pthA-NLS转化糖橙获得抗溃疡病种质及椪柑转chit42基因的研究
|
摘要
柑橘在世界农业经济中占有重要地位,但其产业发展一直受到柑橘各种病害的制约。栽培措施与农药对柑橘病害的防治能力有限,且化学农药还会造成严重的环境污染,抗病育种成为解决柑橘病害的必然选择。柑橘常规育种受到育种周期长、珠心胚干扰、性器官败育、遗传上高度杂合等因素影响而进程缓慢,同时物种间生殖隔离亦阻碍了常规育种进程。转基因技术的应用与抗病相关基因的克隆为柑橘抗病育种提供了新的思路。本研究采用农杆菌介导法,以柑橘溃疡病致病基因pthA的核定位信号(NLS)和抗真菌病害基因chit42为外源目的基因,分别将其转入到糖橙和椪柑中,通过嫁接使抗性芽再生成苗。采用PCR和RT-PCR等技术对转基因材料进行鉴定分析;通过离体接种病菌实验对转基因植株的抗病能力进行评价。主要研究结果如下:
1.构建了PBI121-NLS植物表达载体;用农杆菌介导法将pthA-NLS基因导入到糖橙中,并经PCR,RT-PCR和Southern blot杂交分析获得9个转基因株系;转基因植株叶片离体接种柑橘溃疡病菌,结果表明转基因植株对柑橘溃疡病菌有很强的抗性。
2.建立了椪柑实生苗上胚轴节间茎段的离体再生体系,结果表明:茎段在MS基本培养基中的萌芽率最高,可以达到83.3%,且萌芽时间短,多为单芽,芽体健壮,嫁接成活率高;抗生素筛选试验表明卡拉霉素(Km)100mg/l时可以有效控制逃逸体和嵌合体的产生;将抗性芽通过嫁接成苗,取新叶提取DNA,通过PCR初步鉴定,18株抗性植株中有3株为转基因植株。
3.采用了7种消毒方法对椪柑成年态节间茎段消毒,结果表明“改良消毒法”可有效降低其污染率和死亡率,分别可降至12.2%和27.2%;椪柑成年态节间茎段在MS+1mg/l BA+0.5 mg/l NAA中的不定芽萌芽率最高,达12.8%:选用5 mm以下的茎尖可明显提高嫁接成活率,不过,使用1 mm左右茎尖进行微芽嫁接嫁接成活率最高,这可能与柑橘接穗越大携带的内生菌越多有关。通过农杆菌介导法将目的基因chit42转入到椪柑中,获得12棵抗性芽,PCR初步检测表明获得1棵转基因植株。
Citrus is an important fruit crop for agricultural economy in the world, and the production is inhibited by citrus disease which severely occurres worldwide. Agronomic practice and bactericide can only solve the problem in some context, and some bactericide can cause heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against the bacterial pathogens. Conventional disease resistance breeding is difficult because of polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis; as well as limitation for reproduction barrier among different species. Resistant genes cloning and transgenic approach offers a potentially bactericide-free and environment-friendly solution for disease pathogen control. In this study, pthA-NLS gene and Chit42 gene were introduced into Citrus sinensis (Linn.) Osbeck and Ponkan (Citrus reticulata Blanco) by Agrobacterium-medialed transformation. Shoot-tip grafting was performed to complete the regeneration system.PCR and RT-PCR were used to analyze the transgenic plants.Disease resistance test indicated that resistance was improved on some transgenic strains. And the resistance mechanism of pthA-NLS in citrus was discussed. The main results are as follows:
1. The plasmid of 'pthA-NLS' was constructed and transfered to Citrus sinensis (Linn.) Osbeck by Agrobacterium-mediated. Nine transgenic strains were obtained through PCR, RT-PCR and Southern blot analysis. Disease resistance test indicated that resistance to canker was obviously improved on some transgenic strains.
2. An efficient regeneration system by direct organogenesis from epicotyl of Ponkan (Citrus reticulata Blanco) in vitro was set up. Seven methods were tested for regeneration, MS without any hormone resulted in the most effective one, with the rates of bud formation efficiency to 83.3% and much stronger buds were got in shortest time .100mg/l Km was used to control chimeric buds. Shoot-tip grafting was performed to complete the regeneration system. Three trangenic strains were confirmed from 18 strains through PCR analysis.
3. Seven methods were tested for the sterilization of mature materials, and the combination of pre-sampling and post-sampling disinfection (innovation method) resulted in the most effective one, with the rates of the contamination and death reduced to 12.2% and 27.2%, respectively. After sterilization with the innovation method, the percentage of the alive internodal segments rates raised to 30.6%. Furthermore,the medium supplemented with 1 mg/l BA and 0.5 mg/l NAA was the best one. The regeneration frequency and bud formation efficiency reached to 12.8%. Shoot-tip grafting was performed to complete the regeneration system. It was found that the buds smaller than 5 mm in length gave higher graft success than that with longer shoots, and the best results were obtained with 1 mm buds. This probably due to more endo-fungi existed in the bigger shoots than in the shorter ones.One buds of 12 was confirmed to transfenic strain by PCR analysis.
1.构建了PBI121-NLS植物表达载体;用农杆菌介导法将pthA-NLS基因导入到糖橙中,并经PCR,RT-PCR和Southern blot杂交分析获得9个转基因株系;转基因植株叶片离体接种柑橘溃疡病菌,结果表明转基因植株对柑橘溃疡病菌有很强的抗性。
2.建立了椪柑实生苗上胚轴节间茎段的离体再生体系,结果表明:茎段在MS基本培养基中的萌芽率最高,可以达到83.3%,且萌芽时间短,多为单芽,芽体健壮,嫁接成活率高;抗生素筛选试验表明卡拉霉素(Km)100mg/l时可以有效控制逃逸体和嵌合体的产生;将抗性芽通过嫁接成苗,取新叶提取DNA,通过PCR初步鉴定,18株抗性植株中有3株为转基因植株。
3.采用了7种消毒方法对椪柑成年态节间茎段消毒,结果表明“改良消毒法”可有效降低其污染率和死亡率,分别可降至12.2%和27.2%;椪柑成年态节间茎段在MS+1mg/l BA+0.5 mg/l NAA中的不定芽萌芽率最高,达12.8%:选用5 mm以下的茎尖可明显提高嫁接成活率,不过,使用1 mm左右茎尖进行微芽嫁接嫁接成活率最高,这可能与柑橘接穗越大携带的内生菌越多有关。通过农杆菌介导法将目的基因chit42转入到椪柑中,获得12棵抗性芽,PCR初步检测表明获得1棵转基因植株。
Citrus is an important fruit crop for agricultural economy in the world, and the production is inhibited by citrus disease which severely occurres worldwide. Agronomic practice and bactericide can only solve the problem in some context, and some bactericide can cause heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against the bacterial pathogens. Conventional disease resistance breeding is difficult because of polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis; as well as limitation for reproduction barrier among different species. Resistant genes cloning and transgenic approach offers a potentially bactericide-free and environment-friendly solution for disease pathogen control. In this study, pthA-NLS gene and Chit42 gene were introduced into Citrus sinensis (Linn.) Osbeck and Ponkan (Citrus reticulata Blanco) by Agrobacterium-medialed transformation. Shoot-tip grafting was performed to complete the regeneration system.PCR and RT-PCR were used to analyze the transgenic plants.Disease resistance test indicated that resistance was improved on some transgenic strains. And the resistance mechanism of pthA-NLS in citrus was discussed. The main results are as follows:
1. The plasmid of 'pthA-NLS' was constructed and transfered to Citrus sinensis (Linn.) Osbeck by Agrobacterium-mediated. Nine transgenic strains were obtained through PCR, RT-PCR and Southern blot analysis. Disease resistance test indicated that resistance to canker was obviously improved on some transgenic strains.
2. An efficient regeneration system by direct organogenesis from epicotyl of Ponkan (Citrus reticulata Blanco) in vitro was set up. Seven methods were tested for regeneration, MS without any hormone resulted in the most effective one, with the rates of bud formation efficiency to 83.3% and much stronger buds were got in shortest time .100mg/l Km was used to control chimeric buds. Shoot-tip grafting was performed to complete the regeneration system. Three trangenic strains were confirmed from 18 strains through PCR analysis.
3. Seven methods were tested for the sterilization of mature materials, and the combination of pre-sampling and post-sampling disinfection (innovation method) resulted in the most effective one, with the rates of the contamination and death reduced to 12.2% and 27.2%, respectively. After sterilization with the innovation method, the percentage of the alive internodal segments rates raised to 30.6%. Furthermore,the medium supplemented with 1 mg/l BA and 0.5 mg/l NAA was the best one. The regeneration frequency and bud formation efficiency reached to 12.8%. Shoot-tip grafting was performed to complete the regeneration system. It was found that the buds smaller than 5 mm in length gave higher graft success than that with longer shoots, and the best results were obtained with 1 mm buds. This probably due to more endo-fungi existed in the bigger shoots than in the shorter ones.One buds of 12 was confirmed to transfenic strain by PCR analysis.
引文
[1]孙宝国.香精概论.北京:化学工业出版社版社,1996:104-109
[2]邓秀新.世界柑橘品种改良的进展.园艺学报,2005,32(6):1140-1146
[3]李宁,汪其怀,付仲文.美国转基因生物安全管理考察报告.农业科技管理,2005,24(5):13-17
[4]Kobayashi S,Uchimiya H.Expression and integration of a foreign gene in orange(Citrus sinensis L.Osbeck) protoplasts by direct DNA transfer.Jpn.J.Genet.,1989,64:91-97
[5]贺红,潘瑞炽,韩美丽,李耿光.枳壳外植体离体再生及农杆菌介导的遗传转化.云南植物研究,1998,20(4):459-463
[6]贺红,李耿光.根癌农杆菌对枳壳遗传转化的影响因素.中国中药杂志,1999,24(3):140-142
[7]Endo T,Shimada T,Fujii H,Kobayashi Y,Araki T,Omura M.Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange(Poncirus trifoliata L.Raf.).Trans.Res.,2005,14:703-712
[8]何永睿,邹修平,彭爱红,徐忠强,许兰珍,彭祝春,陈善春.根癌农杆菌介导的柑橘遗传转化技术.热带作物学报,2004,25(1):11-16
[9]Cervera M,Juárez J,Navarro A,Pina J A,Durán-Vila N,Navarro L,Pe(?)a L.Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage.Trans.Res.,1998,7:51-59
[10]Mendes B M J,Boscariol R L,Fiiho F A A M,Almeida W A B.Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange.Pesq.agropec,bras.,Brasilia,2002,37:955-961
[11]Ghorbel R,Dominguez A,Navarro L,Pe(?)a L.High efficiency genetic transformation of sour orange(Citrus aurantium L.) and production of transgenic trees containing the coat protein gene of citrus tristeza virus,Tree Physiol.,2000,20:1183-1189
[12]Ll D D,Shi W,Deng X X.Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene.Plant Cell Rep.,2002,21:153-156
[13]陈善春.张进仁,黄自然,高峰,陈凤珍,隆有庆.根癌农杆菌介导柞蚕抗菌肽D基因转化柑橘的研究.中国农业科学,1997,30(3):7-13
[14]Ghorbel R,Juárez J,Navarro L,Pe(?)a L.Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants.Theor.Appl.Genet.,1999,99:350-358
[15]Ghorbel R,López C,Fagoaga C,Moreno P,Navarro L,Flores R,Pe(?)a L.Transgenic citrus plants expressing the Citrus Tristeza Virus p23 protein exhibit viral-like symptoms.Mol.Plant Pathol,2001 a,2:27-36
[16]Guo,W W,Duan Y X,Oiivares-Fuster O,Wu Z C,Arias C R,Burns J K,Grosser J W.Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene.Plant Cell Rep.,2005,24:482-486
[17]Pe(?)a L,Pérez R M,Cervera M,Juárez J,Navarro L.Early events in Agrobacterium-mediated genetic transformation of citrus explants.Ann.Bot.,2004,94:67-74
[18]Cervera M,Juárez J,Navarro L,Pe(?)a L.Genetic transformation of mature citrus plants.Methods Mol.Biol.,2005,286:177-187
[19]何永睿,邹修平等.根癌农杆菌介导的柑橘遗传转化技术.热带作物学报,2004,25(1):11-15.
[20]李卫,陈亮,蔡得田,等.柑橘基因转化新方法的研究.植物学报,1997,39(8):782-784
[21]Leandro Pena.Transgenic Plant Methods and Protocols.Human Press.
[22]石玮,李尔栋,邓秀新,等.根癌农杆菌介导绿色荧光蛋白基因转化印度酸橘的研究.园艺学报,2002,29(2):109-112.
[23]Lucca P.Effective selection and regeneration of transgenic rice plants with mannose as selective agent.MolecularBreeding,2001,7:43-49.
[24]Costa MGC,Otoni W C,Moore GA.An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi(Macf.) and Production of transgenic plants containing carotenoid biosynthetic genes.Plant Cell Rep,2002,21(4):365-373
[25]Wong W S,Li GG,Ning W,et al.Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-1-carboxylate synthase RNA.Plant Science,2001,161(5):969-977.
[26]Gentile A,Deng Z N,Malfa S L,ea al.Morphological and physiological effects of rolABC genes into citrus genome.Acta Hortic.,2004,632:235-242
[27]罗赛男,张家银,等用单性结实基因defH9-iaaM转化糖橙的研究.湖南农业大学学报,2008,34(2)
[28]冯鹄竣,熊兴耀,邓子牛,张家银.利用DefH9-iaaM5基因转化沙田柚的的初步研究,2006年柑橘年会论文.
[29]谢玉明,郭琛,张家银等.农杆菌介导phyB基因转化枳壳研究.湖南农业大学学报自然科学版,2008,34(1):68-72.
[30]Pena L,Martin-Trillo M,Juarez J,et al.Constitutive expression of Arabidopsis LEAFY or APETAI genes in citrus reduces their generation time.Nature Biotech,2001,19:263-267
[31]Molinari H B C,Marur C J,Filho J C B,et al.Osmotic adjustment in transgenic citrus rootstock Carrizo citrange(Citrus sinensis Osb.x Poncirus trifoliate L.Raf.)overproducing praline.Plant Sci,2004b,167(6):1375-1381.
[32]Cervera M,Ortega C,Navarro A,et al.Generation of transgenic citrus plants with the tolerance-to-salinity gene HAL2 from yeast.J Hortic Sci Biotechnol,2000,75:26-30.
[33]罗赛男,杨莉,等.转录因子TERF1和ITSRF1转入糖橙的研究.2007年中国柑橘学会年会论文
[34]胡蓉,魏泓,陈善春,何永睿.甲型肝炎病毒农壳蛋白融合基因植物表达载体的构建与遗传转化柑橘的研究.遗传,2004,26(4):425-431.
[35]Kobayashi S,Nakamura Y,Kaneyoshi J.Transformation of kiwifruit and trifoliate orange(Poncirus trifoliata) with a synthetic gene encoding the human epidermal growth factor(hEGF).J Jpn SocHort Sci,1996,64(6):763-769.
[36]DEW it PJGM.Molecular characterization of gene for gene systems in plant fungus interactionsand the application of avirulence genes in controlof plant pathogens.Annu Rev Phytopathol,1992,30:391-418
[37]郑秀芳,张勇,缪为.植物抗真菌病害基因工程的研究策略.甘肃科学学报,2002,13(1):63-66
[38]Gutierrez M A,Luth D,Moore G A.Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange(Citrus auranitium L.)plants expressing the coat protein gene of citrus tristeza virus.Plant Cell Rep,1997,16:745-753.
[39]Dominguez A,Guerri J,Cambra M,et al.Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus.Plant Cell Rep,2000,19:427-433.
[40]Yang Z N,lngelbrecht I L,Louzada E,et al.Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red(Citrus paradise Macf.).Plant Cell Rep,2000,19(12):1203-1211.
[41]Iwanami,T,Shimizu T,Ito T,Hirabayashi T.Tolerance to citrus mosaic virus in transgenic trifoliate orange lines harboring capsid polyprotein gene.Plant Dis.,2004,88:865-868
[42]郊启发,陈大成,黄自然,胡桂兵.人工合成柞蚕抗菌肽D基因转化沙田柚.华南农业大学学报,1999,20(1):103-107
[43]Gentile A.,Deng Z.,La Malfa S.,et al.Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemonplants expressing a Trichoderma harzianum chitinase gene.Plant Breeding,2006,125:1-6.
[45]Hansen G,Chilton,et al.Agrolistic transformation of plant cells integration of T-strands generated in plata. 1996,93:14978-14983.
[46]Dominguez A, Cervera M, Perez R M, Romero J, Fagoaga C, Cubero J, Lopez M M, Juarez J, Navarro L, Pena L. Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol. Breed., 2004, 14:171-183
[47]Pena L, Perez R M, Cervera M, Juarez J, Navarro L. Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann. Bot., 2004, 94:67-74
[48]Molinari H B C, Bespalhok J C,Kobayashi A K,et al. Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo(Citrus paradise Macf. xPoncirus trifoliate L.Raf.)using thin epicotyl sections.Scientia Horticulturae,2004a,99:379-385.
[49]Cervera M,Pina J A,Juarez J,et al. Agrobacterium-mediated transformation of citrange:factors affecting transformation and regeneration.Plant Cell Rep, 1998c, 18:271-278.
[50]Cervera M,Lopez M,Navarro L. Virulence and supervirulence of Agrobacterium tumefaciens in woody fruit plants.Physiol Mol Plant Pathol, 1998a,52:67-78.
[51]Bond J E,Roose M L. Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange.Plant Cell Rep, 1998,18:229-234.
[52]Kazuhito A,Hideaki S,Shozo O.Efficient transformation of Ara2 bidopsis thalianaxomparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains.Plant Cell Rep, 1992,12:7-11.
[53]Kaneyoshi (Hiramatsu) J, Kobayashi S, Nakamura Y, Shigemoto N, Doi Y. A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.). Plant Cell Rep., 1994, 13:541-545
[54]Pena L, Cervera M, Juarez J, Navarro A, Pina J A, Navarro L. Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep., 1997, 16:731-737
[55]Luth D, Moore G. Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tiss. Org. Cult., 1999, 57:219-222
[56]Ghorbel R, Malfa S L, Lopez M M, Petit A, Navarro L, Pena L. Additional copies of virG from pTiBo542 provide a super-transformation ability to Agrobacterium tumefaciens in citrus. Physiol. Mol. Plant Pathol., 2001, 58(3): 103-110.
[58]Bolar J. P.,Norelli J.L.,Wong K W, et al. Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology,2000,90:72～77
[59]Kikkert J.R.,Ali GS., Wallace P.G., et al. Expression of a fungal chitinase in Vitis vinifera L. 'Merlot'and 'Chardonnay'plants produced by biolistic transformation . Acta Horticultural, 2000,528:297～303
[60]Meyger G.D., Bigirimana J., Elad Y.,et al. Induced systemic resistance in biocontrol of Botrytis cinerea by Trichoderma harzianum T39. European Journal of Plant Pathology, 1998, 104(3):279~286.
[61]Nzojiyobiri Jean-Berchmans,徐同,宋凤鸣,等.哈茨木霉NF9菌株对水稻的诱导抗病性.中国生物防治,2003,19(3):l11～114
[62]Lorito, M., Woo, S.L., Garcia Fernandez.I., et al. Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proceedings of the National Academy of Science of USA, 1998,95,7860～7865
[63]Bolar J.P., Aldwinkle H.S., Harman G.E., et al. Endochitinase transgenic McIntosh apple lines have increased resistance to scab.Phytopathology, 1997,87 (Supplement)：S10
[64] Lorito M., Scala. F. Invited review microbial genes expressed in transgenic plants to improve disease resistance. Journal of Plant Pathology, 1999,81(2): 73～88
[65]Bolar J.P., Norelli J.L., Harman GE., et al. Expression of fungal chitinolytic enzymea in transgenic apples confers high levels of resistance to scab. Plant biotech and In Vitro biology in the 21 century . Netherland,1999 :465～468
[66]Gentile A., Deng ZN., Malfa La S., et al. Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breeding, 2006,125,1-6
[67]Yang Y, Dean W, Gabrie D W. Intragenic recombination of a single plant pathogen gene provides a mechanism for the evolution of new host specificities. Journal of Bacteriology, 1995,9:4963～4968
[68]Swarup, S, Yang Y, Kingsley M T. A Xanthomonas. citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.Mol.Plant-Microbe Interact.1992,5:204-213
[69]Yang Y,Feyter,D R,Gabriel D W,Host-specific symptoms and increased release of Xanthomonas cirri and X.Campestris pv.malvacearum from leaves are determined by the 102p tandem repeats of pthA and avrb6,respectively,Mol.Plant-Microbe Interaction,1994,9:105-113
[70]Yang Y,Feyter,D R,Gabriel D W,Host-specific symptoms and increased release of Xanthomonas cirri and X.Campestris pv.malvacearum from leaves are determined by the 102p tandem repeats of pthA and avrb6,respectively,Mol.Plant-Microbe Interaction,1994,9:105-113
[71]陈杰忠.果树栽培学各论,南方本.中国农业出版社,北京.2003.
[72]段艳欣.根癌农杆菌介导的柑橘转化体系优化与转LFY、APl基因植株的培育.2006年博士论文
[73]李东栋.柑橘不同品种对根癌农杆菌介导遗传转化的反应差异.分子植物育种,2005,3(1):52-56.
[74]Huang L-C.Huang B-L.Rhim S,et al.Genetically engineering citrus with insecticidal gene Bacillus thuringiensis subsp.Tenebrionis.Proceedings of a symposium on enhancing competitiveness of fruitindustry.Taichung,tai-Wan,March 1997:20-21.
[75]李红叶.柑橘黄龙病、溃疡病的病原认识和学名的变化[J].植物险疫,1999,13(4):240-242.
[76]张其.柑橘溃疡病发生与防治的研究综述.世界农业,1988,(7):30-32.
[77]A.K.Das.Citrus canker-A review.J.Appl.Hort.,2003,5(1):52-60
[78]J.Cubero,J.H.GrahamGenetic.Relationship among Worldwide Strains of Xanthomonas Causing Canker in Citrus Species and Design of New Primers for Their Identification by PCR.Applied and environmental microbiology.2002,4:1257-1264
[79]陈杰忠.果树栽培学各论,南方本.中国农业出版社,北京.2003.
[80]段艳欣.根癌农杆菌介导的柑橘转化体系优化与转LFY、APl基因植株的培育.2006年博士论文.
[81]Hidaka T,Omura M..Transformation of citrus protoplasts by electroporation[J].J Jpn Soc Hortic Sci,1993,62:371-376.
[82]Huang L-C.Huang B-L.Rhim S,et al.Genetically engineering citrus with the insecticidal gene from Bacillus thuringiensis subsp.Tenebrionis.Proceedings of a symposium on enhancing competitiveness of fruitindustry.Taichung,tai-Wan,March 1997:20-21.
[83]D.D.Li,W.Shi,X.X.Deng.Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene.Plant Cell Report,2002,21:153-156.
[84]Murashige T,Skoog F.A revised medium for rapid growth and bioassay with tobacco tissue cultures.Physiol Plant,1962,15:473-497.
[85]Murashige T and Tucker D P H.Growth factor requirements of Citrus tissue culture.Proc 1st Int Citrus Symp,1969,3:1155-1161
[86]DengZ.N,Gentile A,Nicolosi E,etal.ldentification of in vivo and in vitro lenmon mutants by RAPD makers(J).Hort.Sci.70(1):117-125
[87]黄家权,邬志荣,孙中海.影响椪柑上胚轴再生不定芽的几个生理因素.植物生理学通讯,2005,41(1):37-40.
[88]张家银,郭琛,谢玉明等.椪柑成年态节间茎段再生体系的建立.湖南农业大学学报,2008,34(2)
[89]Kobayashi S,Uchnimiya H.Expression and integration of a foreign gene in orange(Citrus sinensis Osb.) protoplasts by direct DNA transfer.The Japannese journal of genetics,1989,64(2):91-97.
[90]Vardi A,Bleichman S,Aviv D.Genetic transformation of Citrus protoplasts and regeneration of transgenic plants.Plant Science,1990,69:199-206.
[91]高峰,华学军,范六云,等.用根癌农杆菌离体转化柑橘.中国柑橘,1990,19(4):3-4.
[92]Almeida W.A.B.;Mourao Filho F.A.A.;Pino L.E.,et al.Genetic transformation and plant recovery from mature tissues of Citrus sinensis L.Osbeck.Plant Science,2003,164:203-211.
[93]姜玲,万蜀渊,王映红等.柑橘茎尖嫁接操作方法的改进及研究.华中农业大学学报,1995,14(4):381-385.
[94]罗永兰,张至元,喻珺.柑橘内生细菌的分离与鉴定.湖北农业科学,2006.45(6):773-775.
[95]王子成,李忠爱,邓秀新.柑橘成年态茎段外植体消毒方法研究.河南大学学报,2005,35(2):57-60
[96]Huang J Q,Yin L Y,Y Xiao-hong,et al.In vitro plant regeneration from the mature tissue of navel orange(Citrus sinensis L.Osbeck) by direct organogenesis.Agricultural Sciences in China,2005,4(3):236-240.
[97]放小平,胡新喜,郭琛,等.用rolB基因转化大红甜橙的初步研究.湖南农业大学学报:自然科学版,2005,31(6):623-626.
[98]谢玉明,邓子牛,郭琛,等.农杆菌介导甜橙遗传转化的初步研究.湖南农业大学学报:自 然科学版,2005,31 (6):627-630.
[2]邓秀新.世界柑橘品种改良的进展.园艺学报,2005,32(6):1140-1146
[3]李宁,汪其怀,付仲文.美国转基因生物安全管理考察报告.农业科技管理,2005,24(5):13-17
[4]Kobayashi S,Uchimiya H.Expression and integration of a foreign gene in orange(Citrus sinensis L.Osbeck) protoplasts by direct DNA transfer.Jpn.J.Genet.,1989,64:91-97
[5]贺红,潘瑞炽,韩美丽,李耿光.枳壳外植体离体再生及农杆菌介导的遗传转化.云南植物研究,1998,20(4):459-463
[6]贺红,李耿光.根癌农杆菌对枳壳遗传转化的影响因素.中国中药杂志,1999,24(3):140-142
[7]Endo T,Shimada T,Fujii H,Kobayashi Y,Araki T,Omura M.Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange(Poncirus trifoliata L.Raf.).Trans.Res.,2005,14:703-712
[8]何永睿,邹修平,彭爱红,徐忠强,许兰珍,彭祝春,陈善春.根癌农杆菌介导的柑橘遗传转化技术.热带作物学报,2004,25(1):11-16
[9]Cervera M,Juárez J,Navarro A,Pina J A,Durán-Vila N,Navarro L,Pe(?)a L.Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage.Trans.Res.,1998,7:51-59
[10]Mendes B M J,Boscariol R L,Fiiho F A A M,Almeida W A B.Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange.Pesq.agropec,bras.,Brasilia,2002,37:955-961
[11]Ghorbel R,Dominguez A,Navarro L,Pe(?)a L.High efficiency genetic transformation of sour orange(Citrus aurantium L.) and production of transgenic trees containing the coat protein gene of citrus tristeza virus,Tree Physiol.,2000,20:1183-1189
[12]Ll D D,Shi W,Deng X X.Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene.Plant Cell Rep.,2002,21:153-156
[13]陈善春.张进仁,黄自然,高峰,陈凤珍,隆有庆.根癌农杆菌介导柞蚕抗菌肽D基因转化柑橘的研究.中国农业科学,1997,30(3):7-13
[14]Ghorbel R,Juárez J,Navarro L,Pe(?)a L.Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants.Theor.Appl.Genet.,1999,99:350-358
[15]Ghorbel R,López C,Fagoaga C,Moreno P,Navarro L,Flores R,Pe(?)a L.Transgenic citrus plants expressing the Citrus Tristeza Virus p23 protein exhibit viral-like symptoms.Mol.Plant Pathol,2001 a,2:27-36
[16]Guo,W W,Duan Y X,Oiivares-Fuster O,Wu Z C,Arias C R,Burns J K,Grosser J W.Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene.Plant Cell Rep.,2005,24:482-486
[17]Pe(?)a L,Pérez R M,Cervera M,Juárez J,Navarro L.Early events in Agrobacterium-mediated genetic transformation of citrus explants.Ann.Bot.,2004,94:67-74
[18]Cervera M,Juárez J,Navarro L,Pe(?)a L.Genetic transformation of mature citrus plants.Methods Mol.Biol.,2005,286:177-187
[19]何永睿,邹修平等.根癌农杆菌介导的柑橘遗传转化技术.热带作物学报,2004,25(1):11-15.
[20]李卫,陈亮,蔡得田,等.柑橘基因转化新方法的研究.植物学报,1997,39(8):782-784
[21]Leandro Pena.Transgenic Plant Methods and Protocols.Human Press.
[22]石玮,李尔栋,邓秀新,等.根癌农杆菌介导绿色荧光蛋白基因转化印度酸橘的研究.园艺学报,2002,29(2):109-112.
[23]Lucca P.Effective selection and regeneration of transgenic rice plants with mannose as selective agent.MolecularBreeding,2001,7:43-49.
[24]Costa MGC,Otoni W C,Moore GA.An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi(Macf.) and Production of transgenic plants containing carotenoid biosynthetic genes.Plant Cell Rep,2002,21(4):365-373
[25]Wong W S,Li GG,Ning W,et al.Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-1-carboxylate synthase RNA.Plant Science,2001,161(5):969-977.
[26]Gentile A,Deng Z N,Malfa S L,ea al.Morphological and physiological effects of rolABC genes into citrus genome.Acta Hortic.,2004,632:235-242
[27]罗赛男,张家银,等用单性结实基因defH9-iaaM转化糖橙的研究.湖南农业大学学报,2008,34(2)
[28]冯鹄竣,熊兴耀,邓子牛,张家银.利用DefH9-iaaM5基因转化沙田柚的的初步研究,2006年柑橘年会论文.
[29]谢玉明,郭琛,张家银等.农杆菌介导phyB基因转化枳壳研究.湖南农业大学学报自然科学版,2008,34(1):68-72.
[30]Pena L,Martin-Trillo M,Juarez J,et al.Constitutive expression of Arabidopsis LEAFY or APETAI genes in citrus reduces their generation time.Nature Biotech,2001,19:263-267
[31]Molinari H B C,Marur C J,Filho J C B,et al.Osmotic adjustment in transgenic citrus rootstock Carrizo citrange(Citrus sinensis Osb.x Poncirus trifoliate L.Raf.)overproducing praline.Plant Sci,2004b,167(6):1375-1381.
[32]Cervera M,Ortega C,Navarro A,et al.Generation of transgenic citrus plants with the tolerance-to-salinity gene HAL2 from yeast.J Hortic Sci Biotechnol,2000,75:26-30.
[33]罗赛男,杨莉,等.转录因子TERF1和ITSRF1转入糖橙的研究.2007年中国柑橘学会年会论文
[34]胡蓉,魏泓,陈善春,何永睿.甲型肝炎病毒农壳蛋白融合基因植物表达载体的构建与遗传转化柑橘的研究.遗传,2004,26(4):425-431.
[35]Kobayashi S,Nakamura Y,Kaneyoshi J.Transformation of kiwifruit and trifoliate orange(Poncirus trifoliata) with a synthetic gene encoding the human epidermal growth factor(hEGF).J Jpn SocHort Sci,1996,64(6):763-769.
[36]DEW it PJGM.Molecular characterization of gene for gene systems in plant fungus interactionsand the application of avirulence genes in controlof plant pathogens.Annu Rev Phytopathol,1992,30:391-418
[37]郑秀芳,张勇,缪为.植物抗真菌病害基因工程的研究策略.甘肃科学学报,2002,13(1):63-66
[38]Gutierrez M A,Luth D,Moore G A.Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange(Citrus auranitium L.)plants expressing the coat protein gene of citrus tristeza virus.Plant Cell Rep,1997,16:745-753.
[39]Dominguez A,Guerri J,Cambra M,et al.Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus.Plant Cell Rep,2000,19:427-433.
[40]Yang Z N,lngelbrecht I L,Louzada E,et al.Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red(Citrus paradise Macf.).Plant Cell Rep,2000,19(12):1203-1211.
[41]Iwanami,T,Shimizu T,Ito T,Hirabayashi T.Tolerance to citrus mosaic virus in transgenic trifoliate orange lines harboring capsid polyprotein gene.Plant Dis.,2004,88:865-868
[42]郊启发,陈大成,黄自然,胡桂兵.人工合成柞蚕抗菌肽D基因转化沙田柚.华南农业大学学报,1999,20(1):103-107
[43]Gentile A.,Deng Z.,La Malfa S.,et al.Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemonplants expressing a Trichoderma harzianum chitinase gene.Plant Breeding,2006,125:1-6.
[45]Hansen G,Chilton,et al.Agrolistic transformation of plant cells integration of T-strands generated in plata. 1996,93:14978-14983.
[46]Dominguez A, Cervera M, Perez R M, Romero J, Fagoaga C, Cubero J, Lopez M M, Juarez J, Navarro L, Pena L. Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol. Breed., 2004, 14:171-183
[47]Pena L, Perez R M, Cervera M, Juarez J, Navarro L. Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann. Bot., 2004, 94:67-74
[48]Molinari H B C, Bespalhok J C,Kobayashi A K,et al. Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo(Citrus paradise Macf. xPoncirus trifoliate L.Raf.)using thin epicotyl sections.Scientia Horticulturae,2004a,99:379-385.
[49]Cervera M,Pina J A,Juarez J,et al. Agrobacterium-mediated transformation of citrange:factors affecting transformation and regeneration.Plant Cell Rep, 1998c, 18:271-278.
[50]Cervera M,Lopez M,Navarro L. Virulence and supervirulence of Agrobacterium tumefaciens in woody fruit plants.Physiol Mol Plant Pathol, 1998a,52:67-78.
[51]Bond J E,Roose M L. Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange.Plant Cell Rep, 1998,18:229-234.
[52]Kazuhito A,Hideaki S,Shozo O.Efficient transformation of Ara2 bidopsis thalianaxomparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains.Plant Cell Rep, 1992,12:7-11.
[53]Kaneyoshi (Hiramatsu) J, Kobayashi S, Nakamura Y, Shigemoto N, Doi Y. A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.). Plant Cell Rep., 1994, 13:541-545
[54]Pena L, Cervera M, Juarez J, Navarro A, Pina J A, Navarro L. Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep., 1997, 16:731-737
[55]Luth D, Moore G. Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tiss. Org. Cult., 1999, 57:219-222
[56]Ghorbel R, Malfa S L, Lopez M M, Petit A, Navarro L, Pena L. Additional copies of virG from pTiBo542 provide a super-transformation ability to Agrobacterium tumefaciens in citrus. Physiol. Mol. Plant Pathol., 2001, 58(3): 103-110.
[58]Bolar J. P.,Norelli J.L.,Wong K W, et al. Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology,2000,90:72～77
[59]Kikkert J.R.,Ali GS., Wallace P.G., et al. Expression of a fungal chitinase in Vitis vinifera L. 'Merlot'and 'Chardonnay'plants produced by biolistic transformation . Acta Horticultural, 2000,528:297～303
[60]Meyger G.D., Bigirimana J., Elad Y.,et al. Induced systemic resistance in biocontrol of Botrytis cinerea by Trichoderma harzianum T39. European Journal of Plant Pathology, 1998, 104(3):279~286.
[61]Nzojiyobiri Jean-Berchmans,徐同,宋凤鸣,等.哈茨木霉NF9菌株对水稻的诱导抗病性.中国生物防治,2003,19(3):l11～114
[62]Lorito, M., Woo, S.L., Garcia Fernandez.I., et al. Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proceedings of the National Academy of Science of USA, 1998,95,7860～7865
[63]Bolar J.P., Aldwinkle H.S., Harman G.E., et al. Endochitinase transgenic McIntosh apple lines have increased resistance to scab.Phytopathology, 1997,87 (Supplement)：S10
[64] Lorito M., Scala. F. Invited review microbial genes expressed in transgenic plants to improve disease resistance. Journal of Plant Pathology, 1999,81(2): 73～88
[65]Bolar J.P., Norelli J.L., Harman GE., et al. Expression of fungal chitinolytic enzymea in transgenic apples confers high levels of resistance to scab. Plant biotech and In Vitro biology in the 21 century . Netherland,1999 :465～468
[66]Gentile A., Deng ZN., Malfa La S., et al. Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breeding, 2006,125,1-6
[67]Yang Y, Dean W, Gabrie D W. Intragenic recombination of a single plant pathogen gene provides a mechanism for the evolution of new host specificities. Journal of Bacteriology, 1995,9:4963～4968
[68]Swarup, S, Yang Y, Kingsley M T. A Xanthomonas. citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.Mol.Plant-Microbe Interact.1992,5:204-213
[69]Yang Y,Feyter,D R,Gabriel D W,Host-specific symptoms and increased release of Xanthomonas cirri and X.Campestris pv.malvacearum from leaves are determined by the 102p tandem repeats of pthA and avrb6,respectively,Mol.Plant-Microbe Interaction,1994,9:105-113
[70]Yang Y,Feyter,D R,Gabriel D W,Host-specific symptoms and increased release of Xanthomonas cirri and X.Campestris pv.malvacearum from leaves are determined by the 102p tandem repeats of pthA and avrb6,respectively,Mol.Plant-Microbe Interaction,1994,9:105-113
[71]陈杰忠.果树栽培学各论,南方本.中国农业出版社,北京.2003.
[72]段艳欣.根癌农杆菌介导的柑橘转化体系优化与转LFY、APl基因植株的培育.2006年博士论文
[73]李东栋.柑橘不同品种对根癌农杆菌介导遗传转化的反应差异.分子植物育种,2005,3(1):52-56.
[74]Huang L-C.Huang B-L.Rhim S,et al.Genetically engineering citrus with insecticidal gene Bacillus thuringiensis subsp.Tenebrionis.Proceedings of a symposium on enhancing competitiveness of fruitindustry.Taichung,tai-Wan,March 1997:20-21.
[75]李红叶.柑橘黄龙病、溃疡病的病原认识和学名的变化[J].植物险疫,1999,13(4):240-242.
[76]张其.柑橘溃疡病发生与防治的研究综述.世界农业,1988,(7):30-32.
[77]A.K.Das.Citrus canker-A review.J.Appl.Hort.,2003,5(1):52-60
[78]J.Cubero,J.H.GrahamGenetic.Relationship among Worldwide Strains of Xanthomonas Causing Canker in Citrus Species and Design of New Primers for Their Identification by PCR.Applied and environmental microbiology.2002,4:1257-1264
[79]陈杰忠.果树栽培学各论,南方本.中国农业出版社,北京.2003.
[80]段艳欣.根癌农杆菌介导的柑橘转化体系优化与转LFY、APl基因植株的培育.2006年博士论文.
[81]Hidaka T,Omura M..Transformation of citrus protoplasts by electroporation[J].J Jpn Soc Hortic Sci,1993,62:371-376.
[82]Huang L-C.Huang B-L.Rhim S,et al.Genetically engineering citrus with the insecticidal gene from Bacillus thuringiensis subsp.Tenebrionis.Proceedings of a symposium on enhancing competitiveness of fruitindustry.Taichung,tai-Wan,March 1997:20-21.
[83]D.D.Li,W.Shi,X.X.Deng.Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene.Plant Cell Report,2002,21:153-156.
[84]Murashige T,Skoog F.A revised medium for rapid growth and bioassay with tobacco tissue cultures.Physiol Plant,1962,15:473-497.
[85]Murashige T and Tucker D P H.Growth factor requirements of Citrus tissue culture.Proc 1st Int Citrus Symp,1969,3:1155-1161
[86]DengZ.N,Gentile A,Nicolosi E,etal.ldentification of in vivo and in vitro lenmon mutants by RAPD makers(J).Hort.Sci.70(1):117-125
[87]黄家权,邬志荣,孙中海.影响椪柑上胚轴再生不定芽的几个生理因素.植物生理学通讯,2005,41(1):37-40.
[88]张家银,郭琛,谢玉明等.椪柑成年态节间茎段再生体系的建立.湖南农业大学学报,2008,34(2)
[89]Kobayashi S,Uchnimiya H.Expression and integration of a foreign gene in orange(Citrus sinensis Osb.) protoplasts by direct DNA transfer.The Japannese journal of genetics,1989,64(2):91-97.
[90]Vardi A,Bleichman S,Aviv D.Genetic transformation of Citrus protoplasts and regeneration of transgenic plants.Plant Science,1990,69:199-206.
[91]高峰,华学军,范六云,等.用根癌农杆菌离体转化柑橘.中国柑橘,1990,19(4):3-4.
[92]Almeida W.A.B.;Mourao Filho F.A.A.;Pino L.E.,et al.Genetic transformation and plant recovery from mature tissues of Citrus sinensis L.Osbeck.Plant Science,2003,164:203-211.
[93]姜玲,万蜀渊,王映红等.柑橘茎尖嫁接操作方法的改进及研究.华中农业大学学报,1995,14(4):381-385.
[94]罗永兰,张至元,喻珺.柑橘内生细菌的分离与鉴定.湖北农业科学,2006.45(6):773-775.
[95]王子成,李忠爱,邓秀新.柑橘成年态茎段外植体消毒方法研究.河南大学学报,2005,35(2):57-60
[96]Huang J Q,Yin L Y,Y Xiao-hong,et al.In vitro plant regeneration from the mature tissue of navel orange(Citrus sinensis L.Osbeck) by direct organogenesis.Agricultural Sciences in China,2005,4(3):236-240.
[97]放小平,胡新喜,郭琛,等.用rolB基因转化大红甜橙的初步研究.湖南农业大学学报:自然科学版,2005,31(6):623-626.
[98]谢玉明,邓子牛,郭琛,等.农杆菌介导甜橙遗传转化的初步研究.湖南农业大学学报:自 然科学版,2005,31 (6):627-630.