苦苣苔科植物非洲堇之病毒誘導基因靜默技術

Abstract

非洲堇 (African violet, Saintpaulia ionantha) 為苦苣苔科之多年生草本植物，具有自然突變種 (輻射對稱花) 與野生種 (兩側對稱花) 兩種花型。本實驗室比較兩種非洲堇花型之對稱性基因表現模式，結果發現CYC (CYCLOIDEA) 基因的過量表現，可能是使花朵由兩側對稱呈現輻射對稱的重要原因，但其對於非洲堇表徵變化上扮演的角色無法明確得知。本研究的目的為應用病毒誘導基因靜默 (VIGS, virus induced gene silencing) 技術，建立於非洲堇系統上，以供非洲堇花對稱性基因功能研究。我們使用CymMV (cymbidium mosaic virus) 及TRV (tobacco rattle virus) 所衍生的兩種VIGS系統，利用靜默將造成植物產生白化表徵的SiPDS (Saintpaulia ionantha phytoene desaturase) 基因作為標記，並選擇與花部對稱性相關的SiCYC基因 (Saintpaulia ionantha cycloidea, cycloidea) 作為欲研究之目標基因，構築出pCymMV-SiPDS、pTRV2-SiCYC、pTRV2-SiPDS三種載體，感染非洲堇扦插苗。由於我們所使用的TRV病毒載體須以農桿菌作為感染媒介，因此我們另將pCambia-3130載體轉入GV3101、GV2260及LBA4404三種不同品系的農桿菌中感染植物，利用載體上的GUS序列，來研究不同品系農桿菌對於非洲堇感染之效率。結果三種品系農桿菌感染非洲堇兩天後，經GUS染色，發現無任何GUS蛋白表現，這可能是由於農桿菌對於非洲堇扦插苗的感染率低，且檢測樣本數不足所致；而以CymMV及TRV病毒載體感染一個月後，非洲堇無任何靜默表徵，植物體以RT-PCR檢測，亦無本實驗所感染的病毒RNA存在。然而本實驗所使用之非洲堇品系中，無論有無感染處理的非洲堇，皆可檢測出人為TRV2病毒序列，但卻無病毒RNA移動所必須之TRV1病毒序列存在，這表示人為TRV2序列可能原本即存在於本實驗所使用之非洲堇品系DNA中，造成植物難以再度感染TRV病毒。後續實驗應使用無病毒序列之原生種非洲堇，並以較易感染農桿菌的組培苗作為實驗材料。

African violet (Saintpaulia ionantha) is perennial Gesneriaceae plants. Its flowers show two different type of symmetry: zygomorphy (wild type) and actinomorphy (perolic type) types. In precious studies on gene expression pattern for flower symmetry, we found CYC(CYCLOIDEA) gene is a candidate in controlling flower development for zygomorphy. However, its function and phenotypic effect have never been investigated. The aim of this study is therefore to establish a VIGS system in African violet. With VIGS system, we can further explore the function of floral symmetry gene such as CYC. To establish the system, we start by using SiPDS (Saintpaulia ionantha phytoene desaturase) as a reporter gene and then flower symmetric SiCYC (Saintpaulia ionantha cycloidea) as our interest gene. We use two virus vectors, CymMV (cymbidium mosaic virus) and TRV (tobacco rattle virus), produce three constructs: pCymMV-SiPDS、pTRV2-SiCYC and pTRV2-SiPDS. TRV vector was transformed into agrobacterium for infection. In order to compare the efficiency of different Agrobacterium strains, we transformed pCambia-3130 into GV3101, GV2260 and LBA4404 three different agrobacterium strains. And use GUS as a reporter gene to detect the efficiency of different agrobacterium strains. The result shows that 2 day post infecttion with three strains of agrobacterium carrying GUSintron-pCambia-3130, there was no GUS activity in leaves. This may due to the low infection rate of agrobacterium to Africa plantlet, and our limited sample size. 1 month after infect with TRV and CymMV virus vector, there was no silencing phenotypes. When using RT-PCR detect CymMV virus RNA, no CymMV virus RNA was detected in the plant. TRV infection also failed to induce VIGS. However, surprisingly we found artificial TRV2 sequence in both infected and non-infected plants. But there was no TRV1 detectable in the plants. Since the detected TRV2 was different from our construct, and TRV2 can not move without TRV1, the TRV2 sequence may have already inserted into plant genome before our experiment, and cause the failure of our infection. In the future works, it should be cautious to confirm non-TRV2 insertion strain for establishing African violet VIGS system. No TRV2 insert primitive Africa violet may be the proper material for investigation.