一個參與在病毒誘導水楊酸相關抗性之轉錄因子PhaTF13之分子選殖與鑑定

Abstract

蘭花屬於Orchidaceae科，其成員超過35000種，是被子植物中最大的科之一，然而蘭花在病原菌入侵的情形下，如何引發抗性反應還有其機制仍有許多部分有待研究。實驗室先前以能夠引發病毒(Cymbidium mosaic virus)誘導性基因靜默(VIGS)之病毒載體─pCymMV-Gateway攜帶187個預測的Phlaenodopsis aphrodite var. Formosa轉錄因子，篩選會參與在病毒誘導水楊酸相關抗性反應之基因，由此發現PhaTF13會影響P. aphrodite中pathogenesis related gene 1的同源性基因PhaPR1的表現。本實驗選殖PhaTF13基因，透過Rapid Amplification of cDNA Ends (RACE)取得其完整的序列及正確的轉錄起始、終止點。再經由序列比對、演化分析發現PhaTF13蛋白帶有兩個轉錄因子常見的鋅指型功能區塊(zinc finger domain)且和其他單子葉植物之相似蛋白歸為同一群。為了更確切分析此轉錄因子在水楊酸相關抗性反應中所扮演的角色，利用短暫表現小片段短夾型RNA (hairpin RNA, hpRNA)造成基因靜默現象以降低PhaTF13之表現量，並偵測蘭花中的水楊酸相關之抗性指標基因PhaNPR1、PhaPR1，來針對PhaTF13進行功能性分析，確認PhaTF13位於PhaPR1及PhaNPR1的上游。並且發現在健康P. aphrodite植株中，PhaTF13只有在蕊柱及葉片中有些微表現，而在CymMV病毒感染的P. aphrodite植株中，PhaTF13的表現量在全株中皆明顯上升。最後構築PhaTF13 open reading frame融合綠色螢光蛋白(green fluorescence protein, GFP) 的表現載體(PhaTF13::GFP及GFP::PhaTF13)，在蘭花的原生質體(protoplast)中觀察其表現的區域(subcellular localization)，顯示PhaTF13蛋白主要在細胞核內表現。

Orchidaceae consists of at least 35000 species and is among one of the largest families within angiosperm; however, how they trigger plant defense and response to pathogens remains largely unsolved. In our previous experiments using a Cymbidium mosaic virus (CymMV) induced gene silencing system to screen 187 predicted Phalaenopsis aphrodite var. Formosa transcription factors on virus induced salicylic acids (SA) related plant defense response allowed us to identify PhaTF13 involved in the expression of P. aphrodite homolog of pathogenesis related gene 1, PhaPR1. To identify the transcription start site and stop site of PhaTF13, we used Rapid Amplification of cDNA Ends (RACE) to obtain the 5’- and 3’-end sequences. Phylogenetic analysis revealed that PhaTF13 protein contains conserved A20/AN1 zinc finger domain and is grouped together with other monocotyledons homologs. To more specifically analyze the role of PhaTF13 in SA-related plant defense, we delivered two 21-nt hairpin RNA into P. aphrodite by agroinfiltration, and the data suggested that knockdown of PhaTF13 reduced the expression of PhaPR1 and PhaNPR1. The PhaTF13 only slightly expressed in column and leaf but induced to higher levels afterz CymMV infection in all P. aphrodite parts. Expression of PhaTF13::GFP or GFP::PhaTF13 in P. aphrodite protoplasts revealed that the PhaTF13 is mainly localized in nucleolus.