摘要
十字花科(Cruifrae)芸薹属(Brassica genus)包含了一些重要的油料作物和蔬菜作物,其中有6个物种被广泛种植。甘蓝型油菜(Brassica.napus L.)是全球继大豆和棕榈油之后第三大油料作物。随着人类需求的增加和新科技手段的不断涌现,油菜品种改良正呈多元化发展,植物转基因技术在油菜遗传改良中广泛应用,推动了油菜遗传育种工程技术进行一次新的革命。转基因技术已经成为向油菜导入新型外源基因的重要手段,转基因抗除草剂油菜、抗虫油菜、高油脂含量油菜、油菜新型不育系等已大规模投入商业化生产,抗病、抗环境逆境胁迫(干旱、盐碱、高温和低温)等新型油菜育种材料的创制已成为重要研究课题。
本研究主要目标是,立足西北地区油菜品种,筛选最佳转化受体,建立农杆菌介导的油菜高效转化体系,获得CMO和BADH抗盐耐旱基因的油菜转化体,为利用基因工程改良油菜奠定基础。
研究了甘蓝型油菜花茎外植体高效诱导不定芽的再生体系,结果表明:以含有2,4-D(0.5~1.0mg/L)的培养基对外植体进行预培养,以及在分化培养基中加入AgNO_3(2~6mg/L),可显著降低外植体褐化坏死的频率,提高了不定芽的发生频率及其再生能力;6BA(2.5mg/L)与NAA(0.1mg/L)配合使用有利于提高不定芽发生频率;再生的不定芽90%可长根。
研究了油菜未成熟胚胎离体培养条件下的几种再生方式。结果表明,油菜幼胚离体培养以三种方式实现再生:①幼胚早熟萌发,在茎顶端分生组织区依次产生多个额外子叶,表明幼胚外植体仍保持着胚性发育特点;②子叶近轴端和下胚轴表面直接产生类胚结构,继代后部分类胚结构可发育成体细胞胚,其它则由于无茎顶端分生组织,最终不能形成完整植株;③外植体细胞脱分化形成愈伤组织,继代后胚性愈伤组织产生单生的体细胞胚,最终可发育成完整的小植株。2,4—D是影响外植体再生方式的重要因素,它抑制了幼胚早熟萌发。在无2,4—D的培养基上,幼胚早熟萌发,主要以第一种方式实现再生;在2,4—D诱导下,外植体主要以第二或第三种方式再生。不同基因型间在外植体再生方式和体胚诱导率上存在差异。
以30个甘蓝型油菜品种(系)的子叶和下胚轴为外植体,研究了子叶和下胚轴的再生能力和再生方式,以及在抗菌素(羧苄青霉素和头孢霉素)和筛选剂(卡那霉素)胁迫下,各基因型子叶和下胚轴外植体的再生能力。结果表明,油菜子叶和下胚轴外植体主
The genus Brassica includes many economically important vegetable, condiment and oilseed crops. Within this genus, Brassica napus L. is an important oil crop, ranking third only to soybean and palm oil in global production. Canola oil is widely used as a cooking oil, salad oil, and for the production of margarine. There are several studies on Canola transformation with respect to the introduction of various new traits such as modified oil composition, herbicide tolerance, altered protein composition and insect resistance. The key objective of this work was to develop an efficient Agrobacrerium-mediated transformation system for Canola to facilitate crop improvement through genetic engineering. Transformation has been carried out using various explants, such as stem internodes, stem segments, cotyledonary petioles and hypocotyl segments. The efficiency of A. tumefticiens mediated transformation technique in oilseed rape is influenced by cultivar, donor plant age and explant type. An increase in the transformation efficiencies is desirable in order to decrease the amount of resources needed to produce transgenic plants, and to potentially provide a higher baseline for subsequent transformation of other canola varieties.Excised peduncle segments of three Brassica napus L. cultivars were used to optimize conditions for high-frequency shoot regeneration. The presence of 2,4-D(0.5-1.0 mg/L) in pre-cultured medium markedly enhanced the subsequent frequency of responding explants and the number of shoots/responding explant. The use of AgNO_3 was a prerequisite for efficient shoot regeneration due to it strongly increased morphogenesis and decrease the frequency of explants death. BA (2.5mg/L) in combination with a low concentration of NAA(0.1mg/L) gave optimum shoot bud differentiation. The regenerated shoots could be rooted at a frequency of 95%.The well-established tissue culture system to regenerate plants from a single transformed cell is one of the main prerequisites for crop plant genetic transformation. Somatic embryos provide a tool for efficient transformation because of their excellent ability to regenerate into full plants. This paper describes the different patterns of plant regeneration from excised immature embryo of Brassica napus L.. When immature embryos were placed on MS
hormone-free medium with 1% sucrose, they precociously germinated and produced 2-5 extra cotyledons. This indicated that immature embryos explant contain some cells that retain embryogenic competence. While immature embryos were cultured on MS media supplemented with various concentration of 2,4-D and 3% sucrose, somatic embryos and embryo-like structures were obtained by directly or indirectly. Upon transfer to MS hormone-free medium, most of the somatic embryos developed into plantlets, while the most of embryo-like structures could not due to lacking defined shoot meristems. However, the response to tissue culture is highly genotype dependent.Although genetic transformation protocols are now available for most of the major crop species, the protocols are applicable within each species to only a few genotypes/varieties that regenerate in vitro at high frequency. Cotyledons and hypocotyls of 30 different varieties of Brassica napus from the present varieties, used in the North-west of China, and plant breeding lines were tested for their regeneration response. The result indicated that regeneration in B. napus is highly variable and genotype specific. The frequency of regeneration among the varieties(or lines) are verity different , from 27.5% to 100% in Cotyledon, and from 33.4 to 100%. While under the stress of Cb(600mg/L) or Cef(500mg/L), which usually used as selector agent, the frequency of regeneration were decreased in different extent, indicating that both Cb and Cef can inhibit the regeneration of shoots. Two varieties were excluded as the acceptor of transformation because of the far low frequency of regeneration under the stress of the selective agent. Kanamycin was confirmed as an efficient selective agent for the transformation of Brassica napus, and the maximum transformation efficiency was very different among the different genotype of B. napus.Cotyledon and hypocotyl explants of 9 varieties (lines) of B. napus, from above test, were used to investigate the competence of regeneration of these varieties under the stress of Agrobacterium, for evaluating them, and establish an efficient Agrobacterium-based transformation experiments. The result indicated that the 9 varieties could be divided into three types according to the response to the infection with Agrobacterium: resistant type (R);supper susceptive type (SS) and middle type (M). The R type could not form regeneration shoot owing to hypersusceptibility response at the cut of the explant;the SS type could not also form regeneration shoot owing to propagate so far on the surfer of the explant;and only the M type could form regeneration shoot, but as low frequency. The frequency of regeneration shoot can be enhanced by optimizing the co-cultivate condition: using filter paper as co-cultivate support, result in not only avoiding vitrification of explant, but also prolong the time of co-cultivate, thus the cells (include transformed cells) on the explant could have enough time to regenerate. The use of AgNO3 (3~6mg/L) was a prerequisite for
efficient shoot regeneration under selective conditions, but the mount is dependented on genotype. The hypocotyl explant was more easy death infected by Agrobacterium than cotyledon explant.Glycinebetaine is an important quaternary ammonium compound that is produced in response to salt and other osmotic stresses in many organisms. In several higher plants, the synthesis of glycinebetaine requires the catalysis of choline monooxygenase (CMO) protein and betaine aldehyde dehydrogenase (BADH) protein. We transformed the BADH gene, cloned from Atriplex hortensis and controlled by two 35S promoters of the cauliflower mosaic virus, into two Brassica napus lines of breeding, PI 37 and 487, using Agrobacterium tumefaciens strain AGL1 carrying a binary vector pBin438, with cotyledon and hypocotyl regeneration system. Polymerase chain reaction analyses demonstrated that the BADH gene had integrated into the genome of B. napus. Transgenic B. napus plants showed the levels of activity of BADH were similar to or higher than wild-type plants leaves after exposure to salt stress. Observations on rooting development under the salt stress with concentrations up to 0.5% suggested that the transgenic plants exhibited tolerance to salt stress than wild-type plants. Several transformed lines resistance to Km, from CM9-transformation, were achieved from two lines of Brassica napus. The transgenic plants exhibited tolerance to salt stress than wild-type plants.In plant a transformation method to produce transgenic Brassica napus plants. The procedure included Agrobacterium-mediated inoculation of plants at various development stages along with a vacuum infiltration step. The flowering stage appeared to be the most receptive stage for transformation and production of transgenic plants. The floral-dip method for Agrobacterium-medisAed transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. Application of Agrobacterium to pollen recipient plants yielded transformants. Agrobacterium strains with T-DNA carrying BADH and CMO gene under the control of 35S. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.
引文
[1] 刘后利主编.油菜的遗传和育种[M].上海:上海科学技术出版社,1985
[2] 傅廷栋.中国油菜生产和品种改良的现状与前景[J].安徽农学通报,2000,6(1):2-8
[3] Cardoza V, Stewart C. N. Agrobacterium-mediated Transformation of Canola, Curtis I.S. (ed.), Transgenic Crops of the World -essential Protocols[M]. Netherlands: Kluwer Academic. 2004, 379-387
[4] Cardoza V, Stewart CN. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants[J].Plant Cell Rep, 2003,21: 599-604
[5] 官春云.油菜转基因育种研究进展[J].中国工程科学,2002,vol.(8)4:34-39
[6] 官春云.油菜的转基因育种[J].中国油料作物学报,2005,27(1):97-103
[7] Damgaard O, Jensen LH, Rasmussen OS. Agrobacterium tumefeciens-mediated of Brassica napus winter cultivars[J]. Transgenic Research, 1997, 6: 279-288.
[8] Khanl M R, Rashi H, Ansar M, Chaudry Z. High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus) [J]. Plant Cell, Tissue and Organ Culture, 2003, 75: 223-231
[9] Bhojwani S S, Razdan M K. Studies in plant science 5, plant tissue culture: theory and practice[M]. A revised Edition. Amsterdam: Elsevier Science, 1996
[10] Halperin W. In vitro embryogenesis: Some historical issues and unresolved problems, In: Thorpe T (ed.) In vitro Embryogenesis in Plants[M]. Dordrecht: Kluwer Academic Publishers, 1995. 1-16.
[11] Gautheret R.J. Plant tissue culture: A history[J]. Bet Mag. 1983, 96: 393-410
[12] 韩碧文主编.植物生长与分化[M].北京:中国农业大学出版社,2002
[13] Kuriyama H, Fukuda H. Regulation of Tracheary Element Differentiation[J]. J Plant Growth Regul, 2000, 20: 35-51
[14] 王关林,方宏筠主编.植物基因工程[M].北京:科学出版社,2002
[15] 肖尊安主编.植物生物技术[M].北京:化学工业出版社,2005
[16] 谢从华,柳俊主编.植物细胞工程[M].北京:高等教育出版社,2005
[17] M. Sugiyama, Organogenesis in vitro[J]. Curr. Opin. Plant Biol, 1999, 2: 61-64
[18] 许智宏.植物生物技术[M].上海:上海科学技术出版社,1998
[19] 黄坚钦.植物细胞的分化与脱分化[J].浙江林学院学报,2001,18(1):89-92
[20] 崔克明.植物细胞分化的启动控制和分化过程的阶段性[J].生命科学,1997,9(2):49-54
[21] Tomal Werner, Vaclav Motyka, Miroslav Strnad, and Thomas Schmulling. Regulation of plant growth by cytokinin[J]. PNAS, 2001, 98: 10487-10492
[22] David W. S, Mok, Machteld C Mok. Cytokinin Metabolism and Axtion, Annu. Rev. Plant Physiol[J]. Plant Mol. Biol,2001,52:89-118
[23] Georg Haberer and Joseph J. Kieber. Cytokinins. New Insights into a Classic Phytohormone[J].Plant Physiol, 2002, 128: 354-362
[24] Negrel J, Javelle F, Paynot M. Biochemical Basis of Resistance of Tobacco Callus Tissue Cultures to Hydroxyphenylethylamines[J]. Plant Physiol. 1993, 103: 329-334
[25] Abe M, Katsumata H, Komeda Y, etc. Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[J]. Development. 2003,130: 635-643.
[26] Takahashi, Y., Kuroda, H., Tanaka, T, Machida, Y., Takebe, I. and Nagata, T. Isolation of an auxin-regulated gene cDNA expressed during the transition from GO to S phase in tobacco mesophyli protoplasts[J]. Proc. Natl. Acad. Sci. USA, 1989, 86, 9279—9283
[27] Blanc G, Michaux-Ferriere N, Teisson C, Larder L, Carron MP. Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis[J]. Plant Cell, Tissue and Organ Culture, 1999, 59, 103-112
[28] 黄璐,卫志明.不同基因型玉米的再生能力和胚性与非胚性愈伤组织DNA的差异[J].植物生理学报,1999,25(4):332-338
[29] 王海波,魏景芳,葛亚新,等.小麦愈伤组织状态调控与原生质体培养[J].中国农业科学 1996,29(6):8-14
[30] 曹清波,余毓君.小麦幼胚愈伤组织的细胞类型、功能与内生胚状体[J].华中农业大学学报, 1992, 11(1):26-30
[31] 葛台明,余毓君.小麦Ⅱ型胚性愈伤组织的获得及培养特性研究[J].湖北民族学院学报(自然科学版)1997,15(6):1-4
[32] 汪丽虹,崔凯荣,白志根,王亚馥.石刁柏非胚性细胞和胚性细胞的超微结构及ATP酶的定位[J].兰州大学学报(自然科学版),1996,32(4):123-127
[33] 王亚馥,崔凯荣,汪丽虹,王仑山.小麦体细胞胚发生的超微结构研究[J].植物学报,1994. 36(6):418—422
[34] 范吕发,郭骁才,贾敬芬,牛天堂.植物细胞中淀粉代谢与离体形态发生途径决定的关系[J].华北农学报 2000,15(4):52-5
[35] Kende H. Zeevaart J AD. The Five "Classical" Plant Hormones[J]. The Plant Cell. 1997, 9: 1 197-1210
[36] Zimmerman J L Somatic Embryogenesis: A Model for. Early Development in Higher Plants[J]. The Plant Cell, 1993, 5: 1411-1423
[37] Williams E.G. and G. Maheswaran, somatic embryogenesis: factors influence co-ordinated behaviour of cells as an embryogenic group[J]. Annals of Botany, 1986, 57: 443-462
[38] 周俊彦.植物体细胞在组织培养中产生的胚状体Ⅱ.影响植物胚状体发生和发育的因素[J].植物生理学报,1982,8(1):91-99.
[39] Ainsley, P. J. & A. R Aryan, 1998: Efficient plant regeneration system for immature embryos of triticale (Triticosecale Wittmack) [J]. Plant Growth Regul. 24: 23-30
[40] 崔凯荣,戴若兰主编.植物体细胞胚发生的分子生物学[M].北京:科学出版社,2000
[41] 王萍,王罡,季静,吴颖.大豆体细胞胚增殖保存与萌发植株体系的建立[J].植物学报,2004,46 (2): 154-158
[42] 陈金慧,施季森,诸葛强等.植物体细胞胚胎发生机理的研究进展[J].南京林业大学学报(自然科学版), 2003,27(1): 75-80
[43] 李杉,邢更妹,崔凯荣,王亚馥.植物体细胞胚发生中ATP酶活性时空分布动态与内源激素的变化[J].植物学通报2001,18(3):308-317
[44] 侯丙凯,腾世云.小麦体细胞胚胎发生过程的研究[J].山东大学学报(自然科学版),1994, 29(4):459-462
[45] Sharp W R, Sondahl M R, Caldas L S, etc. The physiology of in vitro asexual embryogenesis [J]. Hort Rev, 1980, 2: 268-310
[46] Elisa Pilion, Mario Terzi, Barbara Baldan, etc. A protocol for obtaining embryogenic cell lines from Arabidopsis[J]. The Plant Journal,1996, 9(4): 573-577
[47] Tzfira T, Rhee Y, Chen M H, etc. Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls[J]. Annu Rev Microbiol, 2000, 54: 187-219
[48] Tzfira T, Vaidya M, Citovsky V. Involvement of targeted proteolysis in plant genetic transformation by Agrobaeterium[J]. Nature, 2004, 431: 87-92
[49] Tzfira T, Citovsky V. From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobaeterium-plant cell interaction[J]. Mol Plant Pathol, 2000, 1: 201-212
[50] Schr6der G, Lanka E. The mating pair formation system of conjugative plasmids versatile secretion machinery for transfer of proteins and DNA[J]. Plasmid, 2005,54:1-25
[51] Thomas C M, Nielsen M. Mechanisms Of, And Barriers To, Horizontal Gene Transfer Between Bacteria[J]. Nature Reviews Microbiology, 2005, (3): 711-721
[52] Christie P J. Type Ⅳ secretion: the Agrobacterium VirB/D4 and related conjugation systems[J]. Biochimiea et Biophysica Acta 1694. 2004, 219-234
[53] Gelvin SB. Gene exchange by design[J]. Nature, 2005, 433: 583-584
[54] Gelvin S B. Agrobacterium and plant genes involved in T-DNA transfer and integration[J]. Annu Rev Plant Physiol Plant Mol Biol, 2000, 51: 223-256.
[55] Gelvin S B. The introduction and expression of transgenes in plants[J]. Curr Opin Biotechnol, 1998, 9: 227-232
[56] Gelvin SB Agrobaeterium-Mediated Plant Transformation: the Biology behind the "Gene-Jockeying" Tool[J]. Microbioi. Mol. Biol. Rev. 2003;67(1): 16-37
[57] Tzfira T, Citovsky V. The Agrobactedum-Plant Cell Interaction. Taking Biology Lessons from a Bug[J]. Plant Physiol. 2003, 133: 943-947
[58] Binns, A.N. T-DNA of Agrobacterium tumefaciens: 25 years and counting[J]. Trends Plant Sci. 2002. 7: 231-233.
[59] Zupan J, Muth T R, Draper O, etc. The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights[J]. Plant J, 2000, 23: 11-28.
[60] Bundock P, den Dulk-Ras A, Beijersbergen A, etc. Transkingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J]. EMBO J. 1995, 14: 3206-3214
[61] Piers KL, Heath JD, Liang X, etc. Agrobacterium tumefaciens-mediated transformation of yeast[J]. Proc. Natl. Acad. Sci. USA 1996, 1613-1618
[62] Roberts RL, Metz M, Monks DE, etc. Purine synthesis and increased Agrobacterium tumefaciens transformation of yeast and plants[J]. Proc. Natl. Acad. Sci. USA 2003, 6634-6639
[63] de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen A.G. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol[J]. 1998, 839-842
[64] Rho HS, Kang S, Lee YH. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea[J]. Mol. Cells 2001, 407-411
[65] Pelczar P, Knick V, Gomez D, etc. Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells[J]. EMBO reports, 2004, 5 (6): 632-637
[66] Kunik T, Tzfira T, Kapulnik Y, etc. Genetic transformation of HeLa cells by Agrobacterium[J]. Proc Natl Acad Sci USA, 2001, 98: 1871-1876
[67] 李卫,董文,周菲,郭光沁,郑国铝.参与在农杆菌介导遗传转化过程中的植物因子研究进展, 中国生物工程杂志,2003,23(12)62-67
[68] 李卫,郭光沁,郑国铝.根癌农杆菌介导遗传转化研究的若干新进展[J].科学通报,2000,45 (8):798-807
[69] 陶均,李玲.农杆菌转化的分子生物学[J].植物生理学通讯,2002,38(6):639-644
[70] Eseobar MA, Dandekar AM. Agrobacterium tumefaciens as an agent of disease[J]. Trends Plant Sci. 2003, 380-386
[71] Wood DW, Setubal JC, Kaul R, etc. The genome of the natural genetic engineer Agrobacterium tumefaciens C58[J]. Science, 2001: 2317-2323
[72] Nair G R, Liu Z, Binns AN. Reexamining the Role of the Accessory Plasmid pAtC58 in the Virulence of Agrobacterium tumefaciens Strain C58[J]. Plant Physiol. 2003, 133: 989-999
[73] Douglas CJ, Halperin W, Nester EW. Agrobacterium tumefaciens mutants affected in attachment to plant cells[J]. J. Bacteriol. 1982, 1265-1275
[74] Zorreguieta A, Geremia RA, Cavaignac S, etc. Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene[J]. Mol. Plant Microbe Interact. 1988, 121-127
[75] Cangelosi G.A, Martinetti G, Leigh JA, etc. Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1, 2-glucan[J]. J. Bacteriol. 1989, 1609-1615
[76] Winans S C. Two way chemical signaling in Agrobacterium-plant interactions[J]. Microbiol Rev, 1992, 56: 12-31
[77] Stachel S E, Zambryski P. VirA and virD control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens[J]. Cell, 1986, 46: 325-333.
[78] Kumar S V, Rajam MV. Polyamines enhance Agrobacterium tumefaciens vir gene induction and T-DNA transfer[J]. Plant Science, 2005, 168: 475-480
[79] Zhu Y, Nam J, Humara JM, Gelvin SB, etc. Identification of Arabidopsis rat mutants[J]. Plant Physiol. 2003, 494-505
[80] Zhu Y, Nam J, Carpita NC, etc. Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene[J]. Plant Physiol. 2003, 1000-1010
[81] Tzfira T, Citovsky V. Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium[J]. Trends in Cell Biology, 2002, 12: 121-129
[82] Pansegrau W, Schoumacher F, Hohn B, etc. Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation[J]. Proc Natl Acad Sci USA, 1993, 90: 11538-11542.
[83] Mysore KS, Bassuner B, Deng XB, etc. Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration[J]. Mol Plant-Microbe Interact, 1998, 11: 668-683
[84] Ziemienowicz A, Tinland B, Bryant J, etc. Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro[J]. Mol Cell Biol, 2000, 20: 6317-6322.
[85] Bako L, Umeda M, Tiburcio AF, etc. The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants[J]. PNAS 2003, 10108-10113
[86] Michielse B, Ram FJ, Hooykaas JJ, etc. Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures. Journal of Bacteriology[J]. 2004, 186: 2038-2045.
[87] Dombek P, Ream W. Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2[J]. J Bacteriol, 1997, 179: 1165-1173.
[88] Duckely M, Hohn B. The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang of T-DNA transfer[J]. FEMS Microbiology Letters, 2003, 223: 1-6.
[89] Deng W, Chen L, Peng WT, Liang X, Sekiguchi S etc. VirEl is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium[J]. Mol Microbiol, 1999, 31: 1795-1807.
[90] Duckely M, Oomen C, Axthelm F, Vetc. The VirElVirE2 complex ofAgrobacterium tumefaciens interacts with single-stranded DNA and forms channels[J]. Molecular Microbiology. 2005, 58 (4): 1130-1142
[91] Vergunst, A C, van Lier M CM, den Dulk-Ras A, Hooykaas, P JJ. Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE 1 [J]. Plant Physiol. 2003, 133: 978-988
[92] Li J, Wolf S G, Elbaum M, Tzfira T. Exploring cargo transport mechanics in the type Ⅳ secretion systems[J]. TRENDS in Microbiology, 2005, 13:295-28
[93] Cascales E, Christie PJ. The versatile bacterial type Ⅳ secretion systems[J]. Nature Review Microbiology. 2003, (1): 137-150
[94] Christie PJ. Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria[J]. J Bacterioi. 1997, 179: 3085-3094
[95] Vergunst AC, Schrammeijer B, den Dulk-Ras A, etc. VirB/D4-dependent protein translocation from Agrobacterium into plant cells[J]. Science 2000, 979-982
[96] Zupan J, Ward D, Zambryski P. Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaeiens to plant cells[J]. Curr Opin Microbiol, 1998, 1: 649-655
[97] Li J, Krichevsky A, Vaidya M, Tzfira T, etc. Uncoupling of the functions of the Arabidopsis VIPI protein in transient and stable plant genetic transformation by Agrobacterium[J]. Proc Natl Acad Sci USA, 2005, 102: 5733-5738
[98] Tao Y M, Rao P K, Bhattacharjee S, Gelvin S B. Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2[J]. Proc Natl Acad Sci USA, 2004, 101: 5164-5169.
[99] Hwang HH, Gelvin SB. Plant Proteins That Interact with VirB2, the Agrobacterium tumefaciens Pilin Protein, Mediate Plant Transformation[J].The Plant Cell, 2004, 16:3148-3167
[100] Ziemienowicz A, Merkle T, Schoumacher F, Hohn B, Rossi L. Import of Agrobacterium T-DNA into plant nuclei. Two distinct functions of VirD2 and VirE2 proteins[J]. Plant Cell, 2001, 13:369-384
[101] Citovsky V, Kapelnikov A, Oliel S, etc. Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro[J]. J Biol Chem, 2004, 279:29528-29533
[102] Tzfira T, Vaidya M, and Citovsky V. Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIPI[J]. PNAS, 2002;99(16): 10435-10440.
[103] Tzfira T, Vaidya M, Citovsky V. VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobaeterium infectivity[J]. EMBO J. 2001, 20, 3596-3607
[104] Lacroix B, Vaidya M, Tzfira T, etc. The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation[J]. The EMBO Journal, 2005, 24, (2): 428-437
[105] Schrammeijer B, den Duik-Ras A, Vergunst AC, etc. Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3[J]. Nucleic Acids Res. 2003, 860-868
[106] Tinland B. The integration of T-DNA into plant genomes[J]. Trend Plant Sci, 1996, 1:178-1841
[107] Schrammeijer B, Risseeuw E, Pansegrau W, etc. Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skpl protein[J]. Curr. Biol. 2001, 258-262
[108] Tzfira T, Li J, Laeroix B etc. Agrobaeterium T-DNA integration: molecules and models[J]. TRENDS in Genetics, 2004, 20, (8): 375-383
[109] Rossi L, Hohn B, Tinland B. Integration of complete transferred DNA units are dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens[J]. Proc Natl Acad Sci USA, 1996, 93: 126-130
[110] Nam J, Matthysse A G, Gelvin S B. Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration[J]. Plant Cell, 1997, 9: 317-333.
[111] Chilton M D, Que Q. Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration[J]. Plant Physiol, 2003, 133: 956-965.
[112] Kumar S, Fladung M. Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration[J]. Plant J, 2002, 31:543-551
[113] 杨继芳,刘明,安利佳.T-DNA整合的研究进展[J].遗传,2004,26(6):991-996
[114] Dudas, A. and Chovanec, M. DNA double-strand break repair by homologous recombination[J]. Murat Res, 2004, 566, 131-167
[115] Gouka RJ, Gerk C, Hooykaas PJ, etc. Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination[J]. Nat. Biotechnol. 1999, 598-601
[116] de Buck S, Jacobs A, van Montagu M, Depicker A. The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration[J]. Plant J, 1999, 20: 295-304.
[117] Brunaud V, Balzergue S, Dubreucq B, etc. T-DNA integration into the Arabidopsis genomc depends on sequences ofpre-insertion sites[J]. EMBO Reports 2002, 3 (12): 1152-1157
[118] Tzfira T, Frankman LR, Vaidya M, Citovsky V. Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates[J]. Plant Physiol. 2003. 133:1011-1023
[119] de Neve M, de Buck S, Jacobs A, Van Montagu M, Depicker A. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs[J]. Plant J, 1997,11: 15-29
[120] van A H, Bundock P, Hooykaas PJJ. Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration[J]. The EMBO Journal, 2001,20, 22:6550-6558
[121] Friesner J, Britt A B. Ku80-and DNA ligase Ⅳ-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration[J]. Plant J, 2003, 34: 427-440
[122] Yi HC Mysorey K S, Gelvin S B. Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation[J]. The Plant Journal, 2002, 32: 285-298.
[123] Mysore KS, Kumar CTR, Gelvin SB. Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation[J]. Plant J, 2000, 21: 9-16.
[124] Mysore K S, Nam J, Gelvin S B. An Arabidopsis historic H2A mutant is deficient in Agrobacterium T-DNA integration[J]. Proc Natl Acad Sci USA, 2000, 97: 948-953.
[125] Kohli A, Leech M, Vain P, etc. Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots[J]. Proc Natl Acad Sci USA, 1998, 95:7203-7208
[126] Ditt RF, Nester EW, Comai L. Plant gene expression response to Agrobacterium tumefaciens[J]. Proc. Natl. Acad. Sci. USA 2001, 10954-10959
[127] Dirt R F, Nester E, Comai L. The plant cell defense and Agrobacterium tumefaciens[J]. FEMS Microbiology Letters, 2005, 247(2): 207-213
[128] Parniske M, Schmidt PE, Kosch K, etc. Plant defense responses of host plants with determinate nodules induced by EPS-defective exoB mutants of Bradyrhizobium japonicum[J]. Mol. Plant Microbe Interact. 1994, 631-638
[129] Gelvin S B. Improving plant genetic engineering by manipulating the host. Trends in Biotechnology, 2003, 21: 95-98.
[130] Felix G, Duran J.D, Voiko S, Boiler T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin[J]. Plant J. 1999, 265-276
[131] Veena J, Hongmei DRW, Geivin S B. Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression[J]. The Plant Journal, 2003, 35 (2): 219-236
[132] Zhu J k. Plant salt tolerance[J]. Trends in Plant sci, 2001, 6:66-71
[133] McCue KF, Hanson AD. Drought and salt tolerance: towards understanding and application[J]. Trends Biotech, 1990, 8: 358-362
[134] Jain RK, Selvaraj G. Molecular genetic improvement of salt tolerance in Plants[J]. Biotechnol Annu Rev, 1997, 3: 245-267
[135] Sakamoto A, Murata N. The Use of Bacterial Choline Oxidase, a Glycinebetaine-Synthesizing Enzyme, to Create Stress-Resistant Transgenic Plants[J]. Plant Physiology, 2001;125(1): 180-188
[136] Sakaraoto A, Alia Norio Murata. Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold[J]. Plant Mol Biol, 1998, 38: 1011-1019
[137] Korstee GJ. The aerobicdecom position of choline by microorganisms[J]. Arch Microbiol, 1970, 71: 235-244
[138] Hanson AD, Rhodes D. ~(14)C Tracer evidence for synthesis of choline and betaine via phosphoryl base intermediates in salinized sugarbeet leaves[J]. Plant Physiol, 1983, 71: 692-700
[139] Russell BL, Rathinasabapathi B, Hanson AD. Osmotic stress induces expression of choline monooxygenase in sugar beet and Amaranth[J]. Plant Physiol, 1998, 116:859-865
[140] Brouquisse R, Weigei P. Evidence for a ferredoxin dependent choline monooxygenase from spina chchloroplaststroma[J]. Plant Physiol, 1989, 90:322-329
[141] Selvaraj G, Jain RK, Olson DJ, etc. Glycinebetaine in oilseed canola and flax leaves: detection by liquid chromatography-continuous flow secondary ion mass spectrometry (LC-CFSIMS) [J]. Phytochemistry, 1995, 38: 1143-1146
[142] 赵博生,衣艳君,刘家尧.外源甜菜碱对干旱/盐胁迫下的小麦幼出生长和光合功能的改善[J].植物学通报,2001,18(3):378-380
[143] 梁峥,骆爱玲.甜菜碱和甜菜碱合成酶[J].植物生理学通讯,1995,31(1):1-8
[144] 侯彩霞,汤章城.细胞相容性物质的生理功能及其作用机制[J].植物生理学通讯,1999,35(1):1-7
[145] 骆爱玲,赵原,李浴春等.甜菜碱脱氢酶在细胞中的定位[J].植物生理学报,1995,21(2):117-122
[146] 刘凤华,郭岩,谷冬梅等.转甜菜碱醛脱氢酶基因植物的耐盐性研究[J].遗传学报,1997, 24(3):54-5841
[147] 林栖凤,李冠一.植物耐盐性研究进展[J].生物工程进展,2000,20(2):20-25.
[148] 侯彩霞,於新建,李荣,等.甜菜碱稳定PSⅡ放氧中心外周多肽机理[J].中国科学(C辑),1998, 28(4):355-361.
[149] 梁峥,赵原,李浴春,等.菠菜甜菜碱醛脱氢酶对甜菜碱醛的氧化[J].植物学报,1991,33(9): 680-686.
[150] 沈义国,杜保兴,张劲松,等.山菠菜胆碱单氧化物酶基因(CMO)的克隆与分析[J].生物工程学报,2001,17(1):1-6.
[151] Rathinasabapathi R, Michael B, Brenda LR etc. Choline monooxygenase, an unusual iron sulfur enzyme catalyzing the first step of glycinebetaine synthesis in plants: prosthetic group characterization and eDNA clonging[J]. Proc Natl Acad Sci USA, 1997, 94: 3454-3458
[152] Nuccio ML, Brendalr, Kurt DN etc. The endogenous choline supply limits glycinebetaine synthesis in tnmsgenic tobacco expressing cholinemonooxygenase[J]. The Plant J, 1998, 16(4): 487-49645
[153] Shen YG, Du BX, Zhang WK, Chen SY. AhCMO, regulated by stresses in Atriplex hortensis, can improve drought tolerance in transgenic tobacco[J]. Theor Appi Genet, 2002, 105: 815-821
[154] 肖岗,张耕耘,刘凤华,等.山菠菜甜菜碱醛脱氢酶基因研究[J].科学通报,1995,40(8):741-745.
[155] 舒卫国,艾万东,陈受宜.菠菜甜菜碱醛脱氢酶基因全序列分析[J].科学通报,1997,42(22): 2441-2445.
[156] 梁峥,马德钦,汤岚,等.菠菜甜菜碱醛脱氢酶基因在烟草中的表达[J].生物工程学报.1997, 13(3):236-240.
[157] 郭北海,张艳敏,李洪杰.甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达[J].植物学报,2000, 42(3):279-283.
[158] 李银心,常凤启,杜立群,等.转甜菜碱醛脱氢酶基因豆瓣菜的耐盐性[J].植物学报,2000,42(5): 480-484.
[159] 刘振林,戴思兰.植物甜菜碱醛脱氢酶基因研究进展[J].西北农林科技大学学报(自然科学版)2004,32(3):104-112
[160] 殷晓军,赵彦修,张慧.甜菜碱的合成及与其相关基因的遗传工程[J].植物生理学通讯.2002, 38(3):299-304
[161] Nicholas S. Plant resistance to environmental stress[J]. Curr Opin Biotech, 1998, 9: 214-219
[162] Tabuchi T, Kawaguchi Y, Azuma T, etc. Similar Regulation Patterns of Choline Monooxygenase. Phosphoethanolamine N-Methyltransferase and S-Adenosyl-l-Methionine Synthetase in Leaves of the Halophyte Atriplex nummularia L[J]. Plant and Cell Physiology 2005 46(3): 505-513
[163] Ciria G, Figueroa Soto. Guillermo Lopez Cervantes etc. Immunolocalization of betaineal dehydedehydrogenase inporcinekidney[J]. Biochem Biophys Res Commun, 1999;258: 732-736
[164] Toshihide N, Sadaki Y, Yasunori Metc. Expression of a betaine aldehyde dehydrogenase gene in rice, a glycine betaine nonaccumu lator, and possible localization of its protein in peroxisomes[J]. The Plant J, 1997, 11(5): 1115-1120
[165] Nuccio ML, McNeil SD, Ziemak M J etc. Cholineimportin to chloroplastslimitsglycine betaine synthesis in tobacco: analysis of Plants engineered with achloroplasticoracyto solicpathway[J]. Metabolism Engineering, 2000, 2(4): 300-31143
[166] Nuccio ML, Rhodes D, McNeil SD etc. Metabolic engineering of Plants for osmotic stress resistance[J]. Curr Opin Plant Biol, 1999, 2: 128-13444
[167] John CC, Hans JB. Genomic approaches to Plant stress tolerance[J]. Curr Opin Plant Biol, 2000, 3: 117-124
[168] Holmstrom KO, Welin B, Mandal A, etc. Production of the Escherichia coli betaine-aidehyde dehydrogenase, an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants[J]. Plant J, 1994, 6: 749-758
[169] Weigel P, Lerma C, Hanson AD. Choline oxidation by intact spinachchloro plasts[J]. Plant Physiol, 1988, 86: 54-60.
[170] Burner M, Lafontaine PJ, Hanson AD. Assay, purification and partical characterization of cholinemonooxygenase from spinach[J]. Plant Physiol, 1995, 108: 581-588.
[171] Arakawa K, Katayama M, Takabe T. Levels of betaine and betaine aldehyde dehydrogenase activity in the green leaves, and etiolated leaves and roots of barley[J]. Plant Cell Physiol , 1990, 31: 797-803
[172] Rathinasabapathi B, McCue KF, Gage DA, etv. Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance[J]. Planta, 1994, 193:155-162.
[173] Nakamura T, Yokota S, Muramoto Y, etc. Expression ofa betaine aldehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes[J].The Plant Journal, 1997, il(5): 1115-1120
[174] Hibino T, Meng Y, Kawamitsu Y, etc. Molecular cloning and function characterization of two kinds of betaine-accumulating mangrove Avicen niamarina(Forsk.)Vierh[J]. Plant Molecular Biology, 2001, 45: 353-363.
[175] Huang J, Hirji R, Adam L, etc. Genetic Engineering of Glycinebetaine Production toward Enhancing Stress Tolerance in Plants: Metabolic Limitations[J]. Plant Physiol, March 2000, 122:747-756
[176] 郭岩,张莉,肖岗,等.甜菜碱醛脱氢酶基因在水稻中的表达及转基因植物的耐盐性研究[J].中国科学(C辑),1997,27(2):151-155
[177] 刘凤华,郭岩,谷冬梅等.转甜菜碱醛脱氢酶基因植物的耐盐性研究[J].遗传学报,1997,24(1): 54-58
[178] Jia G. X, Zhu ZQ, Chang FQ, etc. Transformation of tomato with the BADH gene from Atriplex improves salt tolerance[J]. Plant Cell Rep, 2002, 21: 141-146
[179] Trossat C, Rathinasabapathi B, Hanson AD. Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyzes oxidation of dimethylsulfoniopropionaldehyde and ω -aminoaldehydes[J]. Plant Physioi, 1997, 113:1457-1461
[180] Ishitani M, Nakamura T, Han SY, etc. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid[J]. Plant Moi. Biol, 1995, 27:307-315
[181] Bhattacharya RC, Maheswari M, Dineshkumar V, etc. Transformation of Brassica oleracea var. capitata with bacterial beta gene enhances tolerance to salt stress[J]. Seientia Horticulturae, 2004, 100 (1-4): 215-227
[182] Quan R, Shang M, Zhang H, etc. Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize[J]. Plant Biotechnology Journal, 2004, 2 (6): 477-486.
[183] Hayashi H, Alia M, Deshnium L, etc. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: accumulation of glycine betaine and enhanced tolerance to salt and cold stress[J]. Plant J. 1997, 12:133-142
[184] Alia, Hayashi H, Sakamoto A, Murata N. Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of betaine[J]. Plant J, 1998, 16:155-161
[185] Prasad KVSK, Sharmila P, Pardha SP. Enhanced tolerance of transgenic Brassica juncea to choline confirms successful expression of the bacterial codA gene[J], plant science, 2000, 159:233-242
[186] Prasad KVSK, Pardha S. Enhanced tolerance to photoinhibition in transgenie plants through targeting of glycinebetaine biosynthesis into the chloroplasts[J], plant science, 2004, 166:1197-1212
[187] Park, EJ, Jekni Z, Sakamoto A, etc. Genetic engineering of glyeinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage[J]. The Plant Journal, 2004, 40 (4): 474-487
[188] Suipice R, Tsukaya H, Nonaka H, etc. Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine[J]. The Plant Journal, 2003, 36 (2), 165-176.
[189] Nakamura T, Yokota S, Muramoto Y, etc. Expression of a betaine aldehydrogenase gene in rice, a glycinebetaine non-aecurnulator, and possible localization of its protein in peroxisomes[J]. The Plant Journal, 1997, 11(5): 1115-1120.
[190] Pua EC, Mehra-Palta A, NaKY F etc. Transgenie plants ofBrassica napus L[J]. Bioteclmology, 1987 5:815-817
[191] Moloney MM, Walker JM, Sharma KK, High efficiency transformation of Brassica napus using Agrobacterium vectors[J]. Plant Cell Rep 1989, 8: 238-242
[192] De Block, De Brouwer, Terming, P., Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants[J]. Plant Physiol. 1989, 91: 694-70 i
[193] 石淑稳,周永明,孙学成等.甘蓝型油菜遗传转化体系的研究[J].华中农业大学学报,1998, 17(3):205-210
[194] Sharma K.K., Bhojwani S S, Thorpe T A. Factors affecting high frequency differentiation of shoots and roots from cotyledon explants of Brassica jzmcea[J]. Plant Sci. 1990, 66: 247-253.
[195] Gong H, Pua EC. Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard (Brassicajuncea) in vitro[J], Plant Science, 2004, 167: 1191-1201
[196] Tony W, Petri S, Lidia T etc. Agrobacterium-mediated transformation and stable expression of the green fluorescent protein in Brassica rapa[J]. Plant Physiology and Biochemistry 2003,41: 773-778
[197] Gong H, Pua EC. Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard (Brassica juncea) in vitro[J]. Plant Science, 2004,167: 1191-1201
[198] Pua EC, Deng X, Koha T C. Genotype variability of de novo shoot morphogensis of Brassicu oleraea in vitro in response to ethylene inhibitors and putrescine[J]. J. Plant Physiol. 1999, 155: 598-605.
[199] Wachter R, Fischer K., Gabler R. etc. Ethylene production and ACC-accumulation in Agrobacterium tumefaciens induced plant tumours and their impact on tumour and host stem structure and function[J]. Plant, Cell & Environment, 1999, 22 (10), 1263-1273
[200] 钟蓉.朱峰.油菜的遗传转化及抗溴苯腈转基因油菜的获得[J].植物学报,1997,39(1):22-27.
[201] 李学宝,郑世学,董五辈等.甘蓝型油菜抗虫转基因植株及其抗性分析[J].遗传学报,1999, 26(3):262-268.
[202] 侯丙凯,胡赞民,党本元笛.定点整合抗虫基因到油菜叶绿体基因组并获得转基因植株[J].植物生理与分子生物学学报,2002,28(3):187-192
[203] Xiang Y, Wong W.K.R, Ma M.C etc., Agrobacterium-mediated transformation of Brassica eampestris ssp. parachinensis with synthetic Bacillus thuringiensis crylAb and crylAc genes[J]. Plant Cell Reports, 2000, 19: 251-256
[204] Cho HS, Cao J, Ren JP etc. Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis crylC gene[J]. Plant Cell Reports, 2001,20: 1-7
[205] 唐克轩,徐亚男,李旭峰等.表达雪花莲外源凝集素基因的油菜转基因植株的获得[J].复旦学报.2001,40(6):683-687
[206] Ding LC, Hu CY, Yeh KW, etc. Development of insect-resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato[J]. Plant Cell Reports, 1998, 17: 854-860
[207] 徐淑平,卫志明,黄健秋.根癌农杆菌介导B.t.基因和CpTI基因对花椰菜的转化[J].植物生理与分子生物学学报,2002,28(3):193-199
[208] Eugenie P. Camille, K. Transformation of cauliflower (Brassica oleracea var. botrytis) by transfer of cauliflower mosaic virus genes through combined cocultivation with virulent and avirulent strains of Agrobacterium[J]. plant science 1996, 113: 79-89
[209] 陈军,王兰岚,刘澄清等.用激光微束穿刺法转化甘蓝型油菜小孢子的研究,激光生物学报, 1998,7(2):103-107
[210] 张海燕,田颖川,周奕华等.将商陆抗病毒蛋白(PAP)cDNA导入油菜获得抗病毒转基因植株[J].科学通报,1998,43(23):2534-2537
[211] 王苏燕,叶寅,赵淑珍等.转基因油菜中核酶介导对花椰菜花叶病毒的高度抗病性[J].中国科学(C辑),1997,27(5):426-431
[212] Grison R, Greezes-beset B, Schneicler M, etc. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimaeric chitinase gene[J]. Nature Biotech, 1996, 14: 643-646.
[213] 张丽华,党本元.抗菌核病转基因油菜植株的获得[J].高技术通讯,1999,(12):41-46
[214] 蓝海燕,王长海,张丽华等.导入β-1,3-葡聚糖酶基因及几丁质酶基因的转基因可育油菜及其抗菌核病的研究[J].生物工程学报,2000,16(2):142-146
[215] Zhang HX, Hodson JN, Williams JP. etc. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation[J]. PNAS, 2001, 98(22): 12832-12836
[216] 陈锦清,郎春秀.反义PEP基因调控油菜籽粒蛋白质油脂含量比率的研究[J].农业生物技术学报,1999,7(4):316-320
[217] 石东乔,周奕华,张丽华,刘桂珍等.农杆菌介导的油菜脂肪酸调控基因工程研究[J].高技术通讯,2001(2):1-7
[218] Vesna K, Winnie F, Dennis LB, etc. Improving erucic acid content in rape seed through biotechnology, what can the Arabidopsis FAEI and the yeast SLC1-1 genes contribute[J]. Crop Science, 2001, 41: 739-747
[219] Voelker T A, WorrellAC, Anderson L, etc. Fattyacid biosynthesis redirected to medium chains in transgenic oilseed plants[J]. Science, 1992, 257: 72-74
[220] Knutzon DS, Thompson G A, Radke SE, etc. Modification of seed oil by antisense expression of a stearoyl-acy carrier proteindesaturase gene[J]. Proc Natl Acad Sci USA, 1992,89: 2624-2628
[221] Mariani C, Beuckeleer MD, Truettner J etc. Induction of male sterility in plants by a chimaeric ribonuclease gene[J]. Nature, 1990, 347: 737-741.
[222] Henzi MX, Christey MC, McNeil etc. Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L.) with an antisense 1-aminocyclopropane-1-carboxylic acid oxidase gene[J]. Plant Science 1999, 143: 55-62
[223] Mariani C, Goicoa MA, Mariani Al etc. Achimaeric ribonuolease in hibitor gene restore fertility to male sterile plant[J]. Nature, 1992, 357(4): 384-387
[224] 何业华,熊兴华,官春云等.根癌农杆菌介导TA29-Barnase基因转化甘蓝型油菜的研究,作物学报,2003,29(4):615-620
[225] 彭仁旺,周雪荣,王峻岭等.表达barstar基因及bar基因的转基因油菜的研究[J].遗传学报,1998,25(1):74-79
[226] 叶梁,李枞,宋艳茹.双价、三价种子特异性表达 载体的构建及表达PHB合成相关基因油菜的获得[J].科学通报,2000,45(5):516-521
[227] Bechtold N, Ellis J, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[J]. CR Acad Sci Pads Life Sci, 1993, 316: 1194-1199
[228] Clough S, Bent A. Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana[J]. Plant J, 1998, 16: 735-743
[229] Liu F, Cao MQ, Yao L, etc. In planta transformation of Pakchoi (Brassiea campestris L. ssp Chinesis) by infiltration of adult plants with Agrobacterium[J]. Acta Hortic, 1998, 467: 187-193
[230] 金万梅,巩振辉,宋正旭等.通过植株原位真空渗入遗传转化获得转基因芥菜[J].西北农林科 技大学学报,2003,31(5):39~42.
[231] Wang W C, Menon G, Hansen G. Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants[J]. Plant Cell Reports,2003, 22: 274-28
[232] 徐光硕,饶勇强,陈雁等.用 in planta方法转化甘蓝型油菜[J].作物学报,2004,30(1):1-5
[233] Radke S E, Turner J C, Facciotti D. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens[J]. Plant Cell Rep. 1992, 11: 499~505
[234] Smyth DR, Bowman JL, Meyerowitz EM. Early flower development in Arabidopsis[J]. The Plant Cell. 1990, 2: 755-767
[235] 白书农.植物发育生物学[M].北京:北京大学出版社,2003
[236] 许智宏,卫志明,王熊,等.油菜(Brassica napus L)花茎组织培养中的器官形成[J].实验生物学报,1979,12(4):349~353
[237] 程振东,卫志明,许智宏.根癌农杆菌对甘蓝型油菜的转化及转基因再生[J].植物学报,1994,36(9):657~63
[238] Eapen S, George L. Plant regeneration from peduncle segments of oil seed Brassica species: influence of silver nitrate and silver thiosulfate[J]. Plant Cell, Tissue and Organ Culture. 1997, 51(3): 229~232
[239] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiol Plant, 1962, 15: 473-494
[240] Pua E C, Chi G L. De novo shoot morphogensis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene[J]. Physiol. Plant, 1993, 88: 467~474
[241] Pua E C, Deng X, Koha T C. Genotype variability of de novo shoot morphogensis of Brassicu oleraea in vitro in response to ethylene inhibitors and putrescine[J]. J. Plant Physiol. 1999, 155:598~605
[242] Wachter R, Fischer K, Gabler R, etc. Ethylene production and ACC-accumulation in Agrobacterium tumefaciens induced plant tumours and their impact on tumour and host stem structure and function[J]. Plant, Cell & Environment, 1999, 22(10): 1263~1273
[243] Laux T, Jurgens G. Embryogenesis: a new start in life[J]. Plant Cell, 1997, 9: 989-1000
[244] de Vries SC. Making embryos in plants[J]. Trends Plant Sci., 1998, 3: 451-452
[245] Dumas C, Mogensen HL. Gametes and fertilization: maize as a model system for experimental embryogenesis in flowering plants[J]. Plant Cell, 1993, 5: 1337-1348
[246] Mordhorst AP, Voerman KJ, Hartog MV, etc. Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions[J]. Genetics, 1998, 149: 549-563
[247] Penneil RI, Janniche L, Scofield GN, etc Identification of a transitional cell state in the developmental pathway to carrot somatic embryogenesis[J]. J Cell Biol, 1992, 119: 1371-1380
[248] Toonen MAJ, Verhees JA, Schmidt EDL, etc. AtLTP1 luciferase expression during carrot somatic embryogenesis[J]. Plant J, 1997, 12: 1213-1221
[249] von Recklinghausen IR, lwanowska A, Kieft H, etc. Structure and development of somatic embryos formed in Arabidopsis thaliana pt callus cultures derived from seedlings[J]. Protoplasma, 2000, 211: 217-224
[250] Ikeda-Iwai M, Satoh S, Kamada H. Establishment of a reproducible tissue culture system for the induction of Arabidopsis somatic embryos[J]. J. Exp. Bot., 2002;53(374): 1575-1580
[251] Anna I, Teresa T, Mieczyslaw K, etc. Localization of Phenolic Compounds in the Covering Tissues of the Embryo of Brassica napus L. during Different Stages of Embryogenesis and Seed Maturation[J]. Annals of Botany, 1994, 74;313-320
[252] Bisgrove SR, Crouch ML, Fernandez DE. Chimeric nature of precociously-germinating Brassica napusembryos: mRNA accumulation patterns[J]. Journal of Experimental Botany, 1995, 46(282): 27-33;
[253] Bob B.Buchanan,Gruissem Wilhelm,Russell L.Jones著,瞿礼嘉,顾红雅,白书农,赵进东译.植物生物化学与分子生物学[M].北京:科学出版社,2004
[254] Meinke DW, Franzmann L H, Nickle TC, etc. Leafy Cotyledon Mutants of Arabidopsis[J]. The Plant Cell, 1994,6: 1049-1064,
[255] Donna E. Fernandez. Developmental basis of homeosis in precociously germinating Brassica napus embryos: phase change at the shoot apex[J]. Development, 1997, 124: 1149-1157
[256] Boutilier K, Offringa R, Sharma VK, etc. Ectopie Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth[J]. The Plant Cell, 2002, 14: 1737-1749
[257] Koh WL, Loh CS. Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus[J]. Plant Cell Reports, 2000, 19: 1177-1183
[258] Tamar Lotan, Masa aki Ohto, Kelly Matsudaira Yee, Marilyn A. L. West, Russell Lo, Raymond W. Kwong, Kazutoshi Yamagishi, Robert L. Fischer, Robert B. Goldberg, and John J. Harada (1998). Arabidopsis LEAFY COTYLEDON1 Is Sufficient to Induce Embryo Development in Yegetative Ceils[J]. Cell, 93: 1195-1205
[259] Thomas Laux, Tobias Wu rschum, and Holger Breuninger. Genetic regulation of embryonic pattern formation[J]. The Plant Cell, 2004,16, S190-S202, Supplement
[260] Turgut K. (1998). Efficient shoot regeneration and somatic embryogenesis from immature cotyledons of Brassica napus L.. Plant Breeding, 117(5): 503-504
[261] Wan, Lianglu, Xia, Qun, Qiu, Xiao & Selvaraj, Gopalan. Early stages of seed development in Brassica napus: a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. The Plant Journal, 2002,30 (1): 1-10
[262] Williams E. G. & G. Maheswaran (1986) Somatic embryogenesis: factors influence co-ordinated behaviour of cells as an embryogenic group. Annals of Botany, 57: 443~462
[263] Zobel AM, Kura M, Tykarska T. Cytoplastic and apoplastic location of phenolic compounds in the covering tissue of the Brassica napusradicle between embryogenesis and germination. Annals of Botany, 1989,64: 149-157
[264] Zuo, Jianru,Niu, Qi-Wen,Frugis, Giovanna & Chua,Nam-Hai. The WUSCHEL gene promotes vegetative-to- embryonic transition in Arabidopsis. The Plant Journal, 2002, 30 (3): 349-359
[265] Curtis IS, Nam HG (2001) Transgenic radish (Raphanus sativus L. Iongipinnatus Bailey) by floral-dip method—plant development and surfactant are important in optimizing transformation efficiency. Transgenie Res 10: 363-371
[266] Desfeux C, Clough SJ, Bent AF (2000) Female reproductive tissues are the primary target of Agrobacterium transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895-904
[267] Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB (1996) Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 274: 2060-2063
[268] Ye GN, Stone D, Pang SZ, Creely W, Gonzalez K, Hinchee M (1999) Arabidopsis ovule is the target for Agrobaeterium in planta vacuum infiltration transformation. Plant J 19: 249-257
[269] Jefferson RA, Kavangh TA, Bevan MW. GUS fusions:β-Glu curonidase as a sensitive and versatile gene fusion marker in higher plants[J]. EMBOJ, 1987,13: 3901~3907
[270] Jefferson RA. Assaying chimeric genes in plants: The GUS gene fusion system plant[J]. Mol Bio Rep, 19875: 387-405.
[271] Schneitz K, Hulskamp M, Pruitt RE. Wild- type ovule development in Arabidopsis thaliana. A light microscope study of cleared whole-mout tissue[J]. The Plant Journal, 1995, 7: 731-749
[272] Smyth DR, Bowman JL, Meyerowitz EM. Early flower development in Arabidopsis[J]. The Plant Cell, 1990, 2: 755-767
[273] Bechtold N, Ellis J, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[J]. C R Acad Sci Ⅲ, Sci Vie, 1993,316: 1194-1199
[274] Bechtold N, Jaudeau B, Jolivet S, Maha B, Vezon D, Voisin R, Pelletier G. The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana[J]. Genetics, 2000,155: 1875-1887
[275] Chung M-H, Chen M-K , Pan S-M Floral spray transformation can efficiently generate Arabidopsis transgenic plants[J]. Transgenic Res 2000,9: 471-476