Analysis of the photosynthetic properties and protein patterns in the chlorosis mutant of oryza sativa L., cv. TNG67

C6是台農67號（TNG67）水稻的黃化突變體，在生長過程中，葉子有明顯黃化現象，因此，本實驗在水稻不同的生長時期分別進行光合速率、Rubisco酵素活性及光合色素含量之測定，並進行蛋白質圖譜分析，比對鑑定其差異之蛋白，以了解C6突變體與TNG67水稻的差異。 C6突變體的葉片大約在分蘗期開始明顯變黃，在分蘗盛期有黃葉與綠葉的差異，而且生長高度也較TNG67水稻矮；抽穗時間較TNG67水稻晚7週，所結的稻穗較TNG67水稻小且輕，每穗的穀粒粒數較少，穀粒亦較不充實。 C6突變體黃葉的淨光合作用速率及Rubisco酵素活性都比綠葉及TNG67水稻低；利用色素色層分析所測得的光合色素含量，黃葉的色素含量比綠葉及TNG67低，綠葉的葉綠素a、b含量與TNG67相近，類胡蘿蔔素含量是TNG67的0.3倍。 萃取C6突變體及TNG67水稻分蘗期及幼穗形成期的葉部總蛋白進行二維電泳分析，比較C6突變體及TNG67水稻的蛋白質圖譜之變化。在分蘗期有15個蛋白質點累積量有明顯的差異，在幼穗形成期有17個蛋白質點累積量有明顯的差異，隨著生長發育累積量有明顯改變的蛋白質點有6個；選取14個蛋白質點進行MALDI-Q-TOF MS分析，並與Mascot及ProteinProspector蛋白質資料庫進行蒐尋比對，鑑定出兩個蛋白點的身分，分別是chloroplastic ATP synthase beta chain為C6突變體黃葉中缺乏的蛋白質點，及chloroplastic superoxide dismutase [Cu-Zn]為C6突變體黃葉中累積量有明顯增加的蛋白質點。 經由生理測定及蛋白質體學的技術推測出可能發生突變之蛋白基因，有助於對水稻突變體的特性有更進一步的了解。The material used in this study is the chlorosis mutant of Oryza sativa L. cv. TNG67 called C6. The leaves of C6 mutant become yellow during growth. This experiment measures the photosynthetic rate, the activity of Rubisco and the contents of photosynthetic pigments of the C6 and TNG67 at different stages. The total leaf proteins were separated by 2-D electrophoresis, analyzed by MALDI-Q-TOF MS and identified the protein identifies in the protein databases. The leaves of C6 mutant became yellow at the 14th day after transplanting, and distinguished into yellow and green leaves at tillering stage. The height of C6 mutant was lower than that of TNG67, and the time of panicle initiation was delayed seven weeks. The panicles of C6 mutant were smaller and lighter than that of TNG67. The seed number of each panicle in C6 mutant was less, and the seeds were not fully filled. The net photosynthetic rate and the activity of Rubisco in the yellow leaves of C6 mutant was lower than those of TNG67. The contents of the photosynthetic pigments separated by paper chromatography were lower in the yellow leaves of C6 than C6 green leaves and TNG67 leaves. The chlorophyll a and b contents of the C6 mutant green leaves were similar to TNG67 leaves, but the carotenoid content was only 0.3 folds of TNG67 leaves. The total proteins extracted from the leaves of the C6 mutant and TNG67 at tillering stage and booting stage was separated by 2-D electrophoresis. Analysis of protein profiles showed that there were 15 protein spots exhibiting different accumulation patterns between C6 mutant and TNG67 at tillering stage, and 17 protein spots at booting stage. There were 6 protein spots exhibiting different accumulation patterns during development. Totally, 14 protein spots were analyzed by MALDI-Q-TOF MS, and sreached the protein identities by Mascot and ProteinProspector programs. Two protein spots identified was chloroplastic ATP synthase beta chain which was missing in C6 mutant, and superoxide dismutase [Cu-Zn] which was accumulated in higher level in C6 mutant. Using the physiological properties and proteomics techniques helps us to predict the positions where the mutations occurred. It is a useful way to characterize the mutants generated by chemical mutagenesis.總目錄： 中文摘要……………………………………………………………………I 英文摘要…………………… ……………………………………………II 總目錄…………………………………………………………………… IV 表目錄………………………………………………………………………V 圖目錄………………………………………………………………………VI 壹、前言……………………………………………………………………1 一、植物黃化之因素………………………………………………………2 二、蛋白質圖譜之應用……………………………………………………4 貳、材料與方法……………………………………………………………7 一、植物材料………………………………………………………………7 二、實驗方法………………………………………………………………8 （一）光合作用的測定……………………………………………………8 （二）Rubisco酵素活性的測定………………………………………….8 （三）植物色素之萃取及色層分析…………………………………….10 （四）穗重……………………………………………………………… 11 （五）葉片總蛋白質之萃取…………………………………………… 11 （六）二維電泳………………………………………………………….12 （七）膠體染色及影像分析…………………………………………….13 （八）in-gel digestion……………………………………………… 14 （九）資料庫的鑑定比對……………………………………………….15 參、結果………………………………………………………………………… 16 （一）C6突變體生長特性之觀察……………………………………….16 （二）C6突變體光合作用之測定……………………………………….16 （三）C6突變體光合色素之分析……………………………………….17 （1）粗萃液之光合色素總量之測定…………………………… 17 （2）以色素層析法分析光合色素含量………………………… 18 （四）C6突變體葉部蛋白圖譜之變化………………………………….19 肆、討論………………………………………………………………… 21 伍、參考文獻…………………………………………………………… 55 表目錄： 表一、水稻TNG67與C6穗重之比較..............................25 表二、水稻TNG67及C6的淨光合作用速率(P)、蒸散作用速率(E)、氣孔 導度(GS)、Rubisco酵素活性之比較......................26 表三、水稻TNG67與C6葉片粗萃取液之光合色素含量…………………27 表四、水稻TNG67與C6葉中的色素經植物色素色層分析所回收得到的色 素含量...............................................28 表五、TNG67與C6水稻分蘗期的葉片蛋白質累積量有差異的蛋白質點.. .....................................................29 表六、TNG67與C6水稻幼穗形成期的葉片蛋白質累積量有差異的蛋白質 點...................................................30 表七、隨著生長發育累積量有改變的蛋白質點……………………… 31 表八、取TNG67及C6黃葉幼穗形成期葉片的蛋白質點進行質譜儀分析， 比對鑑定出來的蛋白質點...............................32 圖目錄： 圖一、TNG67水稻在溫室中的生長發育時期………………………… 7 圖二、攜帶式光合作用儀外觀（LCA3）………………………………8 圖三、分光光度計（SHIMADZU UV-1201）的外貌................9 圖四、一維電泳immobiline strip tray的外觀.................12 圖五、TNG67與C6水稻在幼苗期的生長狀況.....................36 圖六、TNG67與C6水稻在分蘗期的生長狀況.....................37 圖七、TNG67與C6水稻在幼穗形成期的生長狀況.................38 圖八、TNG67與C6水稻在插秧完後第3天到第100天植株高度之比較.39 圖九、TNG67與C6水稻種子的外觀.............................40 圖十、TNG67與C6水稻種子去除穎殼的形態.....................41 圖十一、TNG67及C6水稻在不同的生長時間下葉片粗萃取液中的葉綠素 a、b及類胡蘿蔔素含量比較..........................42 圖十二、取TNG67與C6水稻幼苗期之葉片做植物色素色層分析，光合色 素溶於石油醚的狀況................................43 圖十三、取TNG67與C6水稻幼苗期之葉片做植物色素色層分析，將色素 濃縮後點於濾紙上，置於四氯化碳溶劑中觀察其分離的狀況. ..................................................44 圖十四、取TNG67與C6水稻分蘗期之葉片做植物色素色層分析，將色素 濃縮後點於濾紙上，置於四氯化碳溶劑中觀察其分離的況.. ..................................................45 圖十五、取TNG67與C6綠葉水稻幼穗形成期之葉片做植物色素色層分析， 將色素濃縮後點於濾紙上，置於四氯化碳溶劑中觀察其分離的 狀況..............................................46 圖十六、取TNG67與C6黃葉水稻幼穗形成期之葉片做植物色素色層分析， 將色素濃縮後點於濾紙上，置於四氯化碳溶劑中觀察其分離的 狀況..............................................47 圖十七、不同時期的水稻TNG67與C6的葉綠素a、葉綠素b及類胡蘿蔔素 吸收光譜變化的情形................................48 圖十八、TNG67與C6水稻幼苗期之葉組織利用色素色層分析回收的光合色 素含量之比較…………………………………………………50 圖十九、TNG67與C6水稻分蘗期之葉組織利用色素色層分析回收的光合色 素含量之比較…………………………………………………51 圖二十、TNG67與C6水稻幼穗形成期之葉組織利用色素色層分析回收的光 合色素含量之比較……………………………………………52 圖二十一、TNG67與C6水稻分蘗期葉部總蛋白的二維電泳圖.......53 圖二十二、TNG67與C6水稻幼穗形成期葉部總蛋白的二維電泳圖...5