Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92228
標題: SWATH-based comparative proteomics analysis of mutant in Oryza stativa
利用 SWATH 質譜定量技術於分析影響水稻突變株之蛋白質體研究
作者: 簡筱楨
Siao-Zhen Chien
關鍵字: 蛋白質體;葉片顏色;水稻;非標定;proteomics;color of leaf;Oryza stativa;label free
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摘要: 
Photosynthesis is a process to convert light energy into chemical energy, which is stored in carbohydrate molecules in plant leaf. One of important biomolecules for photosynthesis is chlorophyll. Chlorophyll a and chlorophyll b are key members of the chlorophyll, the chlorophyll a/b ratio has an enormous impact on leaf color, and the ratio in general plants is 3. In this study, the abnormal leaf colors, yellow or white, were founded in the mutants of Oryza sativa, SA0405, SA0407 and SA0408, although abnormal leaves, they could still survive and inherited to next generation. Proteins are the final pigment of central dogma, they can strongly describe the phenotype of organism. Protein quantity analysis distribute label and label free. Label free is cheaper, fast and simple than label..Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is novel for label free. Therefore, ultra-performance liquid chromatography mass spectrometry (LC-MS) coupled with SWATH of rice analysis were applied on the identification and quantification of the difference proteins, and then it analyzed with gene ontology analysis to find out the biological pathways. We compared TNG67 with SA0405, SA0407 and SA0408 at tillering and ripening stage. The tillering results showed that total of 324, 376, 373 proteins were quantified successfully in SA0405, SA0407 and SA0408, respectively. The ripening results showed that total of 396, 404, 219 proteins were quantified successfully in SA0405, SA0407 and SA0408, respectively. The SA0407 agronomic traits is similar to SA0408. Therefore to investigate SA0407 and SA0408. According to these results, we found that 7-hydroxymethyl chlorophyll a reductase (HCAR) may lead to leaf color abnormal in SA0407 and SA0408 of tillering and ripening. In addition, the down-regulated expression of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MEP cytidylyltransferase) in SA0408 of tillering and ripening might affect the growth of chloroplast. Finally, the rice proteomic by SWATH analysis platform is successful to understand affect leaf color and chloroplast development of rice mutant In future, the development marker genes of three novel proteins can identify chloroplast development stage and help us to understand the mechanism between photosynthesis and rice yield.

光合作用將光能轉換成化學能,轉換成碳水化合物並儲存於葉片,其中葉綠素扮演重要的角色。葉綠素有葉綠素a (chlorophyll a) 與葉綠素b (chlorophyll b),Chl a/Chl b代表葉片顏色,正常比值為3。SA0405、SA0407 和 SA0408 為經疊氮化鈉誘變之白色或黃色水稻葉色突變株,雖然外在性狀與正常植株 TNG67 明顯不同,但仍舊可以完成整個生長週期,將此性狀傳至下一代。蛋白質位於中心法則末端之角色,最能解釋生物體之外表性狀之功能性。質譜定量分析分為標定以及非標定分析兩種,而非標定定量技術較標定定量技術便宜、快速且簡單,目前以非標定定量技術之 SWATH (Sequential window acquisition of all theoretical fragment ion spectra) 分析平台最為新穎。因此,本實驗為利用超高液相層析儀搭配質譜儀,建立水稻 SWATH 分析平台,進行水稻之蛋白質身分鑑定及定量,並找出具有兩倍以上差異之蛋白質。於此,將 TNG67 各別與 SA0405、SA0407 和 SA0408 於最高分蘗期及成熟期樣品進行比較。最高分蘗期之 SA0405、SA0407、SA0408 各別與 TNG67 比較,共同挑選出 324、376、373 個可定量蛋白質;成熟期之 SA0405、SA0407、SA0408 各別與 TNG67 比較,共同挑選出396、414、219個可定量蛋白質。其中 SA0407 和 SA0408 於農藝性狀較相似,因此先探討 SA0407 和 SA0408。根據定量結果於SA0407,MEP (methylerythritol phosphate) 路徑沒有顯著下降,SA0407 和 SA0408 下游葉綠素合成 7-hydroxymethyl chlorophyll a reductase (HCAR) 顯著下降,進而影響 Chl a和 Chl b之比例,導致外觀性狀與 TNG67 有所不同;此外 SA0408 最高分蘗期及成熟期 MEP路徑之 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MEP cytidyltransferase) 皆有顯著下降,可能經由突變,使得此酵素合成異常,導致葉綠體發育受阻。綜合以上結果,本實驗成功透過水稻蛋白質體之 SWATH 分析平台,了解水稻突變株於葉片顏色與葉綠體發育之影響,並期望於未來利用上述之蛋白質發展標記基因鑑定葉綠體之發育,以及協助了解光合作用和水稻產量之關係。
URI: http://hdl.handle.net/11455/92228
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