驗證草莓NPR1-like基因於抗炭疽病所扮演之角色與建立草莓之病毒誘導基因靜默技術

Validation of the role of NPR1-like genes in defense against anthracnose in strawberry and development of the virus-induced gene silencing technique

舒麟傑(Lin-Jie Shu)

指導教授 ： 鍾嘉綾

共同指導教授 ： 林乃君(Nai-Chun Lin)

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.11049

摘要

草莓是全世界重要的水果之一，然而每年因病蟲的危害所造成的損失是草莓生產上最重要的限制因子，其中草莓炭疽病對於草莓幼苗的危害相對嚴重。本實驗室先前研究發現，將兩種栽培種草莓處理水楊酸後，其抗炭疽病能力會增加;針對水楊酸處理過的草莓葉片進行次世代定序，並分析轉錄體後發現，許多基因的表現受到水楊酸誘導而呈顯著變化，其中部分基因可能與草莓抗炭疽病有關，本研究選擇了草莓的 NPR1 (non-expressor of pathogenesis related genes-1) - like基因群作為進行功能性基因體的候選基因。目前已知NPR1蛋白為在阿拉伯芥水楊酸抗病網絡上極關鍵的調控因子，為了要驗證NPR1-like基因否也是草莓抗炭疽病的關鍵，挑選了三個具潛力的NPR1-like基因Fagene20070、Fagene28768與Fagene28770做進一步探討。在栽培種草莓桃園三號中，Fagene20070表現量皆會受到水楊酸的處理而提升，Fagene28768與Fagene28770則不顯著；然而將桃園三號接種炭疽病菌後Fagene28768與Fagene2877 被大量誘導，但Fagene20070表現量無明顯改變。將Fvgene20070轉殖到阿拉伯芥野生型與npr1突變株中，藉此驗證該基因是否與阿拉伯芥的NPR1功能相同。結果發現，處理水楊酸後，過量表現 Fagene20070 並不會誘導 NPR1 下游之 PR1 的表現，反而使其表現量下降。在野生阿拉伯芥中過量表現 Fagene20070 會降低其對Pseudomonas syringae pv. tomato strain DC3000的抗性; 然而此現象在 npr1突變株中並不會，揭示Fvgene20070在阿拉伯芥上是一NPR1之負調控者的角色。為了建立一個可以對草莓基因功能性分析高通量之篩選平台，初步使用了真空抽氣接種、機械接種與基因槍等方法並搭配不同之接種原、緩衝液配方與環境條件，都未能使TRV成功感染草莓。因此未來需要尋求其他接種原、草莓寄主與不同的方法來成功建立在草莓上的病毒誘導基因靜默系統。

關鍵字

草莓 ； 阿拉伯芥 ； 水楊酸抗病路徑 ； 草莓炭疽病 ； NPR1-like 基因 ； 病毒誘導基因靜默 ； Tobacco rattle virus

並列摘要

Strawberries (Fragaria spp.) are one of the most popular fruits in the world due to their delicious flavor and nutritional value but the presence of strawberry pest threats their production. Anthracnose of strawberry, caused by Colletotrichum gloeosporioides, is a devastating disease which poses serious threat to strawberry production. This hemibiotrophic fungal pathogen is known to induce salicylic acid (SA)-mediated defense signaling pathway in strawberry. A previous study in our lab showed that SA treatment increased the resistance of strawberry cultivar Taoyuan no.3 against anthracnose. An RNA-seq transcriptomic analysis of leaves from SA-treated strawberry leaves further revealed a large number of genes potentially involved in SA defense network in strawberry. Among numerous candidate genes, three putative NPR1 (non-expressor of pathogenesis related genes-1)-like genes, Fagene20070, Fagene28768 and Fagene28770, were chosen for further study. In strawberry cultivar Taoyuan no.3, the expression level of Fagene20070 in SA-treated plants was higher than that in the control plants but not Fagene28768 and Fagene28770. In contrast, inoculation of C. gloeosporioides significantly induced the expression of Fagene28768 and Fagene28770, but not that of Fagene20070 in Taoyuan 3. Furthermore, we introduced 35Spro:Fvgene20070 into the wild-type and npr1 mutant of Arabidopsis to investigate whether they can complement the function of the Arabidopsis npr1 gene. The expression level of AtPR1, SA-pathway marker gene, in wild-type background transgenic lines were less than wild-type after Arabidopsis treated with SA and AtPR1 in npr1 transgenic lines were also suppressed. The transgenic lines were inoculated with Pseudomonas syringae pv. tomato (Pst) strain DC3000 to investigate the resistant ability of transgenic lines. Fvgene20070 decreased the resistant ability against Pst DC3000 in wild-type Arabidopsis; however, it did not affect npr1 mutant. Accordingly, gene20070 was determined to suppress the NPR1-medited SA signaling pathway against DC3000. In addition, TRV-based virus-induced gene silencing (VIGS) was performed to develop a technique for functional genetics. Nevertheless, TRV can’t successfully propagate in strawberry no matter we operated three different inoculation methods paired with various kinds of inoculums, inoculation buffers and environmental conditions. After all, other virus vectors, different strawberry hosts or inoculation methods should be adjusted to establish the VISG system on strawberry in the future.

並列關鍵字

Strawberry ； Arabidopsis ； Salicylic acid-mediated defense signaling pathway ； Strawberry anthracnose ； NPR1-like genes ； Virus-induced gene silencing ； Tobacco rattle virus

參考文獻

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