綠豆(Vigna radiata L. v. 2937)屬於不耐寒植物，本實驗室為瞭解低溫逆境對葉片細胞核基因之影響，以28℃照光生長葉片之poly(A)+ mRNAs，建立一cDNA library，再以差異式篩選法(differential screening)篩選受低溫明顯抑制之基因，共篩選出13個不同的基因；本論文即是針對其中一個rubisco activase（rca）基因進行更深入的探討。首先，在南方氏雜合分析（Southern blot analysis）中可以發現，rca在綠豆中為單套基因；利用北方雜合分析及西方雜合分析方法（Northern hybridization and Western blot analysis）分析rca基因的表現情形，在28℃常溫及16小時光照的光週期下，發現其mRNA的表現受到概日韻律之調控；且蛋白質的累積量亦是隨著mRNA表現量上升而增加；而綠豆rca基因概日韻律之控制，在10℃的低溫逆境下即發生改變；在相同的光週期下，除了表現量逐漸下降之外，概日韻律的週期亦逐漸變長，失去一定的規則。但低溫並不會影響其蛋白質生成的轉譯作用，RCA蛋白質之累積即使在低溫下亦隨著其mRNA表現量的增減而隨之改變。
確定低溫下rca基因的確是在轉錄作用受到抑制後，為進一步瞭解此基因在低溫下被調控的可能機制，故利用genome walking的方式選殖出一段位於綠豆rca啟動子區域，長1,231 bp之DNA片段；經序列比對後，發現數種可能調控基因表現的區域，分別為：光誘導、組織專一性表現、概日韻律調控、醣類回饋抑制、缺水及ABA誘導等五類可能存在之調控區。但實驗證明的結果，在ABA及缺水逆境處理下的綠豆幼苗植株，其rca基因並未因上述處理而有表現量大增之情形。

The mungbean (Vigna radiata L. v. 2937) is a chilling-sensitive plant. A cDNA library was constructed from poly(A)+ mRNAs of 28℃-light-grown leaves for screening cold-suppressed nuclear genes. One of the 13 cold-suppressed nuclear genes obtained by differential screening was rubisco activase (rca) gene. Southern blot analysis suggested the presence of a single rca gene in the mungbean genome. The expression of leaf rca mRNA was found to be regulated by circadian rhythm under 28℃ and 16L/8D photoperiod. In addition, the accumulation of RCA protein was positively propotional to the rca mRNA expression level. However, the rhythm in mungbean leaves was interrupted by 10℃ chilling treatments. Under the 16L/8D photoperiod, the rca mRNA expression was not only mistiming, but depressed by chilling stress. Even so, the RCA protein was translated as long as the mRNA existed during the 10℃ chilling treatment.
The promoter region (1,231 bp) of rca gene was cloned by the genome walking method. Six possible regulatory motifs were found in this cis-acting regulatory DNA element. They included light-inducible, tissue-specific, circadian control, sugar repression, sugar starvation, ABA and water stress response elements. But the significant increase of rca gene expression was found in the treatments of ABA and water stress.

簡寫表-------------------2
中文摘要-----------------4
英文摘要-----------------6
第一章 前言------------7
第二章 材料與方法-----16
第三章 結果-----------35
第四章 討論-----------41
參考文獻----------------48
圖表--------------------54

龔政哲 (1997) 豌豆白化幼苗冷溫誘導蛋白質基因之研究：基因篩選、結構及其表現的分析。國立臺灣大學植物學研究所博士論文
楊敏宗 (2001) 低溫逆境抑制綠豆幼苗葉片細胞核基因之表現及質體發育。國立臺灣大學植物學研究所博士論文
Allen DJ, Ort DR (2001) Impact of chilling temperatures on phoyosynthesis in warm-climate plants. Trends Plant Sci 2001 6: 36-42
Baker SS, Wilhelm KS, Thomashow MF (1994) The 5''-region of Arabidopsis thaliana cor15a has cis-actingelements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24:701-713
Beuf L, Kurano N, Miyachi S (1999) Rubisco activase transcripts (rca) abundance increases when tha marine unicellular green alga Chlorococcum littorale is grown under high-CO2 stress. Plant Mol Biol 41: 627-635
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254
Emaus D, Wilson JM (1983) ABA levels and effects in chilled and hardened Phaseolus vulgaris. J Exp Bot 34: 1000-1006
Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR (1988) An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene.Proc Natl Acad Sci USA 85:7089-7093
Grossman A, Bartlet S, Chua NH (1980) Energy-dependent uptake of cytoplasmically synthesized polypeptides by chloroplasts. Nature 285: 625-628
Guy CL (1990) Cold acclimation and freezing stress tolerence: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol 41: 187-223
Hammond ET, Andrews TJ, Woodrow IE (1998) Regulation of ribulose-1,5-bisphosphate carboxylase / oxygenase by carbamylation and 2-carboxyarabinitol 1-phosphate in tobacco: insights from studies of antisense plants containing reduced amounts of Rubisco activase. Plant Physiol 118: 1463-1471
Hwang YS, Karrer EE, Thomas BR, Chen L, Rodriguez RL (1998) Three cis-elements required for rice alpha-amylase Amy3D expression during sugar starvation Plant Mol Biol 36:331-341
Kreps JA, Kay SA (1997) Coordination of plant metabolism and development by the circadian clock. Plant Cell 9: 1235-1244
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685
Levitt J (1980) Response of plants to environmental stress. Vol. 1. Chilling, freezing and high temperature stresses. 2nd ed. New York: Academic Press
Liu Z, Taub CC, McClung CR (1996) Identification of an Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) minimal promoter regulated by light and the circadian clock. Plant Physiol 112: 43-51
Lorimer GH (1981) The carboxylation and oxygenation of ribulose 1,5-bisphosphate: The primary events in photosynthesis and photorespiration. Annu Rev Plant Physiol. 32: 349-383
Lyon JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24: 445-466
Martino-Catt S, Ort DR (1992). Low temperature interrupts circadian reguation of transcriptional activity in chilling-sensitive plants. Proc Natl Acad Sci USA 89: 3731-3735.
McWilliam JR, Kramer PJ, Musser RL (1982) Temperature induced water stress in chilling sensitive plants. Aust J Plant Physiol 9: 343-352
Nishida I, Murata M (1996) Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Annu Rev Plant Physiol Plant Mol Biol 47: 541-568
Orozco BM, Ogren WL (1993) Localization of light-inducible and tissue -specific regions of the spinach ribulose bisphosphate caboxylase /oxygenase (rubisco) activase promoter in transgenic tobacco plants. Plant Mol Biol 23: 1129-1138
Piechulla B, Merforth N, Rudolph B (1998) Identification of tomato Lhc promoter regions necessary for circadian expression Plant Mol Biol 38:655-662
Pla M, Vilardell J, Guiltinan MJ, Marcotte WR, Niogret MF, Quatrano RS, Pages M (1993) The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28. Plant Mol Biol 21:259-266
Preisig-Muller R, Kindl H (1992) Sequence analysis of cucumber cotyledon ribulosebisphospate carboxylas/oxygenase activase cDNA. Biochem Biophys Acta 1171: 206-206
Raison JK (1973) The influence of temperature-induced phase changes on kinetics of respiratory and other membrane-associated enzymes. J Bioenerg 4: 258-309
Rinki A (1992) Temporal organization of chilling resistence in cotton seedlings: Effects of low temperature and relative humidity. Planta 187: 517-522
Rinki A (1991) Temperature-induced phase shifting of circadian rhythms in cotton seedlings as related to variations in chilling resistence. Planta 185: 407-414
Robinson SP, Portis AR Jr (1988) Release of nocturnal inhibitor, carboxyarabinitol-1-phosphate from ribulose bisphosphate carboxylase / oxygenase by Rubisco activase. FEBS Lett 233: 413-416
Rokka A, Zhang L, Aro EM, (2001) Rubisco activase: an enzyme with a temperature-dependent dual function? The Plant J 25: 463-471
Sabine JR, Raymond EZ (1991) Organization and expression of two tandemly oriented genes encoding ribulosebisphophate carboxyase/oxygenase activase in Barley. J Biol Chem 266: 4677-4685.
Salvucci ME, Ogren WL (1996) The mechanism of Rubisco activase: insights from studies of properties and structure of the enzyme. Photosynth. Res.
Salvucci ME, Werneke JM, Ogren WL, Portis AR Jr (1987) Purificaiton and species distribution of Rubisco activase. Plant Physiol 84: 930-936 47: 1-11
Salvucci ME, Portis AR Jr, Ogren W L (1985) A soluble choloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo. Photosynth Res 7: 193-201
Somerville CR, Portis AR Jr (1982) A mutant of Arabidopsis thaliana which lacks activation of RuBP carboxylase in vivo. Plant Physiol 70: 381-387
Stitt M, Lilley RM, Heldt HW (1982) Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts. Plant Physiol 70: 971-977
Streusand VJ, Portis AR Jr (1987) Rubisco activase mediates ATP-dependent activation of ribulose bisphosphate carboxylase. Plant Physiol 85: 154-152
Terzaghi WB, Cashmore AR (1995) Light-regulated transcription Annu Rev Plant Physiol Plant Mol Biol 46:445-474
Theg SM, Bauerle C, Olsen LJ, Seiman BR, Keegstra K (1989) Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J Biol Chem 264: 6730-6736
Thomashow MF (2001) So what’s new in the field of plant cold acclimation? Lots! Plant Physiol 125: 89-93
Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Physiol Plant Mol Biol 50: 571-599
To KY, Suen DF, Chen SCG (1999). Molecular characterization of ribulose-1,5-bisphosphate caboxylase/oxygenase activase in rice leaves. Planta 209: 66-76.
Toyofuku K, Umemura T, Yamaguchi J (1998) Promoter elements required for sugar-repression of Ramy3D gene for alpha-amylase in rice. 428: 275-280
Wang Z-Y, Portis AR Jr (1992) Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase / oxygenase and its enhancement by ribulose-1,5-bisphosphate caboxylase/oxygenase activase-mediated hydrolysis of ATP. Plant Physol. 99: 1348-1353
Werneke JM, Orgen WL (1989) Structure of an Arabidopsis thaliana cDNA encoding Rubisco activase. Nucleic Acids Res 17: 2871
Werneke JM, Chatfield JM, Orgen WL (1989) Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis. Plant Cell 1: 815-826
Werneke JM, Zielinski RE, Orgen WL (1988) Structure and expression of spinach leaf coda encoding ribulose bisphosphate carboxylase / oxygenase activase. Proc Natl Acad Sci USA 85: 787-791
Wilson JM (1976) The mechanism of chill- and drought-hardening of Phaseolus vulgaris leaves. New phytol 76: 257-270
William ME, Foster R, Chua N-H (1992) Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4: 485-496
Yokoi S, Higashi SI, Kishitanni S, Murata N, Toriyama K (1998) Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Mol Breed 4: 269-275
Zang N, Kallis RP, Ewy RG, Portis AR Jr (2002) Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of larger Rubisco activase isoform. Proc Natl Acad Sci USA 99: 3330-3334
Zang N, Portis AR Jr (1999) Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f. Proc Natl Acad Sci USA 96: 9438-9443
Zilian Z, Setsuko K (2000) Molecular cloning and characterization of cDNAs encoding two isforms of ribulose-1,5-bisphosphate caboxylase / oxygenase activase in rice (Oryza sativa L.). J. Biochem. 128: 383-389