根癌农杆菌介导ubiC基因转化甘蓝的研究
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摘要
为获得转ubiC基因甘蓝，对植物基因工程载体的构建、甘蓝的组织培养以及基因转化条件进行了研究。通过PCR方法从大肠杆菌基因组中扩增得到了ubiC基因，扩增产物克隆到pUC118载体，转化大肠杆菌JM109。提取质粒，连接重组到经相同酶切的Ti质粒pBI121上，构建了植物表达载体pBI121-ubiC，并将其转入根癌农杆菌LBA4404中。同时，采用甘蓝无菌苗的下胚轴为外植体，以MS为基本培养基，分别用直接分化再生系统和愈伤再生系统诱导不定芽的分化，建立了高效稳定的再生系统，不定芽分化频率达80％以上。利用根癌农杆菌介导将ubiC基因转入甘蓝，通过预培养、侵染、共培养、脱菌培养、筛选转化体等步骤，最后获得抗性愈伤组织。
The constuction of plant expression vector,tissue culture of cabbage and gene transformation conditions were studied to obtain ubiC transformants.ubiC gene was obtained from the Escherichia coli.by PCR amplication and was ligated to pUC118 vector. Subsequently, the recombined plasmid was transformed to the strain JM109 of Escherichia coli., which was picked up and was ligated to pBI121. As a result, the plant expression vector of pBI121-ubiC was constructed successfully. The hypocotyl segments of cabbage were used as the explants and cultured on a MS medium supplemented with plant hormone, which was established a cabbage regenerated system.The differentiation rate of adventitious buds was above 80%. In this study,ubiC gene was transformed to cabbage through Agrobacterium mediated system. And kanamycin-resistant callus tissue was obtained by pre-culture, infection co-culture and selection.
The constuction of plant expression vector,tissue culture of cabbage and gene transformation conditions were studied to obtain ubiC transformants.ubiC gene was obtained from the Escherichia coli.by PCR amplication and was ligated to pUC118 vector. Subsequently, the recombined plasmid was transformed to the strain JM109 of Escherichia coli., which was picked up and was ligated to pBI121. As a result, the plant expression vector of pBI121-ubiC was constructed successfully. The hypocotyl segments of cabbage were used as the explants and cultured on a MS medium supplemented with plant hormone, which was established a cabbage regenerated system.The differentiation rate of adventitious buds was above 80%. In this study,ubiC gene was transformed to cabbage through Agrobacterium mediated system. And kanamycin-resistant callus tissue was obtained by pre-culture, infection co-culture and selection.
引文
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[39] Yazaki K, Tanaka H, Matsuoka et al. Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transfomants. Plant Cell Report, 1998,18:214-219
[40] Sommer S, Khle A, Yazaki K, Shimomura K, et al.Genetic engineering of shikonin biosynthesis. Hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene. Plant Molcular Biology, 1999, 39:683-693
[41] Boehm R, Sommer S, Li S-M, et al. Genetic engineering of shikonin biosynthesis: Expression of the bacterial ubiA gene in Lithospermum erythrorhizon. Plant&Cell Physiology, 2000, 41:911-919
[42] 徐克勋．精细有机化工原料中间体手册．北京：化学工业出版社，1998
[43] 矢崎一史.Secondary metabolic engineering toward plant molecular breeding for pathogen and insect resistance. 1998,56(9): 39-40
[44] Sankawa U, Ebizuka Y, Miyazaki et al. Antitumor activity of shikonin and its derivatives.Chemical Pharmaceutical Bulletin, 1977,25:2392-2395
[45] 郭勇．生物制药技术．北京：中国轻工业出版社，2000
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[47] 韩迎山．植物细胞培养生产天然产物的研究．生物化学与生物物理进展，1989，16(1)：19-22
[48] Fujita Y, Hara Y, Suga C, et al. Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. A new medium for the produxtion of shikonin derivatives. Plant Cell Report, 1981, 1:61-63
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[51] 周立刚，郑光植，王世林等．滇紫草愈伤组织培养与紫草素产生．云南植物研究，1992，13(3)：315-320
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[55] 贺红，冼建春，肖省娥等．溪黄草离体培养和快速繁殖．中草药，2001，32(3)：255-256
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[2] James C. Global review of commercialized transgenic crops. Ithaca NY: International service for the Acquisition of Agro-Biotech Application, 1998
[3] 倪德祥，邓志龙．转基因植物及应用现状．上海农业学报，1993，9(1)：91-96
[4] 薜庆善．体外培养的原理与技术．科学出版社，2001，999-1019
[5] 伊华林．我国经济植物组织培养概况．湖北农学院学报，2001，21(3)：279-284．
[6] 李子银，胡会庆．农杆菌介导的植物遗传转化发展．生物工程进展，1998，18(1)：22-26
[7] 方宏筠，李大力，王关林等．转豇豆胰蛋白酶抑制剂抗虫甘蓝植株的获得．植物学报，1997，39(10)：940-945
[8] Berthomieu P, Jouanin L. Transformation of rapid cycling cabbage (Brassica oleracea var. capitata) with Agrobacterium tumefaciens. Plant Cell Report, 1992,11:334-338
[9] Horeau N. A comparative study of in vitro shoot regeneration from cotyledonand root explants of four varieties of Brassica oleracea L. Current Science, 1988,57(24): 1349-1351
[10] 王关林，方宏筠．植物基因工程原理与技术．北京：科学出版社，1998
[11] 官春云，李栒，李文彬．芸薹属植物的生物工程．长沙：湖南科学技术出版社，1999，68-76
[12] 程振东，卫志明，许智宏．芸苔属作物的遗传转化．植物生理学通讯，1992，8(3)：161-164
[13] 薛红卫，卫志明，许智宏．通过PEG法转化甘蓝获得转基因植株．植物学报，1997，39(1)：28-33
[14] Ding. Development of insect-resistant transgenic cauliflower plants expressing he trypsin inhibitor gene isolated from local sweet potato. Plant Cell Report.1998, 17:854-860.
[15] 姜翌．甘蓝转生长素基因系自交后代叶球和根系的发育特征．西北植物学报．1998，18(2)：223-228．
[16] 张七仙，敖光明．根癌农杆菌介导的甘蓝高效稳定的遗传转化系统的建立及对CpTI基因转化的研究．农业生物技术，2001，9(1)：72-75
[17] 李大力，王丹，杨树林等．水蛭肽基因的克隆及其转化甘蓝．生物技术，2002，12(3)：4-5
[18] Ohlrogge J. Plant metabolic engineering: Are we ready for phase two? Current Opinion in Plant Biology, 1999,2:121-122
[19] 李育阳．基因表达技术．北京：科学出版社，2001
[20] Franken E, Teuschel U, Hain. Recombinant proteins from transgenic plants. Current Opinion in Biotechnology, 1999,13:1090-1093
[21] Owen MRL, Pen J. Transgenic Planst:A Production System for Industrial and Pharmaceutical Proteins. John Wiley & Son Inc.,1996,261-272
[22] Mor TS, Gomez-Lim MA, Palmer KE. edible vaccines-a concept coming of age. Trends in Microbiology, 1998,6(11):449-453
[23] Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature, 1989,342:76-78
[24] Steinbuchel A, Fuchtenbusch B. Bacterial and other biological systems for polyester production. Trends in Biotechnology, 1998,16:419-427
[25] Hooykaas GMS, Hooykaas PJJ, Schilperoort RA. Expression of Ti plastid genes in monocotyledonous plants infected with Agrobacterium turnefaciens. Nature,1984, 311:763-764
[26] 陶余敏．农杆菌转化单子叶植物的可能性及问题．植物生理学通讯，1992，28(6)：402-406
[27] Rainri DM, Botino P, Gordon MP, et al. Agrobacterium-mediated transformation of rice. Biotechnology, 1990,8:33-38
[28] Graves ACF, Goldman SL. The transformation of Zea mays seedling with Agrobacterium tumefaciens:detection of T-DNA specific enzyme activities. Plant Molecular Biology, 1986,7:43-50
[29] Chen M, Fry JE, Pang SI, et al. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiology, 1997,115:971-980
[30] Cohen A, Meredith CR Agrobacterium-mediated transformation of Lilium Acta Horticulturae, 1992,325:611-618
[31] Hernalsteens JP, Toong LP, Schell J, et al. An Agrobacterium transformation cell culture from the monocot Asparagus officinalis. EMBO J, 1984,3:3039-3401
[32] Graves ACF, Goldman SL. Agrobacteriurn tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA encoded genes. J Bacteriol, 1987,169:1745-1746
[33] 傅荣昭，孙勇如，贾士荣．植物遗传转化技术手册．中国科学技术出版社，1994
[34] Nichols Bp, Green TM. Cloning and sequencing of Escherichia coli.: ubiC and urification of chorismate lyase. J Bacterial, 1992, 174:7352-7359
[35] Siebert M, Bechthold A, Melzer M, et al. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli. FEBS Letters, 1992,307(3):347-350
[36] Siebert M, Severin K, Heide L. Formation of 4-hydroxybenzoate in Escherichia coli.: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase. Microbiology, 1994, 140:897-904
[37] Siebert M, Sommer S, Li S-M, et al. Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco. Plant Physiology,1996,112: 811-819
[38] Sommer S, Heide L. Expression of bacterial chorismate pyruvate-lyase in tobacco: Evidence for the presence of chorismate in the cytosol. Plant & Cell Physiology, 1998, 39:1240-1244
[39] Yazaki K, Tanaka H, Matsuoka et al. Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transfomants. Plant Cell Report, 1998,18:214-219
[40] Sommer S, Khle A, Yazaki K, Shimomura K, et al.Genetic engineering of shikonin biosynthesis. Hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene. Plant Molcular Biology, 1999, 39:683-693
[41] Boehm R, Sommer S, Li S-M, et al. Genetic engineering of shikonin biosynthesis: Expression of the bacterial ubiA gene in Lithospermum erythrorhizon. Plant&Cell Physiology, 2000, 41:911-919
[42] 徐克勋．精细有机化工原料中间体手册．北京：化学工业出版社，1998
[43] 矢崎一史.Secondary metabolic engineering toward plant molecular breeding for pathogen and insect resistance. 1998,56(9): 39-40
[44] Sankawa U, Ebizuka Y, Miyazaki et al. Antitumor activity of shikonin and its derivatives.Chemical Pharmaceutical Bulletin, 1977,25:2392-2395
[45] 郭勇．生物制药技术．北京：中国轻工业出版社，2000
[46] 宁文，曹日强．硬紫草细胞悬浮培养和紫草宁极其衍生物的形成．生物工程学报，1994，10(1)：76-80
[47] 韩迎山．植物细胞培养生产天然产物的研究．生物化学与生物物理进展，1989，16(1)：19-22
[48] Fujita Y, Hara Y, Suga C, et al. Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. A new medium for the produxtion of shikonin derivatives. Plant Cell Report, 1981, 1:61-63
[49] 徐敏源，刘骅，陈彤等．理化因素对新疆紫草培养细胞紫草宁形成的影响．浙江农业大学学报(自然科学版)．1998(24)：34-36
[50] 于树宏，李玲，何晓明．利用细胞培养技术提高次生代谢物的产量．生物学通报，1998，33(7)：40-42
[51] 周立刚，郑光植，王世林等．滇紫草愈伤组织培养与紫草素产生．云南植物研究，1992，13(3)：315-320
[52] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning .2nd. New York, Cold Spring Harbor Laboratory, 1989
[53] 奥斯伯，金斯顿，塞德曼等．精编分子生物学实验指南．北京：科学出版社，1998
[54] 袁晓东，李晶泉，王红伟等．超忠实性PCR用DNA聚合酶．遗传．1999，21(6)：47-48
[55] 贺红，冼建春，肖省娥等．溪黄草离体培养和快速繁殖．中草药，2001，32(3)：255-256
[56] 崔渤．经济植物的组织培养与快速繁殖．北京：农业出版社，1985，84-88
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