探討雙股RNA對蘭花啟動子之甲基化及對轉錄之影響

Abstract

蘭花為台灣重要外銷花卉，根據農委會部統計，去年出口值已突破7,200萬美元，其中蝴蝶蘭 (Phalaenopsis sp.)是台灣最具競爭力之蘭花產業然而在學術方面，因蘭花為非模式物種，生長緩慢，且蘭花轉基因植株之建構困難，造成研究上的障礙。經前人在煙草上研究之證實，在啟動子上因甲基化所產生的基因靜默之性狀是可以被遺傳的，因此本實驗擬使用雙股RNA誘發蝴蝶蘭啟動子產生甲基化，並探討其發生甲基化的情形對轉錄之影響，提供建立基因剔除轉基因植株系統的新方法之基礎。本實驗將蝴蝶蘭系統性抗病途徑中之指標基因pathogen-related protein 1 (PR1) 之啟動子分為12個片段作為標的，利用實驗室已建構之東亞蘭嵌紋病病毒載體系統，將PR1片段藉農桿菌打入蘭花葉片，在病毒載體複製過程中，會產生PR1啟動子的雙股RNA片段，以引發甲基化 (RNA-directed DNA methylation, RdDM) 導致基因靜默(Transcriptional gene silencing, TGS)。接種後一個月觀察PR1表現量，發現其中五個處理組PR1表現量有明顯下降之趨勢。由於所使用之病毒載體只會被侷限在接種區域中，但在系統葉中仍可發現基因靜默現象，推測基因靜默的訊號是可系統性移動的。進而萃取蘭花genomic DNA，利用亞硫酸氫鹽定序分析其序列，得知在 PR1啟動子區域確實產生甲基化，且甲基化程度和基因靜默程度具有相關性。過去利用可轉錄區域構築之基因靜默病毒載體，在蘭花上所造成的基因靜默效應僅能維持兩個月，但在此實驗中，持續追蹤至第四個月皆能維持基因靜默之效果，將進一步驗證此等基因靜默是否可藉遺傳將此性狀傳下去下一代。

Phalaenopsis orchids have successfully gained a significant market share in Taiwan. Exports of Phalaenopsis orchids earned Taiwan $72 million in 2012. Though Phalaenopsis sp. are important agricultural products, they are non-model organisms with difficulties for studies because of their large genome size, low transformation efficiency, and long regeneration time. Its long life cycle makes functional analysis hard to achieve through stable transgenic orchids. Previously, virus-induced gene silencing (VIGS) using Cymbidium mosaic virus (CymMV) recovers after 8 weeks of inoculation in flower stalks, and therefore leads to difficulty in gene functional analysis during vegetative phase. In this study, the same VIGS vector was used to conduct a silencing system with Phalaenopsis pathogen-related gene 1 (PR1) promoter. PR1 promoter was divided into 12 fragments and cloned into the VIGS vector separately. In the process of virus replication, dsRNA of PR1 promoter fragments were generated then induced RNA-directed DNA methylation (RdDM) to cause transcriptional gene silencing (TGS). After 1 month of inoculation by Agrobacteria, 5 of the 12 treatments showed repression of PR1 gene. Although the virus vector was restricted in inoculation area, the silencing signal can move systemically and cause reduction on PR1 gene expression. Further analyzing the bisulfite sequencing of Phalaenopsis genomic DNA, the homologous PR1 promoter region was discovered methylated. The degree of DNA methylation is related to the gene silencing level. And the silencing efficiency can last 4 months long in inoculation leaf.