Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/29189
標題: D型胺基酸氧化酵素基因(daao)作為甘藍葉綠體基因轉殖的篩選標誌基因之研究
Using D-Amino Acid Oxidase Gene (daao) as the Selectable Marker Gene for Cabbage Chloroplast Transformation
作者: 朱宛茹
Chu, Wan-Ru
關鍵字: D-amino acid oxidase
D型胺基酸氧化酵素
selectable marker gene
cabbage
篩選標誌基因
甘藍
出版社: 園藝學系所
引用: 小松謙一、杉浦弘吉、松田昭生、山本敬三。1988。Production of 7-aminocephalosporanic acid and its derivatives. Jpn. Kokai Tokyo Koho (in Japanese) JP 6307488. 李易輯。2009。D型胺基酸氧化酵素基因(daao)作為甘藍之農桿菌基因轉殖法的篩選標誌基因之研究。國立中興大學園藝學研究所碩士論文。 林志輝、鄭榮宗、陳良築。2004。葉綠體基因轉殖。植物基因轉殖之原理與應用-第九章:111-120。植物生物技術教學資源中心主編。 林依萱。2007。共同轉移Bt基因到結球白菜葉綠體之研究。國立中興大學園藝學研究所碩士論文。 許家言。2006。小白菜組織培養再生與基因轉殖之研究。國立中興大學園藝學研究所博士論文。 陳立德。2005。基因槍法共同轉殖基因到甘藍葉綠體之研究。國立中興大學園藝學研究所碩士論文。 張隆武。1996。D-胺基酸氧化酵素基因、蘇力菌殺蟲晶體蛋白基因及抗凍蛋白基因轉移至甘藍與結球白菜之研究。國立中興大學園藝研究所碩士論文。 曾夢蛟、劉程煒。2004。甘藍之基因轉殖技術。植物基因轉殖與分子檢測技術-第二章:十字花科作物-第三節: p.57-74。植物生物技術教學資源中心主編。 黃群益。1999。共同轉移蘇力菌殺蟲晶體蛋白、D型氨基酸氧化酵素、轉酮醇酵素、熱休克蛋白等基因至甘藍之研究。國立中興大學園藝研究所碩士論文。 詹富智、林靜宜。2006。無篩選標示基因轉基因植物之開發及其最新發展。農業生技產業季刊(5): 25-29。財團法人生物技術開發中心。 劉程煒。2003。水稻農桿菌基因轉殖系統與甘藍及水稻葉綠體基因轉殖系統之建立及應用。國立中興大學園藝研究所博士論文。 Alonso, J., J. L. Barredo, B. Diez, E. Mellado, F. Salto, J. L. Garcia, and E. Cortes. 1998. D-amino acid oxidase gene from Rhodotorula gracilis(Rhodospoidium toruloides) ATCC 26217. Microbiol. 144: 1095-1101. Bennett, P. M., C. T. Livesey, D. Nathwani, D. S. Reeves, J. R. Saunders, and R. Wise. 2004. An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J. Antimicrob. Chemother. 53: 418-431. Bock, R. and M. S. Khan. 2004. Taming plastids for a green future. Trends Biotechnol. 22(6): 311-317. Bogorad, L. 2000. Engineering chloroplasts: An alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18(6): 257-263. Daniell, H and S. Varma. 1998. Chloroplast-transgenic plant: panacea-no! gene containment-yes! Nat. Biotechnol. 16: 602. Daniell, H. 1999. GM crops: Public perception and scientific solution. Trends Plant Science. 4: 467-469. Daniell, H., B. Muthukumar and S. B. Lee. 2001. Marker free transgenic plants: engineering the chloroplast genome without use of antibiotic selection. Curr. Genet. 39: 109-116. Daniell, H., M. S. Khan, and L. Allison. 2002. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7(2): 84-91. Daniell, H., S. Kumar and N. Dufourmantel. 2005. Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology. 23(5): 238-245 D’Aniello A., G. D’Onofrio, M. Pischetola, G. D’Aniello, A. Vetere, L. Petrucellina, G. H. Fishefl. 1993. Biological role of D-amino acid oxidase and D-aspartate oxidase. J. Biochemistry 36: 26941-26949. Darbanil, B., A. Eimanifar, C. N. Stewart, Jr, and W. N. Camargo. 2007. Methods to produce marker-free transgenic plants. Biotechnol. J. 2: 83-90. Ebinuma, H., K. Sugita, E. Matsunaga, and M. Yamakado. 1997. Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc. Natl. Acad. Sci. USA 94: 2117-2121. Erikson, O., M. Hertzberg, and T. Nasholm. 2004. A conditional marker gene allowing both positive and negative selection in plants. Nat. Biotechnology 22: 455-458. FDA. 1992. Statement of policy: foods derived from new plant varieties. US. Fed. Regist. 57(104): 22984-23005. Fernández-San Millán, A., A. Mingo-Castel, M. Miller, and H. Daniell. 2003. A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol. J. 1: 71-79. Fisher, N., O. Stampacchia, K. Redding and J. D. Rochaix. 1996. Selectable marker recycling in the chloroplast. Mol. Gen Genet. 251(3): 373-380. Haldrup, A., S. G. Petersen, and P. F. Okkels. 1998. Positive selection: A plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Rep. 18: 76-81. Heifetz, P. B. 2000. Genetic engineering of the chloroplast. Biochimie. 82: 655-666. Hou, B. K., Y. H. Zhou, L. H. Wan, Z. L. Zhang, G. F. Shen, Z. H. Chen and Z. M. Hu. 2003. Chloroplast transformation in oilseed rape. Transgenic Research 12: 111-114. Inaba, Y., K. Mizukami, N . Hamada-Sato, T. Kobayashi, C . Imada, and E. Watanabe. 2003. Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase. Biosens. Bioelectron. 19: 423-431. James, C. 2008. Global status of commercialized biotech/GM crops: 2008. ISAAA Brief. 39. Kok, E. J., H. P. Noteborn, and H. A. Kuiper. 1994. Food safety assessment of marker genes in transgenic crops. Trends Food Sci. Tech. 5(9): 294-298. König, A. 2003. A framework for designing transgenic crops-science, safety and citizen''s concerns. Nat. Biotechnol. 21(11): 1274-1279. Komari, T., Y. Hiei, Y. Saito, N. Murai, T. Kumashiro, 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. Komarnytsky, S., A. Gaume, A. Garvey, N. Borisjuk, and I. Raskin. 2004. A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots. Plant Cell Rep. 22: 765-773. Konno, R. and Yasumura, Y. 1992. D-amino acid oxidase and its physiological function. Int. J. Biochem. 24: 519-524 Koreen, R., A. Peremarti, S. Gómez-Galera, S. Naqvi, M. Moralejo, P. Muñoz, T. Capell, and P. Christou. 2007. Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgen. Res. 16(3): 261-280. Lim, H. T., H. J. Lee, E. J. Park and Y. N. Song. 2000. Factors influencing on the high efficient plant regeneration and genetic transformation in Brassica vegetable crops. 3rd ISHS International Symopsium on Brassicas-12th Crucifer Genetics Workshop (help at Horticulture Research International),Wellesbourne, cv 35 9EF, UK. 5th-9th,Sep., 2000. Liu, Cheng-Wei, Chin-Chang Lin, Jeremy J. W. Chen, and Menq-Jiau Tseng. 2007. Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep. 26(10): 1733-1744. Liu, Cheng-Wei, Chin-Chung Lin, Jinn-Chin Yiu, Jeremy J. W. Chen and Menq-Jiau Tseng. 2008. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor. Appl. Genet. 117: 75-88. Lutz, K. D., J. E. Knapp, and P. Maliga. 2001. Expression of bar in the plastid genome confers herbicide. Plant Physiol. 125: 1585-1590. MacKenzie, D. and Morven M. 2002. Who’s afraid of GM feeds? Feed Mix. 10(3): 16-19. Maliga, P.. 2002 Engineering the plastid genome of higher plants. Curr. Opin. Plant Biol. 5: 164-172. Maliga, P. 2003. Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21(1): 20-28. Mattevi, A., M. A. Vanoni, F. Todone, M. Rizz, A. Teplyakov, A. Coda, M. Bolognesi and B. Curti. 1996. Crystal structure of D-amino acid oxidase:a case of active site mirrorimage convergent evolution with flavocytochrome b2. Proc. Natl. Acad. Sci. USA 93: 7496-9501. Miki, B. and S. Mchugh. 2004. Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol. 107(3): 193-232. Pilone, M. S. 2000. D-amino acid oxidase: new findings. Cell. Mol. Life. Sci. 57: 1732-1747. Reddy, V. S., S. Leelavathi, A. Selvapandiyan, R. Raman, F., V. Giovanni, R. Shukla and K. Bhatnagar. 2002. Analysis of Chloroplast transformed tobacco plant with cryIa5 under rice psbA transcriptional elements reveal high level expression of Bt toxim without imposing yield penalty and stable inheritance of transplastome. Molecular Breeding. 9: 259-269. Rogers, S. G., H. Klee, M. Byren, R. B. Horsch, and R. T. Fraey. 1988. Improved vector for plant transformation: expression cassette vectors and new selectable marker. Method in Enzymology. Academic Press. Sentheshanmuganathan, S. and W. J. Nickerson. 1962. Transaminase and D-amino acid oxidase of Trigonopsis variabilis. J. gem. Microbiol. 27:465-471. Serizawa. N., K. Nakagawa, T. Haneishi, S. Kamimura, and A. Naito. 1980. Enzymatic conversion of cephamycin C by D-amino acid oxidase from Trigonopsis variabilis. J Antibiot 33(6):585-90. Su, N., Y. M. Wu, B. Y. Sun and G. F. Shen. 2001. A new way of plant genetic engineering chloroplast transformation. Biotechnology Information 4: 9-13. Sugita, K., E. Matsunaga, and H. Ebinuma. 1999. Effective selection system for generating marker-free rransgenic plants independent of sexual crossing. Plant Cell Rep. 18:941-947. Sundar, I. K. and N. Sakthivel. 2008. Advances in selectable marker genes for plant transformation. J. Plant Physiol. 165(16): 1698-1716. Srivastava, V. and D.W. Ow. 2004. Marker-free site-specific gene integration in plants. Trends Biotechnol. 22, 627–629. Szwajcer-Dey, E., J. R. Miller, S. Kovacevic and K. Mosbach. 1990. Characterization of a D-amino acid oxidase with high activity against cephalosporin C from the yeast from Trigonopsis variabilis. Biochem. International. 20: 1169-1178. Tishkov, V. I. and Khoronenkova S. V. 2005. D-amino acid oxidase: structure, catalytic mechanism, and practical application. Biochemistry (Mosc.) 70: 40-54. Trost, E. M. and L. Fischer. 2002. Minimization of by-product formation during D-amino acid oxidase catalyzed racemate resolution of D/L-amino acids. J. Mol. Catal. B:Enzymatic 19-20: 189-195. Umhau, S., L. Pollegioni, G. Molla, K. Diederichs, W. Wolfram, M. S. Pilone, and S. Ghisla. 2000. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. Proc. Natl. Acad. Sci. USA 97: 12463-12468. Yang, Z. H., H. Jin, P. Plaha, B. T. Woong, G. B. Jiang, C. Woo, H. D. Ynu, Y. P. Lim and H. Y. Lee. 2004. An improved plant regeneration protocol using cotyledonary explants from inbred lin of Chinensis cabbage (Brassica rapa ssp. pekinensis). J. Plant Biotechnology. 6(4): 235-239. Ye, G. N., P. T. J. Hajdukiewicz, D. Broyles, D. Rodriguez, C. W. Xu, N. Nehra and J. M. Staub. 2001. Plastid-expressed 5-enolpyruvylshikimate-3- phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J. 25(3): 261-270.
摘要: 在作物基因轉殖過程中,利用篩選標誌基因(selectable marker genes)可 以快速且有效率的區分出轉殖與非轉殖細胞。然而在獲得轉殖植株後,篩選標誌基因便無特殊用途,並可能產生環境生態以及生物安全性等方面的疑慮。因此,目前被廣泛應用於轉殖作物篩選上的耐抗生素及抗除草劑標誌基因,將面臨減少或無法使用的命運。植物葉綠體基因轉殖具有增加轉殖基因大量表現、不會造成基因污染、基因靜默及插入位置效應、及較細胞核基因轉移穩定等優點;因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因,因此對非抗抗生素基因的篩選標誌基因的需求,更具必要性及急迫性。 D型胺基酸氧化酵素(D-amino acid oxidase, DAAO)是一種以FAD做為輔基的黃素蛋白,可以催化D型胺基酸的脫氨氧化反應。由於大部份D型組態的胺基酸,不能被植物代謝,且易對植物產生毒害。因此本研究以甘藍為材料,建立利用daao基因作為葉綠體基因轉殖之篩選標誌基因的系統。 本研究是將以分離自三角酵母(Trigonopsis variabilis)中的daao基因及耐抗生素之aada基因為篩選標誌基因、並以egfp及gus為報導基因,所構築之甘藍葉綠體基因轉殖載體,藉由基因槍法轉殖至甘藍的葉綠體。本研究之目的為:(一)、探討以 daao 基因作為甘藍葉綠體基因轉殖的篩選標誌之可行性,(二)、比較daao基因及aada基因作為甘藍葉綠體基因轉殖的篩選標誌之優劣。 將已構築之pMT91-ED(egfp-daao)、pMT91-GD(gus-daao)、pMT91-EDA (egfp-daao-aada)、以及pMT91-GDA (gus-daao-aada)等四種載體,利用基因槍法將其轟擊至`初秋´甘藍之下胚軸或葉片的葉綠體。再生培殖體經200~500 ppm D-alanine或20~50 ppm spectinomycin持續篩選,可以獲得再生植株。轉殖植株葉片以PCR及RT-PCR分析之結果顯示,daao及aada篩選標誌基因及egfp及gus報導基因已存在於轉殖甘藍之基因組中,並表現其mRNA。GFP綠色螢光及GUS活性染色分析的結果也顯示egfp及gus等報導基因亦可在轉殖甘藍中順利表現綠色螢光及藍色反應。本研究結果顯示以daao基因來作為甘藍之葉綠體基因轉殖的篩選標誌基因是可行的。本研究已初步完成建立以D-alanine來篩選甘藍之葉綠體基因轉殖系統。由於甘藍培殖體對spectinomycin之忍受毒害濃度的高敏感性,因此發展D-alanine的篩選系統,在植株外觀的篩選比Spectinomycin篩選的優點更顯而易見。
In the process of transgenic crops, the use of selectable marker genes can be quickly and efficiently distinguish transgenic and non-transgenic cells. However, the antibiotic and herbicide resistance marker genes have generated a number of environmental, ecological, and biological aspects of safety concerns. Therefore, the development of antibiotic-free selectable marker genes is of outstanding importance for commercialization of transgenic crops. Expression of foreign genes via plastid genomes not only dramatically enhances the level of expression and absence of epigenetic effects, but also prevents out cross of the introduced foreign genes via pollen grains. Thus, transformation of the plastid genome is a new and attractive alternative to engineering the nuclear genome. D-amino acid oxidase (DAAO) catalyzes the oxidative deamination of D-amino acids to produce the corresponding keto acids, NH3 and H2O2. Most of the D-configuration amino acids cannot be metabolized by plants, and deterioration the growth of plants. In this study, the possibility of using D-amino acid oxidase gene (daao) as an antibiotic-free selectable marker gene in cabbage plastid gene transformation is studied. The objectives of this study are to develop and establish the daao gene as the antibiotic-free selectable marker for cabbage plastid transformation, and to compare the transformation efficiency between using aada (aminoglycoside-3’- adenyl transferase) and daao genes as the selectable marker. A set of plasmid vectors that contain daao gene cloned from the yeast (Trigonopsis variabilis) as antibiotic-free selectable marker was constructed by Dr. M. T. Yang’s laboratory. One of the two plant reporter genes, egfp and gus, was chosen as target gene in the constructed vectors in this study. The selectable marker (daao, aada) and reporter genes (egfp, gus) were cloned as a cassette. All of the constructed genes were driven by the Prrn promoter and terminated by T-psbA terminator. Four cabbage plastid transformation vectors, pMT91-ED (egfp-daao), pMT91-GD (gus-daao), pMT91-EDA (egfp-daao-aada), and pMT91-GDA (gus-daao-aada) were transferred into the hypocotyls or leaves of cabbage chloroplast via particle gun mediated transformation. The regenerated plantlets were induced and selected by 200 to 500 ppm D-alanine or 20 to 50 ppm spectinomycin。 The results of PCR and RT-PCR analysis indicated that transformed genes (daao, aada, gus, and egfp) were integrated into the plastid genome of transplastomic cabbage plants, and expressed its mRNA. GFP fluorescence and gus histochemical staining analyses showed the emission of green fluorescence and blue-color reaction were presented in the egfp or gus gene transformed transplastomic cabbage plants, respectively. Our results indicated that the system of using daao gene as selection marker for plastid transformation, which has several advantages over the conventional used aada gene, offers new possibilities for non-antibiotics selectable marker in commercially important crops.
URI: http://hdl.handle.net/11455/29189
其他識別: U0005-2008201015102400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2107201014143400
Appears in Collections:園藝學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.