- 學位論文
於俄氏草中使用查爾酮合酶作為靜默標記基因開發胡瓜嵌紋病毒介導之病毒誘導基因靜默系統
Development of Cucumber Mosaic Virus-Mediated Virus-Induced Gene Silencing using Chalcone synthase as a silencing marker in Titanotrichum oldhamii
摘要
俄氏草 (Titanotrichum oldhamii) 屬苦苣苔科一員，是研究花序反轉的潛在物種，花序反轉是指植物花朵（序）之分生組織反轉回營養分生組織的過程。在部分物種中，包括俄氏草，用於無性繁殖的珠芽就是由反轉後的組織產生。先前的研究已經發現了一些與花序反轉相關的候選基因，我們對於潛在的基因調控和發育機制感到好奇。然而對於非模式植物如 俄氏草，透過傳統的永久性轉殖系統來進行基因功能性分析仍然是耗時且具有挑戰性。據過往文獻報導，病毒誘導的基因靜默 (Virus-Indiced Gene Silencing, VIGS) 是一種可在相對較短的時間內使目標基因靜默，並且在植物體上表現性狀的有效方法。而查爾酮合成酶 (Chalcone synthase, CHS) 在花青素合成路徑上扮演重要的角色，由於其在花瓣上可見的靜默性狀，查爾酮合成酶是研究花序上 VIGS 的合適標記基因。又先前研究已發現胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV) 可以系統性感染俄氏草並且引發基因靜默。因此，我們將 ToCHS cDNA 部分片段插入 CMV 的 VIGS 載體中，並通過農桿菌（菌株 EHA105）浸潤方式接種到約 5 個月大的俄氏草中。發現新出現的花在接種後約 14 天顯示出預期的 CHS 靜默性狀——花青素顏色褪色，褪色的花朵在接種後 20 至 30 天間大量出現。再來，我們利用吸光值測量花青素含量，以及通過 RT-qPCR 檢測 ToCHS 基因表現量，我們發現在接種後 20 至 30 天間的靜默效益顯著。結合此研究結果與 Ko (2018) 的結果，顯示在俄氏草中基於 CMV 的 VIGS 時期應長達約一個月，始於接種後第二週到第六週間，且在接種後第三週到第五週間有強烈的靜默效益。總結，我們通過靜默 ToCHS 成功地在俄氏草的花瓣上觀察到靜默性狀，本研究是首次對苦苣苔科植物繁殖組織進行 VIGS 的研究論文。這項 VIGS 研究不僅可以幫助我們未來在俄氏草中花序反轉的研究工作，而且還可以為苦苣苔科植物提供另一個成功的 VIGS 案例。
並列摘要
Titanotrichum oldhamii (Gesneriaceae) is a potential specie for studying inflorescence reversion, which is a process that allows floral meristem reverse to vegetative meristem. In some species including T. oldhamii, the asexual reproductive bulbils are produced by the reverse tissues. Previous studies have found some candidate genes associated with inflorescence reversion. We are curious about the underlying gene regulation and developmental mechanisms. However, functional analysis through the genetic transformation in non-model plants such as T. oldhamii remains time-consuming and challenging. Virus-induced gene silencing (VIGS) has been reported to be an efficient approach to silence target genes in a relatively short time and cause the phenotype on the plant body. Chalcone synthase (CHS) plays a significant role in the biosynthesis of anthocyanin, which is a suitable marker gene for studying VIGS on the inflorescences owing to the visible silencing phenotype on petals. In previous works, we found that cucumber mosaic virus (CMV) could systematically infect T. oldhamii and induce VIGS. Therefore, we inserted partial ToCHS cDNA into the CMV VIGS vector and inoculated it to five months old T. oldhamii by Agrobacterium (strain EHA105) infiltration. The newly formed flowers first showed reduced anthocyanin pigmentation at 14 days post-inoculation (DPI), and the silencing flowers largely appeared during 20-30 DPI. The phenotype was associated with CHS silencing. The expression level of ToCHS examined by RT-qPCR and the anthocyanin content analysis by measuring absorbance showed that the silencing efficiency was significant between 20-30 DPI. Integrating the results of this study and Ko (2018), showed that the period of CMV-based VIGS in T. oldhamii was around one month. The silencing period occurred between two to six weeks post-inoculation (WPI) and had strong silencing efficiency between three to five WPI. In conclusion, we succeeded to observe the silencing phenotype in petals of T. oldhamii by silencing ToCHS as a marker gene, and this study is the first VIGS study on the reproductive tissue of the Gesneriaceae plant. This VIGS study not only can help our future works in inflorescence reversion studies in T. oldhamii but also serves another successful VIGS case within Gesneriaceae plants.