNA的偏好性及对其靶基因进行GO功能注释和富集分析，初步解析小立碗藓AGO蛋白介导基因沉默的调控机制及其与高等植物的异同。ChIP-seq技术分析了与小立碗藓AGO蛋白结合的DNA，分析了其与DNA区域结合的特性并对相关基因进行GO富集分析，为研究AGO蛋白的生物学功能提供了基础。双突变体Ppago6ago9在原丝体向茎叶体转化过程中与野生型的表型有差异，分析发现PpAGO6/9可能通过调控miRNA及生长素等发育相关基因的表达影响此过程。获得了候选miRNA1-5（数据未发表，用代号表示）的STTMmiR1-5株系15个及其靶基因的敲除株系17个。发现STTMmiR1株系拟叶的发育存在缺陷，PpmiRNA1可能参与拟叶发育过程；STTMmiR2株系影响茎叶体发育，导致多细胞阶段茎叶体顶端重新形成原丝体,说明PpmiRNA2参与调控小立碗藓由原丝体向茎叶体转变过程；STTMmiR4株系正常拟叶上能够发育形成原丝体，表明PpmiRNA4可能参与原丝体再生或发育过程。同时对STTMmiR1-5株系从原丝体到茎叶体转化过程进行观察，发现STTMmiR2和STTMmiR5株系与野生型有差异并进行研究。此外，我们还获得了大量与该项目相关的数据，发现了调节苔藓再生、茎叶体生长、衰老、孢子发育等的基因并进行研究，为进一步深入研究奠定了很好的基础。