(100.25.42.117) 您好!臺灣時間:2021/04/21 17:29
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:呂俊賢
研究生(外文):Chun-Hsien Lu
論文名稱:植物熱休克蛋白質HSP101的應用性研究
論文名稱(外文):Application of Plant Heat Shock Protein, HSP101
指導教授:張清俊張清俊引用關係
指導教授(外文):Ching-Chun Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:112
中文關鍵詞:篩選標誌植物耐熱性熱休克蛋白質101
外文關鍵詞:selection markerHSP101plant thermotolerance
相關次數:
  • 被引用被引用:3
  • 點閱點閱:110
  • 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:0
  • 收藏至我的研究室書目清單書目收藏:0
植物中具有一受熱誘導而大量表現之蛋白質HSP101,其主要功能為促使變性後聚集在一起的蛋白質分開,使之可以在其他的熱休克蛋白質分子幫忙下重新摺疊或進行分解,因此為植物耐熱所必須。我們利用CaMV 35S啟動子,將水稻的hsp101基因以農桿菌基因轉殖法轉入菸草,並持續性地大量表現於菸草之中,以期望能獲得高耐熱性的菸草植株。利用PCR與南方墨點法的偵測方式,得到五株轉殖植株。以西方墨點法分析其T1子代之HSP101表現量之後,觀察到其中兩株為高表現量之轉殖植株。經由耐熱性測試後,我們發現於菸草中持續性大量表現HSP101,確實可以使菸草在不需要熱誘導的情形下,提升耐熱性以抵抗突然來襲的熱緊迫環境。
利用HSP101的抗熱性質,我們試著發展一套新的植物基因轉殖之篩選策略,也就是利用hsp101基因當做篩選標誌,搭配適當的溫度條件作為篩選壓力,可以在利用農桿菌做為基因轉殖方法,進行菸草葉圓盤基因轉殖過程中,篩選出轉殖成功之癒傷組織。同時也初步觀察到利用熱所篩選出的癒傷組織,有較高之外源基因的表現量。應用熱篩選策略可以免除使用抗生素的花費,並解決抗藥性基因汙染環境的疑慮,所篩選出的基因轉殖植物預期地將同時具有較高的基礎耐熱性質。
Among heat-shock proteins, hsp101 appears to play a major and specific role in acquired thermotolerance of plant. The function of HSP101 is to promote the resolubilization of protein aggregates, and reactivate protein activity in cooperation with other molecular chaperones as a refolding machine under heat stress. Therefore, HSP101 is essential for plant survival under severe heat stress. We had transformed tobacco with rice hsp101 gene through Agrobacterium-mediated transformation. Stable integration and expression of the transgene into tobacco genome was demonstrated by PCR, Southern and western blot analyses. We found that transgenic plants with overexpressing rice HSP101 confer thermotolerant ability.
The selection markers currently used for plant transformation have raised some concerns such as environmental safety, antibiotic resistance and food allergenicity. To take advantage of the function of HSP101, we are trying to develop hsp101 gene as a selection marker, with high temperature as the selection pressure during the process of plant transformation. We demonstrated that transformants could be obtained by this novel selection method after Agrobacterium-mediated transformation. Our preliminary results show that transformants selected with high temperature expressing higher GUS level than traditional kanamycin selection method. We expect that transgenic plants selected through this method will confer thermotolerant ability.
第一章、前言…………………………………………………………………1
第二章、文獻探討……………………………………………………………3
1. 植物的耐熱性是由多因子所控制………………………………………3
1.1 膜脂質飽和度……………………………………………………………3
1.2 活性氧化物的代謝………………………………………………………4
1.3 熱休克蛋白質……………………………………………………………4
2. HSP101為植物獲得性耐熱性所必須……………………………………9
3. HSP101具有轉譯活化子的性質可提升轉譯效率………………………11
4. 篩選標誌…………………………………………………………………12
4.1 植物基因轉殖常用的篩選標誌………………………………………13
4.2 目前篩選標誌所帶來的問題…………………………………………13
4.3 解決問題的方式有移除篩選標誌與開發新的篩選標誌……………14
4.3.1 移除基因轉殖植物中的篩選標誌…………………………………14
4.3.2 新篩選標誌的開發與利用…………………………………………17
第三章、材料與方法………………………………………………………20
1. 實驗材料…………………………………………………………………20
2. 實驗方法…………………………………………………………………20
2.1 構築植物基因轉殖載體………………………………………………20
2.2 菸草基因轉殖…………………………………………………………24
2.3 製備兔子抗HSP101免疫血清…………………………………………25
2.4 南方墨點法……………………………………………………………26
2.5 西方墨點法……………………………………………………………27
2.6 調查未轉殖之菸草的耐熱性…………………………………………27
2.7 分析基因轉殖菸草的耐熱性…………………………………………28
2.8 構築熱篩選試驗載體…………………………………………………28
2.9 調查未轉殖之菸草葉圓盤的耐熱性…………………………………30
2.10 熱篩選測試……………………………………………………………31
2.11 大腸桿菌耐熱測試……………………………………………………31
第四章、結果………………………………………………………………33
1. 獲得兔子抗水稻HSP101之免疫血清……………………………………33
2. 獲得持續性大量表現水稻HSP101之基因轉殖菸草品系………………33
3. 未轉殖菸草耐熱性之調查………………………………………………34
4. Oshsp101菸草轉殖植株之基礎耐熱性測試結果………………………35
5. 未轉殖菸草葉圓盤耐熱性之調查………………………………………35
6. 熱篩選測試………………………………………………………………36
7. 大腸桿菌耐熱測試………………………………………………………38
第五章、討論………………………………………………………………39
1. 於菸草中持續性大量表現HSP101可抵抗突然來襲的熱緊迫環境……39
2. 於大腸桿菌中誘導大量表現HSP101對其耐熱性並無顯著的影響……39
3. 熱篩選策略可避免抗藥性基因之使用並使轉殖植株具有耐熱性質…40
4. 熱篩選策略可篩選出有價值的外源基因高表現量之轉殖植株………41
5. 熱篩選策略同時可提供外源基因提升轉譯效率之效果………………41
第六章、參考文獻…………………………………………………………43
Agarwal M, Sahi C, Katiyar-Agarwal S, Agarwal S, Young T, Gallie DR, Sharma VM, Ganesan K, Grover A (2003) Molecular characterization of rice hsp101: complementation of yeast hsp104 mutation by disaggregation of protein granules and differential expression in indica and japonica rice types. Plant Mol Biol 51: 543-553.

Al-Khatib K, Paulsen, G.M. (1999) High-temperature effects on photosynthetic processes in temperate and tropical cereals. Crop Sci. 39: 119-125.

Bernstein JA, Bernstein IL, Bucchini L, Goldman LR, Hamilton RG, Lehrer S, Rubin C, Sampson HA (2003) Clinical and laboratory investigation of allergy to genetically modified foods. Environ Health Perspect 111: 1114-1121.

Celerin M, Gilpin AA, Schisler NJ, Ivanov AG, Miskiewicz E, Krol M, Laudenbach DE (1998) ClpB in a cyanobacterium: predicted structure, phylogenetic relationships, and regulation by light and temperature. J Bacteriol 180: 5173-5182.

Chen DH, Ronald, P.C. (1999) A rapid DNA minipreparation method suitable for AFLP and other PCR applications. Plant Mol Biol Reporter 17: 53-57.

Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci U S A 88: 10558-10562.

Daniell H (1999) GM crops: public perception and scientific solutions. Trends Plant Sci 4: 467-469.

Daniell H, Muthukumar B, Lee SB (2001) Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet 39: 109-116.

Daniell H, Wiebe PO, Millan AF (2001) Antibiotic-free chloroplast genetic engineering - an environmentally friendly approach. Trends Plant Sci 6: 237-239.

De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19: 71-74.

Ebmeier A, Allison L, Cerutti H, Clemente T (2004) Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218: 751-758.

Erikson B, Hertzberg M, Nasholm T (2004) A conditional marker gene allowing both positive and negative selection in plants. Nat Biotechnol 22: 455-458.

Fitter AH, Hay, R.K.M., (1987) Environmental Physiology of Plants. Academic Press, London.

Gallie DR (2002) The 5'-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res 30: 3401-3411.

Gallie DR, Fortner D, Peng J, Puthoff D (2002) ATP-dependent hexameric assembly of the heat shock protein Hsp101 involves multiple interaction domains and a functional C-proximal nucleotide-binding domain. J Biol Chem 277: 39617-39626.

Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18: 1151-1155.

Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94: 73-82.

Goldsbrough AP, Lastrella, C.N., Yoder, J.I., (1993) Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology 11: 1286-1292.

Gurley WB (2000) HSP101: a key component for the acquisition of thermotolerance in plants. Plant Cell 12: 457-460.

Haldrup A, Petersen SG, Okkels FT (1998) The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol Biol 37: 287-296.

Harndahl U, Hall RB, Osteryoung KW, Vierling E, Bornman JF, Sundby C (1999) The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress. Cell Stress Chaperones 4: 129-138.

Heifetz PB (2000) Genetic engineering of the chloroplast. Biochimie 82: 655-666.

Hohn B, Levy AA, Puchta H (2001) Elimination of selection markers from transgenic plants. Curr Opin Biotechnol 12: 139-143.

Hong SW, Vierling E (2000) Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc Natl Acad Sci U S A 97: 4392-4397.

Joersbo M, Donaldson, I., Kreiberg, J., Petersen, S.G., Brundstedt, J., Okkels, F.T., (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4: 111-117.

Katiyar-Agarwal S, Agarwal M, Grover A (2003) Heat-tolerant basmati rice engineered by over-expression of hsp101. Plant Mol Biol 51: 677-686.

Kim BH, Schoffl F (2002) Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins. J Exp Bot 53: 371-375.

Klaus S.M., et al. (2004) Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat Biotechnol 22: 225-229.

Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10: 165-174.

Koscianska E, Wypijewski K (2001) Electroporated intact BY-2 tobacco culture cells as a model of transient expression study. Acta Biochim Pol 48: 657-661.

Kunkel T, Niu QW, Chan YS, Chua NH (1999) Inducible isopentenyl transferase as a high-efficiency marker for plant transformation. Nat Biotechnol 17: 916-919.

Lee JH, Schoffl F (1996) An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic Arabidopsis thaliana. Mol Gen Genet 252: 11-19.

Lee YR, Nagao RT, Key JL (1994) A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance. Plant Cell 6: 1889-1897.

Ling J, Wells DR, Tanguay RL, Dickey LF, Thompson WF, Gallie DR (2000) Heat shock protein HSP101 binds to the Fed-1 internal light regulator y element and mediates its high translational activity. Plant Cell 12: 1213-1227.

Link, V, Sinha AK, Vashista P, Hofmann MG, Proels RK, Ehness R, Roitsch T (2002) A heat-activated MAP kinase in tomato: a possible regulator of the heat stress response. FEBS Lett 531: 179-183.

Lubaretz O, Zur Nieden U (2002) Accumulation of plant small heat-stress proteins in storage organs. Planta 215: 220-228.

Lutz Nover KB, Pascal Doring, Shravan Kumar Mishra, Arnab Ganguli, and Klaus-Dieter Scharf (2001) Arabidopsis and the Hsf world: how many heat stress transcription factors do we need? Cell Stress Chaperones 6: 177-189.

Ma JK (2000) Genes, greens, and vaccines. Nat Biotechnol 18: 1141-1142.

Maestri E, Klueva N, Perrotta C, Gulli M, Nguyen HT, Marmiroli N (2002) Molecular genetics of heat tolerance and heat shock proteins in cereals. Plant Mol Biol 48: 667-681.

Malik MK, Slovin JP, Hwang CH, Zimmerman JL (1999) Modified expression of a carrot small heat shock protein gene, hsp17. 7, results in increased or decreased thermotolerancedouble dagger. Plant J 20: 89-99.

Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107: 193-232.

Mishra SK, Tripp J, Winkelhaus S, Tschiersch B, Theres K, Nover L, Scharf KD (2002) In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato. Genes Dev 16: 1555-1567.

Morimoto RI, Tissieres, A., Georgopoulos, C., (1994) The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Murakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K (2000) Trienoic fatty acids and plant tolerance of high temperature. Science 287: 476-479.

Nieto-Sotelo J, Kannan KB, Martinez LM, Segal C (1999) Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp100 protein homologue. Gene 230: 187-195.

Nieto-Sotelo J, Martinez LM, Ponce G, Cassab GI, Alagon A, Meeley RB, Ribaut JM, Yang R (2002) Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth. Plant Cell 14: 1621-1633.

Panchuk, II, Volkov RA, Schoffl F (2002) Heat stress- and heat shock transcription factor-dependent expression and activity of ascorbate peroxidase in Arabidopsis. Plant Physiol 129: 838-853.

Plater ML, Goode D, Crabbe MJ (1996) Effects of site-directed mutations on the chaperone-like activity of alphaB-crystallin. J Biol Chem 271: 28558-28566.

Queitsch C, Hong SW, Vierling E, Lindquist S (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12: 479-492.

Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234: 49-59.

Sanmiya K, Suzuki K, Egawa Y, Shono M (2004) Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants. FEBS Lett 557: 265-268.

Schirmer EC, Glover JR, Singer MA, Lindquist S (1996) HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem Sci 21: 289-296.

Shi WM, Muramoto Y, Ueda A, Takabe T (2001) Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana. Gene 273: 23-27.

Stone P (2001) The effects of heat stress on cereal yield and quality. In: A.S. Basra(Ed.) Crop Responses and Analysis to Temperature Stress,. Food Products Press, Binghamton, NY, pp.243-291.

Sung DY, Guy CL (2003) Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences. Plant Physiol 132: 979-987.

Sung DY, Kaplan F, Lee KJ, Guy CL (2003) Acquired tolerance to temperature extremes. Trends Plant Sci 8: 179-187.

Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31: 1174-1179.

Wehmeyer N, Vierling E (2000) The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance. Plant Physiol 122: 1099-1108.

Wells DR, Tanguay RL, Le H, Gallie DR (1998) HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. Genes Dev 12: 3236-3251.

Wenck A, Pugieux C, Turner M, Dunn M, Stacy C, Tiozzo A, Dunder E, van Grinsven E, Khan R, Sigareva M, Wang WC, Reed J, Drayton P, Oliver D, Trafford H, Legris G, Rushton H, Tayab S, Launis K, Chang YF, Chen DF, Melchers L (2003) Reef-coral proteins as visual, non-destructive reporters for plant transformation. Plant Cell Rep 22: 244-251.

Wu FS, Feng, T.Y. (1999) Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells. Plant Cell Reports 18: 381-386.

Yoder JI, Goldsbrough, A.P., (1994) Transformation systems for generating marker-free transgenic plants. Biotechnology 12: 263-267.

You SJ, Liau CH, Huang HE, Feng TY, Prasad V, Hsiao HH, Lu JC, Chan MT (2003) Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation. Planta 217: 60-65.

Zhu YJ, Agbayani R, Moore PH (2004) Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation. Plant Cell Rep 22: 660-667.

Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18: 442-445.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
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