地錢 AGO1 中 miR159 的鑑定揭⽰其與 miR319 的差異調控功能

Abstract

微型 RNA（microRNA, miRNA）是一類長度約 20-24 個核苷酸的小分子非編碼RNA，能調控基因表現，參與植物生長發育及協助適應環境等。miR159 與 miR319 屬於植物中高度保守的 miR159/319 家族，兩者的序列高度相似，但在開花植物中調控不同的功能。過去研究已在苔蘚植物中證實 miR319 的存在，然而 miR159 的缺失，導致 miR159/319 家族在早期陸生植物中的演化歷程與功能分化仍不清楚。本研究重新分析已發表之小 RNA 資料庫及 MpAGO1 免疫沉澱中的小RNA資料庫，並從地錢（Marchantia polymorpha）中成功鑑定出 miR159。該 miRNA 顯著累積於 MpAGO1 中，顯示其在地錢中的調控功能性。序列比對、前驅物二級結構分析及系統發育分析結果進一步證實此 miRNA 屬於保守的 miR159 家族。本研究為 miR159 首次於苔類植物中之報導，擴展了目前 miR159 在陸生植物中的已知分布範圍。為探討 Mpo-miR159 與 Mpo-miR319 的生物學功能，本研究進一步進行目標基因預測及 RNA 降解體 (degradome) 分析。結果顯示 MpR2R3-MYB21 與 MpRKD 為地錢 miR159 及 miR319 調控網絡中的重要目標基因。為進一步驗證其功能，本研究利用 CRISPR/Cas9 系統建立 mir159 與 mir319 突變株。結果顯示，Mpmir319age 突變株的 gemmae 形成數量明顯減少，而 Mpmir319bge 突變株幾乎無法形成正常 gemma cup 及 gemmae。另一方面，Mpmir159ge 突變株則出現頂端分生組織分裂異常及 gemma cup 側壁發育缺陷等表型。此外，降解體分析發現 MpR2R3-MYB21 的切割訊號在 mir159ge 突變株中明顯下降，而在 mir319age 突變株中僅輕微下降，顯示 Mpo-miR159 為 MpR2R3-MYB21 的主要調控因子。相較之下，MpRKD 的切割訊號僅於mir319age 突變株中下降，而在mir159ge 與mir319bge 突變株中則無明顯變化，揭示 Mpo-miR319a 為 MpRKD 的主要調控因子。啟動子報導基因分析則顯示，MIR319a、MIR319b、MpR2R3-MYB21 與 MpRKD 主要表現於 gemmae 及 gemma cup 中，而 MIR159 則表現於發育中的頂端分生組織區域。綜合上述結果，Mpo-miR159 與 Mpo-miR319 在地錢營養生長及無性繁殖過程中具有不同但部分重疊的調控功能。本研究不僅首次在苔類植物證實 miR159 的存在，也提供了 miR159/319 家族於早期陸生植物演化及功能分化的重要證據。

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in post-transcriptional gene regulation in plants. Among conserved plant miRNAs, miR159 and miR319 belong to closely related miRNA families that share high sequence similarity but regulate distinct developmental processes in flowering plants. Previous studies have identified miR319 in bryophytes, whereas miR159 has long been considered absent from this lineage, leaving the evolutionary history and functional diversification of the miR159/319 family unclear in early-diverging land plants. In this study, we identified Mpo-miR159 in the liverwort Marchantia polymorpha through the reanalysis of published small RNA datasets and MpAGO1-IP small RNA libraries. Mpo-miR159 was strongly enriched in MpAGO1, supporting its role as a functional miRNA. Sequence comparison, precursor structure analysis, and phylogenetic analysis further demonstrated that Mpo-miR159 belongs to the conserved miR159 family and revealed a close evolutionary relationship between the miR159 and miR319 families. This finding provides the first evidence for the presence of miR159 in a liverwort. Functional analyses using CRISPR/Cas9-generated mutants revealed distinct developmental phenotypes. mir319age mutants produced fewer gemmae within gemma cups, whereas mir319bge mutants failed to form normal gemma cups and gemmae. mir159ge mutants displayed abnormal apical meristem development, including altered meristem division and defective gemma cup formation. To investigate the functions of Mpo-miR159 and Mpo-miR319, target prediction and degradome analyses were performed. The results identified MpR2R3-MYB21 and MpRKD as major targets of Mpo-miR159 and Mpo-miR319, respectively. Degradome analysis showed that cleavage of MpR2R3-MYB21 was markedly reduced in mir159ge mutants and only slightly reduced in mir319age mutants, suggesting that Mpo-miR159 is the major regulator of MpR2R3-MYB21. In contrast, cleavage of MpRKD was reduced in mir319age mutants but not in mir159ge or mir319bge mutants, indicating that Mpo-miR319a contributes mainly to MpRKD regulation. Additionally, promoter-reporter analyses revealed that MIR319a, MIR319b, MpR2R3-MYB21, and MpRKD were expressed in gemmae and gemma cups, whereas MIR159 expression was mainly observed in developing apical regions. Together, these findings demonstrate that functional diversification of the miR159/319 family had already occurred in early-diverging land plants. The identification of Mpo-miR159 extends the known distribution of miR159 to liverworts and provides new insights into the evolution and conservation of the miR159/319 regulatory network during land plant evolution.