( 您好!臺灣時間:2021/03/05 03:52
字體大小: 字級放大   字級縮小   預設字形  


研究生(外文):Chin-Fu Cheng
論文名稱(外文):Constitutive Expression of a Plant Ferredoxin-like Protein (pflp) in Transgenic Rice Enhances Capacity of Photosynthetic Carbon Assimilation
指導教授(外文):Mang-Jye Ger
外文關鍵詞:crop yieldphotosynthesisplant ferredoxin – like protein (PFLP)
  • 被引用被引用:0
  • 點閱點閱:266
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:18
  • 收藏至我的研究室書目清單書目收藏:0
光合作用主要是植物吸收光能後將二氧化碳固定成各種醣類,例如: 作為能量利用的葡萄糖與果糖,作為運輸用的蔗糖與儲存用的澱粉。第一型硫鐵蛋白廣泛分布在植物綠色組織中,其主要在電子傳遞鏈中扮演傳遞電子的角色。從甜椒萃取之植物硫鐵蛋白(Plant Ferredoxin-Like Protein,PFLP)經蛋白質序列比對後發現,其序列與阿拉伯芥、蕃茄、水稻及菠菜之第一型硫鐵蛋白有高度同源性。先前研究發現pflp轉殖植物在病原菌感染後可大幅產生過氧化物(Reactive oxygen species, ROS),進而誘導過敏反應產生。由於PFLP屬於光合作用第一型硫鐵蛋白,此暗示過表現植物硫鐵蛋白於轉殖植物中可能經由加強光合作用與糖類代謝來增加抵抗病原菌的能力。為了探討上述機制的可能性,花椰菜鑲嵌病毒35S啟動子(CaMV 35S promoter)被用來大量表現PFLP蛋白於水稻中,並且分析轉殖水稻的光合作用效率。經由反轉錄聚合酶鏈式反應及西方墨點法確認pflp轉基因以成功表現並累積PFLP蛋白於轉殖水稻中。實驗結果顯示轉殖水稻具有較高的電子傳遞能力與氣體交換速率。在醣類含量方面,轉殖水稻均具有高含量的果糖,葡萄糖,蔗糖與澱粉。而轉殖水稻中醣類合成相關基因轉錄活性大多高於野生型水稻2至4倍。上述結果證實轉殖水稻含有較高之醣類累積是導因於其具有較高電子傳遞效率、較高氣體交換速率與較強醣類合成相關基因表現。在產量分析方面,轉殖水稻無論是在單株實粒重、單株分穗數或種子內澱粉含量均高於野生種水稻。此外,以病原菌接種植株後,偵測氣體交換速率變化,發現轉殖株在此期間可維持氣體交換速率;而野生型之氣體交換速率則明顯的下降。綜合上述結果,本篇論文指出於水稻中持續表現pflp可增強光合作用碳固定效率,並進而提升水稻產量。
Photosynthesis is the major metabolic pathway that converts carbon dioxide (CO2) into organic compounds, such as fructose, glucose, sucrose, and starch in plant. Ferredoxin-I (Fd-I) protein, known as an electron carrier in photosynthesis, generally exists in green tissue. The protein sequence of the plant ferredoxin – like protein (PFLP), isolated from sweet peppers, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Previous studies reported that constitutive expression of pflp in transgenic plants exhibit host resistance to virulent bacterial pathogens. The disease resistance of transgenic plants result from the induction of reactive oxygen species (ROS) generation. It is possible that PFLP could enhance photosynthetic efficiency. In order to assess this possibility, pflp transgenic rice was generated and studied their capacity of photosynthetic carbon assimilation. In this report, two transgenic lines of rice (pflp-1 and pflp-2) showed that the pflp transgenic rice exhibit highly photosynthesis efficiency. As comparing the photosynthetic gas exchange rate, the pflp-1 and the pflp-2, respectively, exhibited 1.3- and 1.2-fold higher than the wild type (WT). The capacity of photosynthetic electron transport of two transgenic lines are significantly higher than WT. Additionally, the fructose, glucose, sucrose and starch contents in the pflp-1 and pflp-2 transgenic lines were higher than those of the WT. The transcript activity of carbohydrate metabolism related genes in transgenic lines were higher than those that of WT. In crop yield, on grain weight per plant the pflp-1 and the pflp-2 showed 1.7- and 1.3-fold higher than WT. As for the number of panicles per plant, the pflp-1 and the pflp-2 are increased 1.4- and 1.5-fold higher than the WT. In addition, we demonstrated that the transgenic rice maintain normal photosynthetic gas exchange rate on the period of pathogen infected. Taken altogether; we demonstrate that constitutive expression of pflp in transgenic rice could enhance capacity of photosynthetic carbon assimilation and increase crop yield.
Table of contents
List of figures
Chinese abstract
English abstract
Chapter I Introduction
Chapter II Materials and methods
2.1 Plant materials and growth condition
2.2 Genomic DNA extraction and Genomic DNA PCR2.3 RNA extraction, Reverse transcription PCR and Quantitative reverse transcription
2.4 Determination the content of chlorophyll, protein and western blot analysis
2.5 Gas exchange rate and chlorophyll fluorescence measurement
2.6 Soluble sugars and starch contents assay
2.7 Crop yield assay
2.8 Bacterial blight inoculation
2.9 Statistical
Chapter III Results
3.1 Constitutive expression of pflp in transgenic rice
3.2 PFLP can increase the capacity of photosynthetic electron transport
3.3 PFLP can enhance rate of photosynthetic gas exchange rate in pflp transgenic lines

3.4 Highly contents of soluble sugar and starch accumulate in transgenic rice results from relative high expression pattern of carbohydrate metabolism related gene3.5 Analysis of crop yield in pflp transgenic lines
3.6 pflp transgenic lines maintain photosynthetic gas exchange rate after a bacterial pathogen infection
Chapter IV Discussion
Chapter V Reference
Chapter VI Supplemental table
Akihiro T, Mizuno K, Fujimura T (2005) Gene expression of ADP-glucose pyrophosphorylase and starch
contents in rice cultured cells are cooperatively regulated by sucrose and ABA. Plant Cell Physiol 46: 937–946
Arnon DI (1971) The light reactions of photosynthesis. Proc Natl Acad Sci 68: 2883-2892
Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of
excess photons. Annu Rev Plant Physiol Plant Mol Biol 50: 601–639
Baker NR (2008) Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annu Rev Plant Biol
59: 89–113
Balmer Y, Vensel WH, Cai N, ManieriW, Schu¨rmann P, Hurkman WJ, Buchanan BB (2006) A
complete ferredoxin-thioredoxin system regulates fundamental processes in amyloplasts. Proc Natl Acad Sci 103: 2988–2993
Baroli I, Price GD, Badger MR, Caemmerer S (2008) The contribution of photosynthesis to the red light
response of stomatal conductance. Plant Physiol 146: 737–747
Biemelt S, Sonnewald U (2006) Plant–microbe interactions to probe regulation of plant carbon metabolism.
Journal of Plant Physiol 163: 307-318
Bonfig KB, Schreiber U, Gabler A, Roitsch T, Berger S (2006) Infection with virulent and avirulent P.
syringae strains differentially affects photosynthesis and sink metabolism in arabidopsis leaf. Planta 225: 1–12
Brettel K, Leibl W (2001) Electron transfer in photosystem I. Biochim.Biophys. Acta. 1507:100–114
Bukhov N, Carpentier R (2004) Alternative Photosystem I-driven electron transport routes: mechanisms
and functions. Photosynth Res 82: 17–33
Chan YL, Lin KH, Sanjaya, Liao LJ, Chen WH, Chan MT (2005) Gene stacking in Phalaenopsis orchid
enhances dual tolerance to pathogen attack. Transgenic Res14:279–288
Chen WH , Tseng YC , Liu YC , Chuo CM , Chen PT, Tseng KM, Yeh YC , Ger MJ , Wang HL
(2008) Cool-night temperature induces spike emergence and affectsphotosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite. Plant Cell Rep 27: 1667-1675
Chida H, Nakazawa A, Akazaki H, Hirano T, Suruga K, Ogawa M, Satoh T, Kadokura K, Yamada S, Hakamata W, Isobe K, Ito T, Ishii R, Nishio T, Sonoike K, Oku T (2007) Expression of the algal
cytochrome c6 gene in arabidopsis. Enhances photosynthesis and growth. Plant Cell Physiol. 48:948–95
Cho Jl, Ryoo N, Eom JS, Lee DW, Kim HB, Jeong SW, Lee YH, Kwon YK, Cho MH, Bhoo SH, Hahn TR, Park YI, Hwang I, Sheen J, Jeon JS (2009) Role of the rice hexokinases osHXK5 and osHXK6 as
glucose sensors Plant Physiol 149: 745–759
Dayakar BV, Lin HJ, Chen CH, Ger,MJ ,Lee BH, Pai CH, Chow D, Huang HE, Hwang SY, Chung MC , Feng TY (2003) Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying
harpinpss-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS). Plant Molecular Biol 51: 913–924
Dunford RP, Durrant MC, Catley MA, Dyer T (1998) Location of the redox-active cysteines in
chloroplast sedoheptulose-1,7-bisphosphatase indicates that its allosteric regulation is similar but not
Identical to that of fructose-1,6-bisphosphatase. Photosynth Res 58: 221–230
Ehness R, Ecker M, Godt DE, and Roitsch T (1997) Glucose and stress independently regulate source and
sink metabolism and defense mechanisms via signal transduction pathways involving protein phosphorylation. The Plant Cell 9: 1825-1841
Farquhar GD, Caemmerer SV, Berry JA (1980) A Biochemical Model of Photosynthetic CO2
assimilation in leaf of C3 Species. Planta 149:78-90
Farquhar GD, Sharkey TD (1982) Stomatal conductance and photosynthesis. Ann Rev of Plant Physiol
33: 317–345
Feng L, Wang K , Li Y, Tan Y, Kong J , Li H, Li YS, Zhu Y (2007) Overexpression of SBPase enhances
photosynthesis against high temperature stress in transgenic rice plants. Plant Cell Rep 26: 1635–1646
Fu Y, Ballicora MA, Leykam JF, Preiss J (1998) Mechanism of reductive activation of potato tuber
ADP-glucose pyrophosphorylase. J Biol Chem 273: 25045–25052
Geigenberger P, Kolbe A, Tiessen A (2005) Redox regulation of carbon storage and partitioning in
response to light and sugars. J Exp Bot 56: 1469–1479
Gibson SI (2000) Plant sugar-response pathways Part of a Complex Regulatory Web. Plant Physiol
Hanke GT, Hase T (2008) Variable photosynthetic roles of two leaf-type ferredoxins in arabidopsis, as
revealed by RNA interference. Photochem Photobiol 84: 1302–1309
Holtgrefe S, Bader KP, Horton P, Scheibe R, Schaewen A ,Backhause JE (2003) Decreased content of
leaf ferredoxin changes electron distribution and limits photosynthesis in transgenic potato plants. Plant Physiol 133:1768–1778
Huang HE, Ger MJ, Yip MK, ChenCY ,Pandey AK, Feng TY (2004) A hypersensitive response was
induced by virulent bacteria in transgenic tobacco plants overexpressing a plant ferredoxin-like protein (PFLP). Mol Plant Pathol. 64: 103–110
Ishimaru K (2003) Identification of a locus increasing rice yield and physiological analysis of its function.
Plant Physiol 133:1083–1090
Jacquot JP, Lopez JJ, Miginiac MM, Lemaire S, Cherfils J, Chueca A, Lopez GJ (1997) Cysteine-153
is required for redox regulation of pea chloroplast fructose-1,6-bisphosphatase. FEBS Lett. 401:
Joliot P, Joliot A (2006) Cyclic electron flow in C3 plants. Biochim et Biophys Acta. 1757: 362–368
Joliot P, Joliot A (2002) Cyclic electron transfer in plant leaf. Proc Natl Acad Sci 99: 10209–10214
Kern J, Renger G (2007) Photosystem II: Structure and mechanism of the water:plastoquinone
oxidoreductase. Photosynth Res 94:183–202
Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant
development. Cur Opin in Plant Biol 7:235–246
Lawson T (2009) Guard cell photosynthesis and stomatal function. New Phytologist 181: 13–34
Lee SK, Hwang SK, Han M, Eom JS, Kang HG, Han Y, Choi SB, Cho MH, Bhoo SH, An G, Hahn TR, Okita TW, Jeon JS (2007) Identification of the ADP-glucose pyrophosphorylase isoforms essential for
starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.) Plant Mol Biol 65:531–546
Lee SK, Jeon JS , Bornke F, Voll L, Cho JI, Goh CH, Jeong SW, Park YI, Kim SJ, Choi SB, Miyao A,
Hirochika H, An G, Cho MH, Bhoo SH, Sonnewald U, Hahn TR (2008) Loss of cytosolic
fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth
retardations in rice (Oryza sativa). Plant, Cell and Environment 31:1851–1863
Lefebvre S, Lawson T, Zakhleniuk OV, Lloyd JC, Raines CA (2005) Increased
sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and
growth from an early stage in development. Plant Physiol 138: 451–460
Li X, Xing J, Gianfagna TJ, Janes HW (2002) Sucrose regulation of ADP-glucose pyrophosphorylase
subunit genes transcript levels in leaves and fruits. Plant Sci 162: 239–244
Liau CH, Lu JC, Prasad V, Hsiao HH, You SJ, Lee JT, Yang NS, Huang HE, Feng TY, Chen WH Chan MT (2003) The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot
disease in Oncidium orchid. Transgenic Res 12: 329–336
Lichtenthaler HK (1987) Chlorophylls and cartenoids. In methods in enzymology Academic Press Inc.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative
PCR and the 2∆∆CT method. Methods 25: 402–408
Lunn JE and MacRae E (2003) New complexities in the synthesis of sucrose. Curr Opin Plant Biol.6:
Miyagawa Y, Tamoi M, Shigeoka S (2001) Overexpression of acyanobacterial fructose-1,
6-/sedohetulose-1, 7-bisphosphatase in tobacco enhances photosynthesis and growth. Nat
Biotechnol 19:965–969
Miyake C, Yokota A (2000) Determination of the rate of photoreduction of O2 in the water–water cycle in
watermelon leaves and enhancement of the rate by limitation of photosynthesis. Plant Cell Physiol 41: 335–343
Munekage Y, Hashimoto M, Miyake C, Tomizawa K, Endo T, Tasaka M, Shikanai T (2004)
Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 429: 579–582
Nagai YS, Sakulsingharoj C , Edwards GE , Satoh H, Greene TW ,Blakeslee B ,Okita TW (2009)
Control of starch synthesis in cereals: metabolite analysis of transgenic rice expressing an up-regulated cytoplasmic ADP-glucose pyrophosphorylase in developing seeds. Plant Cell Physiol 50: 635–643
Noguchi K, Yoshida K (2008) Interaction between photosynthesis and respiration in illuminated leaves.
Mitochondrion 8: 87–99
Ort DR, Baker NR (2002) A photoprotective role for O(2) as an alternative electron sink in
photosynthesis? Curr Opin Plant Biol 5: 193–198
Orzechowski S (2008) Starch metabolism in leaves Acta Biochimica Polonica 55: 435–445
Reynolds M, Foulkes MJ, Slafer GA, Berry P, Parry MAJ, Snape JW, Angus WJ (2009) Raising yield
potential in wheat. J Exp Bot 60: 1899–1918
Rumeau D, Peltier G, Cournac L (2007) Chlororespiration and cyclic electron flow around PSI during
photosynthesis and plant stress response. Plant, Cell and Environment 30: 1041–1051
Scafaro AP, Haynes PA, Atwell BJ (2010) Physiological and molecular changes in Oryza meridionalis
Ng., a heat-tolerant species of wild rice. J Exp Bot 61: 191–202
Scholes JD, Lee PJ, Horton P, Lewis DH (1994) Invertase understanding changes in the photosynthetic
and carbohydrate metabolism of barley leaves infected with powdery mildew. New Phytologist 126:213–222.
Schurmann P, Buchanan BB (2008) The ferredoxin/thioredoxin system of oxygenic photosynthesis.
Antioxid Redox Signal. 10: 1236-1274
Sun J, Gibson KM, Kiirats O, Okita TW, Edwards GE (2002) Interactions of nitrate and CO2
enrichment on growth ,carbohydrates, and rubisco in arabidopsis starch mutants. Significance of starch and hexose. Plant Physiol 130:1573–1583
Sun J, Okita TW, and Edwards GE (1999) Modification of carbon partitioning, photosynthetic capacity
and O2 sensitivity in arabidopsis plants with low ADP-glucose pyrophosphorylase activity. Plant Physiol 119: 267–276
Swaminathan MS (2007) Can science and technology feed the world in 2025? Field Crops Res 104: 3-9
Swarbrick PJ, Schulze LP, Scholes JD (2006) Metabolic consequences of susceptibility and resistance in
barley leaves challenged with powdery mildew. Plant, Cell and Environment 29: 1061–1076
Tang K , Sun X , Hu Q , Wu A, Lin CH,Lin HJ , Twyman RM, Christou P , Feng TY (2001)
Transgenic rice plants expressing the ferredoxin-like protein (AP1)from sweet pepper show enhanced resistance to Xanthomonas oryzae pv. oryzae. Plant Sci 160: 1035–1042
Tsuchiya K, Mew TW, Wakimoto S (1982) Bacteriological and pathological characteristics of wild types
and induced mutants of Xanthomonas oryzae pv. oryzae. Phytopathology 72:43–46.
Voss I, Koelmann M, Wojtera J, Holtgrefe S, Kitzmann C, Backhausen JE, Scheibe R (2008)
Knockout of major leaf ferredoxin reveals new redox-regulatory adaptations in arabidopsis thaliana. Physiol Plant.133: 584–598
Wang CY, Chiou CY, Wang HL, Krishnamurthy R, Venkatagiri S ,Tan J, Yeh KW (2008)
Carbohydrate mobilization and gene regulatory profile in the pseudobulb of oncidium orchid during
the flowering process. Planta 227: 1063–1077
Wang HL, Lee PD, Chen WL, Huang DJ, Su JC (2000) Osmotic stress-induced changes of sucrose
metabolism in cultured sweet potato cells. J Exp Bot 51:1991-1999
Weschke W, Panitz R, Sauer N, Wang Q, Neubohn B, Weber H, Wobus U (2000) Sucrose transport into
barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation. Plant j 21: 455-467
Wong SC, Cowan IR, Farquhar HD (1985) Leaf conductance in relation to rate of CO2 assimilation.
Plant Physiol 78: 826-829
Xiao W, Sheen J, Jang JC (2000) The role of hexokinase in plant sugar signal transduction and growth
and development. Plant Mol Biol 44: 451–461
Yamamoto H, Kato H , Shinzaki Y, Horiguchi S , Tomizawa Toshiharu KI, Miyake C(2006)
Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants-Stimulation of
CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco. Plant
Cell Physiol. 47: 1355–1371
Yip MK, Huang HE, Ger MJ, Chiu SH, Tsai YC, Lin CI, Feng TY (2007) Production of soft rot
resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants. Plant Cell Rep 26: 449–457
Zhang Q (2007) Strategies for developing green super rice. Proc Natl Acad Sci 104: 16402–16409
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔