地錢細胞色素P450受tasiRNA及微型核酸雙調控機制研究及大岩桐花瓣原生質體暫時性表達系統之建立

Abstract

植物的微型核酸 (miRNAs) 在後轉錄層級調控目標基因的表現，控制著植物生長發育的過程。其中，保守性的miRNAs在植物發育的所有方面幾乎都發揮了至關重要的作用。本論文的研究展示保守的miR390在基礎陸地植物地錢中的分子調控機制及功能並建立了開花植物中花瓣原生質體暫時性轉殖系統，為研究miR390調控開花相關基因提供了一個技術平台。在第二章中，研究顯示在地錢中miR390透過裁切TAS3轉錄本產生轉導性短干擾RNAs (tasiRNAs)，從中找到一個大量表現的tasiRNA，tasi78A，並預測到其目標基因為一個細胞色素P450基因，MpCYP78A101。同時還預測到MpCYP78A101受到另一個miRNA, miR11700共同調控。透過暫時性轉殖系統驗證了tasi78A及miR11700負向調控MpCYP78A101，並且可能通過調控生長素訊息來影響地錢的無性生殖器官孢芽 (gemma) 以及有性生殖器官的生長發育。第三章則是建立了開花植物苦苣苔屬中大岩桐的花瓣原生質體暫時性轉殖系統，將切成條狀的花瓣浸泡在1.5%纖維素酶 (cellulase) 和 0.4% 離析酶 (macerozyme) 的酵素溶液中六小時，可獲得高產量的花瓣原生質體，並達到41.4%的轉殖效率。本論文對於保守的miR390從基礎陸地植物到開花植物在演化上、基因調控機制及功能性將有更全面的了解。

Plant microRNAs (miRNAs) regulate target gene expression at the post-transcriptional level and control the growth and development of plants. Among them, conserved miRNAs play key roles in plant development, with functions spanning almost all aspects. This study unveils the molecular regulatory mechanisms and functions of the conserved miR390 in the basal land plant, Marchantia polymorpha. Moreover, a petal protoplast transient transformation system in flowering plant was established, providing a technical platform for investigating miR390 regulation of flowering-related genes. In Chapter 2, we demonstrated that miR390 generates trans-acting short-interfering RNAs (tasiRNAs) by cleaving TAS3 transcripts in M. polymorpha. Among these tasiRNAs, one highly expressed tasiRNA, tasi78A, was identified, and its target gene was predicted to be a cytochrome P450 gene, MpCYP78A101. Moreover, we further predicted that MpCYP78A101 is co-regulated by another miRNA, miR11700. Using a transient transformation system, we validated that tasi78A and miR11700 negatively regulate MpCYP78A101 and potentially affect the development of asexual reproductive organs (gemmae) and sexual organs through auxin signaling. In Chapter 3, we established a petal protoplast transient transformation system in the flowering plant Gloxinia (Sinningia speciosa). By immersing petal strips in an enzyme solution containing 1.5% cellulase and 0.4% macerozyme for 6 hours, we obtained high yields of petal protoplasts and achieved a transformation efficiency of 41.4%. This study provides a further understanding of the evolution, gene regulatory mechanisms, and functional roles of conserved miR390 from basal land plants to flowering plants.