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姓名 蕭保元(Pao-Yuan Shiao) 查詢紙本館藏 畢業系所 生命科學系 論文名稱 持續表現阿拉伯芥色氨酸合成酶可以幫助抵抗逆境
(Over-expression of Arabidopsis Tryptophan Synthase β Chain gene can Enhance Tolerance to stresses)相關論文 檔案 [Endnote RIS 格式]
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摘要(中) 環境逆境常會誘導植物基因大量表現，這些基因可能是訊息傳導或是抵抗機制的酵素或是逆境反應的基因等，以使植物產生各種可能的抵抗機制而存活。本實驗以北點墨法發現一個會受到硫酸銅所誘導的基因tryptophan synthase β-chain 1 (TSB1)，位於色氨酸(tryptophan)合成路徑，負責將絲氨酸(serine)和吲哚(indole)合成色氨酸之步驟。經由農桿菌轉殖技術將TSB1基因大量表現於阿拉伯芥，來瞭解這個受銅誘導表現的基因功能，並觀察TSB1轉殖株與逆境的耐受性之關係。在TSB1轉殖株與未轉殖之wild type的逆境分析中發現，轉殖株對於硫酸銅(CuSO4)、氯化鎘(CdCl2)、氯化鈉(NaCl)及paraquat之耐受性高於未轉殖的wild type。並發現一位於色氨酸衍生物的代謝路徑下游的基因aldehye dehydrogenase 3 (ALDH3)在轉殖植物中RNA的表現量大於未轉殖的wild type。根據文獻參考，推測TSB1轉殖株的逆境耐受性的增加可能與ALDH3基因參與的代謝途徑相關。為瞭解之間的相關性，利用GC/MS分析TSB1轉殖株與未轉殖的wild type色胺酸含量及其代謝途徑中的衍生物。結果顯示在轉殖植物中除色氨酸含量增加外，ALDH3的產物5-hydroxyindole acetoaldehyde (HIAA)含量也有增加。上述這些結果建議TSB1轉殖株對逆境耐受性的增加有可能是受到ALDH3這個基因表現量增加，進而影響TSB1轉殖株對逆境的耐受性。 摘要(英) Environmental stresses activate plant genes expression that may act as signal or defense enzymes and/or responsive genes to enhance plant to tolerant or resistant to various stresses. A gene, tryptophan synthase β-chain 1(TSB1) induced by 100 μM CuSO4 is responsible for the final step of tryptophan synthesis pathway; TSB1 converts the indole and serine to tryptophan. To study the function of TSB1, we create a TSB1 over- expressed transgenic Arabidopsis by Agrobacterium tumefaciens. TSB1 transgenic plants were tolerance to copper, cadmium, salinity and paraquat comparing to wild type plant. The mRNA transcripts of aldehye dehydrogenase 3 (ALDH3), one synthesis enzyme in the down streaming of tryptophan-derived metabolism pathway, were increased in TSB1 transgenic plants than wild-type plants. The results imply that the metabolism pathway of ALDH3 may be contributed to the tolerance to various stresses in TSB1 transgenic plants. The results of GC/MS analysis show that the concentrations of tryptophan and HIAA which is the product of the ALDH3 enzyme, are higher than those of wild type. These results suggest that increased expression of ALDH3 in TSB1 transgenic plant may increase tolerance to stresses. 關鍵字(中) ★ 色胺酸
★ 色胺酸合成酶β鏈
★ 對逆境的耐受性關鍵字(英) ★ tolerance to stresses
★ tryptophan
★ tryptophan sytnase β chain論文目次 目 錄
中文摘要 ------------------------------------------------------------ Ⅰ
英文摘要 ------------------------------------------------------------ Ⅱ
目錄 ---------------------------------------------------------------- Ⅲ
圖表目錄 ------------------------------------------------------------ Ⅵ
縮寫與全名對照表 ---------------------------------------------------- Ⅶ
壹、緒論 ------------------------------------------------------------ 1
一、前言 ------------------------------------------------------------ 1
二、色胺酸合成酶β鏈基因-------------------------------------------- 1
三、重要的色胺酸衍生物--------------------------------------------- 2
四、重金屬對植物體的毒害------------------------------------------- 3
五、植物抵禦過量金屬毒害之策略------------------------------------- 4
六、研究目的------------------------------------------------------- 5
貳、材料與方法 ------------------------------------------------------ 6
ㄧ、阿拉伯芥AtTSB1基因選殖----------------------------------------- 6
1、阿拉伯芥total RNA 之純化------------------------------------------ 6
2、RNA 電泳分析------------------------------------------------ 6
3、反轉錄聚合酶連鎖反應---------------------------------------------- 7
4、純化PCR產物片段--------------------------------------------- 8
5、載體與基因的建造-------------------------------------------- 9
6、製備大腸桿菌的勝任細胞-------------------------------------- 9
7、細菌的轉型作用---------------------------------------------- 10
8、快速萃取質體DNA -------------------------------------------- 10
9、從載體切除基因片段------------------------------------------ 11
10、製備Agrobacteria的勝任細胞--------------------------------- 11
11、農桿菌的轉型作用 ------------------------------------------ 12
二、阿拉伯芥AtTSB1基因植物的轉殖----------------------------------- 12
12、植物的轉型作用--------------------------------------------- 12
13、轉殖植株的篩選--------------------------------------------- 13
14、阿拉伯芥DNA之純化------------------------------------------ 13
15、南方氏點墨法----------------------------------------------- 14
三、非生物性因素處理----------------------------------------------- 16
四、色胺酸及其衍生物含量的測定------------------------------------- 17
參、結果 ------------------------------------------------------------ 19
一、阿拉伯芥植物中的TSB1基因的表現在銅處理受到誘導----------------- 19
二、轉殖載體的構築------------------------------------------------- 19
三、阿拉伯芥轉殖及轉殖植物的篩選----------------------------------- 19
四、以南方氏墨點法分析TSB1轉殖植株之插入的基因數目----------------- 19
五、以北方點墨法分析TSB1轉殖植株基因表現情形----------------------- 20
六、非生物性處理測試轉殖植株之耐受性------------------------------- 20
七、其他與逆境有關的基因是否會受到TSB1表現而影響------------------- 22
八、色胺酸及其衍生物含量的測定------------------------------------- 22
肆、討論 ------------------------------------------------------------ 24
伍、圖表------------------------------------------------------------- 28
陸、參考文獻 -------------------------------------------------------- 47
附錄 --------------------------------------------------------------- 50
圖 表 目 錄
圖一、阿拉伯芥AtTSB1基因之RT-PCR結果--------------------------------- 28
圖二、北方點墨法分析 TSB1在100 μM硫酸銅處理下
經過不同楚裡時間點TSB1表現的情形------------------------------- 30
圖三、南方氏墨點法分析轉植株插入的基因數目--------------------------- 29
圖四、北方點墨法分析 TSB1轉殖株與未轉殖wild type TSB1基因表現情形---- 31
圖五、硫酸銅(CuSO4)處理下wild-type與TSB1轉殖株生長情形 ------------- 32
圖六、氯化鎘(CdCl2)處理下wild-type與TSB1轉殖株生長情形--------------- 34
圖七、paraquat處理下wild-type與TSB1轉殖株生長情形 ------------------- 36
圖八、氯化鈉(NaCl)處理下wild-type與TSB1轉殖株生長情形 --------------- 37
圖九、乾旱處理下wild-type與TSB1轉殖株生長情形 ----------------------- 39
圖十、北方點墨法分析 TSB1轉殖株與未轉殖wild type各種基因表現情形----- 40
圖十一、色氨酸含量分析圖--------------------------------------------- 42
圖十二、HIAA含量分析圖----------------------------------------------- 44
表一、Wild type 和TSB1轉殖株在處理或不處理100 μM CuSo4之
內生性色氨酸和HIAA的含量表------------------------------------- 46參考文獻 尤曉莉 (2002) 小葉浮萍之色胺酸合成酶β鏈基因選殖與色胺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
劉秀玲 (2004) 於逆境下酵母菌褪黑激素合成酶之功能分析。國立中央大學碩士論文
Azmitia EC (2001) Modern views on an ancient chemical: serotonin effects on cell proliferation, maturation, and apoptosis. Brain Res Bull 56:413-424
Barczak AJ, Zhao J, Pruitt KD, Last RL (1995) 5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana. Genetics 140:303-313
Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159-182
Edlund A, Eklof S, Sundberg B, Moritz T, Sandberg G (1995) A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues. Plant Physiol 108:1043-1047
Foreman J, Demidchik V, Bothwell JH, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JD, Davies JM, Dolan L (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422:442-446
Harumi T, Matsushima S (2000) Separation and assay methods for melatonin and its precursors. J Chromatogr B Biomed Sci Appl 747:95-110
Kirch HH, Nair A, Bartels D (2001) Novel ABA- and dehydration-inducible aldehyde dehydrogenase genes isolated from the resurrection plant Craterostigma plantagineum and Arabidopsis thaliana. Plant J 28:555-567
Muller A, Weiler EW (2000) Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana. Planta 211:855-863
Murphy A, Taiz L (1995) Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance. Plant Physiol 109:945-954
Radwanski ER, Barczak AJ, Last RL (1996) Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana. Mol Gen Genet 253:353-361
Radwanski ER, Last RL (1995) Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. Plant Cell 7:921-934
Rao MV, Dubey PS (1993) Response of hydrogen peroxide scavenging system in two soybean cultivars exposed to SO(2): experimental evidence for the detoxification of SO(2) by enhanced H2O2 scavenging components. Environ Pollut 82:99-105
Reiter RJ, Tan DX, Manchester LC, Lopez-Burillo S, Sainz RM, Mayo JC (2003) Melatonin: detoxification of oxygen and nitrogen-based toxic reactants. Adv Exp Med Biol 527:539-548
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
Shikanai T, Muller-Moule P, Munekage Y, Niyogi KK, Pilon M (2003) PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. Plant Cell 15:1333-1346
Siraki AG, O'Brien PJ (2002) Prooxidant activity of free radicals derived from phenol-containing neurotransmitters. Toxicology 177:81-90
Sriprang R, Hayashi M, Ono H, Takagi M, Hirata K, Murooka Y (2003) Enhanced accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase. Appl Environ Microbiol 69:1791-1796
Sunkar R, Bartels D, Kirch HH (2003) Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance. Plant J 35:452-464
van Hoof NA, Hassinen VH, Hakvoort HW, Ballintijn KF, Schat H, Verkleij JA, Ernst WH, Karenlampi SO, Tervahauta AI (2001) Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene. Plant Physiol 126:1519-1526
Vijayalaxmi, Reiter RJ, Tan DX, Herman TS, Thomas CR, Jr. (2004) Melatonin as a radioprotective agent: a review. Int J Radiat Oncol Biol Phys 59:639-653
Xie G, Forst C, Bonner C, Jensen RA (2002) Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants. Genome Biol 3:RESEARCH0004
Zhao J, Last RL (1995) Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana. J Biol Chem 270:6081-6087
Zhao J, Williams CC, Last RL (1998) Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor. Plant Cell 10:359-370指導教授 詹明才、黃雪莉
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