博碩士論文 90224004 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:34 、訪客IP:18.207.238.169
姓名 黃雅琳(Ya-Ling Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 蕃茄根部受銅逆境之基因調控
(Gene regulation of copper stress in Lycopersicon esculentum (cv CL5915) root tissue)
相關論文
★ 陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究★ 鄰苯二酚加氧酵素的熱穩定性提昇研究
★ Triton X-100 分解菌之分離和分解酵素之特性研究★ Triton X-100加氧酵素之純化與定性
★ Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究★ Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學:以二維電泳法分析等電點4-8之蛋白質表現
★ Pseudomonas nitroreducens TX1之具耗氧活性之麩胺酸合成酶之單離★ 人類細胞株生產含多種亞型的 干擾素-a之蛋白質體學研究
★ 辛基苯酚之分解:分解菌和生物復育之菌相研究★ 分解辛基苯酚聚氧乙基醇之耗氧酵素(二氫硫辛醯胺脫氫酶)的純化與定性
★ AtNPR1轉殖番茄之性狀分析及抗病機制研究★ Pseudomonas putida TX2分解辛基苯酚聚氧乙基醇及其具雌激素活性代謝物之研究
★ 以功能性蛋白質體學研究Pseudomonas nitroreducens TX1生長於辛基苯酚聚氧乙基醇之代謝與逆境反應★ 以功能性蛋白質體學研究Pseudomonas putida TX2生長於 辛基苯酚聚氧乙基醇與辛基苯酚之代謝與逆境反應
★ 以功能性基因體學研究細菌異化辛基苯酚 聚氧乙基醇及抗逆境之基因★ Pseudomonas nitroreducens TX1中二氫硫辛醯胺脫氫酶分解辛基苯酚聚氧乙基醇之研究
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
摘要(中) 銅對於植物的生長為一必須的微量金屬元素,其對許多酵素之功能上為一無可替代的輔因子。過量銅濃度下,在生物體內會造成Fenton反應,促使活性氧化物生成,進一步對生物體造成傷害。為了避免傷害的造成,植物體中必然有解毒機制存在。直至目前為止,尚未有針對於過量銅對植物體基因表現上造成的改變之研究報告。在本論文中,使用反向相減式微陣列、中研院生農所蕃茄基因微陣列及康乃爾蕃茄基因微陣列,篩選受過量銅影響表現之基因。我們發現在過量銅處理下,共有22個無重複之基因(tentative consensus)受到抑制,而有72個基因受到誘導。在銅逆境下會誘導大部分的ROS清除者,包含glutathione S-transferase/peroxidase、ascorbate free radical reductase及cytochrome P450,顯示銅逆境與氧化性傷害具有高度之相關性。除此之外heat shock proteins也受到過量銅的誘導,象徵對銅逆境的耐受性增加。而Tonoplast instrinsic protein delta type及vacuolar H+ ATPase B subunit在銅逆境下則受到抑制,這可能表示液泡在受過量銅處理下會有所變化。
摘要(英) Copper is an essential micro-nutrient for normal plant growth and behaves as an un-replaceable enzyme cofactor. Copper, under excess condition in organisms, will enhance the production of reactive oxygen species (ROS) due to Fenton reaction. These ROS will further damage the cells. To avoid this situation, there must be some detoxification mechanisms in plants. So far, the global gene research of the response to excess copper in plants hasn’t been reported. We used the reverse subtracted cDNA microarray, IBS Tomato-Array and CGEP Tom1-Array to survey genes which response to excess copper in tomato roots. We found that 22 non-redundant tentative consensus were down-regulated under copper stress, and 72 ones been up-regulated. According to the nature of these genes, we suggest that copper stress has strong correlation with oxidative stress. These up-regulation genes most are ROS scavengers, such as glutathione S-transferase/peroxidase, ascorbate free radical reductase and cytochrome P450. Moreover, some heat shock proteins were also up-regulated which implicates the elevated tolerance to copper. Tonoplast instrinsic protein delta type and vacuolar H+ ATPase B subunit were down-regulated under excess copper. This indicates the activity in vacuole could altered in response to copper stress.
關鍵字(中) ★ 銅逆境 關鍵字(英) ★ copper stress
論文目次 目錄
中文摘要------------------------------------------------------------------------Ⅰ
英文摘要 ----------------------------------------------------------------------Ⅱ
目錄 ----------------------------------------------------------------------------Ⅲ
『圖』目錄 -------------------------------------------------------------------Ⅴ
『表』目錄--------------------------------------------------------------------Ⅵ
『附錄』目錄-----------------------------------------------------------------Ⅶ
縮寫與全名對照表-----------------------------------------------------------Ⅷ
第一章 緒論
一、 蕃茄簡介 ----------------------------------------------------------1
二、 銅在植物中所扮演的角色 -------------------------------------1
三、 銅在植物內的恆定機制-----------------------------------------2
四、 文獻中已知受過量銅影響之基因-----------------------------3
五、 過量銅離子濃度對植物生理的影響--------------------------5
六、 研究動機-----------------------------------------------------------5
第二章 材料與方法
第一節 實驗材料
一、 種子的來源---------------------------------------------------------7
二、 蕃茄的種植與過量銅濃度處理---------------------------------7
第二節 實驗方法
一、 訊息核糖核酸之純化 --------------------------------------------8
二、 反向相減式雜交法 -----------------------------------------------9
三、 建立反向相減式cDNA基因庫--------------------------------15
四、 cDNA微陣列技術 ----------------------------------------------17
五、 基因定序與分析--------------------------------------------------21
六、 北方墨點法--------------------------------------------------------21
第三章 實驗結果
第一節 過量銅反向相減式基因庫微陣列之製作
一、 製作過量銅反向相減式基因庫-----------------------------------26
二、 製作微陣列晶片-----------------------------------------------------26
第二節 受過量銅所影響之基因篩選
一、 受過量銅所抑制(down-regulation)之基因----------------------28
1. 在Sub-Array中之分析結果---------------------------------------28
2. 在IBS Tomato-Array中之分析結果-----------------------------30
3. 在CGEP Tom1-Array中之分析結果 ---------------------------30
二、 受過量銅所誘導(up-regulation)之基因--------------------------31
1. 在IBS Tomato-Array中之分析結果-----------------------------32
2. 在CGEP Tom1-Array中之分析結果 ---------------------------33
第四章 討論-----------------------------------------------------------------35
第五章 建議-----------------------------------------------------------------42
參考文獻 ---------------------------------------------------------------------43
圖目錄
圖一、製備蕃茄根部組織反向相減式雜交法之品質控制-------------48
圖二、受過量銅抑制之蕃茄根部細胞的基因片段大小之確定-------49
圖三、北方墨點法分析GADPH基因在蕃茄根部受銅逆境下之表現-----------------------------------------------------------------------------50
圖四、Sub-Array 圖像Data 1-----------------------------------------------51
圖五、Sub-Array 圖像Data 2-----------------------------------------------52
圖六、Sub-Array Data1之內控制基因訊號分佈圖,及所有基因在以內控制基因校正後之四重複平均訊號值---------------------------53
圖七、Sub-Array Data2之內控制基因訊號分佈圖,及所有基因在以內控制基因校正後之四重複平均訊號值---------------------------54
圖八、兩次Sub-Array資料間之相關性分析-----------------------------55
圖九、Sub-Array中,受銅抑制之基因片段大小 ------------------------56
圖十、北方墨點法實驗結果-------------------------------------------------57
圖十一、CGEP Tom1-Array受過量銅處理之基因訊號分佈圖--------58
圖十二、Sub-Array中所得之TC,以其比率之平均值,對應於IBS Tomato-Array及CGEP Tom1-Array的比率平均值----------------59
圖十三、楊明華之過量銅正向相減式基因庫之基因訊號分佈-------60
圖十四、在IBS Tomato-Array及CGEP Tom1-Array共有之基因訊號表現倍率對應圖---------------------------------------------------------61
表目錄
表一、Modefied Hoagland 水耕培養液-----------------------------------62
表二、本研究所選用引子(primer)之序列及其長度---------------------63
表三-1~2、Sub-Array之受過量銅抑制的基因序列比對結果----64~65
表四、IBS Tomato-Array中受過量銅影響之基因分析總表 ---------66
表五-1~2、IBS Tomato-Array中受過量銅抑制基因表現小於0.5之基因---------------------------------------------------------------------67~68
表六、於CGEP Tom1-Array受過量銅抑制而基因表現小於0.5之基因 ------------------------------------------------------------------------69
表七、本論文中所有受抑制(down-regulation)之基因TC統整--------70
表八-1~5、IBS Tomato-Array中受過量銅誘導,表現倍率大於2之基因---------------------------------------------------------------------71~75
表九-1~5、於CGEP Tom1-Array受過量銅誘導,表現倍率大於2之基因------------------------------------------------------------------76~80
表十-1~3、IBS-Array及CGEP Tom1-Array受過量銅誘導兩倍以上之已知功能TC總表-------------------------------------------------81~83
附錄
附錄一、植物調控金屬的恆定機制四個步驟----------------------------84
附錄二、(a)酵母菌中已知之銅運輸機制
(b)目前已知植物內與酵母菌相對應之銅運輸功能表------85
附錄三、製備相減式cDNA雜交法實驗流程-----------------------------86
附錄四、北方墨點法實驗流程 --------------------------------------------87
附錄五、免疫探知作用呈色反應-------------------------------------------87
附錄六-1~5、Sub-Array中所有定序基因之Blast結果總表-------88~92
參考文獻 尤曉莉(2002) 小葉浮萍之色胺酸合成酶β鏈基因選殖與色安酸及其衍生物受銅影響之化學分析. 國立中央大學碩士論文.
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.
楊明華(2002) 利用cDNA微陣列技術探討蕃茄根部組織受過量銅誘導之基因. 國立中央大學碩士論文.
Babu TS, Marder JB, Tripuranthakam S, Dixon DG, Greenberg BM (2001) Synergistic effects of a photooxidized polycyclic aromatic hydrocarbon and copper on photosynthesis and plant growth: evidence that in vivo formation of reactive oxygen species is a mechanism of copper toxicity. Environ Toxicol Chem 20: 1351-1358
Balandin T, Castresana C (2002) AtCOX17, an Arabidopsis homolog of the yeast copper chaperone COX17. Plant Physiol 129: 1852-1857
Banzet N, Richaud C, Deveaux Y, Kazmaier M, Gagnon J, Triantaphylides C (1998) Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells. Plant J 13: 519-527
Broun P, Tanksley SD (1996) Characterization and genetic mapping of simple repeat sequences in the tomato genome. Mol Gen Genet 250: 39-49
Budiman MA, Mao L, Wood TC, Wing RA (2000) A deep-coverage tomato BAC library and prospects toward development of an STC framework for genome sequencing. Genome Res 10: 129-136
Clemens S (2001) Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212: 475-486
Culotta VC, Howard WR, Liu XF (1994) CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J Biol Chem 269: 25295-25302
Desikan R, S AH-M, Hancock JT, Neill SJ (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol 127: 159-172
Droppa M, Masojidek, J., Rozsa, Z., Wolak, A., Jorvath, L. I., Farkas, T., Horvath, G. (1987) Characteristics of Cu deficiency-induced inhibition of photosyntheic electron transport in spinach chloroplasts. Biochim Biophys Acta 891: 75-84
Elfstrand M, Sitbon F, Lapierre C, Bottin A, von Arnold S (2002) Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants. Planta 214: 708-716
Evans KM, Gatehouse JA, Lindsay WP, Shi J, Tommey AM, Robinson NJ (1992) Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: implications for PsMTA function. Plant Mol Biol 20: 1019-1028
Fernandes AR, Peixoto FP, Sa-Correia I (1998) Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress. Arch Microbiol 171: 6-12
Foley RC, Liang ZM, Singh KB (1997) Analysis of type 1 metallothionein cDNAs in Vicia faba. Plant Mol Biol 33: 583-591
Gallego SM, Benavides MP, Tomaro ML (1996) Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Science 121: 151-159
Gaxiola RA, Fink GR, Hirschi KD (2002) Genetic manipulation of vacuolar proton pumps and transporters. Plant Physiol 129: 967-973
Giritch A, Ganal M, Stephan UW, Baumlein H (1998) Structure, expression and chromosomal localisation of the metallothionein-like gene family of tomato. Plant Mol Biol 37: 701-714
Glerum DM, Shtanko A, Tzagoloff A (1996) Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase. J Biol Chem 271: 14504-14509
Gralla EB, Thiele DJ, Silar P, Valentine JS (1991) ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene. Proc Natl Acad Sci U S A 88: 8558-8562
Gross C, Kelleher M, Iyer VR, Brown PO, Winge DR (2000) Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem 275: 32310-32316
Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 53: 1-11
Henriques FS (1989) Effects of copper deficiency on the photosynthetic apparatus of sugar beet. Plant Physiol 135: 453-458
Henriques FS, Fernandes JC (1991) Biochemical, physiological, and structural effects of excess copper in plants. The Botanical Review 57: 246-273
Himelblau E, Amasino RM (2000) Delivering copper within plant cells. Curr Opin Plant Biol 3: 205-210
Himelblau E, Mira H, Lin SJ, Culotta VC, Penarrubia L, Amasino RM (1998) Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis. Plant Physiol 117: 1227-1234
Hirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S, Alonso JM, Dailey WP, Dancis A, Ecker JR (1999) RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis. Cell 97: 383-393
Horvath G, Melis, A., Hideg, E., Droppa, M., Vigh, I., (1987) Role of lipids in the organization and function of photosystem II studied by homogeneous catalytic hydrogenation of thylakoid membranes in situ. Biochim Biophys Acta 891: 68-74
Hsieh HM, Liu WK, Chang A, Huang PC (1996) RNA expression patterns of a type 2 metallothionein-like gene from rice. Plant Mol Biol 32: 525-529
Jauh GY, Phillips TE, Rogers JC (1999) Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell 11: 1867-1882
Johnson CM, Stout PR, Boroger TC, Carlton AB (1957) Comparative chlorine requirements of different plant species. Plant and Soil 8: 337-353
Kampfenkel K, Kushnir S, Babiychuk E, Inze D, Van Montagu M (1995) Molecular characterization of a putative Arabidopsis thaliana copper transporter and its yeast homologue. J Biol Chem 270: 28479-28486
Lewis S, Donkin ME, Depledge MH (2001) Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors. Aquat Toxicol 51: 277-291
Li Y, Trush MA (1993) DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation. Carcinogenesis 14: 1303-1311
Lipman CB, McKinney G (1931) Proof of the essential nature of copper for higher green plants. Plant Physiology 6: 593-599
Maitani T, Kubota H, Sato K, Yamada T (1996) The Composition of Metals Bound to Class III Metallothionein (Phytochelatin and Its Desglycyl Peptide) Induced by Various Metals in Root Cultures of Rubia tinctorum. Plant Physiol 110: 1145-1150
Maksymiec W (1997) Effect of copper on cellular processes in higher plants. Photosynthetica 34: 321-342
Mira H, Martinez N, Penarrubia L (2002) Expression of a vegetative-storage-protein gene from Arabidopsis is regulated by copper, senescence and ozone. Planta 214: 939-946
Mira H, Martinez-Garcia F, Penarrubia L (2001a) Evidence for the plant-specific intercellular transport of the Arabidopsis copper chaperone CCH. Plant J 25: 521-528
Mira H, Vilar M, Perez-Paya E, Penarrubia L (2001b) Functional and conformational properties of the exclusive C-domain from the Arabidopsis copper chaperone (CCH). Biochem J 357: 545-549
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405-410
Neumann D, Lichtenberger O, Gunther D, Tschiersch K NL (1994) Heat-shock proteins induce heavy-metal tolerance in higher plants. Planta 194: 360-367
Ooi C, Rabinovich E, Dancis A, Bonifacino J, Klausner R (1996) Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis. EMBO J. 15: 3515-3523
Patsikka E, Kairavuo M, Sersen F, Aro EM, Tyystjarvi E (2002) Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll. Plant Physiol 129: 1359-1367
Pena MM, Lee J, Thiele DJ (1999) A delicate balance: homeostatic control of copper uptake and distribution. J Nutr 129: 1251-1260
Sambrook J, Russell DW (2001) Molecular Cloning.
Sancenon V, Puig S, Mira H, Thiele DJ, Penarrubia L (2003) Identification of a copper transporter family in Arabidopsis thaliana. Plant Mol Biol 51: 577-587
Sandmann G, Boger P (1980) Copper-mediated lipid peroxidation process in photosynthetic membranes. Plant Physiol 66: 797-800
Schafer HJ, Greiner S, Rausch T, Haag-Kerwer A (1997) In seedlings of the heavy metal accumulator Brassica juncea Cu2+ differentially affects transcript amounts for gamma-glutamylcysteine synthetase (gamma-ECS) and metallothionein (MT2). FEBS Lett 404: 216-220
Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53: 1351-1365
Sommer AL (1931) Copper as an essential for plant growth. Plant Physiology 6: 339-345
Sun W, Van Montagu M, Verbruggen N (2002) Small heat shock proteins and stress tolerance in plants. Biochim Biophys Acta 1577: 1-9
Thiele DJ (1988) ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol Cell Biol 8: 2745-2752
Valentine JS, Gralla EB (1997) BIOCHEMISTRY: Enhanced: Delivering Copper Inside Yeast and Human Cells. Science 278: 817-818
van der Zaal EJ, Droog FN, Boot CJ, Hensgens LA, Hoge JH, Schilperoort RA, Libbenga KR (1991) Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression. Plant Mol Biol 16: 983-998
Winge DR (2002) Copper metalloregulation of gene expression. Adv Protein Chem 60: 51-92
Wintz H, Vulpe C (2002) Plant copper chaperones. Biochem Soc Trans 30: 732-735
Zhou J, Goldsbrough PB (1994) Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell 6: 875-884
Zhu H, Shipp E, Sanchez RJ, Liba A, Stine JE, Hart PJ, Gralla EB, Nersissian AM, Valentine JS (2000) Cobalt(2+) binding to human and tomato copper chaperone for superoxide dismutase: implications for the metal ion transfer mechanism. Biochemistry 39: 5413-5421
Zhu Z, Labbe S, Pena MM, Thiele DJ (1998) Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor. J Biol Chem 273: 1277-1280
指導教授 詹明才、黃雪莉
(Ming-Tsair Chan、Shir-Ly Huang)
審核日期 2003-7-18
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明