鄰苯二甲酸丁酯苯甲酯對空心菜與萵苣生理及蛋白質體之影響

Effects of Benzyl Butyl Phthalate on the Physiology and Proteome of Water Celery and Lettuce

黃瀚慶(Han-Ching Huang)

指導教授 ： 王一雄 ； 顏瑞泓

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02793

摘要

本研究是探討環境荷爾蒙〈Endocrine disruptor〉中，近年逐漸受到各國高度關切的鄰苯二甲酸丁酯苯甲酯〈Benzyl butyl phthalate, BBP〉對葉菜類作物的影響。基於台灣日益嚴重的河川環境污染問題，本研究選用台灣種植普遍、食用人口多且需引用河水或地下水灌溉的空心菜〈Water celery〉及萵苣〈Lettuce〉作為試驗植物。
研究結果發現，在濃度為100 mg L-1的BBP處理下，空心菜生長至28天時，植體外觀發生矮化現象，植體鮮重與乾重均減少超過50％，並且較控制組減少一片成熟葉片。此外，BBP不會造成空心菜的葉綠素濃度下降，卻導致葉片中有植物逆境指標物─脯胺酸的累積。
在空心菜蛋白質體〈Proteomic analysis〉與質譜儀分析〈Mass spectrometry analysis〉的結果中發現，BBP會導致一含有DEAD-box helicases的蛋白質表現量增加，由此結果可以推論，BBP會直接或間接造成空心菜細胞核中的DNA受損，迫使空心菜增加該蛋白的表現量來修復受損的DNA。此外，BBP造成GAI-like protein 1表現量增加，使得空心菜細胞中吉貝素〈Gibberellin〉的含量相對較少，可能就是空心菜植體生長矮小的主要原因。而(-)-linalool synthase的增加，顯示植體增加沉香醇〈Linalool〉的合成，推測此種萜類〈Terpenoids〉的合成為空心菜的一種防禦反應。
在萵苣的部分，研究結果發現，在濃度為100 mg L-1的BBP處理下，萵苣生長至42天時，植體外觀發生矮化現象，植體鮮重與乾重也有下降的趨勢。而隨著BBP處理濃度的增加，各實驗組會較控制組減少一至二片成熟葉片。此外，BBP不會造成萵苣的葉綠素濃度下降，亦不會導致萵苣葉片中脯胺酸累積的情況發生。
在萵苣蛋白質體〈Proteomic analysis〉與質譜儀分析〈Mass spectrometry analysis〉的結果中發現，BBP造成兩種蛋白質含量降低，分別是S-adenosylmethionine synthetase 1與replication protein-related。此二蛋白質在細胞體中負責S-adenosylmethionine的合成與DNA的修復，由此結果推論，BBP的處理會影響萵苣細胞中的轉甲基反應〈Transmethylation〉與各種DNA代謝相關的正常生理反應，這可能就是造成萵苣生長受阻的原因。另外，BBP誘導F-box protein At2g17036與Magnesium-chelatase subunit chlI的表現量上升，此二蛋白質皆負責細胞受環境脅迫時特定的訊息傳導，藉由特定的調控機制使細胞得以應付外在逆境。因此可以推論，萵苣經BBP處理後增加的這兩種酵素，可能就是對BBP逆境作出的防禦機制。
本研究結論指出，在經過BBP處理的水耕系統中，空心菜與萵苣的生長情形及外觀型態，皆會因不同濃度的BBP處理而產生顯著的改變。另外，由植物蛋白質體學方面的分析結果發現，BBP可能經由影響空心菜與萵苣正常蛋白質的功能，進而造成其外觀型態、生長速率與生理代謝上的各種改變。

關鍵字

鄰苯二甲酸丁酯苯甲酯 ； 空心菜 ； 萵苣 ； 蛋白質體

並列摘要

The effects of the endocrine disruptor, benzyl butyl phthalate (BBP), on the growth of leaf vegetable water celery (Ipomoea aquatica Forsk.) and lettuce (Lactuca sativa Linn.) were investigated. The results showed that biomass and mature leaves of water celery decreased upon treating with 100 mg L-1 BBP in hydroponic culture for 28 days. Beside dwarf, accumulation of proline were also found in BBP-treated water celery. But there was no decrease of chlorophyll in samples treated with any concentration of BBP.
The proteome of the leaf tissue was analyzed using 2-dimentional gel electrophoresis (2-DE) and mass spectrometry (MS). Amount the protein spots with different expression between the normal control and the 100 mg L-1 BBP-treated water celery, one protein spot identified having DEAD-box helicases region increased by BBP treated. This protein is believed to increase in expression in response to DNA damage as a repairing mechanism, and, therefore, it might be DNA damage how BBP affected water celery. The other two protein spots with increased expression on the leaf of treatment with BBP were identified as GAI-like protein 1 and (-)-linalool synthase. Increased expression of GAI-like protein 1 should be responsible for the dwarf of plants, and synthesis of linalool could be a defense mechanism of water celery against BBP.
Besides, the results of lettuce also showed the decrease of biomass and mature leaves upon treating with 100 mg L-1 BBP in hydroponic culture for 42 days, and the plants were dwarfed by BBP. But there is neither proline accumulation nor chlorophyll decrease in BBP-treated lettuce.
As the results of proteome analysis of lettuce leaf tissue, four protein spots showed different expression between the normal control and the 100 mg L-1 BBP-treated lettuce: two decreased and two increased in treated plants. The protein spots decreased were identified as S-adenosylmethionine synthetase 1 and replication protein-related, and they are responsible for transmethylation and DNA repairing respectively. Loss of these two function in cell could be the way BBP interfered with plant growth. Then, the two protein spots increased in BBP-treated lettuce were identified as F-box protein At2g17036 and Magnesium-chelatase subunit chlI. The increase of these protein are defense mechanisms, and plant cells could be adjusted in response to changing environment by their regulation.
This study indicated that the growth and morphology of water celery and lettuce showed a significant dose-response relationship upon treatment with BBP in a hydroponic culture system. Furthermore, BBP also affects the proteome formation as well as the physiology and the morphology of water celery and lettuce during growth.

並列關鍵字

Benzyl butyl phthalate ； water celery ； lettuce ； proteome

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