摘要
本論文之原點為小葉浮萍相減式cDNA 基因庫中受過量銅誘導
的基因-色胺酸合成酵素β鏈，色胺酸合成酵素β鏈是植物體合成色
胺酸過程中最後一個關鍵酵素，色胺酸在植物細胞中是唯一會轉化為
可吸收光能之物質的胺基酸，其下游的衍生物（tryptophan derivatives）
中較為熟知的有葉綠素（chlorophyll）、生長激素（auxin, IAA）、血清
素（serotonin）以及褪黑激素（melatonin）。其中，生長激素IAA 是
具有多功能的植物賀爾蒙，在細胞中作為訊息傳遞者或是基因的調節
者；褪黑激素則是首先發現於人類松果體之神經傳導物質，與光週期
有極高的相關性，並且具有清除自由基之能力。在植物界中，目前熟
知褪黑激素為種子中抵禦過氧化傷害之重要物質。
本論文利用5’端快速基因放大技術（5’-RACE）取得色胺酸合成
酵素β鏈之cDNA 全長，進一步取得基因上游之啟動子，以利日後分
析基因調節之機制，另外由南方氏點墨法判斷出色胺酸合成酵素β鏈
基因在浮萍基因組中應屬於一個中小型的基因家族（gene family）。
本論文同時使用高效液相層析儀追蹤浮萍在過量銅環境中，於不
同時間點下色胺酸與其重要衍生物之含量變化，證實色胺酸合成酵素
β鏈之基因表現與色胺酸及其重要衍生物之產生具有正相關性外，高
效液相層析儀同步自由基清除者偵測系統（HPLC on-line free radical
scavenger detection system）更有助於釐清此類衍生物在小葉浮萍中之
生理功能。

Abstract
Our previous study has identified several copper responsive genes in
duckweed, Lemna minor. One of the genes encodes tryptophan synthase
β-chain (TSB) which catalyzes the last step of tryptophan synthesis
pathway in plants. The strong expression of TSB under excess of copper
may indicate that the elevation of tryptophan synthesis and its subsequent
metabolic intermediates could respond to the toxicity imposed by this
metal. Tryptophan is a precursor of the phyto-hormone, auxin (IAA)
which mediates multiple signals in plants for development, gravitropism
and stress responses. Several tryptophan derivatives are also intrinsic
light-adsorbing compounds such as chlorophyll, serotonin and melatonin.
Melatonin was first discovered in pineal grain of the vertebrates that
governs the diurnal and photo-periodic response. Subsequently, it was
identified as a free radical scavenger in other organisms such as bacteria
and plants. This thesis correlated the timing of gene expression of TSB
and the accumulation profiles of the tryptophan and its derived
compounds such as IAA and melatonin in order to unfold the
physiological response to excess of copper. Genomic Southern analysis
showed TSB is a small- to mid-sized gene family. The full-length cDNA
was obtained by 5’-RACE and its upstream region was isolated using
genomic walking. Time course analysis for tryptophan and its derivatives
IAA and melatonin by HPLC showed reasonable correlation between
gene expression of TSB and the accumulation of the metabolites. The
further application by HPLC on-line free radical scavenger detection
system can assist the functional assays for such metabolites.

陸、參考文獻
吳岳隆（2001）利用相減式雜交法鑑定小葉浮萍受巴拉刈與銅誘導之基因.
國立中央大學碩士論文.
Ayres, R.U.（1992）Toxic Heavy Metals：Materials Cycle Optimization. Proc.
Natl. Acid. Sci. 89：815-20.
Azmitia, E.C. （2001）Modern View on an Ancient Chemical：Serotonin
Effects on Cell Proliferation, Maturation, and Apoptosis. Brain Res. Bull.
413-24.
Bittle, D. Dalton, T. L, S. Samson, A. Gedamu, L. and Andrews, G.K.
（1998）The DNA Binding Activity of Metal Response Element- binding
Transcription Factor-1 Is Activated in vivo and in vitro by Zinc, but Not by
Other Transition Metals. J. Biol. Chem. 273：7127-33.
Buzby, J.S. Yamada, T. and Tobin, E.M.（1990）A Light-Regulated DNA-
Binding Activity Interacts with a Conserved Region of a Lemna gibba
rbcS Promoter. Plant Cell 2：805-814.
Chang, W.C. and Hsing, Y.I.（1979）The Use and Tissue Culture Research of
Duckweeds. Natl. Sci. Counc Monthly. 7：753-64.
Cobbett, C.S.（2000）Phytochelatin Biosynthesis and Function in Heavy-metal
Detoxification. Cur. Opin. Plant Biol. 3：211-16.
Daniel , K.G. Ellen, G.S. and Thomas, L.R.（2000）The Role of Free
Indole-3-acetic Acid (IAA) Levels, IAA Transport, and Sucrose Transport
in the High Temperature Inhibition of Primary Root Development in Pea
(Pisum sativum L. cv. Alaska). J. Plant Growth Regul. 19：347-58.
Degenhardt, J. and Tobin, E.M.（1996）A DNA binding Activity for One of
Two Closely Defined Phytochrome Regulatory Elements in an Lhcb
Promoter Is More Abundant in Etiolated Than in Green Plants. Plant Cell
8：31-41.
Desikan, R. Mackerness, S.A.H. Hancock, J.T. and Neill, S.J.（2001）
Regulation of the Arabidopsis Transcriptom by Oxidative Stress. Plant
Physiol. 127：159-72.
Edlunt, A. Eklöf, S. Sundberg, B. Moritz, T. and Sandberg, G.（1995）
A Microscal Technique for Gas Chromatography-Mass Spectrometry
Measurements of Picogram Amounts of Indole-3 Acetic Acid on Plant
Tissues. Plant Physiol. 108：1043-7.
Glawischnig, E. Tomas, A. Eisenreich, W. Spiteller, P. Bacher, A.
and Gierl, A.（2000）Auxin Biosynthesis in Maize Kernels. Plant
Physiol. 123：1109-19.
Hoenicke, K. Simat, T.J. Steinhart, H. KöHLER, H.J. and Schwab, A.
（2001）Determination of Free and Conjugated Indole-3-Acidic Acid,
Tryptophan, and Tryptophan Metabolites in Grape Must and Wine. J.
Agric Food Chem. 49：5494-501.
Hooley, R.（1998）Auxin Signaling：Horming in with Trageted Genetics. Plant
Cell 10：1581-3.
Johnson, C. Glover, G. and Arias, J. （2001）Regulation of DNA binding and
trans-Activation by a Xenobiotic Stress-activated Plant Transcription
Factor. J. Biol. Chem. 276：172-8.
Koizumi, S. Suzuki, K. Ogra, Y. Yamada, H. and Otsuka, F.（1999）
Transcriptional Activity and Regulatory Protein binding of
Metal-Responsive Elements of the Human Metallothionein IIA Gene. J.
Biol. Chem. 259：635-42.
Koizumi, S. Suzuki, K. and Otsuka, F.（1992）A Nuclear Factor That
Recognize the Metal-Responsive Elements of the Human Metallothionein
IIA Gene. J. Biol. Chem. 267：18659-64.
Koleva, I.I. Niederländer, H.A.G. and ven Beek, T.A.（2001）Application of
ABTS Radical Cation for Selective On-Line Detection of Radical
Scavengers in HPLC. Anal. Chem. 73：3373-81.
Lasswell, J. Rogg, L.E. Nelson, D.C. Rongey, C. and Bartel, B.（2000）
Cloning and Chacterization of IAR1, a Gene Required for Auxin
Conjugate Sensitivity in Arabidopsis. Plant Cell 12：2395-408.
Limson, J. Nyokong, T. and Daya, S.（1998）The Interaction of Melatonin
and its Precursor with Aluminum, Cadmium, Copper, Iron, Lead, and
Zinc：An Adsorptive Volta metric Study. J. Pineal Res. 24：15-21.
Linder, M.C.（2001）Copper and Genomic Stability in Mammals. Mutation
Research 475：141-52.
Li, Q. Hu, N. Daggett, M.A.F. Chu, W.A. Bittel, D. Johnson, J.A. and
Andrews, G.K.（1998）Participation of Upstream Stimulator Factor（USF）
in Cadmium-Induction of the Mouse Metallothionein-Ⅰ Gene. Nucleic
Acids Res. 26：5182-89.
Lu, H. and Mcknight, D.T.（1999）Tissue-Specific Expression of the β-
Subunit of Tryptophan Synthase in Camptotheca acuminata, an Indole
Alkaloid-Producing Plant. Plant Physiol. 120：43-51.
Manchester, L.C. Tan, D.X. Reiter, RJ. Park, W. Monis, K. and Qi, W.
（2000）High Levels of Melatonin in the Seeds of Edible Plants Possible
Function in Germ Tissue Protection. Life Science 67：3023-29.
Marklová, E. MakoviCková, H. and Krákorová, I.（2000）Screening for
Defects in Tryptophan Metabolism. J. Chromatogr. A. 870:289-93.
Müller, A. Hillebrand, H. and Weiler, E.W.（1998）Indole-3-Acetic Acid is
Synthesized from L-Tryptophan in Roots of Arabidopsis thaliana. Planta
206：362-369.
Murphy, A. Peer, W.A. and Taiz, L.（2000）Regulation of Auxin Transport by
Aminopeptidases and endogenous flavonoids. Planta 211：315-24.
Okubara, P.A. William, S.A. Doxsee, R.A. and Tobin, E.M.（1993）Analysis
of Genes Negatively Regulated by Phytochrome Action in Lemna gibba
and Identification of a Promoter Region Required for Phytochrome
Responsiveness. Plant Physiol. 101：915-24.
Otsuka, F. Iwamatsu, A. Suzuki, K. Ohsawa, M. Hamer, D.H. and Koizumi,
S. （1994） Purification and Chacterization of a Protein That Binds to Metal
Responsive Elements of the Human Metallothionein IIA Gene. J. Biol.
Chem. 269：23700-07.
Ouyang, J. Shao, X. and Li, J.（2000）Indole-3-glycerol phosphate, a
branchpoint of indole-3-acetic acid biosynthesis from the tryptophan
biosynthetic pathway in Arabidopsis thaliana. Plant J. 24：327-33.
Palmiter, R.D.（1998）The Elusive Function of Metallothioneins. Proc. Natl.
Acid. Sci. 95：8428-30.
Reiter, R.J. Tan, D.X. Cabrera, J. D’Arpa, D. Sainz, R.M. Mayo, J.C. and
Ramos, S.（1999）The Oxidant/Antioxidant Network：Role of Melatonin.
Biol. Signals Recept. 8：56-63.
Schützendübel, A. and Polle, A.（2002）Plant Responses to Abiotic Stresses：
Heavy Metal-Induced Oxidative Stress and Protection by Mycorrhization.
J. Exp. Botany 53：1351-65.
Tan, D.X. Manchester, L.C. Reiter, R.J. Qi, W. Karbownik, M. and Calvo,
J.R.（2000）Significance of Melatonin in Antioxidative Defense System：
Reactions and Products. Biol. Signals Recept. 9：137-59.
Van Tassel, D.L. Roberts, N. Lewy, A. and O’Neill, S.D.（2001）Melatonin in
Plant Organs. J. Pineal Res. 31：8-15.
Wang, W.（1990）Literature Review on Duckweed Toxicity Testing. Environ.
Res. 52：7-22.
Werner, T.（1999）Models for prediction and recognition of eukaryotic
promoters. Mammalian Genome 10: 168-75.
Xie, G. Forst, C. Bonner, C. and Jensen, RA（2001）Significance of two
distinct types of tryptophan synthase beta chain in Bacteria, Archaea and
higher plants. Genome Biology 3：RESEARCH0004.
Yoo, H.Y. Chang, M.S. and Rho, H.M.（1999）Heavy Metal Mediated
Activation of the Rat Cu/Zn Superoxide Dismutase Gene via a Metal-
Responsive Element. Mol. Gen. Genet. 262：310-13.
Yoshida, T.（1996）Determination of Reduced and Oxidized Glutathione in
Erythrocytes by High-Performance Liquid Chromatography with
Ultraviolet Absorbance Detection. J. Chromatogr. B 687：157-64.
Zawilska, J.B. and Nowak, J.Z.（1999）Melatonin：from Biochemistry to
Therapeutic Applications. Pol. J. Pharmacol. 51：3-23.
Zhao, J. Williams, C.C. and Last, R.L.（1998）Induction of Arabidopsis
Tryptophan Pathway Enzymes and Camalexin by Amino Acid Starvation,
Oxidative Stress, and an Abiotic Elicitor. Plant Cell 10：359-70.