利用 Marchantia polymorpha 葉狀體轉殖技術研究高溫影響 miR11707-MpAGO1 之調控功能

Abstract

植物微型 RNA (microRNAs; miRNA) 在基因表達調控中扮演關鍵角色。Argonaute (AGO) 蛋白作為核糖核酸誘導沈默複合體 (RNA-induced silencing complex; RISC) 的核心組件，負責介導目標mRNA的裂解或轉譯抑制。在早期陸生植物 Marchantia polymorpha中， miR11707.1 以及miR11707.2 是兩的特有的miRNA，源自於同一前驅物，並且都能被載入 MpAGO1 中，調控 MpAGO1 mRNA。儘管先前的研究已經為 miR11707-MpAGO1 調控模組提供了基礎見解，但對其功能和分子機制的更詳細理解仍有待深入。本研究利用 Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9 (CRISPR-Cas9) 基因編輯技術結合葉狀體轉殖，在 Takaragaike-1 (Tak-1) 和 Takaragaike-2 (Tak-2) 兩種遺傳背景下成功建立了 mir11707ge突變株，有效降低先前配子體轉殖而產生的後代因染色體重組所引起的遺傳背景變異。降解組分析顯示，在 28°C 下， RISC 活性發生改變，並且不同遺傳背景下 mir11707ge突變株也呈現出 miRNA 目標基因的切割差異。在外表型上， mir11707ge 突變體表現出較小的葉狀體和葉狀體邊緣捲曲，其中 mir11707ge/Tak-2 在 28°C 下顯示出更高的敏感性。利用遠紅光誘導，高溫抑制了野生型和 mir11707ge 突變株的有性生殖器官發育，然而在正常溫度下 miR11707 缺失對有性生殖器官誘導沒有顯著影響。啟動子分析證實 MIR11707 在芽孢中廣泛表達，然而 MpAGO1 啟動子活性較低或無法檢測。總而言之，本項研究闡明了 miR11707-MpAGO1 調控在 M. polymorpha 中扮演著的關鍵角色，並暗示了遺傳背景可能影響 RNA 沉默，為早期陸生植物在環境壓力下 RNA 沉默途徑的演化和功能提供了新見解。

Plant microRNAs (miRNAs) play crucial roles in regulating gene expression, with Argonaute (AGO) proteins serving as core components of the RNA-induced silencing complex (RISC), which mediates target mRNA cleavage or translational repression. In the early land plant Marchantia polymorpha, miR11707.1 and miR11707.2 are species-specific miRNAs derived from the same precursor, capable of loading into MpAGO1 for MpAGO1 mRNA cleavage. Although previous studies have provided foundational insights into the miR11707-MpAGO1 regulatory module, a more detailed understanding of its functions and molecular mechanisms remains needed. In this study, we employed Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9 (CRISPR-Cas9) gene editing, combined with thallus transformation, to generate mir11707ge mutants in two genetic backgrounds, Takaragaike-1 (Tak-1) and Takaragaike-2 (Tak-2), thereby effectively minimizing genetic background variation caused by chromosomal recombination, which was observed in previous sporeling transformation mutant progenies. Degradome analysis revealed altered RISC activity at 28°C, characterized by the loss of miR11707-mediated negative regulation of MpAGO1, leading to differential cleavage efficiencies of various miRNA target genes. Phenotypically, mir11707ge mutants displayed smaller thallus size and thallus margin curling, with mir11707ge/Tak-2 showing heightened sensitivity at 28°C. Under far-red light, induction assays showed that at high temperature, sexual organ development was strongly suppressed in both wild-type and mir11707ge mutants, and miR11707 deficiency had no significant effect on sexual organ induction. Promoter analysis confirmed broad expression of MIR11707 in gemmae, while MpAGO1 promoter activity was low or undetectable. Collectively, this study elucidates the critical role of the miR11707-MpAGO1 regulatory module in M. polymorpha and provides evidence that genetic background may influence RNA silencing, offering new insights into the evolution and functional dynamics of RNA silencing pathways in early land plants under environmental stress.