以玉米幼胚为受体转化海藻糖合成酶基因
|
摘要
干旱是玉米生产的主要限制因素之一。玉米自身的水分生理状况也决定了它比其他禾本科作物更易受干旱危害。尽管玉米不同基因型间存在一定的耐旱性差异,但耐旱性受微效多基因控制,遗传机制复杂,传统的遗传改良难度较大。因此,借助转基因方法,将外源耐旱基因转入玉米,可能是有效提高玉米耐旱能力的快捷方法。
近年来耐旱基因研究取得了较快进展,相继克隆了一些重要的耐旱基因,试图提高其耐旱性,但因这些基因耐旱能力不够强,耐旱机制与玉米生理代谢不协调等原因,转基因后代的耐旱性不能达到玉米生产要求(宋松泉等,2000;王关林等,2002;宋凤斌等,2005)。因此需要探索利用耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因,海藻糖合成酶基因TPS1就是其中之一。TPS1合成的海藻糖在干旱条件下通过氢键与氨基酸相连,可以代替水起到防止蛋白质变性和细胞融合的作用(Crowe等,1992)。如果利用海藻糖的抗逆耐旱特性,采用基因工程将其相关酶的基因转入玉米等作物,进而培育耐旱抗逆新品种,这对于植物遗传改良具有重要意义。
常用的玉米转基因方法多以胚性愈伤组织作为外源基因受体,而胚性愈伤组织的诱导和分化再生,严重受到基因型的限制,只有极少数材料能稳定高效诱导胚性愈伤组织,加之胚性愈伤组织培养时间长,操作繁琐,分化再生率不高等原因,严重制约了玉米转基因研究的进展。因此,有必要开展其他转基因方法研究,以提高转化效率。为此,本试验开展了以玉米幼胚为受体、用农杆菌介导法将酿酒酵母海藻糖合成酶基因TPS1转化玉米的研究,结果如下:
1)以幼胚为受体转化外源基因,可缩短转化过程。以幼胚为转化受体,与含外源基因的农杆菌共培养后,不经过愈伤组织诱导和筛选,直接再生植株,减少基因型对愈伤组织为受体的转基因限制,避开组织培养的繁锁过程,缩短转化程序。
2)以幼胚为受体转化外源基因的优化体系。由于幼胚对农杆菌浸染更敏感,尤其是褐化率比愈伤组织更高,造成转基因失败,为此试验通过对幼胚大小和抗褐化剂半胱氨酸(Cys)的浓度筛选,发现长度在1.5～2.0 mm的幼胚更适合农杆菌介导的幼胚转化,并且在农杆菌与幼胚的共培养阶段添加200 mg/L的半胱氨酸,能够有效降低幼胚在农杆菌浸染过程中的褐化率(29.3%),比对照褐化率(53.7%)减少约一倍。
3)通过PCR检测,初步发现外源基因已转入受体。将转基因再生植株移栽后,生长至5叶期,每株取一小块叶片,按5株组成一个混合样,提取DNA,进行PCR特异扩增,有目的条带的再分单株检测,最后得到1株阳性转基因植株。按混样检测法可以提高阳性植株检测效率和准确性。
Drought is the major limiting factor in the production of maize, and maize is much easier suffering from drought than other gramineous plants by the physiology of itself. The drought tolerance which is controlled by microeffect polygene are not the same in different gene type. It is bottlenecks in maize improvement. Therefore, transgenic operation is an useful technology to overcome reproductive isolation among species and utilize drought tolerance exotic genes.
In recent years, a large number of drought tolerance genes are transferred into maize. But the transgenic plants can not fit the damand of maize production, because the anti-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself (Song et al., 2000; Wang et al., 2002; Song et al., 2005 ) . It is very important to make use of hight anti-drought ability of the gene which is more consistant with maize.Trehalos-6-phosphate synthase (TPS) is one of them for trait of adversity tolerance. Trehalose has been shown to stabilise proteins and membranes under stress conditions, especially during desiccation. By replacing water through hydrogen bonding to polar residues, trehalose prevents the denaturation of proteins and the fusion of membranes (Crowe J H et al., 1992) . So it's very significant to transfer the related gene which has the strong ability of drought tolerance into maize.
Abundant study on the genetic transformation of maize take embryonic callus induced from immature embryo as recipient. There were only few materials can highly and stable induce embryonic callus due to genetype. The research of mazie gene transform were restrict in long-playing cultureing, cockamamie manipulateing, low seedling rate.It is necessary to advance gene trasform rate by other recipient.Therefore, maize immature embryos were taken as recipient in this study ,and transformed by Agrobacterium tumefaciens EHA105 with TPS1 gene from saccharomyces cerevisiae.The resulta as following:
1) The transform process is much shorter for takeing immature embryos as recipient. The immature embryos were concultured with Agrobacterium tumefaciens which contained exotic gene. The process of induceing immature embryos into embyronic calli and selected with antibiotic were cut down,bring immature embryos into seedling directly.That make study much shorter and easier than taking embyronic calli as recipient.
2) Optimize the system which takes immature embryos as recipient. Immature embryos is sensitive to agrobacterium tumefaciens,and that make highly brown ratio and fail transform.This study optimize the system by selecting length of immature embryos and concentration of Cys, the results indicated that the 1.5-2.0 mm of immature embryos were better than other length, and adding Cys 200mg/L in coculture can reduce brown ratio (29.3%) effectively, almost half of antitheses (53.7%).
3) Result of specific PCR test showed the exotic gene had been transformed in maize. Taking 5 plants as 1 mix-sample to extract DNA, after transplants grow to 5 leaves period .Then test DNA by specific PCR and repeat test if there was positive. Finally one plant was detected to be positive. It is more efficiently and accurately to test mix-sample.
近年来耐旱基因研究取得了较快进展,相继克隆了一些重要的耐旱基因,试图提高其耐旱性,但因这些基因耐旱能力不够强,耐旱机制与玉米生理代谢不协调等原因,转基因后代的耐旱性不能达到玉米生产要求(宋松泉等,2000;王关林等,2002;宋凤斌等,2005)。因此需要探索利用耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因,海藻糖合成酶基因TPS1就是其中之一。TPS1合成的海藻糖在干旱条件下通过氢键与氨基酸相连,可以代替水起到防止蛋白质变性和细胞融合的作用(Crowe等,1992)。如果利用海藻糖的抗逆耐旱特性,采用基因工程将其相关酶的基因转入玉米等作物,进而培育耐旱抗逆新品种,这对于植物遗传改良具有重要意义。
常用的玉米转基因方法多以胚性愈伤组织作为外源基因受体,而胚性愈伤组织的诱导和分化再生,严重受到基因型的限制,只有极少数材料能稳定高效诱导胚性愈伤组织,加之胚性愈伤组织培养时间长,操作繁琐,分化再生率不高等原因,严重制约了玉米转基因研究的进展。因此,有必要开展其他转基因方法研究,以提高转化效率。为此,本试验开展了以玉米幼胚为受体、用农杆菌介导法将酿酒酵母海藻糖合成酶基因TPS1转化玉米的研究,结果如下:
1)以幼胚为受体转化外源基因,可缩短转化过程。以幼胚为转化受体,与含外源基因的农杆菌共培养后,不经过愈伤组织诱导和筛选,直接再生植株,减少基因型对愈伤组织为受体的转基因限制,避开组织培养的繁锁过程,缩短转化程序。
2)以幼胚为受体转化外源基因的优化体系。由于幼胚对农杆菌浸染更敏感,尤其是褐化率比愈伤组织更高,造成转基因失败,为此试验通过对幼胚大小和抗褐化剂半胱氨酸(Cys)的浓度筛选,发现长度在1.5～2.0 mm的幼胚更适合农杆菌介导的幼胚转化,并且在农杆菌与幼胚的共培养阶段添加200 mg/L的半胱氨酸,能够有效降低幼胚在农杆菌浸染过程中的褐化率(29.3%),比对照褐化率(53.7%)减少约一倍。
3)通过PCR检测,初步发现外源基因已转入受体。将转基因再生植株移栽后,生长至5叶期,每株取一小块叶片,按5株组成一个混合样,提取DNA,进行PCR特异扩增,有目的条带的再分单株检测,最后得到1株阳性转基因植株。按混样检测法可以提高阳性植株检测效率和准确性。
Drought is the major limiting factor in the production of maize, and maize is much easier suffering from drought than other gramineous plants by the physiology of itself. The drought tolerance which is controlled by microeffect polygene are not the same in different gene type. It is bottlenecks in maize improvement. Therefore, transgenic operation is an useful technology to overcome reproductive isolation among species and utilize drought tolerance exotic genes.
In recent years, a large number of drought tolerance genes are transferred into maize. But the transgenic plants can not fit the damand of maize production, because the anti-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself (Song et al., 2000; Wang et al., 2002; Song et al., 2005 ) . It is very important to make use of hight anti-drought ability of the gene which is more consistant with maize.Trehalos-6-phosphate synthase (TPS) is one of them for trait of adversity tolerance. Trehalose has been shown to stabilise proteins and membranes under stress conditions, especially during desiccation. By replacing water through hydrogen bonding to polar residues, trehalose prevents the denaturation of proteins and the fusion of membranes (Crowe J H et al., 1992) . So it's very significant to transfer the related gene which has the strong ability of drought tolerance into maize.
Abundant study on the genetic transformation of maize take embryonic callus induced from immature embryo as recipient. There were only few materials can highly and stable induce embryonic callus due to genetype. The research of mazie gene transform were restrict in long-playing cultureing, cockamamie manipulateing, low seedling rate.It is necessary to advance gene trasform rate by other recipient.Therefore, maize immature embryos were taken as recipient in this study ,and transformed by Agrobacterium tumefaciens EHA105 with TPS1 gene from saccharomyces cerevisiae.The resulta as following:
1) The transform process is much shorter for takeing immature embryos as recipient. The immature embryos were concultured with Agrobacterium tumefaciens which contained exotic gene. The process of induceing immature embryos into embyronic calli and selected with antibiotic were cut down,bring immature embryos into seedling directly.That make study much shorter and easier than taking embyronic calli as recipient.
2) Optimize the system which takes immature embryos as recipient. Immature embryos is sensitive to agrobacterium tumefaciens,and that make highly brown ratio and fail transform.This study optimize the system by selecting length of immature embryos and concentration of Cys, the results indicated that the 1.5-2.0 mm of immature embryos were better than other length, and adding Cys 200mg/L in coculture can reduce brown ratio (29.3%) effectively, almost half of antitheses (53.7%).
3) Result of specific PCR test showed the exotic gene had been transformed in maize. Taking 5 plants as 1 mix-sample to extract DNA, after transplants grow to 5 leaves period .Then test DNA by specific PCR and repeat test if there was positive. Finally one plant was detected to be positive. It is more efficiently and accurately to test mix-sample.
引文
Bell W, Sun W, Hohmann S,et al. Composition and Functional Analysis of the Saccharomyces cerevisiae Trehalose Synthase Complex[J]. The Journal of Biological Chemistry. 1998, 33311-33319
Birch G G., Wolfrom M L and Tyson R S. Advances in Carbohydrate Chemistry. Academic Press NewYork.. 1963, 18(40): 201-225
Boue SM,Cater CH,Ehrlich KC,Cleveland TE.Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus.Journal of Agricultural Food and Chemistry. 2000, 48: 2167-2172
Bronwyn R F, Shou H X, et al. Agrobacterium tumefaciens-Mediated Transformation of Maize Embryos Using a Standard Binary Vector System. Plant Physiol[J]. 2002, 129: 13-22
Capell T, Escobar C, Liu H, et al. Overexpression of the oat arginine decarboxylase cDNA in transgenic rice(Oryza satira L. )affects normal development patterns in vitro and results in putresine accumulation in transgenic plants.TheorAppl,Genet. 1998, 97: 246-254
Cotter D., Anita J. Martel, and Paul MacDonald.Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone Appl Microbiol. 1975, 29: 115-117
Crowe JH,Crowe LM,Chapman D.Preservation of membranes in an hydrobiotic organism:the role of trehalose[J].Science. 1984, 223(4637): 701-703
Crowe JH, Carpenter JF, Crowe LM. The role of vitrification in anhydrobiosis. Annu Rev Physiol. 1998, 60: 73-103
Crowe J H, Hoekstra FA, Crowe LM. Anhydrobiosis[J]. Annu Rev Physio. 1992, 54: 579
Deng W, Chen L, Liang X, et al. VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-bindingprotein VirE2 in Agrobacterium. Mol Microbiol. 1999,31(6): 1795-1807
Du Juan, Zhu Zhen, Li Wan-chen. Over-expression of Exotic Superoxide Dismutase Gene MnSOD and Increase in Stress Resiatance in Maize.Journal of plantphysiology and molecular biology. 2006, 32(1): 57-63
Elbein AD. The metabolism of α,α-trehalose[J].Adv Carbohydr Chem Biochem. 1974, 30: 227
Frame, B R, H Shou, R K Chikwamba Z Zhang, C Xiang, T M Fonger, S E K Pegg, B Li, D S.Nettleton D. Pei& K. Wang,. Agrobactefium tumefaciens mediated transform ation of maize embros using a standard binary vetor system. Plant. 2002, 129(1): 13-22
Fromm ME, Taylor LP, and Walbot, V.Nature (London) . 1986, 319, 791-793.
Fromm ME, Morrish F, Armstrong C, et al.Bio / Technology. 1990, 8, 833-839.
Fullner KJ, Lara JC, Nester EW . Pilus assembly by Agrobacterium T-DNA transfer genes. Science. 1996, 273: 1107-1109
Gelvin S B. Agrobacterium VIrE2 proteins can form a complex T strands in the plant cytoplasm. J Bacteriol. 1998,180(16): 4300-4302
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adans TR, et al. Plant Cell1. 990, 2, 603-618.
Graves ACF, Goldman SL, Plant.Mol.Biol. 1986, 7: 43-50.
Green, J. L., and Angell, C. A., 1989, Phase Relations and Vitrification in Holmstrom K O, Mantyla E, Welin B, et al. Drought tolerance in tobacco[J]. Nature. 1996, 379: 683-684.
Grimsley N, Hohn T, Davies JW, et al.Nature (London) . 1987, 325, 177-179
Haldrup A., Petersen S G ., Okkeles F. T . Positive selection: a plant sdection principle based in the food industry. Plant Cell Report. 1998, 18: 76—81.
Hansen.G., Evidence for Agrobacterium-induced apoptosis in maize cell. Mol.Plant-Microbe Interact. 2000. 3 (6): 649-657.
Harding TS. History of trehalose , its discovery and method of preparation [J]. Sugar. 1923,25: 476
HieiY T, Komari. Stableinheritance of transgenesin rice plan tstransform ed by Agrobacterium tumefaciens. Third international Rice Genetics Symposium. 1995, 10: 16-20 \
Hinchee M A W, Conner-Ward D V, Newell C A. Productionof transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technol. 1988, 6: 915-922
Holmstrom KO, Mantyla E, WelinB, et.al.. Drought tolerance in tobacco.Nature. 1996, 379: 683-684
Holmstrom K O. Engineering plant adaptation to water stress[J]. Acta universitatis Agriculture sueciae Agraia. 1998, 84: 49
Ishida, Y., H. Satto, S. Ohta, Y. Hiei, T. Komari& T. Kumashiro, High eficiency transformation of maize(Zea mays L.) mediated by Agroboneterium Tumefiwier. Nature Biotech. 1996,14(6): 745-750
Jin S, Song Y, Pan S。 Nester E W . Characterisation ofa vi mutation that confers constitutive virulence gen e expression in Agrobacterium tumefaciem. Molecular Microbiology, 1993, 7: 555-562
Jin Su, Qingxi Shen, Tuan-Hua David Ho, Ray Wu, Dehydration stress regulated transgene expression in stably transformed rice plants, Plant physio1. 1998, 117 : 913-922
Kishor P B K, Hong Z, Miao G H, et al. Over expression Of [delta]-Pyroline-5-Carboxylate synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 1995, 108(4): 1387-1394
Lee L Y , Gelvin S B, Kado C I. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export. [J] Bacteriol. 1999 ,181(1): 186-196
Liang Z, Ma D Q, Tang L, et al. Expression of the spinach betaine dehyde dehydrogenase (BADH) gene in transgenic tobacco plants.Chinese Journal of Biotechnology. 1997, 13: 153-159
Mayerhofer R, Koncz-Kalman Z, Nawrath C, et al. T-DNA integration: a model of illegitimate recombination in plants.EMBOJ. 199, 10(3): 679-704
Ohta Y, Proc. Natl. Acad. Sci.U.SA. 1986, 83, 715-719.
Olhoft PM. Somera DA.L-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cptyledonary-node cells. Plant Cell Reports. 2001, 20: 706-711.
Rasco-Gaunt S, LiuD, LiC P, OohertyA, etal. Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize .Plant Cell Reports[J]. 2003, 21(6): 569-576
Richard-Forget FC,Groupy PM,Nicols JJ.Cysteine as an inhibitor of enzymatic browning:kinetic studies[J].Journal of Agricultural Food and Chemistry. 1992, 40: 2108-2113
Sahi S V, Chilton M D, ChiltonW S. Corn metabolites afect growth and virulence of Agrobacterinm tumefaciem . Proc Nail Acad Sci USA [J]. 1990, 27: 3879-3883
Salas M G, Park S H, Srivatanakul M , et al. Temperature influence on stable T-DNA integration in plant cells. 2001, 20(8): 701-705
Santos M A,et al. Methods of obtaining maize totipotent tissues. Ⅰ. Seedling segments culture. Plant Seitett[J], 1984, 33: 309-316
Sato S, Xing A, Ye X, et al. Production of-linolenic acid and stearidonic acid in seeds of marker-free transgenic soybean . Crop Sci. 2004, 44: 646-652
Sidorenko L, Bruce W , Maddock S, et al. Functional an alysis of two matrix attachment region(MAR)elements in transgenic maize plants. Transgenic search[J]. 2003, 12(2): 137-154
Sola-Penna, M., and Meyer-Fernandes, J. R. Arch. Biochem. Biophys. 1998, 360: 10-14
Songstad D D, Peterson W L, Armstrong C L.Establishment of friable ernbryogenic(typeⅡ)callus from immature tassels of Zea mays(Poaceae). Am J Bot[J]. 1992, 79: 761-764
Sundberg C, Meek L, Carroll K, et al. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. Bacteriol[J]. 1996, 178(4): 1207-1212
Sundberg C D, Rean W. The Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domainsrequired for single-stranded DNA binding and cooperative interaction. J Bacteriol.1999,181(21):6850-6855
Thevelein JM.Regulation oftrehalose mobilisation in fungi..Microbiol Rev.1984,48:42-59
Timasheff S N.The control of protein stability and association by weak interactions with water:how do solvents affect these processes? Annu Rev Biophys Biomol Struct.1993,22:67-97
Tu J,One I,Zhang Q,et al.Transgenic rice variety IRF-2 with Xa21 is resistant to bacterial blight.TheorApplGenet.1998,97:31-36
Wan Y C,Widhdm JM,LemauxPG,etal.Type I callus as abombardment target for generating fertile transgenic maize(Zea mays L.).Planta[J].1995,196:7-14
Wingler,A.,The function oftrehalose biosynthesis in plants.Phytochemistry,.2002,60(5):437-440
Yang FY.Climate condition assessment for maize production.china climate impact assessment,Beijing:Meteorology Press.2004,73-79
Yeo ET,Kwn HB,Han SE et.al,Genetic engineering of drought resistant introduction of the trehalose-6-phosphonte synthase(TPS 1)gene from saccharamyces cerevisiae.Mol Cells.2000,10(3)::263-268
Zambryskip,Joos H,Genetell O C,Ieemans J,Vanmontag U W,Schell J.Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity[J].EMBO.1983,2:2143-2145.
Zhao ZY,Gu W,Cai T,Tagliani LA,Hondred DA,Bond D,Krell S,Rudert ML,Bruce WB,Pierce DA Molecular analysis of TO plants transformed by Agrobacterium and comparison of
Agrobacterium-mediated transformation with bombardment transformation in maize.Maize Genet Coop Newslett.1998,72:34-37
Zhao Z Y,Gu W N,Cai T S,et al.Highthroughput genetic tranmsformation mediated by Agrobacterinm tumefaciens in maize.Molecular Breeding[J].2002,8(4):323-333
Zhu B,Su J,Chang M C,et al.Overexpression of a △1-pyrroline-5- carboxylate synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice.Plant Sci.,1998,139:41-48
Adams,《酵母遗传学方法实验指南》[M].北京:科学出版社.2000,84.
白云凤,王国英,苟明月等。农杆菌介导玉米转基因的影响因素研究.中国生态农业学报.2006,14(3):174-175
丁群星,谢友菊,戴景瑞.用子房注射法将Bt毒蛋白基因导入玉米的研究.中国科学(B辑).1993,3(7):707-713
付光明,苏乔,吴畏等.转BADH基因玉米的获得及其耐盐性.辽宁师范大学学报,2006,29(3):344-347
付凤玲,张莉萍,朱祯.玉米优良自交系转基因受体系统建立及转化后的筛选与再生.四川农业大学学报[J].2000,18(2):97-98.
郭北海,张艳敏,李洪杰等.甜菜碱醛脱氢酶(BADH)基因转化小麦及表达.植物学报[J],2000,42(3):279-283
巩健,杨芳.单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究.生物技术.2007,17(3):2-5
黄璐,卫志明.农杆菌介导的玉米遗传转化[J]。实验生物学报.,1999,25:116-124
胡建广,祁喜涛,根癌农杆菌介导超甜玉米自交系幼胚转化研究.广东农业科学.2004,11:32-34
冀俊丽,盛长忠,石明等.通过负压花粉管法将耐盐基因HVAl转人小麦的研究.麦类作物学报.2002,22(2):10-13
刘录祥,赵林妹,梁欣欣等.基因枪法获得逆境诱导转录因子DREBlA转基因小麦的研究.中国生物工程杂志,2003,23(11):53-56
刘伟华,赵秀振.梁虹等.枯草杆菌果聚糖蔗糖酶基因转化小麦的研究中国农业科学.2006,39(2):231-236
宋松泉,林忠平,傅家瑞.植物脱水耐性的分子基础.林忠平等编.走向21世纪的植物分子生物学.北京:科学出版社.2000,242-252
宋凤斌,王晓波.玉米非生物逆境生理生态[M].北京:科学出版社.2005,10-120
吴亮其 范战民 郭蕾等.通过转δ-OAT基因获得抗盐抗旱水稻.科学通报.2003,48(9)2050-2055
吴家道,黄忠祥.玉米体细胞培养中胚状体的发生.植物生理学通讯.1985,(2):13-17
王关林,方宏筠.植物基因工程(第二版)[M].北京:科学出版社.,2002,61-73
王自章,张树珍,杨本鹏等.甘蔗根癌农杆菌介导转化海藻糖合酶基因获得抗渗透胁迫能力增强植株.中国农业科学[J].2003,36(2):140-146
王萍,郭永来,高世庆等..基因枪法将GmDREB基因导入大豆的研究.大豆科学.2007,26(3):315-318
徐香玲,高晶,刘伟华等.Ti质粒介导的Bt,k-S内毒素蛋白基因转化大豆的初步研究.大豆科学.1997,16(1):6-11
薛哲勇,支大英,夏光敏等.根癌农杆菌介导AtNHXI基因转化小麦.山东大学学报(理学版),2003,38(1):106-109
尹青女,吕惠颖,朱保葛等.农杆菌介导法热激转录因子-8基因转入大豆.分子植物育种.2004, 2(6):783-787
尹小燕,杨爱芳,张可炜等.转AtNHX1基因玉米的生产及耐盐性分析.植物学报.2004,7:52-57
杨建波,许智宏,卫志明等.影响根癌农杆菌附着禾谷类作物培养细胞的因素.实验生物学报[J],1993,26(1):1-9
朱明.海藻糖的功能及其应用.粮食科技与经济.2003,5:74-76.
张荣,王国英,张晓红等.农杆菌介导的玉米遗传转化的建立[J].农业生物技术学报,2001,9(1):45-48
张世煌.我国玉米种业现状与发展战略.种子科技[J].2006,6:9-11
张艳贞,王罡,胡汉桥等.农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究.遗传[J],2002,24(1):35-39
张荣,王国英,张晓红等.农杆菌介导的玉米遗传转化的建立[J].农业生物技术学报,2001,9(1):45-48
Birch G G., Wolfrom M L and Tyson R S. Advances in Carbohydrate Chemistry. Academic Press NewYork.. 1963, 18(40): 201-225
Boue SM,Cater CH,Ehrlich KC,Cleveland TE.Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus.Journal of Agricultural Food and Chemistry. 2000, 48: 2167-2172
Bronwyn R F, Shou H X, et al. Agrobacterium tumefaciens-Mediated Transformation of Maize Embryos Using a Standard Binary Vector System. Plant Physiol[J]. 2002, 129: 13-22
Capell T, Escobar C, Liu H, et al. Overexpression of the oat arginine decarboxylase cDNA in transgenic rice(Oryza satira L. )affects normal development patterns in vitro and results in putresine accumulation in transgenic plants.TheorAppl,Genet. 1998, 97: 246-254
Cotter D., Anita J. Martel, and Paul MacDonald.Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone Appl Microbiol. 1975, 29: 115-117
Crowe JH,Crowe LM,Chapman D.Preservation of membranes in an hydrobiotic organism:the role of trehalose[J].Science. 1984, 223(4637): 701-703
Crowe JH, Carpenter JF, Crowe LM. The role of vitrification in anhydrobiosis. Annu Rev Physiol. 1998, 60: 73-103
Crowe J H, Hoekstra FA, Crowe LM. Anhydrobiosis[J]. Annu Rev Physio. 1992, 54: 579
Deng W, Chen L, Liang X, et al. VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-bindingprotein VirE2 in Agrobacterium. Mol Microbiol. 1999,31(6): 1795-1807
Du Juan, Zhu Zhen, Li Wan-chen. Over-expression of Exotic Superoxide Dismutase Gene MnSOD and Increase in Stress Resiatance in Maize.Journal of plantphysiology and molecular biology. 2006, 32(1): 57-63
Elbein AD. The metabolism of α,α-trehalose[J].Adv Carbohydr Chem Biochem. 1974, 30: 227
Frame, B R, H Shou, R K Chikwamba Z Zhang, C Xiang, T M Fonger, S E K Pegg, B Li, D S.Nettleton D. Pei& K. Wang,. Agrobactefium tumefaciens mediated transform ation of maize embros using a standard binary vetor system. Plant. 2002, 129(1): 13-22
Fromm ME, Taylor LP, and Walbot, V.Nature (London) . 1986, 319, 791-793.
Fromm ME, Morrish F, Armstrong C, et al.Bio / Technology. 1990, 8, 833-839.
Fullner KJ, Lara JC, Nester EW . Pilus assembly by Agrobacterium T-DNA transfer genes. Science. 1996, 273: 1107-1109
Gelvin S B. Agrobacterium VIrE2 proteins can form a complex T strands in the plant cytoplasm. J Bacteriol. 1998,180(16): 4300-4302
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adans TR, et al. Plant Cell1. 990, 2, 603-618.
Graves ACF, Goldman SL, Plant.Mol.Biol. 1986, 7: 43-50.
Green, J. L., and Angell, C. A., 1989, Phase Relations and Vitrification in Holmstrom K O, Mantyla E, Welin B, et al. Drought tolerance in tobacco[J]. Nature. 1996, 379: 683-684.
Grimsley N, Hohn T, Davies JW, et al.Nature (London) . 1987, 325, 177-179
Haldrup A., Petersen S G ., Okkeles F. T . Positive selection: a plant sdection principle based in the food industry. Plant Cell Report. 1998, 18: 76—81.
Hansen.G., Evidence for Agrobacterium-induced apoptosis in maize cell. Mol.Plant-Microbe Interact. 2000. 3 (6): 649-657.
Harding TS. History of trehalose , its discovery and method of preparation [J]. Sugar. 1923,25: 476
HieiY T, Komari. Stableinheritance of transgenesin rice plan tstransform ed by Agrobacterium tumefaciens. Third international Rice Genetics Symposium. 1995, 10: 16-20 \
Hinchee M A W, Conner-Ward D V, Newell C A. Productionof transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technol. 1988, 6: 915-922
Holmstrom KO, Mantyla E, WelinB, et.al.. Drought tolerance in tobacco.Nature. 1996, 379: 683-684
Holmstrom K O. Engineering plant adaptation to water stress[J]. Acta universitatis Agriculture sueciae Agraia. 1998, 84: 49
Ishida, Y., H. Satto, S. Ohta, Y. Hiei, T. Komari& T. Kumashiro, High eficiency transformation of maize(Zea mays L.) mediated by Agroboneterium Tumefiwier. Nature Biotech. 1996,14(6): 745-750
Jin S, Song Y, Pan S。 Nester E W . Characterisation ofa vi mutation that confers constitutive virulence gen e expression in Agrobacterium tumefaciem. Molecular Microbiology, 1993, 7: 555-562
Jin Su, Qingxi Shen, Tuan-Hua David Ho, Ray Wu, Dehydration stress regulated transgene expression in stably transformed rice plants, Plant physio1. 1998, 117 : 913-922
Kishor P B K, Hong Z, Miao G H, et al. Over expression Of [delta]-Pyroline-5-Carboxylate synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 1995, 108(4): 1387-1394
Lee L Y , Gelvin S B, Kado C I. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export. [J] Bacteriol. 1999 ,181(1): 186-196
Liang Z, Ma D Q, Tang L, et al. Expression of the spinach betaine dehyde dehydrogenase (BADH) gene in transgenic tobacco plants.Chinese Journal of Biotechnology. 1997, 13: 153-159
Mayerhofer R, Koncz-Kalman Z, Nawrath C, et al. T-DNA integration: a model of illegitimate recombination in plants.EMBOJ. 199, 10(3): 679-704
Ohta Y, Proc. Natl. Acad. Sci.U.SA. 1986, 83, 715-719.
Olhoft PM. Somera DA.L-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cptyledonary-node cells. Plant Cell Reports. 2001, 20: 706-711.
Rasco-Gaunt S, LiuD, LiC P, OohertyA, etal. Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize .Plant Cell Reports[J]. 2003, 21(6): 569-576
Richard-Forget FC,Groupy PM,Nicols JJ.Cysteine as an inhibitor of enzymatic browning:kinetic studies[J].Journal of Agricultural Food and Chemistry. 1992, 40: 2108-2113
Sahi S V, Chilton M D, ChiltonW S. Corn metabolites afect growth and virulence of Agrobacterinm tumefaciem . Proc Nail Acad Sci USA [J]. 1990, 27: 3879-3883
Salas M G, Park S H, Srivatanakul M , et al. Temperature influence on stable T-DNA integration in plant cells. 2001, 20(8): 701-705
Santos M A,et al. Methods of obtaining maize totipotent tissues. Ⅰ. Seedling segments culture. Plant Seitett[J], 1984, 33: 309-316
Sato S, Xing A, Ye X, et al. Production of-linolenic acid and stearidonic acid in seeds of marker-free transgenic soybean . Crop Sci. 2004, 44: 646-652
Sidorenko L, Bruce W , Maddock S, et al. Functional an alysis of two matrix attachment region(MAR)elements in transgenic maize plants. Transgenic search[J]. 2003, 12(2): 137-154
Sola-Penna, M., and Meyer-Fernandes, J. R. Arch. Biochem. Biophys. 1998, 360: 10-14
Songstad D D, Peterson W L, Armstrong C L.Establishment of friable ernbryogenic(typeⅡ)callus from immature tassels of Zea mays(Poaceae). Am J Bot[J]. 1992, 79: 761-764
Sundberg C, Meek L, Carroll K, et al. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. Bacteriol[J]. 1996, 178(4): 1207-1212
Sundberg C D, Rean W. The Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domainsrequired for single-stranded DNA binding and cooperative interaction. J Bacteriol.1999,181(21):6850-6855
Thevelein JM.Regulation oftrehalose mobilisation in fungi..Microbiol Rev.1984,48:42-59
Timasheff S N.The control of protein stability and association by weak interactions with water:how do solvents affect these processes? Annu Rev Biophys Biomol Struct.1993,22:67-97
Tu J,One I,Zhang Q,et al.Transgenic rice variety IRF-2 with Xa21 is resistant to bacterial blight.TheorApplGenet.1998,97:31-36
Wan Y C,Widhdm JM,LemauxPG,etal.Type I callus as abombardment target for generating fertile transgenic maize(Zea mays L.).Planta[J].1995,196:7-14
Wingler,A.,The function oftrehalose biosynthesis in plants.Phytochemistry,.2002,60(5):437-440
Yang FY.Climate condition assessment for maize production.china climate impact assessment,Beijing:Meteorology Press.2004,73-79
Yeo ET,Kwn HB,Han SE et.al,Genetic engineering of drought resistant introduction of the trehalose-6-phosphonte synthase(TPS 1)gene from saccharamyces cerevisiae.Mol Cells.2000,10(3)::263-268
Zambryskip,Joos H,Genetell O C,Ieemans J,Vanmontag U W,Schell J.Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity[J].EMBO.1983,2:2143-2145.
Zhao ZY,Gu W,Cai T,Tagliani LA,Hondred DA,Bond D,Krell S,Rudert ML,Bruce WB,Pierce DA Molecular analysis of TO plants transformed by Agrobacterium and comparison of
Agrobacterium-mediated transformation with bombardment transformation in maize.Maize Genet Coop Newslett.1998,72:34-37
Zhao Z Y,Gu W N,Cai T S,et al.Highthroughput genetic tranmsformation mediated by Agrobacterinm tumefaciens in maize.Molecular Breeding[J].2002,8(4):323-333
Zhu B,Su J,Chang M C,et al.Overexpression of a △1-pyrroline-5- carboxylate synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice.Plant Sci.,1998,139:41-48
Adams,《酵母遗传学方法实验指南》[M].北京:科学出版社.2000,84.
白云凤,王国英,苟明月等。农杆菌介导玉米转基因的影响因素研究.中国生态农业学报.2006,14(3):174-175
丁群星,谢友菊,戴景瑞.用子房注射法将Bt毒蛋白基因导入玉米的研究.中国科学(B辑).1993,3(7):707-713
付光明,苏乔,吴畏等.转BADH基因玉米的获得及其耐盐性.辽宁师范大学学报,2006,29(3):344-347
付凤玲,张莉萍,朱祯.玉米优良自交系转基因受体系统建立及转化后的筛选与再生.四川农业大学学报[J].2000,18(2):97-98.
郭北海,张艳敏,李洪杰等.甜菜碱醛脱氢酶(BADH)基因转化小麦及表达.植物学报[J],2000,42(3):279-283
巩健,杨芳.单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究.生物技术.2007,17(3):2-5
黄璐,卫志明.农杆菌介导的玉米遗传转化[J]。实验生物学报.,1999,25:116-124
胡建广,祁喜涛,根癌农杆菌介导超甜玉米自交系幼胚转化研究.广东农业科学.2004,11:32-34
冀俊丽,盛长忠,石明等.通过负压花粉管法将耐盐基因HVAl转人小麦的研究.麦类作物学报.2002,22(2):10-13
刘录祥,赵林妹,梁欣欣等.基因枪法获得逆境诱导转录因子DREBlA转基因小麦的研究.中国生物工程杂志,2003,23(11):53-56
刘伟华,赵秀振.梁虹等.枯草杆菌果聚糖蔗糖酶基因转化小麦的研究中国农业科学.2006,39(2):231-236
宋松泉,林忠平,傅家瑞.植物脱水耐性的分子基础.林忠平等编.走向21世纪的植物分子生物学.北京:科学出版社.2000,242-252
宋凤斌,王晓波.玉米非生物逆境生理生态[M].北京:科学出版社.2005,10-120
吴亮其 范战民 郭蕾等.通过转δ-OAT基因获得抗盐抗旱水稻.科学通报.2003,48(9)2050-2055
吴家道,黄忠祥.玉米体细胞培养中胚状体的发生.植物生理学通讯.1985,(2):13-17
王关林,方宏筠.植物基因工程(第二版)[M].北京:科学出版社.,2002,61-73
王自章,张树珍,杨本鹏等.甘蔗根癌农杆菌介导转化海藻糖合酶基因获得抗渗透胁迫能力增强植株.中国农业科学[J].2003,36(2):140-146
王萍,郭永来,高世庆等..基因枪法将GmDREB基因导入大豆的研究.大豆科学.2007,26(3):315-318
徐香玲,高晶,刘伟华等.Ti质粒介导的Bt,k-S内毒素蛋白基因转化大豆的初步研究.大豆科学.1997,16(1):6-11
薛哲勇,支大英,夏光敏等.根癌农杆菌介导AtNHXI基因转化小麦.山东大学学报(理学版),2003,38(1):106-109
尹青女,吕惠颖,朱保葛等.农杆菌介导法热激转录因子-8基因转入大豆.分子植物育种.2004, 2(6):783-787
尹小燕,杨爱芳,张可炜等.转AtNHX1基因玉米的生产及耐盐性分析.植物学报.2004,7:52-57
杨建波,许智宏,卫志明等.影响根癌农杆菌附着禾谷类作物培养细胞的因素.实验生物学报[J],1993,26(1):1-9
朱明.海藻糖的功能及其应用.粮食科技与经济.2003,5:74-76.
张荣,王国英,张晓红等.农杆菌介导的玉米遗传转化的建立[J].农业生物技术学报,2001,9(1):45-48
张世煌.我国玉米种业现状与发展战略.种子科技[J].2006,6:9-11
张艳贞,王罡,胡汉桥等.农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究.遗传[J],2002,24(1):35-39
张荣,王国英,张晓红等.农杆菌介导的玉米遗传转化的建立[J].农业生物技术学报,2001,9(1):45-48