一個番茄鋅指蛋白基因之鑑認與功能分析

identification and functional study of a tomato zinc finger protein gene

趙鐸駿(Chun-To Chao)

指導教授 ： 詹明才

共同指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00923

摘要

植物生長會遭受非生物性與生物性逆境的影響，其中缺水逆境(water deficit, WD)與青枯病菌(Ralstaonia solanacearum)造成的細菌性萎凋(bacterial wilt, BW)是限制作物正常的重要因子。BW與WD皆會造成植物缺乏水分，而關於植物對抗這些逆境之相關機制研究尚需探討。本論文針對WD/BW microarray分析所選出之基因群，進ㄧ步用virus-induced gene silencing (VIGS)確認其中基因是否參與番茄抗青枯病機制。利用半定量PCR檢測已證實在番茄抗病品系中，目標基因經過VIGS後，其RNA含量確實下降；再進一步將經過基因靜默之番茄植株接種青枯病菌後，植株內之病菌量顯著比對照組植物高，且可造成青枯病萎凋病徵。利用半定量PCR測試也證實其中ㄧ些基因的RNA表現量確實會受青枯病菌感染而上升。為了瞭解各基因之確切功能，本研究進一步針對SlZFP進行功能性研究。結果發現SlZFP的RNA表現量會受水楊酸及乙烯所抑制，而SlZFP之綠色螢光重組蛋白在阿拉伯芥原生質體中的表現位置是位於細胞骨架(cytoskeleton)。此外，多個過量表現SlZFP或SlZFP-GFP的阿拉伯芥或菸草轉基因品系也已建立。環境逆境測試結果顯示：轉殖SlZFP可以提升阿拉伯芥對鹽害逆境之抗性，對軟腐病之反應未顯著改變，但卻提早青枯病之發病的時間。從這些結果推測，位於細胞骨架上的蛋白SlZFP的表現量可能必須維持在平衡的狀態方能維持植物最佳之抗病反應，而SlZFP在植物病害-逆境反應之相抵觸交互作用中可能具某種的功能。透過本研究所得結果，使得我們對植物對抗缺水逆境與病害機制有更多的了解，且預期可能對研擬青枯病之有效防治管理策略將有所啟發。

關鍵字

青枯病 ； 缺水逆境 ； 鋅指蛋白 ； 細胞骨架 ； 植物荷爾蒙

並列摘要

Plant constantly encounters environmental stresses, including abiotic and biotic factors. Water deficit (WD) and bacterial wilt (BW, caused by Ralstaonia solanacearum or Rs) are very important factors limiting crop production worldwide. The nature of BW shares commonness with that of WD. However, information on plant defense response to these stresses is far from sufficient. To elucidate plant stress defense mechanisms, this study aimed to study functions of a group of genes previously selected from tomato WD/BW microarray analyses. First, the genes were subjected to VIGS assays to study their roles in tomato BW defense response. The transcript accumulation of the test genes in stembases of a silenced BW-resistant tomato cultivar (Hawaii 7996, H7996) was reduced at various levels. Further BW bioassays revealed that silencing of a few genes led to a significant increase of R. solanacearum growth and BW symptom development. Additionally, The expression of a few of these genes in a H7996 was enhanced in response to R. solanacearum infection, further suggesting their involvement in tomato BW response. Moreover, functional study was performed on SlZFP. Its expression at transcriptional level was reduced by salicylic acid and ethephon treatment. Localization assay showed that SlZFP:GFP recombinant proteins colocalized on cytoskeleton with the microtubule marker protein in Arabidopsis protoplasts. Transgenic Arabidopsis and Nicotiana benthamiana plants containing 35S::SlZFP or 35S::SlZFP-GFP have been generated. Compared to the control plants, the transgenic Arabidopsis lines conferred enhanced tolerance to salinity, similar response to Erwinia carotovora subsp. carotovora, and increased BW development. These results suggest that the level of SlZFP might need to be fine-tuned in order to achieve the optimal disease defense response. This protein might play some role in the possible antagonistic interaction in plant responses to abiotic and biotic stresses. This study is expected to pave the way not only for elucidating mechanisms and determinants involved in plant stress defense responses, but also potentially benefit the establishment of useful disease control means.

並列關鍵字

bacterial wilt ； water deficit ； zinc finger protein ； cytoskeleton ； phytohormone

參考文獻

陳永義 (2008). 番茄對抗青枯病菌之分子機制研究。國立台灣大學植物科學研究所碩士論文。

Abdrakhamanova, A., Wang, Q.Y., Khokhlova, L., and Nick, P. (2003). Is microtubule disassembly a trigger for cold acclimation? Plant Cell Physiol 44, 676-686.

Agarwal P.K., Agarwal P., Reddy M.K., Sopory S.K. (2006). Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 25: 1263–1274.

Apel, K., and Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373-399.

Arnon, D.I. (1949). Copper Enzymes in Isolated Chloroplasts - Polyphenoloxidase in Beta-Vulgaris. Plant Physiology 24, 1-15.