番茄對抗青枯病菌之 分子機制研究

Molecular defense mechanism of tomato against bacterial wilt

陳永義(Yong-Yi Chen)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02149

摘要

植物遭受到生物性逆境後會經由一系列的訊息傳遞產生防禦機制，有效對抗不同病菌。青枯病（bacterial wilt）為細菌性維管束病害，造成全球許多作物的嚴重致死萎凋病。但目前對於植物抗青枯病菌 (Ralstonia solanacearum) 的抗病機制研究相當不足，尤其茄科植物是青枯病菌主要的天然宿主，其分子抗病機制研究卻很有限。所以本研究選擇番茄（Solanum lycopersicum）抗病品系H7996為對象，探討茄科作物對抗青枯病菌的分子防禦機制。首先利用Tobacco Rattle Virus (TRV)-based vector 進行virus-induced gene silencing (VIGS)，找出最適合抗青枯病番茄品系H7996的基因靜默條件，以有效地篩選青枯病菌防禦基因。以農桿菌注射在子葉後觀察植株不同部位出現基因靜默的時間點。結果發現第五天於番茄地基部開始出現基因靜默現象，在根部是第七天開始發生，在新生葉則是在第十天開始發生。基因靜默後第十天效果可擴散到全株，故選擇此時間點作青枯病試驗的接種病菌時間點。此外，本研究亦發現利用TRV-VIGS進行多基因靜默時，將欲靜默的基因片段同時建構在一個載體的方法比利用混合農桿菌法的靜默效果佳。其次，利用上述建立之TRV-VIGS系統探討番茄青枯病的抗病訊息傳導，結果顯示防禦相關的Mitogen-activated protein kinases (MAPKs) 和水楊酸(salicylic acid)相關訊息傳遞基因皆參與番茄抗青枯病機制，這是首次在茄科作物真正瞭解水楊酸與MAPK相關基因參與抗青枯病的訊息傳導路徑。最後，針對實驗室前人選自番茄microarray分析之特定青枯病菌誘導性基因群，利用VIGS初步判定其是否參與抗病反應，結果發現其中七個基因可能參與番茄抗青枯病機制，並對其中SlCLL 和SlRLI2基因的特性進行進一步研究。這是第一篇探討番茄青枯病抗病訊息傳遞路徑的研究，也鑑認出一些新的番茄抗病防禦基因，值得未來更深入研究。

關鍵字

番茄 ； 青枯病菌 ； 水楊酸 ； VIGS ； MAPK

並列摘要

Bacterial wilt (BW), caused by Ralstonia solanacearum, is a devastating vascular disease of many economically important crops worldwide. However, information on plant defense mechanism to the infection of this soil-borne bacterium is limited. In this study, virus-induced gene silencing (VIGS) was employed to decipher signaling pathways involved in tomato resistance to this pathogen. Firstly, factors involved in Tobacco rattle virus (TRV)-based VIGS system were optimized for efficient gene functional study in tomato. Spatial and temporal analysis showed that gene silencing in 9-day-old tomato plant initiates 5 days post inoculation (DPI) in stembases, 7 DPI in roots and 9 DPI in young leaves. Analysis involving multi-gene silencing revealed that a chimeric construction approach is more efficient as compared to mixing multiple Agrobacterium strains carrying individual target genes. By employing the optimized system, this study further revealed silenced salicylic acid-related signaling transduction pathways and mitogen-associated protein kinase cascades play role in tomato BW-resistance. This is the first report elucidating the intertwined defense network governing resistance to bacterial wilt in tomato. Finally, previous microarray analysis led to the identification of a group genes differentially expressed specifically in H7996. Using the VIGS system, this study further confirmed a few of these genes do play a role in tomato defense to R. solanacearum. Two of these genes, SlCLL and SlRLI2, were subjected to further characterization. Together these studies enlighten molecular mechanism of tomato BW resistance and genes involved.

並列關鍵字

Solanum lycopersicum ； Ralstonia solanacearum ； SA ； VIGS ； MAPK

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