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

丁在松、趙明、荊玉祥、李良璧及匡廷云 (2007)。玉米 pepc基因過表達對基因水稻光合速率的影響。作物學報33，717-722。
王榮富、張云華、于江龍及焦德茂 (2007)。轉pepc基因水稻耐光抑制和光氧化的特性。安徽農業大學學報34，320-323。
李季航、向珣朝、何立斌及李平 (2005)。水稻亚種間雜交F1光合特徵研究。植物學通報22，432-438。
李霞、焦德茂及戴傳超 (2005)。轉pepc基因水稻對光氧化逆境的影響。作物學報31，408-413．
李霞、焦德茂、劉蔚及周月兰 (2007)。轉植玉米pepc基因水稻農藝性狀及生理特性。江蘇農業學報23，87-92。
林建吏 (2002)。C4光合基因轉殖水稻的氣體交換能力及C4光合酵素性之分析。國立中興大學植物學系碩士論文。
黃三光及曾經洲 (2002)。基因改造作物的優點與潛藏的危機。行政院農業委員會藥物毒物試驗所。
黃郁容 (2008)。〝C4 水稻〞之遺傳工程：表達玉米PCK於水稻葉綠體以提高CO2濃度來促進其光合作用。國立嘉義大學農業生物技術研究所碩士論文。
陳玥妗 (2009)。表達玉米磷酸烯醇丙酮羧化酶以促進蝴蝶蘭之光合作用與生長。國立嘉義大學農業生物技術研究所碩士論文。
楊仕枚 (2007)。藍綠菌CO2濃縮機制促進水稻的光合作用與產量。國立嘉義大學農業生物技術研究所碩士論文。
Adams, K. L., Cronn, R., Percifield R. and Wendel, J. F. (2002) Genes duplication by polyploidy shows unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proc. Natl. Acad. Sci. 100, 4649-4654.

Agarie, S., Miura, A., Sumikura, R., Tsukamoto, S., Nose, A., Arima,
S., Matsuoka, M. and Miyao-Tokutomi, M. (2002) Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plants. Plant Sci. 162, 257-265.

Aoyaki, K. and Bassham, J.A. (1986) Appearance and accumulation of C4 carbon pathway enzymes in developing wheat leaves. Plant Physiol. 80, 334–340.

Asai, N., Nakajima, N., Tamaoki, M., Kamada, H. and Kondo, N. (2000) Role of malate synthesis mediate by phosphoenolpyruvate carboxylase in guard cells in the regulation of stoamal movement. Plant cell Physiol. 41, 10-15.

Bandyopadhyay, A., Data, K., Zhang, J., Yang, W., Raychaudhuri, S., Miyao, M. and Datta, S. K. (2007) Enhance photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize. Plant Sci. 172, 1204-1209.

Badger, M. R. and Price, G. D. (2003) CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J. Expt. Bot. 54, 609-22.

Berner, R. A. (1993) Paleozoic atmospheric CO2: importance of solar radiation and planteEvolution. Science 261, 68-70.

Beerling, D. J., Osborne, C. P. and Chaloner, W. G. (2001) Evolution of leaf-form in land plants linked to atmospheric CO2 decline in the Late Palaeozoic era. Nature 410, 352-4.

Black, C. C. (1973) Photosynthetic carbon fixation in relation to net CO2 uptake. Ann. Rev. Plant Physiol. 24, 253-286.

Borland, A. M. and Taybi, T. (2004) Synchronization of metabolic processes in plants with crassulacean acid metabolism. J. Expt. Bot. 55, 1255-1265

Brown R. H. and Bouton J. H. (1993) Physiology and genetics of interspecific hybrids between photosynthetic types. Ann. Rev. Plant Physiol. Plant Mol. Biol. 44, 435-456.

Cerling, T. E., Ehleringer, J. R. and Harris, J. M. (1998). Carbon dioxide starvation, the development of C4 ecosystems, and mammalian evolution. Phil. Trans. Roy. Soc. London B. Biol. Sci. 353, 159-70.

Chollet, R., Vidal, J. and O'Leary, M. H. (1996) Phosphoenolpyruvate
carboxylase: a ubiquitous, highly regulated enzyme in plants. Ann. Rev.
Plant Physiol. Plant Mol. Biol. 47, 273-298.

Chen X., Zhang, X., Liang, R., Zhang, L., Yang, F. and Cao, M. (2004) Expression of the intact C4 type pepc gene cloned from maize in transgenic winter wheat. Chinese Science Bul. 49, 2137-2143.

Cheng, S., Cao, L., Zhuang, J., Chen, S., Zhan, X., Fan, Y., Zhu, D. and Min, S. (2007) Super hybrid rice breeding in china: achievements and prospects. J. Plant Biol. 49, 805-810.

Cheng, S., Zhuang, J., Fan, Y., Du, J. and Cao, L. (2007) Progress in
research and development on hybrid rice: a super-domesticate in China.
Ann. Bot. 100, 959-66.

Cotelle, V., Pierre J. and Vavasseur, A. (1999) Potential strong regulation of guard cell phosphoenolpyruvate carboxylase through phosphorylation. J. Expt. Bot. 50, 777-783

Cua, L. D. (1951) Fertile teraploids of japonica x indica in rice. The Japan Academy 27, 43-48.

Drincovich, M. F., Casati, P., Andreo, C. S., Chessin, S. J., Franceschi, V. R., Edwards, G. E. and Ku, M. S. B. (1998) Evolution of C4 photosynthesis in Flaveria species. Plant Physiol. 117, 733-744.

Endo, T., Mihara, Y., Furumoto, T., Matsumura, H., Kai, Y. and Izui, K. (2008). Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity. J. Exp. Bot. 59, 1811-8.

Evenson, R. E. and Gollin, D. (2003) Assessing the impact of the green revolution, 1960 to 2000. Science 300, 758-62.

Fukayama, H., Tsuchida, H., Agarie, S., Nomura, M., Onodera, H., Ono, K., Lee, B., Hirose, S., Toki, S., Ku, M. S. B., Makino, A., Matsuoka, M. and Miyao, M. (2001) Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant physiol. 127, 1136-1146.

Furumoto, T., Hata, S. and Izui, K. (1999) cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme. Plant Mol. Biol. 41, 301–311.

Gehlen, J., Panstruga, R., Smets, H., Merkelbach, S., Kleines, M., Porsch, P., Fladung, M., Becker, I., Rademacher, T., Hausler, R. E. and Hirsch, H. J. (1996) Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum. Plant
Mol. Biol. 32, 831-48.

Gupta, S. K., Ku, M. S. B., Lin, J. H., Zhang, D. and Edward, G. E. (1994) Light/dark modulation of photoenolpyruvate carboxylase in C3 and C4 species. Photosyn. Res. 42,133-143
Griffiths, S., Sharp, R., Foote, T. N., Bertin, I., Wanous, M., Reader, S., Colas, I. and Moore, G. (2006) Molecular characterization of Ph1 as a major chromosome pairing locus in polyploidy wheat. Nature 439, 749-752.

Hatch, M. D. (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim. Biophys. Acta. 895, 81-106.

Häusler, R.E., Hirsch, H.J., Kreuzaler, F. and Peterhansel, C. (2002) Overexpression of C4-cycle enzymes in transgenic C3 plants: a biotechnological approach to improve C3-photosynthesis. J. Exp. Bot. 53, 591-607.

Hudspeth, R. L., Glaekin , C. A., Bonner, J. and Grula, J. W. (1986) Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase: expression of different genefamily members in leaves and roots. Pro. Natl. acad. Sci. USA 83, 2884-2888.

Hudspeth, R. L., Grula, J. W., Dai, Z., Edwards, G. E. and Ku, M. S. B. (1992) Expression of maize phosphoenolpyruvate carboxylase in transgenic tobacco : effects on biochemistry and physiology. Plant Physiol. 98, 458-464.

Hudspeth, R. L., Grula, J. W., Dai, Z., Edwards, G. E. and Ku, M. S. B. (1992) Expression of maize phosphoenolpyruvate carboxylase in transgenic tobacco. Plant Physiol. 98, 458-464.

International rice genome sequencing project (2005) The map-based sequence of the rice genome. Nature 436, 793-800.

Ishmaru, K., Ohkawa, Y., Ishige, T., Tobias, D. J. and Ohsugi, R. (1998) Elevated pyruvate, orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene. Plant Physiol. 103, 340-346.

Ji, B. H., Tan, H. H., Zhou, R., Jiao, D. M. and Shen, Y. G. (2005) Promotive effect of low concentration of NaHSO3 on photophosphorylation and photosynthesis in phosphoenolpyruvate carboxylase transgenic rice leaves. J. Integrative Plant Biol. 48, 178-186

Jiao, D., Huang, X., Li, X., Chi, W., Kuang, T., Zhang, Q., Ku, M. S. B. and Cho, D. (2002) Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes. Photosyn. Res. 72, 85-93.

Jiao, D., Kuang, T., Li X., Ge Q., Huang, X., Hao, N. and Bai, K. (2003) Physiological characteristics of the primitive CO2 aonaentrating mechanism in PEPC transgenic rice. Science in China. 46, 438-446.

Jordan, D. B. and Ogren, W. L. (1984) The CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase dependence on ribulose bisphosphate concentration, pH and temperature. Planta 161, 308-313

Kebeish, R., Niessen, M., Thiruveedhi ,K., Bari, R., Hirsch, H. J., Rosenkranz, R., Stabler, N., Schonfeld, B., Kreuzaler, F. and Peterhansel, C. (2007) Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Nature
Biotech. 25, 593-9.

Poetsh, W., Hermans, J. and Westhoff, P. (1991) Multiple cDNA of phosphoenolpyruvate carboxylase in C4 dicot Flaveria trineriva. FEBS Lett. 292, 133-136.

Keys, A. J., Bird, I. F., Cornelius, M.J., Lea, P.J., Wallsgrove, R. M. and Miflin, B. J. (1978) Photorespiratory nitrogen cycle. Nature 275, 741-743.

Khush, G. S. (2001) Green revolution: the way forward. Nature Rev. Genet. 2, 815-22.

Ku, S. B. and Edwards, G. E. (1977) Oxygen inhibition of photosynthesis - temperature dependence and relation to O2/CO2 solubility ratio. Plant Physiol. 59, 986-990.

Ku, S. B., Shieh, Y. J., Reger, B. J. and Black, C. C. (1981) Photosynthetic characteristics of portulaca grandiflora, a succulent C4 dicot. Plant Physiol. 68, 1073-1080.

Ku, M. S. B., Agarie, S., Nomura, M., Fukayama, H., Tsuchida, H., Ono, K., Hirose, S., Toki, S., Miyao, M. and Matsuoka, M. (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nature Biotech. 17, 76-80.

Ku, M. S. B., Cho, D., Li, X., Jiao, D. M., Pinto, M., Miyao, M. and Matsuoka, M. (2001) Introduction of genes encoding C4 photosynthesis enzymes into rice plants: physiological consequences. In: Rice Biotechnology: Improving Yield, Stress Tolerance and Grain Quality.
Novaritis Foundation. 236, 100-116. Chichester.

Ku, M. S. B., Kano-Murakami, Y. and Matsuoka, M. (1996). Evolution and expression of C4 photosynthesis genes. Plant Physiol. 111, 949-57.

Kubo, T. and Yoshimura, A. (2002). Genetic basis of hybrid breakdown in a Japonica/Indica cross of rice, Oryza sativa L. Theor. Appl. Genet. 105, 906-911

Leegood, R. C., von Caemmerer S. and Osmond, C. B. (1996) Metabolite transport and photosynthetic regulation in C4 and CAM plants. In DT Dennis, DH Turpin, DD Layzell, DK Lefebvre, eds, Plant Metabolism. Longman, London, pp 341–369.

Leegood, R. C. (2002) C4 photosynthesis: principles of CO2 concentration and prospects for its introduction into C3 plants. J. Expt. Bot. 53, 581-590.

Lepiniec, L., Keryer, E., Philippe, H., Gadal, P. and Cretin, C. (1993) Sorghum phosphoenolpyruvate carboxylase gene family: structure, regulation and molecular evolution. Plant Mol. Biol. 21, 487-502.

Li, J., Xiang, X., Zhou, H., He, L., Zhang, K., and Li, P. (2006) Photosynthetic characteristics and heterosis in transgenic hybrid rice with maize phosphoenolpyruvate carboxylase (pepc) gene. Rice Sci. 13, 185-192.

Masterson, J. (1994). Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science 264, 421-424.

Magnin, N. C., Cooley, B. A., Reiskind, J. B. and Bowes, J. (1997) Regulation and localization of key enzymes during the induction of Kranz-less, C4-type photosynthesis in Hydrilla verticillata. Plant Physiol. 115, 1681-1689.

Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K. and Kai, Y. (2002). Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure 10, 1721-30

Matsuoka, M. and Minami, E. (1989). Complete structure of the gene for phosphoenolpyruvate carboxylase from maize. Eur. J. Biochem. 181, 593-8.

Matsuoka, M. and Sanada, Y. (1991). Expression of photosynthetic genes from the C4 plant, maize, in tobacco. Mol. Gen. Genet. 225, 411-9.

Miyagawa, Y. Tamoi, M., and Shigeoka, S. (2001) Overexpression of cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth. Nature Biotech. 19, 965-969.

Matsuoka, M., Furbank, R. T., Fukayama, H. and Miyao, M. (2001) Molecular engineering of C4 photosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 297-314.

Moor, P. D. (1982) Evolution of photosynthetic pathways in plowering plants. Nature 295, 647-648.
Nakamura, A. (2004) Breeding and prevalence of japonica hybrid rice. Plant Physiol. 80, 334-340.

Narasimman, R. and Rangasamy, S. R. (1993) Comparison of fertility between the F1, F2 and another derived lines in the crosses of indica/ japonica and japonica/ indica in rice (Oryza sativa L.). Euphutyca 66, 19-25.

Normile, D. (2006). Agricultural research. Consortium aims to supercharge rice photosynthesis. Science 313, 423.

Ogren, W. L. (1984) Photorespiration: pathways, regulation and modification. Ann. Rev. Plant Physiol. 35, 415-442.

Osborn, T. C., Pires, J. C., Birchler, J. A., Auger, D. L., Chen, Z. J., Lee, H. S., Comai, L., Madlung, A., Doerge, R. W., Colot, V. and Martienssen, R. A. (2003). Understanding mechanisms of novel gene expression in polyploids. Trends Genet. 19, 141-7.

Osmond, C. B. (1978) Crassulacean acid metabolism: a curiosity in context . Annu. Rev. Plant Physiol. 29, 379-414.

Raines, C. A. (2006) Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle. Plant, Cell and Environ. 29, 331-339.

Rashid, H., Yokoi, S., Toriyama, K. and Hinata, K. (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Report. 15, 727–730.

Reiskind, J. B., Madsen, T.V., van Ginkel L.C. and Bowes, G. (1997) Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentration mechanism in Hydrilla, a submerged monocot. Plant Cell Environ. 20, 211-220.

Song, Z. J., Du, C. Q., Dai, B. C., Chen, D. L., Chen, J. G. and Cai, D. T. (2007) Studies on the growth habits and characteristics of two polyploidy indica- japonica hybrid rice with powerful heterosis. Agricultural Science in China 3, 265-274.

Surridge, C. (2002). Agricultural biotech: the rice squad. Nature 416, 576-578.

Suzuki, S., Murai, N., Burnell, J. N. and Arai, M. (2000) Change in photosynthetic carbon flow to transgenic rice plant that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides. Plant Physiol. 124, 163-172 .

Suzuki, S., Murai, N., Kasaoka, K., Hiyoshi, T., Imaseki, H., Burnell, J. N. and Arai, M. (2006) Carbon metabolism in transgenic rice plants that express phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase. Plant Sci. 170, 1010-1019.

Takeuchi, Y., Akagi, H., Kamasawa, N., Osumi, M. and Honda, H. (2000) Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme. Planta 211, 265-274.

Taniguchi, Y., Ohkawa, H., Masumoto, C., Fukuda, T., Tamai, T., Lee, K., Sudoh, S., Tsuchida, H., Sasaki, H., Fukayama, H. and Miyao, M. (2007) Overproduction of C4 photosynthetic enzyme in transgenic rice plants: an approach in introduce the C4-like photosynthetic pathway into rice. J. Expt. Bot. 59, 1799-1809.

Toh, H., Kawamura, T. and Izui, K. (1994) Molecular evolution of phosphoeno/ pyruvate carboxylase. Plant Cell Environ. 17, 31 -43

Trevanion, S. J., Brooks, A. L. and Leegood, R. C. (1995) Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons of Cucurbita pepo L. Planta 196, 653–658.

Tsuchida, H., Sasaki, H., Fukayama, H. and Miyao, M. (2008)
Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice. J. Exp. Bot. 59, 1799-809.

Voznesenskaya, E.V., Franceschi, V. R., Kiirats, O., Freitag, H. and Edwards, E. (2001) Kranz anatomy is not essential for terrestrial C4 plant photosynthesis. Nature 414, 543-546.

Wang, D. Z., Jiao, D.M., Wu, S., Li, L.L., Chi, W., Wang, S. H., Li, C. Q., Luo, T. C. and Wang X. F. (2002) Breeding for parents of hybrid rice with maize pepc gene. Agricultural Science in China 1, 1067-1073.

Westhoff, P. and Gowik, U. (2004) Evolution of C4 Phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria. Ann. Bot. 93, 13-23.

Whitney, S. M., Baldet, P., Hudson,G. S. and Andrews, T. J. (2001)
Form I Rubisco form non-green-algae are expressed abundantly but not assembled in tobacco chloroplast. Plant J. 26, 535-547.

Wong, C. K. (1989) A new approach to chromosome doubling for haploid rice plants. Theor. Appl. Genet. 77, 149-151.

Yu, J., Hu, S., Wang, J., Wong, G. K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X., Cao, M., Liu, J., Sun, J., Tang, J., Chen, Y., Huang, X., Lin, W., Ye, C., Tong, W., Cong, L., Geng, J., Han, Y., Li, L., Li, W., Hu, G., Li, J., Liu, Z., Qi, Q., Li, T., Wang, X., Lu, H., Wu, T., Zhu, M., Ni, P., Han, H., Dong, W., Ren, X., Feng, X., Cui, P., Li, X., Wang, H., Xu, X., Zhai, W., Xu, Z., Zhang, J., He, S., Xu, J., Zhang, K., Zheng, X., Dong, J., Zeng, W., Tao, L., Ye, J., Tan, J., Chen, X., He, J., Liu, D., Tian, W., Tian, C., Xia, H., Bao, Q., Li, G., Gao, H., Cao, T., Zhao, W., Li, P., Chen, W., Zhang, Y., Hu, J., Liu, S., Yang, J., Zhang, G., Xiong, Y., Li, Z., Mao, L., Zhou, C., Zhu, Z., Chen, R., Hao, B., Zheng, W., Chen, S., Guo, W., Tao, M., Zhu, L., Yuan, L. and Yang, H. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79-92

Yanagisawa, M. (2007) production and physiological characteristics of transgenic rice simultaneously overexpressing PEP carboxylase, pyruvate, Pi dikinase and NADP-malic enzyme. National Chiayi University master thesis.

Zhang Q., Jiao D., Ling L.L, Zhang Y. H. and Huang X. Q. (2005) Studt of the protective effects in PEPC transgenic rice. Agricultural Science in China 2, 94-100

International food policy research institute; IFPRI (2002) Green revolution. http://www.ifpri.org/