利用基因編輯與體外RISC分析研究蘚苔植物與被子植物微型核酸調控機制

Abstract

基因沉默是個植物中為抵禦微生物或面對環境改變時常見的處變機制。Dicer-like 4 (DCL4) 是植物體中負責切割核糖核酸並產生可與Argonaute 1蛋白 (AGO1)結合之短干擾核糖核酸 (short interfering RNA, siRNA)，反式作用干擾小核糖核酸 (trans-acting siRNA)，或病毒小核糖核酸(viral small RNA)。Hyponastic Leaves 1 (HYL1) 蛋白是個雙股核糖核酸連接蛋白，是小分子核糖核酸(microRNA, miRNA)生產路徑中一個重要的調節蛋白。AGO1為核糖核酸誘導沉默複合物(RNA-induced silencing complex, RISC)的重要組合物之一，已被證明在地錢中會被MIR11707所調控。常間回文重複序列叢集關聯蛋白(Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9, CRISPR-Cas9）基因編輯技術在此研究中被應用於變異地錢及阿拉伯介中相關的調節者。另外，葡萄球菌核酸酶样tudor结构域蛋白1 (Tudor-SN, TSN)，捲曲葉蛋白(Curly leaf, CLF)，和類異染色質蛋白1 (Like Heterochromatin Protein 1, LHP1)皆為植物調節機制中的重要調節者，TSN與信使核糖核酸(messenger RNA, mRNA)的去帽路徑相關，CLF和LHP1則與阿拉伯介的開花相關。因此，我們假設這些蛋白質可以它們的專有方式影響AGO1的功能。協同性蛋白水解酶 (helper-component proteinase; HC-Pro) 是個來自馬鈴薯Y病毒屬的RNA沉默抑制蛋白，它可以影響被感染的植物體内核糖核酸誘導沉默複合物的活動。爲了瞭解HC-Pro和AGO1之間的聯係，我們的前輩把P1/HC-ProTu 轉植株中的自噬蛋白ATG8a去除。在研究的下一步，我們把這個轉植株進行了與Col-0的雜交，由此間接把ATG8a恢復。此轉植株預計可以恢復ATG8a的AGO1降解的功能。除此之外，我們也成功製造出TSN，CLF和LHP1基因的變異植株。在此論文中，我們利用體外核糖核酸誘導沉默複合物活動測定(in vitro RISC activity assay)研究了AGO1蛋白質在各個轉植株及變異植株中的活動。作爲地錢中的模範植物，Marchantia polymorpha在近期植物學研究一直是非常重要的研究模本。利用CRISPR-Cas9的技術，通過把向導RNA (guide RNA, gRNA)片段插入載體並轉入地錢中，我們將DCL4, HYL1和MIR11707由Tak-1中變異去除。我們將會觀察各變異植株的表性特徵。

Gene silencing is a typical pathway in plants for defending from viruses and environmental changes. Dicer-like 4 (DCL4) is a dicer protein in plant responses to produce short-interfering RNA (siRNA) for trans-acting siRNA (tasiRNA) and viral small RNA that could be loaded into Argonaute 1 (AGO1) for gene silencing, whereas Hyponastic Leaves 1 (HYL1) is a double strand RNA binding protein that is one of the regulators in the microRNA (miRNA) biogenesis pathway. As an essential component in the RNA-induced silencing complex (RISC), AGO1 is also known to be regulated by miR11707 in Marchantia polymorpha. To further investigate the roles of these regulators, CRISPR-Cas9 gene editing has been used to generate knocked-out mutant in Arabidopsis and M. polymorpha plants in this study. Moreover, Tudor-SN (TSN), Curly Leaf (CLF), and Like Heterochromatin Protein1 (LHP1) proteins were shown to be important in plant regulatory pathways, whereas TSN related to uncapping of mRNA, CLF, and LHP1 are both related to the flowering of Arabidopsis thaliana. So, we hypothesized that they affect AGO1 function in their specific ways. Helper component-proteinase (HC-Pro) is a viral RNA silencing suppressor from potyvirus that will disrupt the RISC activity in the virus-infected plant. To study the relation between HC-Pro and AGO1, our senior produced a P1/HC-Pro transgenic line that ATG8a is being knocked out simultaneously. As the study's next step, they cross this transgenic line with Columbia (Col-0) to recover the atg8a mutant activity. It is expected that, in this transgenic line, the degradation of AGO1 shall be recovered compared to the atg8a knocked-out line. We had also successfully mutated TSN, CLF, and LHP1 genes in A. thaliana Col-0 ecotype. In this thesis, we studied AGO1 protein levels and in vivo RISC activity assay in all transgenic lines to confirm the AGO1 activity in those lines. As a model plant in liverwort, M. polymorpha has been a critical study model in the plant-related study. With CRISPR-Cas-9, we have knocked out DCL4, HYL1, and MIR11707 genes by inserting a guide RNA (gRNA) fragment in a vector and transferring the vectors into M. polymorpha plants. We will be studying their phenotypic symptoms when these regulators are knocked-out successfully.