臺灣博碩士論文加值系統

根據光合作用機制，植物可分為C3、C4及CAM三個主要類型。由於C4具有CO2濃縮機制，C4植物較C3植物適應於高光、高溫及乾旱下生長而有較高的光合與生長效率，相反的C3植物因光呼吸作用旺盛不能有效固定CO2。水稻是C3植物的一種,無法透過光合作用有效利用CO2將它轉化為碳水化合物與產量。要增加C3作物的光合率與生長有2個可能的途徑：(1)減少光呼吸作用，或(2)表達有效率的C4光合機制。光呼吸作用因Rubisco的特性在C3與C4植物是一致的，不易改變，後者則希望藉由遺傳工程及基因改造將額外的C4固碳途徑導入C3作物，使它有效進行羧合反應濃縮CO2以抑制或減緩光呼吸作用，進而提高光合效率及產能。
近年來，由於分子生物學和基因改造技術快速發展，許多C4光合作用的關鍵基因紛紛從玉米、高梁及莧菜中被分離出來，並重新建構表現於C3作物中。玉米的磷酸烯醇丙酮酸羧化酶(phosphoenolpyruvate carboxylase；PEPC)在C4 植物光合作用的羧合反應中扮演重要的角色，透過轉殖已被表達於水稻中，並有效的提升水稻的光合作用及產量(Ku et al., 1999, Jiao et al., 2001, Ku et al., 2000)。目前作物育種學家也希望能利用傳統的雜交方式將重要的轉殖基因導入優良水稻品種以培育出更優良的品種。所獲得之雜交品種除了含玉米的C4光合特性外，也表現出雜交優勢。
本試驗將表達玉米PEPC的日本稉稻(PC/K)與大陸的秈稻(93-11)雜交，為了提高稔實率同時將雜交而得之F1植株以秋水仙素倍增其染色體(四倍體)。經過篩選獲得高度表達玉米PEPC的轉殖雜交水稻之二倍體與四倍體進行分生與光合生理分析，Southern blot分析確認玉米PEPC基因在轉殖雜交稻基因組中以一個拷貝數存在，northern blot與western immuno-blot也分別檢測到PEPC之mRNA與蛋白質之表達；在溫室條件下栽種分析雜交稻的光合率、生長與增量，結果發現轉殖雜交稻與親本(PC/K及93-11)比較，在飽和光及飽和二氧化碳條件下有較高的光合率，而且對光與二氧化碳的利用效率也比較高。這些結果顯示表達玉米PEPC可以提高水稻的光合效率與生長，為〝C4 rice〞的基因工程奠定了基礎。

According to photosynthetic mechanism, higher plants can be divided into three types, namely C3, C4 and CAM plants. C4 plants exhibit higher photosynthetic efficiency than C3, especially under high light, warm temperature and drought conditions, due to the C4 CO2 concentration mechanism. Rice is a C3 plant and exhibits a wasteful carbon metabolism called photopiration and thus has a lower photosynthetic capacity and productivity. Two approaches can be taken to increase rice photosynthesis and productivity : (1) to reduce photorespiration, or (2) to increase the carboxylation efficiency through the expression of the C4 pathway. The first approach maybe considered difficult as Rubisco from both C3 and C4 plants possess the same property. Several laboratories have attempted to use the second approach to improve C3 crops.
With the rapid development of molecular biology and transgenic technology in recent years, several genes encoding the key C4 photosynthesis enzymes derived from maize, sorghum or amaranth, have been isolated, cloned, and expressed in C3 crops. Phosphoenolpyruvate carboxylase (PEPC) is known to play the key role in C4 photosynthesis by catalyzing the initial fixation of atmospheric CO2 in C4 plants. Maize PEPC gene has been previously overexpressed in transgenic rice of japonica background, which exhibited enhanced photosynthesis and productivity (Ku et al., 1999, Jiao et al., 2001, Ku et al., 2000). It is of great interest to introduce this trait to other rice cultivars via conventional hybridization in order to combine both heterosis and maize C4 photosynthetic trait in the hybrids.
In this study, PEPC transgenic rice derived from a japonica cultivar Kitaake (PC/K) was crossed with an indica cultivar 93-11. Furthermore the chromosomes of the F1 hybrid was doubled by Colchicine. After screening, both diploid tetraploid transgenic hybrids with high PEPC activities were obtained for molecular and physiological study. The integration of the maize gene in the transgenic hybrid rice plants was confirmed by Southern blot analysis and its expression was also examined at RNA level by northern blot analysis and at protein level by western immunoblot analysis. Preliminary photosynthetic physiology study with plants grown in the greenhouse showed that transgenic hybrids exhibit higher photosynthetic rates at saturating light and saturating CO2 conditions. In addition, they also have higher light use efficiency and carboxylation efficiency. The results suggest that maize PEPC can improve rice photosynthesis and growth, laying the ground work for engineering〝C4 rice〞.

目錄(Content)
壹、前言…………………………………………………………… 1
貳、前人研究……………………………………………………… 4
一、植物演化及光合機制之分類………………………………… 4
二、C4 Rice 之研究……………………………………………… 9
三、磷酸烯醇式丙酮酸羧化酶的特性與調控…………………… 23
四、PEPC轉殖水稻……………………………………………… 25
五、PEPC轉殖雜交水稻…………………………………………… 28
六、多倍體對生理生長型態之影響……………………………… 31
參、材料與方法…………………………………………………… 34
第一部分 試驗材料…………………………………………………34
一、玉米PEPC基因轉殖載體與轉殖水稻……………………………34
二、轉殖水稻之雜交……………………………………………… 36
第二部份 試驗步驟…………………………………………………37
一、轉殖雜交水稻之PEPC活性測定……………………………… 37
1-1. 轉殖雜交水稻之發芽…………………………………37
1-2. PEPC酵素活性檢測……………………………………………37
二、轉殖雜交水稻分子生物檢測……………………………………39
2-1. PEPC轉殖雜交水稻分析：DNA level…………………………39
2-2. PEPC轉殖雜交水稻分析：RNA level…………………………45
2-3. PEPC轉殖雜交水稻分析：Protein level………………… 48
三、轉殖雜交水稻之生理分析…………………………………… 51
3-1. 光合作用分析…………………………………………………52
3-2. 葉綠素含量之測定……………………………………………54
四、轉殖雜交水稻農藝性狀分析………………………………… 55
4-1. 水稻分櫱數(tillers)與穗數(panicles)之調查………… 55
4-2. 水稻總穀粒重(total grain weight)之調查…………… 56
肆、結果…………………………………………………………… 57
一、轉殖雜交稻二倍體後代PEPC的表達……………………… 57
二、轉殖雜交稻四倍體後代PEPC的表達……………………… 62
三、轉殖雜交水稻PEPC蛋白質的表達量……………………… 67
四、轉殖雜交稻後代玉米pepc mRNA之表達量……………… 69
五、玉米pepc基因在轉殖雜交稻基因組之偵測……………… 71
六、轉殖雜交稻之光合生理反應……………………………… 73
七、農藝姓狀…………………………………………………… 86
伍、討論…………………………………………………………… 99
一、同質結合轉殖雜交水稻之篩選…………………………… 100
二、轉殖雜交稻之玉米pepc基因導入確認…………………… 100
三、轉殖雜交稻之玉米PEPC的 mRNA與蛋白質表達量…………101
四、F4代轉殖雜交水稻之光合生理分析……………………… 102
五、農藝性狀結果分析………………………………………… 104
陸、綜合討論與未來研究方向…………………………………… 107
柒、參考文獻……………………………………………………… 110
捌、附錄…………………………………………………………… 122
附錄一、水稻發芽培養基配方…………………………………… 122
附錄二、酵素活性測定溶液與配方……………………………… 123
附錄三、SDS-PAGE之溶液及膠體配置………………………… 124
附錄四、西方轉漬法之溶液…………………………………… 126
圖目錄(List of Figure)
圖一、玉米C4 PEPC基因的轉殖載體………………………………35
圖二、PC/K x 93-11轉殖雜交水稻二倍體F2代PEPC酵素活性檢測結果………………………………………………………………………59
圖三、PC/K x 93-11轉殖雜交水稻二倍體F3代PEPC酵素活性檢測結果………………………………………………………………………60
圖四、PC/K x 93-11轉殖雜交水稻二倍體F4代PEPC酵素活性檢測結果………………………………………………………………………61
圖五、PC/K x 93-11轉殖雜交水稻四倍體F2代PEPC酵素活性檢測結果………………………………………………………………………64
圖六、PC/K x 93-11轉殖雜交水稻四倍體F3代PEPC酵素活性檢測結果………………………………………………………………………65
圖七、PC/K x 93-11殖雜交水稻四倍體轉F4代PEPC酵素活性檢測結果………………………………………………………………………66
圖八、西方免疫轉漬法(western immuno-bolt)檢測玉米、Kitaake、93-11、PC/K與轉殖雜交水稻二倍體及四倍體之PEPC蛋白質含量結果………………………………………………………………………68
圖九、北方墨點轉漬法 (northern blot) 檢測轉殖雜交水稻玉米PEPC mRNA的表現量…………………………………………………70
圖十、南方墨點轉漬法(Southern blot)檢測轉殖雜交水稻中玉米pepc基因的插入位置與拷貝數………………………………………72
圖十一、(A)轉殖雜交稻(PC/K x 93-11)二倍體與四倍體及其父本(93-11)及(A)母本(PC/K)與野生型植株(K)光合作用光線反應比較………………………………………………………………………74
圖十二、轉殖雜交水稻及其親本(93-11及PC/K)與野生型植株在光飽和點下之光合率比較…………………………………………………76
圖十三、轉殖雜交水稻及其親本(93-11及PC/K)與野生型植株之光利用效率比較……………………………………………………………78
圖十四、(A)二氧化碳飽和下之光合率D (B)葉肉細胞內二氧化碳濃度(Ci) (C)氣孔導度……………………………………………………81
圖十五、轉殖雜交水稻及其親本與野生型植株之二氧化碳利用率………………………………………………………………………84
圖十六、轉殖雜交水稻及其親本與野生型植株之二氧化碳補償點(CO2 compensation point)比較……………………………………85
圖十七、轉殖雜交稻與親本及野生型植株(93-11, K)在不同生長期之表現型(phenotype)比較………………………………………………89
圖十八、轉殖雜交稻與親本及野生型植株葉綠素含量比較………91
圖十九、轉殖雜交稻與親本及野生型植株農藝性狀比較…………93
圖二十、轉殖雜交稻與親本及野生型植株結穗率比較……………96
圖二十一、轉殖雜交稻與親本及野生型植株收穫指數比較………98
圖二十二、利用ictB、PEPC及PCK同時表達於水稻達成”C4水稻
遺傳工程的最終目標……………………………………109
表目錄(List of Table)
表一、轉殖雜交稻與其親本(93-11及PC/K)及野生型水稻(k)在飽和光強度時(2000�n�慆ole CO2 / �慆ole photon)的光合率比較………………………………………………………………………76
表二、轉殖雜交稻與其親本(93-11及PC/K)及野生型水稻(k)對光利用率比較…………………………………………………………………78
表三、轉殖雜交稻與其親本及野生型植株 (A)在飽和二氧化碳濃度時(2000�n�尳/L)的最大光合率，與(B)葉肉細胞內二氧化碳濃度(�n�尳 /L)及(C)氣孔導度之比較…………………………………………………………………………82
表四、轉殖雜交稻與親本及野生型植株在飽和光強度時(2000�n�慆ole/ m2/s )的二氧化碳利用效率(CE)比較…………………………84
表五、轉殖雜交稻與親本及野生型植株二氧化碳補償點比較………85
表六、轉殖雜交稻與親本及野生型植株葉綠素含量比較……………91
表七、轉殖雜交稻與親本及野生型植株農異性狀比較………………94
表八、不同代之轉殖雜交稻與親本及野生型植株結穗率比較………97
表九、轉殖雜交稻與親本及野生型植株收穫指數比較………………98

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