除草劑依滅草對阿拉伯芥及水蚤之鏡像選擇性

Enantioselectivity of herbicide imazapyr to Arabidopsis thaliana and Daphnia pulex

蕭鈺齡(Yu-Ling Hsiao)

指導教授 ： 顏瑞泓

國立臺灣大學/生物資源暨農學院/農業化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.01142

摘要

本研究建立二氮雜戊烯類 (imidazolinones) 除草劑依滅草 (imazapyr) 之鏡像異構物分離方法並研究其消旋體 (racemate) 及其不同鏡像異構物對阿拉伯芥及水蚤之影響。結果發現，利用高效液相層析方法搭配 Chiralcel OJ-H 對掌性管柱，在動相為正己烷/異丙醇 (85/15, v/v)，流速 1.5 mL min-1 之條件下，可得到有效率之依滅草鏡像異構物分離方法。而以逆相 HPLC, GC-MS, 圓二色光譜分析儀及旋光度計鑑定後得到收集依滅草之鏡像異構物 (S,-)-依滅草及 (R,+)-依滅草 (peak 1及 peak 2) 分別有 95% 及 99 % 之純度。
Rac-依滅草及其鏡像異構物對野生型阿拉伯芥之生長影響有顯著的差異。暴露相同濃度除草劑 10 天之野生型阿拉伯芥在外觀上，以 (R,+)-依滅草影響最為顯著，並隨濃度上升影響越明顯。在生理指標方面，發現葉綠素含量也有相同的趨勢。在脯胺酸 (proline) 及丙二醛 (MDA) 含量實驗中，發現施用相同濃度除草劑時，(S,-)-依滅草對野生型阿拉伯芥之影響最小，且 rac-依滅草對野生型阿拉伯芥之影響小於 (R,+)-依滅草。在乙醯乳酸合成酶 (ALS) 活性抑制實驗中，(R,+)-依滅草對野生型阿拉伯芥 ALS (wtALS) 活性抑制效果最顯著，最高濃度下可抑制約 83% wtALS 活性，而在相同濃度之 (S,-)-依滅草只可抑制約 20% wtALS 活性。
此外，依滅草及其鏡像異構物對突變型阿拉伯芥 GH90 之生長也有不同程度影響，其中 (R,+)-依滅草對阿拉伯芥 GH90 的外觀、各項生理指標及抗氧化酵素影響最為顯著；且 (R,+)-依滅草在最高濃度下對突變型阿拉伯芥 GH90 之 ALS (mALS) 活性約可抑制 27%，相同濃度下 rac-依滅草約可抑制 10% mALS 活性，而 (S,-)-依滅草對 mALS 之活性則幾乎沒有抑制能力。
模擬 wtALS 立體模型和依滅草的鏡像異構物結合的模型之後，也可以證實 (R,+)-依滅草比 (S, -)-依滅草更容易和 wtALS 結合；而 mALS 的突變位置則恰位於此結合位置之通道上形成屏障，因而造成抗性。然而，mALS 模型中存在的第二結合位置也顯示出較容易和 (R,+)-依滅草結合，故對具抗性之阿拉伯芥 GH90，依滅草仍具有鏡像選擇性。

關鍵字

二氮雜戊烯類 ； 依滅草 ； 阿拉伯芥 ； 鏡像異構物

並列摘要

In this study, the effects of mobile phase composition and flow rate on separation efficiency were investigated and the enantiomers were also collected to study the enantioselectivity of imazapyr to Arabidopsis thaliana. Successful enantiomeric separation of imidazolinone herbicides by high-performance liquid chromatography (HPLC) equipped with Chiralcel OJ-H column was performed. Imazapyr, one of the imidazolinone herbicides, was analyzed. The pure enantiomers of imazapyr could be obtained by HPLC with mobile phase of n-hexane/2-propanol at 85/15 (v/v) and flow rate at 1.5 mL/min. The separated enantiomers were determined by HPLC, GC-MS, circular dichroism spectropolarimeter and polarimeter. The purity of these eanatiomers were 95 % and 99 %.
The selective effects of the enantiomers of imazapyr on wild type A. thaliana were investigated. The two enantiomers and the racemate had significantly different effects on A. thaliana. After treated with herbicides at the same concentration, significant effect on external appearance showed in the (R,+)-imazapyr treatment, and the higher the concentration treated, the more adverse effects were showed. The chlorophyll content revealed the same trend. The (S,-)-imazapyr treatment has lower proline content than the other treatments and the (R,+)-isomer treatment has the highest proline content. The MDA content of the (R,+)-imazapyr treatment was also higher than other two treatments significantly. Additional, the inhibition of acetolactate synthase from A. thaliana (wtALS) was also enantioselective. (R,+)-imazapyr inhibited the activity of wtALS around 83%. (S,-)-isomer and racemate inhibited the activity of wtALS about 20 and 53%, respectively.
Moreover, the effects of imazapyr and its enantiomers on mutant A. thaliana GH90 were significant whether in appearance, physiological indices or antioxidant enzymes. (S,-)-Imazapyr shows less stress then rac- and (R,+)-imazapyr. The inhibition rate of ALS extracted from mutant A. thaliana GH90 (mALS) shown that (R,+)-imazapyr inhibited the activity about 20%, and (S,-)-isomer was nearly no inhibition ability. As the result, it reveals that the enantiomers of imazapyr had selective effects on A. thaliana both wild type and mutant.
It reveals that there are unfavorable contacts between wtALS and (S,-)-imazapyr by simulation of the wtALS-imazapyr binding complex. The point mutation of mALS just blocked the channel leading to the binding site and that cause the resistance. On the other hand, (R,+)-imazapyr shows more favorable to bind to the second binding site of mALS than (S,-)-imazapyr. That explains the enantioselective of imazapyr toward imazapyr-resistant A. thaliana GH90.

並列關鍵字

imidazolinone herbicides ； imazapyr ； Arabidopsis thaliana ； enantiomers

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