Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97560
標題: 利用SWATH質譜定量技術於水稻葉色突變株之蛋白質體研究
SWATH-based comparative proteomics analysis of leaf color mutant in Oryza sativa
作者: 蔡鴻樹
Hung-Shu Tsai
關鍵字: 水稻;葉色突變株;蛋白質體;oryza sativa;proteomics;SWATH;PAL
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摘要: 
光合作用為整個植物的主要能量來源,會影響植物是否可以存活下去;此作用與葉片的顏色息息相關,當植物葉片轉黃時,會因為無法形光合作用,導致死亡。而水稻葉色突變株 SA0405、SA0407 和 SA0408為台農67號 (TNG67) 經疊氮化鈉誘變之水稻葉色突變株,其外表性狀及生理狀況有明顯不同,株高較矮小且葉片顏色偏黃偏淡,卻能完整生長、抽穗及成熟,但其產生原因尚未明瞭。近年來,質譜發展迅速,其中蛋白質體學技術已應用於許多領域,包含植物性狀研究。2012 年Aebersold 等人以開發出非標定定量技術 Sequential window acquisition of all theoretical fragment ion spectra (SWATH) 之質譜掃描模式,其能偵測特定視窗內的所有母離子,加以撞碎後,收集其所有碎片離子之訊號,對於蛋白質之定量分析是一大幫助。因此,本實驗以非標定定量技術 SWATH 搭配超高效能液相層析儀 (ultra high performance liquid chromatography, UHPLC) 串聯四極棒飛行時間式質譜儀 (quadrupole/time-of-flight mass spectrometry, QTOF MS),探討葉色突變株 SA0405、SA0407 和 SA0408 與 TNG67 葉片之插秧後第 45 天、最高分蘗期、抽穗期與成熟期之蛋白質體表現差異,期望能了解此外表性狀、生理狀況之原因。在 16 個樣品中,總共鑑定到 2408 個蛋白。在這些蛋白中,則以表現量比值 > 1.5或 < 0.67 作為顯著差異標準 (突變株/TNG67)。而藉由 KEGG pathway 處理後,發現突變株中 Phenylalanine ammonia-lyase (PAL) 有大量表現,而 PAL 則會形成類黃酮、苯丙素及木質素等激素的前驅物,因此懷疑是此蛋白之變化導致突變株獨特的性狀。而 PAL 為抗逆境蛋白,在本實驗的結果中發現突變株葉綠體 ribosome 中的 L21 (G23) 及 L27 (G36) 蛋白大量下降。而這兩個蛋白若大量下降時,會影響葉綠體內重要蛋白之合成。因此就此產生逆境。從此結果得知,本實驗成功藉由 SWATH 質譜定量技術,找出葉色突變株與 TNG67 在外表性狀及生理狀況為何有如此差異的原因。

Photosynthesis provided the main energy in plants. Plants could not survive without photosynthesis. Photosynthesis was highly related to the color of leaves. The leaf color mutants SA0405, SA0407 and SA0408 used in this study were induced from a wild-type rice, TNG67, by sodium azide mutagenesis; therefore, TNG 67 and color mutants share the same genetic background. The phenotypes and physiology of these color mutants were obviously different from TNG67. Surprisingly, they have similar photosynthesis efficiency and life cycle. But the molecular mechanisms involved in these phenomenon is not clear. In recent years, mass spectrometry develop rapidly. Proteomics have been applied in many researchs, include the phenotypes and physiology of plant. In 2012, Aebersold etc. have developed a new label-free quantitative proteomics method, Sequential window acquisition of all theoretical fragment ion spectra (SWATH). SWATH data was acquired by repeatedly cycling through sequential mass windows over the whole chromatographic elution range generating a complete recording of all analytes in the sample. Thus, this paper will use SWATH method with ultra high performance liquid chromatography (UHPLC) and quadrupole/time-of-flight mass spectrometry (QTOF MS) to research the proteomics between leaf color mutants and wild-type rice in 45 days after transplant, tillering, spiking, mature period. Expect the results may be helpful in further elucidating the molecular mechanisms between leaf color mutants and wild-type rice. In the 16 samples, 2408 proteins were generated. Proteins with a fold change of > 1.5 or < 0.67 (mutants/TNG67) considered as differentially expressed proteins in this research. After analysis by KEGG pathway, find out the quantity of Phenylalanine ammonia-lyase (PAL) in leaf color mutants bigger than the quantity in wild-type rice. PAL will produce flavonoids, phenylpropanoids, lignins and other enzymes. Thus, we doubt this protein highly related to the phenotypes and physiology of leaf color mutants. PAL is a protein for confrontation in adversity. In our results, we also find L21 and L27 down expression in leaf color mutants, which are the proteins in chloroplast ribosome. If those two proteins have down-expression, the proteins in chloroplast will not be synthesis. This is the reason for adversity. Finally, we use SWATH to find out the relationship between leaf color mutants and wild types rice successfully.
URI: http://hdl.handle.net/11455/97560
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