苹果主栽品种β-1，3-葡聚糖酶基因转化及抗病性状检测
|
摘要
本研究以苹果(Malus domestica Borkh)主栽品种嘠拉(Gala)和富士(Fuji)组培苗为试材,研究了叶片离体再生和叶盘法遗传转化的若干影响因素,优化了苹果叶片离体再生不定芽系统和遗传转化系统,获得了携带β-1,3-葡聚糖酶基因的富士转化株系,并对嘎拉和富士转化株系进行了PCR,PCR-Sourthern和RT-PCR检测,成功获得了有目的基因表达的嘎拉和富士转化株系。进行接种苹果轮纹抗病检测鉴定时,转化株的β-1,3-葡聚糖酶、PAL和PPO活性较非转化株高,具有较强的抗病性。试验结果表明:
1.富士的不定芽再生培养基中直接加入10.0～15.0mg/L SNP可以显著提高其再生效率。
2.农杆菌在不加入抗生素的培养基中进行二次悬浮时,部分农杆菌会发生质粒丢失现象,随悬浮培养时间的延长,丢失率显著增高;质粒丢失程度与目的基因、启动子和农杆菌菌株的组合有关;同时,在0.5 h～6.0 h悬浮时间范围内,质粒丢失主要与菌液浓度有关,而与悬浮时间关系不大;直接吸取一悬液或离心后分别悬浮于细菌培养液和植物培养液等不同的悬浮培养方式下质粒稳定性没有差别。
3.研究了影响农杆菌介导遗传转化的各种因素,优化了遗传转化途径:外植体在蔗糖浓度为60～90g/L的分化培养基上预培养1~2d;农杆菌二次悬浮后OD600值0.3～0.4时在摇床上晃动侵染8min;干燥共培养2d后进行脱菌、选择、筛选。
4.共有12个富士转化株系通过了Kan抗性检测,具体包括三个方面:①转化株在附加Kan50mg/L的继代培养基上能够连续增殖继代,生长正常。②转化株叶片在附加或未加Kan50mg/L的再生培养基上的再生能力无明显差异。③转化株在附加Kan50mg/L的生根培养基中能正常生根。
5.通过Kan抗性筛选的12个富士株系经PCR检测有11个株系呈现阳性,再经PCR-Southern blot检测,证明17个嘎拉转化株系和11个富士转化株系的检测结果全部呈现阳性,证明β-1,3-葡聚糖酶基因已经整合到嘎拉和富士植株基因组中。RT-PCR检测显示,β-1,3-葡聚糖酶基因在17个嘎拉转化株系都有不同量的表达,而只在3个富士转化株系中有少量表达。
6.苹果轮纹病菌在24～28℃较适宜菌丝体生长,24℃时分生孢子囊和分生孢子发生最多;培养基pH5.0以及接种后在黑光灯下培养有利于分生孢子囊和分生孢子产生;菌丝体生长到一定程度后,刮除菌丝体对分生孢子囊和分生孢子的发生有显著促进作用。
7.对部分嘎拉转化株系接种轮纹病菌,对与抗病性相关的生理指标进行检测,结果表明:转化株系的β-1,3-葡聚糖酶活性、PAL和PPO活性均高于非转化株,表现较强抗病性。
In this study, the disease-resistant geneβ-1,3-glucanase was transferred into apple cultivar Gala and Fuji via Agrobacterium tumefaciens strain EHA 105. The adventitious bud regeneration system and genetic transformation system had been modified by studying the effect of explant types and status on the efficiency of regeneration and transformation. Transgenic plants were obtained and examined by PCR, PCR-Sourthern and RT-PCR. After inoculated Apple Ring Spot,the vigor ofβ-1,3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant. The result showed:
1. Adding 10.0～15.0mg/L SNP into culture medium could improve the regeneration rate significantly.
2. Some agrobacterium strains were plasmid instability when cultivated the second time under condition of liquid culture without presenting of antibiotic during genetic transformation via agrobacterium-mediated system, the losing increased along with the culture timing. No correlation was found between the losing and one single factor of target genes, promoters, and agrobacterium strains, but there was a correlation between the losing and combination of target genes, promoters, and agrobacterium strains. The losing was mainly related to the concentration of plasmids but liquid culture timing during the culture of 0.5 h~6.0 h. No difference was found in terms of the plasmid losing when liquid culture was done by taken solution directly from previous culture or centrifugate, and no matter bacteria culture or MS
3. By comparing the factors on agrobacterium-mediated gene transformation, it suggests the optimal protocol for gene transformation in apple would be: at first, the explants should be pre-cultured for one day or two on the inducing medium with 60～90mg/L saccharose; the bacterium concentration for inoculation should be cultivated twice in the medium without presenting of antibiotic, the appropriate bacterium concentration was OD600=0.3～0.4 around, and the co-cultivation time is about 8 minutes, co-cultured on the filter paper less for two days, and then, the explants were transferred onto the inducing medium.
4. There were twelve transgenic Fuji plantlets passed the kanamycin-resistant test. The kanamycin-resistant test of transgenic plantlets was based on the three main aspects:①The transgenic plantlets were able to be subcultured on media supplemented with 50mg/L kanamycin, continuously.②The leaf pieces of the transgenic plantlets showed as same regeneration ability on media containing 50mg/L kanamycin as on media without kanamycin.③The transgenic plantles were able to root normally on media supplemented with 50mg/L kanamycin.
5. Choose the plantlets which pass the kanamycin-resistant test for tested by PCR, the result showed that eleven transgenic plantlets showed specific positive band as same as the plasmid control.And also made PCR-Southern blot for further tests, which proved that seventeen Gala and eleven Fuji also showed specific positive band as same as the plasmid, The result indicated that the Glu gene has been integrated into the gala apple genome. Then through RT-PCR, it proved that the Glu gene has been expressed more or less in seventeen Gala and expressed a little in three Fuji.
6. The mycelia of Apple Ring Spot grew well under 24～28℃condition, and could generated conidiophores most. Adjusted culture medium pH to 5.0 and incubated under black light after inoculation could benefit to the conidiophore’s generation. The removing of mycelia after their growth to some extent had significant promoting effect on the conidiophore’s generation.
7. After inoculated Apple Ring Spot, the vigor ofβ-1, 3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant.
1.富士的不定芽再生培养基中直接加入10.0～15.0mg/L SNP可以显著提高其再生效率。
2.农杆菌在不加入抗生素的培养基中进行二次悬浮时,部分农杆菌会发生质粒丢失现象,随悬浮培养时间的延长,丢失率显著增高;质粒丢失程度与目的基因、启动子和农杆菌菌株的组合有关;同时,在0.5 h～6.0 h悬浮时间范围内,质粒丢失主要与菌液浓度有关,而与悬浮时间关系不大;直接吸取一悬液或离心后分别悬浮于细菌培养液和植物培养液等不同的悬浮培养方式下质粒稳定性没有差别。
3.研究了影响农杆菌介导遗传转化的各种因素,优化了遗传转化途径:外植体在蔗糖浓度为60～90g/L的分化培养基上预培养1~2d;农杆菌二次悬浮后OD600值0.3～0.4时在摇床上晃动侵染8min;干燥共培养2d后进行脱菌、选择、筛选。
4.共有12个富士转化株系通过了Kan抗性检测,具体包括三个方面:①转化株在附加Kan50mg/L的继代培养基上能够连续增殖继代,生长正常。②转化株叶片在附加或未加Kan50mg/L的再生培养基上的再生能力无明显差异。③转化株在附加Kan50mg/L的生根培养基中能正常生根。
5.通过Kan抗性筛选的12个富士株系经PCR检测有11个株系呈现阳性,再经PCR-Southern blot检测,证明17个嘎拉转化株系和11个富士转化株系的检测结果全部呈现阳性,证明β-1,3-葡聚糖酶基因已经整合到嘎拉和富士植株基因组中。RT-PCR检测显示,β-1,3-葡聚糖酶基因在17个嘎拉转化株系都有不同量的表达,而只在3个富士转化株系中有少量表达。
6.苹果轮纹病菌在24～28℃较适宜菌丝体生长,24℃时分生孢子囊和分生孢子发生最多;培养基pH5.0以及接种后在黑光灯下培养有利于分生孢子囊和分生孢子产生;菌丝体生长到一定程度后,刮除菌丝体对分生孢子囊和分生孢子的发生有显著促进作用。
7.对部分嘎拉转化株系接种轮纹病菌,对与抗病性相关的生理指标进行检测,结果表明:转化株系的β-1,3-葡聚糖酶活性、PAL和PPO活性均高于非转化株,表现较强抗病性。
In this study, the disease-resistant geneβ-1,3-glucanase was transferred into apple cultivar Gala and Fuji via Agrobacterium tumefaciens strain EHA 105. The adventitious bud regeneration system and genetic transformation system had been modified by studying the effect of explant types and status on the efficiency of regeneration and transformation. Transgenic plants were obtained and examined by PCR, PCR-Sourthern and RT-PCR. After inoculated Apple Ring Spot,the vigor ofβ-1,3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant. The result showed:
1. Adding 10.0～15.0mg/L SNP into culture medium could improve the regeneration rate significantly.
2. Some agrobacterium strains were plasmid instability when cultivated the second time under condition of liquid culture without presenting of antibiotic during genetic transformation via agrobacterium-mediated system, the losing increased along with the culture timing. No correlation was found between the losing and one single factor of target genes, promoters, and agrobacterium strains, but there was a correlation between the losing and combination of target genes, promoters, and agrobacterium strains. The losing was mainly related to the concentration of plasmids but liquid culture timing during the culture of 0.5 h~6.0 h. No difference was found in terms of the plasmid losing when liquid culture was done by taken solution directly from previous culture or centrifugate, and no matter bacteria culture or MS
3. By comparing the factors on agrobacterium-mediated gene transformation, it suggests the optimal protocol for gene transformation in apple would be: at first, the explants should be pre-cultured for one day or two on the inducing medium with 60～90mg/L saccharose; the bacterium concentration for inoculation should be cultivated twice in the medium without presenting of antibiotic, the appropriate bacterium concentration was OD600=0.3～0.4 around, and the co-cultivation time is about 8 minutes, co-cultured on the filter paper less for two days, and then, the explants were transferred onto the inducing medium.
4. There were twelve transgenic Fuji plantlets passed the kanamycin-resistant test. The kanamycin-resistant test of transgenic plantlets was based on the three main aspects:①The transgenic plantlets were able to be subcultured on media supplemented with 50mg/L kanamycin, continuously.②The leaf pieces of the transgenic plantlets showed as same regeneration ability on media containing 50mg/L kanamycin as on media without kanamycin.③The transgenic plantles were able to root normally on media supplemented with 50mg/L kanamycin.
5. Choose the plantlets which pass the kanamycin-resistant test for tested by PCR, the result showed that eleven transgenic plantlets showed specific positive band as same as the plasmid control.And also made PCR-Southern blot for further tests, which proved that seventeen Gala and eleven Fuji also showed specific positive band as same as the plasmid, The result indicated that the Glu gene has been integrated into the gala apple genome. Then through RT-PCR, it proved that the Glu gene has been expressed more or less in seventeen Gala and expressed a little in three Fuji.
6. The mycelia of Apple Ring Spot grew well under 24～28℃condition, and could generated conidiophores most. Adjusted culture medium pH to 5.0 and incubated under black light after inoculation could benefit to the conidiophore’s generation. The removing of mycelia after their growth to some extent had significant promoting effect on the conidiophore’s generation.
7. After inoculated Apple Ring Spot, the vigor ofβ-1, 3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant.
引文
[1]刘祖棋,张石城.植物抗性生理学[M].北京:中国农业出版社,1994.1-15.
[2]冯斌.异戊烯基转移酶基因转化苹果的研究[D].沈阳:沈阳农业大学, 1998.
[3] Ben JC.Cornelissen Leo SM[J]. Plant Physiol. 1993(11): 709-712.
[4] Brown AG, Janick J, More JN.Advances in Fruit Breeding [M].West LaFayette: Purdure Univ.Press. 1975.3-37.
[5] Kombrink E, Schmelrer E.Ihe hyperneneitive response and its role in local and systemic disease resiatance [J]. Eucupean Journal of Plain Pathology, 2001, 107: 69-78.
[6] Hammenxhmidi R.Phytoaleains: what have we learned after60 years [J]. Annual Review of Phytopetbology, 1999, 37: 285-306.
[7] Martin Heil, Riahard M, Bostock.Induced systemic resistance (ISR) against pathogens in the context induced plant defences [J]. Annals of Botanr, 2002, 89: 503-512.
[8] Vanloon L C, Van Strien E A.The families of pathogenesis related proteins, their activities, and comparative an analysis of PR1 type pmteine [J]. Phyaiol., Mol.Plant Pathol, 1999, 55: 85-97.
[9] SelitmnnikoH C P. Antifungnl pmteins[J]. Appl.Environ. Micro-biol, 2001, 67: 2883-2894.
[10]蓝海燕,陈正华.葡萄糖酶及其在植物中的发育调节和防卫反应[J].生物技术通报,1998, 4: 10-15.
[11] Clearke A E, Stone B A.Chemistry and Bioligy of (1, 3)-β-glucanse [M].Acedemic Press, 1988.
[12] Arlorio M, Ludwig A, Boller T, et al. Inhibition of growth by plant chitittaeea endβ-1,3-glucanases [J]. Protoplaetna, 1992, 171; 34-43.
[13] Ham K S, Wu S C, Darvill A G, et al. Fungal pathoens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis related endo-β-1,3-glucanases [J]. Plant J., 1997, 11: 169-179.
[14] Canihos Y. CET. In vitro effect of a bacterialβ-1,3-glucanase in strptomyces livdans on Bremia actucae[J]. Journal of Turkish Pbytopathology, 1995, 24(3): 115-120.
[15] Jensen L G, et al. Tranagenic barley expressing a protein-engineered, thermostable (1,3-1,4)-β-glucanase during germination [J]. Proceedings of the National Academy of Sciences of the United states of America, 1996, 93(8): 3487一3491.
[16]蓝海燕,王长海,张丽华,等.导入β-1,3-葡聚糖酶及几丁质酶基因的可育油菜及其抗菌核病的研究[J].生物工程学报, 2000, 16(2): 142-145.
[17]冯道荣,许新萍,卫剑文,等.使用双抗真菌蛋白提高水稻杭病性的研究[J].植物学报,1999, 41(11): 1187-1191.
[18]蓝海燕,周顺川,王长海,等.表达β-1,3-葡聚糖酶及几丁质醉基因的转基因可育烟草及其抗菌核病的研究[J].遗传学报, 2000, 27(1): 70-77.
[19] Barker C J, Orlandi E W. Active oxygen in plant path ogenesis[J]. Annu Rev Phytopathol, 1995, 33: 299.
[20] Mehdy M C.Active oxygen species inplant defense against pathogens [J]. P1ant Physio1, 1994, 105:467.
[21] Vine A, Tennaken R, Dixon R et al.H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response [J]. Cell, 1994, 79:583.
[22]谢春艳,宾金华,陈兆平,等.多酚氧化酶及其生理功能[J].生物学通报,1999, 34 (6): 11-3.
[23]欧阳光察,薛应龙.植物苯丙烷类代谢的生理意义及其调控[J].植物生理学通报, 1988, 3: 9-16.
[24]樊自红,沈瑞祥,周仲明.过氧化物酶和苯丙氨酸解氨酶与毛白杨抗锈性的关系[J].植物病理学报,1989, 19 (2): 95-00.
[25]李保聚,李云凤.黄瓜不同抗性品种感染黑星病菌后过氧化物酶和多酚氧化酶的变化[J].中国农业科学, 1998, 31(1)86-88.
[26]胡景江,文建雷.过氧化物酶和多酚氧化酶与杨树溃疡病抗性的关系[J].西北林学院学报,1990, 5(1): 46-1.
[27]毕阳,张维一.感病甜瓜果实的呼吸、乙烯及过氧化物酶变化的研究.植物病理学报[J], 1993, 23(1): 69-3.
[28]沈其益,阎龙飞,李庆基.棉花感染枯萎病后过氧化物同工酶的变化[J].植物学报, 1978, 20 (2): 108-13.
[29]赵军营,常永义,朱建兰.杏树受嗜果刀孢菌侵染后叶片内苯丙氨酸解氨酶活性的变化[J].果树科学, 2000, 17(4): 277-281.
[30] Petig N.Changes in POD and PPO associated with natural and induced resistance of tomato to F.wilt. Physiol[J].Plant Patho1, 1974, 4: 14-50.
[31] Park W M. Peroxidase activity in leaftissue of rice infected by Pyricularia oryzae[J]. Korean J. Plant Patho1. 1985, 1(3): 178-183.
[32]王雅平,吴兆苏,刘伊强.小麦抗赤霉病性的生化研究及其机制的探讨[J].作物学报, 1994, 20(3): 327-33.
[33]庄炳昌,王玉民,谢雪菊.抗性不同大豆品种感染灰斑病后若干生化反应[J].1993, 19 (6): 567-570
[34]李靖,利容千,袁文静.黄瓜感染霜霉病菌叶片中一些酶活性的变化[J].植物病理学报, 1991, 21(4): 277-283.
[35]杨莉,徐昌杰,陈昆松.果树转基因研究进展与产业化展望[J].果树学报, 2003, 20(6): 331-337.
[36] James DJ, Passey AE,Barbara DJ, el al. Cenelic transformation of apple (Malus pumila mill.) using a disarmed Ti-binary veclar [J]. Plant Cell Rep, 1989, 7: 658-661.
[37] Liu Q, Ingersall J, Owens L, el al. Response of transgenic Royal Gala apple (Malus domestica Barh h) schools carrying a modified cecropin MB39 gene, Lo Erwinia amylovara[J]. Plant Cell Rep, 2001, 20: 306-312.
[38]刘庆忠,赵红军,刘鹏,等.抗菌肽MB39基因导入皇家嘎拉苹果及其四倍体植株的培育[J].园艺学报, 2001, 28(5): 392-398.
[39] Norelli JL, Barejsza-Wysacha E, Reynaird JP, el al. Transgenic Royal Gala apple expressing allacin E has increased field resistance la Erwinia amylovara(fire blight)[J]. Acla Hort, 2000, 538: 631-633.
[40] Norelli JL, Bolar JP, Harman CE, el al. Transgennic apple plants expressing chilinases from Trichoderma have increased resistance lacab(Luria inacq ualis)[J]. Acla Hort , 2000, 538: 617-618.
[41] Bolar JP, Norelli JL, Wang KW, et al.Expression of endacilinase from Trichoderma harsianum in transgenic apple increase resistance la apple scab and reduces vigor [J]. Phylopalhol, 2000, 90: 72-77.
[42] Kikkert J R, Hebert Sorle D. Transgenic plantlets of‘Chancellor’grapevine (Vitis sp.) from ballistic transformation of embryogenic cell suspensions. Plant Cell Rep., 1996, 15: 311-16.
[43] Reyniord JP,Mourgurs F, Norelli J, et al. First evident for difference in fire blight resistance among transgenic pear clones expression attacin E gene from Hyalophora cecropia[J]. Plant Sci, 1999, 149: 23-31.
[44] Mourgues F, Brisset MN, Chevreau E, et al. Activily of different antibacterial peptides on Eruina amylovora growth, and evaluation of the phyloloxicily and slahilily of cecropins[J]. Plant Sci, 1998, 139: 83-91.
[45] Pulerka CJ, Bocchetti C, Dang P, et al. Pear transformed with a lytic peptide gene for disease control affects nontargel organism,pear psylla(Homoptera , Psyllidae)[J], J Entomol, 2002, 95: 797-802.
[46] Moffal AS, Crop engineering goes south [J]. Science, 1999, 285: 370-371.
[47]方宏筠,王关林,王火旭,等.抗菌肽基因转化樱桃矮化砧木获得抗根瘤病的转基因植株[J].植物学报, 1999, 41(11): 1192-1198.
[48] Fitch MMM, Manshardt RM,Consalves D, et al. Stable transformation of papaya via microprojectile, bombardment[J]Plant Cell Rep, 1990, 9: 189-194.
[49] Fitch MMM,Manshardt RM,Consalves D, D, et al. Virus resistant papaya plants derived from tissues bombarsed with the coat protein gene of papaya ringspot virus[J], Bio/Technol, 1992, 10: 1466-1472.
[50]李亚新.首例商品化的转基因果树-番木瓜[J].园艺学报, 2000, 28(1) :51.
[51]赵志英,周鹏,曾宪松,等.核酶基因转化番木瓜的研究[J].热带作物学报, 1998, 19(2): 20-25.
[52]汤浩茹,Wallbraun M,任正隆,等.通过农杆菌介导法将哈兹木酶几丁质酶ThEn-42基因导入核桃[J].园艺学报, 2001, 28(1): 12-18.
[53] Finslad K, Marlin RR, Transformation of strawberry for virus ressitance [J].Acta Hort, 1995, 385: 86-90.
[54]张丽丽,等.农杆菌介导β-1,3-葡聚糖酶基因转入嘎拉苹果的研究[D].保定:河北农业大学,2007
[55]王贵章,等.抗真菌病害基因转化苹果(Malus domestica)的研究[D].西安:西北农林科技大学,2006.
[56] Cheng M, Hu T C, Layton J, et al. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat [J]. In Vitro Cellular & Developmental Biology-Plant, 2003, 39: 595-604.
[57]丁莉萍,陈泠,李圣纯,等.根癌农杆菌介导小麦成熟胚遗传转化影响因素的研究[J].麦类作物学报,2007, 27(5): 761-766.
[58]黄培堂,等译.分子克隆实验指南[M].北京:科学出版社, 2002, 27.
[59] Neill S J, Desikan R, Hancock J T. Nitric oxide signaling in plants. Neu Phytologist, 3003, 1: 11-12.
[60]赵晓刚,徐张红,何奕昆,等.NO在植物中的调控作用.植物学报[J], 2004, 21(1): 44-51.
[61]李际红,邢世岩,焦振,等.窄冠白杨组培快繁与再生体系的建立[J].农业生物技术科学, 2008, 24(7): (60-64).
[62]韩小娇,杨洪强,由淑贞,等.平邑甜茶叶片不定芽再生及NO的效应[J],园艺学报, 2008, 35(3): 419-422.
[63]Tun N N, Holk A, Scherer F E. Rapid increase of NO release in plant cell culture induced by cytokinin . FEBS.
[64] ?tv?s K, Pasternak T P, et al.Nitric oxide is required for , and promotes auxin-mediated activation of , cell division and em-bryogenic cel formation but dose not influence cell cycle progression in alfafa cell cultures. The Plant Journal, 2005, 43: 849-860.
[65]师校欣,王斌,杜国强等.根癌农杆菌介导豇豆胰蛋白酶抑制剂基因转入苹果主栽品种[J].园艺学报, 2000, 27(3): 282-284.
[66]施利利,王松文,蔡宝立等.影响农杆菌介导的水稻遗传转化效率的因素[J].天津农学院学报, 2003, 10(4): 53-57.
[67]李达旭,张杰,赵建等.根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织[J].植物生理与分子生物学报, 2006, 32 (1): 45-51.
[68]毕瑞明,贾海燕,封德顺等.农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析[J].生物工程学报, 2006, 22 (3): 431-437.
[69]余叔文.植物生理与分子生物学[M].北京:北京科学出版社,1998.
[70]万钧,覃筱婷,周俊初.三叶草素基因在快生型大豆根瘤菌中的表达[J].高技术通讯, 1996, 11: 50-52.
[71]郭丽红,陈善娜,龚明.玉米幼苗热激诱导抗冷性过程中钙的效应[J].云南大学学报,2003, 25(02): 148-152.
[72]张志宏,景士西,王关林.果树基因工程研究进展[J].果树科学, 1995, 12(3): 188~193.
[73] Jamea DJ.Passey AJ, Barbara DJ, Genetic transformation of apple (Malus punila Mill.) using a disarmed Ti-binary vector [J]. Plant Cell Reports, 1989, 7: 658~661.
[74] Danderkar AM, Urastu SL, Matsuta N. Factors influencing virulence in Agrobacterium-mediated transformation of apple[J]. Acta Hort, 1990, 280: 483~494.
[75] Lambert C, Tepfer D. Use of agrobacterium rhizogens to creat transgenic apple trees having at altered organogenic response to hormones[J]. Theor Apple Genet, 1992, 85: 105~109.
[76]程家胜,鄂超苏,田颖川等.转Bt抗虫基因苹果植株再生[J].中国果树, 1994(4): 14~15.
[77]程家胜, Dandekar AM, Urastu SL.苹果基因转移技术初报[J].园艺学报, 1992, 19(2): 101~104.
[78]潘增光,邓秀新.苹果组织培养再生技术研究进展[J].果树科学, 1998, 15(3): 261~266.
[79]刘北东,等.农杆菌介导几丁质酶基因转化大豆子叶主要影响因素的研究[D].哈尔滨:东北农业大学,2001.
[80]张振霞,刘萍,杜雪玲等.农杆菌介导GUS基因对多年生黑麦草转化的研究[J].广西植物.2002, 27(1): 121-126.
[81]易自力,曹守云,王力,等.提高农杆菌转化水稻频率的研究[J] .遗传学报,2001, 28( 4) : 352- 358.
[82] Ming C, Tianci H,Jeanne L, Chong N L, Oyce E F.Desiccation of plant tissues post-Agrobacterium infection enhancesT-DNA delivery and increases stable transformation efficiency inwheat. Vitro Cell Dev Biol,2003, 39: 595-604.
[83]王艳丽,叶兴国,杜丽璞等.农杆菌敏感小麦基因型的筛选研究[J].麦类作物学报.2005, 25(6): 6-10.
[84]石云鹭,丁在松,张彬等.影响农杆菌介导旱稻转化效率主要因素的研究[J].作物学报,2007, 33(7): 1135-1140.
[85]梁业红,叶兴国,张世煌.适用4种玉米基因型的农杆菌转化方法的探讨[J].作物学报.2007, 33(5): 771-775.
[86]丁莉萍,陈泠,李圣纯等.跟癌农杆菌介导小麦成熟胚遗传转化影响因素的研究[J].麦类作物学报.2007, 27(5): 761-766.
[87]马慧,赵开军,徐正进.农杆菌介导的水稻遗传转化现状及展望[J].中国生物工程杂志,2003, 23(4): 23~31.
[88] Rshid H, Yokoi S, Toriyama K. Transgenic plant production mediated by Agrobacterium in Indian rice [J]. Plant Cell Rep, 1996, 15: 727~730.
[89]洪玉梅,李美娜,乔壮,等.不同培养条件对苹果轮纹烂果病病菌的影响[J].中国果树,2001(2):29-30.
[90]刘晓琳,马丽亚,王勇,等.苹果轮纹病致病菌的生物学特性初探[J].天津农业科学,1998, 4(2): 10-12.
[91]吴桂本,刘传德,王继秋,等.苹果轮纹病和炭疽病病原菌生物学研究[J].中国果树, 2001, (1): 7-11.
[92]薛莲,檀根甲.采后苹果果实轮纹病的研究进展[J].安徽农业科学,2004, 32(6): 1227-1230, 1266.
[93]刘菊花.龙牙百合几丁质酶和β-1,3-葡聚糖酶基因的遗传转化研究[D].长沙:湖南农业大学,2003.
[94] Marjori A M et al. 1995. Plant Physiol, 107: 679-685.
[95]吴刚等.生物技术. 2000. 10(2): 27-32.
[96] Matzke M A et al. 1995. Trends, inGenetic. 11: 1-3.
[97]王关林,方宏筠.植物基因工程(第二版). 2002: 640-646.
[2]冯斌.异戊烯基转移酶基因转化苹果的研究[D].沈阳:沈阳农业大学, 1998.
[3] Ben JC.Cornelissen Leo SM[J]. Plant Physiol. 1993(11): 709-712.
[4] Brown AG, Janick J, More JN.Advances in Fruit Breeding [M].West LaFayette: Purdure Univ.Press. 1975.3-37.
[5] Kombrink E, Schmelrer E.Ihe hyperneneitive response and its role in local and systemic disease resiatance [J]. Eucupean Journal of Plain Pathology, 2001, 107: 69-78.
[6] Hammenxhmidi R.Phytoaleains: what have we learned after60 years [J]. Annual Review of Phytopetbology, 1999, 37: 285-306.
[7] Martin Heil, Riahard M, Bostock.Induced systemic resistance (ISR) against pathogens in the context induced plant defences [J]. Annals of Botanr, 2002, 89: 503-512.
[8] Vanloon L C, Van Strien E A.The families of pathogenesis related proteins, their activities, and comparative an analysis of PR1 type pmteine [J]. Phyaiol., Mol.Plant Pathol, 1999, 55: 85-97.
[9] SelitmnnikoH C P. Antifungnl pmteins[J]. Appl.Environ. Micro-biol, 2001, 67: 2883-2894.
[10]蓝海燕,陈正华.葡萄糖酶及其在植物中的发育调节和防卫反应[J].生物技术通报,1998, 4: 10-15.
[11] Clearke A E, Stone B A.Chemistry and Bioligy of (1, 3)-β-glucanse [M].Acedemic Press, 1988.
[12] Arlorio M, Ludwig A, Boller T, et al. Inhibition of growth by plant chitittaeea endβ-1,3-glucanases [J]. Protoplaetna, 1992, 171; 34-43.
[13] Ham K S, Wu S C, Darvill A G, et al. Fungal pathoens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis related endo-β-1,3-glucanases [J]. Plant J., 1997, 11: 169-179.
[14] Canihos Y. CET. In vitro effect of a bacterialβ-1,3-glucanase in strptomyces livdans on Bremia actucae[J]. Journal of Turkish Pbytopathology, 1995, 24(3): 115-120.
[15] Jensen L G, et al. Tranagenic barley expressing a protein-engineered, thermostable (1,3-1,4)-β-glucanase during germination [J]. Proceedings of the National Academy of Sciences of the United states of America, 1996, 93(8): 3487一3491.
[16]蓝海燕,王长海,张丽华,等.导入β-1,3-葡聚糖酶及几丁质酶基因的可育油菜及其抗菌核病的研究[J].生物工程学报, 2000, 16(2): 142-145.
[17]冯道荣,许新萍,卫剑文,等.使用双抗真菌蛋白提高水稻杭病性的研究[J].植物学报,1999, 41(11): 1187-1191.
[18]蓝海燕,周顺川,王长海,等.表达β-1,3-葡聚糖酶及几丁质醉基因的转基因可育烟草及其抗菌核病的研究[J].遗传学报, 2000, 27(1): 70-77.
[19] Barker C J, Orlandi E W. Active oxygen in plant path ogenesis[J]. Annu Rev Phytopathol, 1995, 33: 299.
[20] Mehdy M C.Active oxygen species inplant defense against pathogens [J]. P1ant Physio1, 1994, 105:467.
[21] Vine A, Tennaken R, Dixon R et al.H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response [J]. Cell, 1994, 79:583.
[22]谢春艳,宾金华,陈兆平,等.多酚氧化酶及其生理功能[J].生物学通报,1999, 34 (6): 11-3.
[23]欧阳光察,薛应龙.植物苯丙烷类代谢的生理意义及其调控[J].植物生理学通报, 1988, 3: 9-16.
[24]樊自红,沈瑞祥,周仲明.过氧化物酶和苯丙氨酸解氨酶与毛白杨抗锈性的关系[J].植物病理学报,1989, 19 (2): 95-00.
[25]李保聚,李云凤.黄瓜不同抗性品种感染黑星病菌后过氧化物酶和多酚氧化酶的变化[J].中国农业科学, 1998, 31(1)86-88.
[26]胡景江,文建雷.过氧化物酶和多酚氧化酶与杨树溃疡病抗性的关系[J].西北林学院学报,1990, 5(1): 46-1.
[27]毕阳,张维一.感病甜瓜果实的呼吸、乙烯及过氧化物酶变化的研究.植物病理学报[J], 1993, 23(1): 69-3.
[28]沈其益,阎龙飞,李庆基.棉花感染枯萎病后过氧化物同工酶的变化[J].植物学报, 1978, 20 (2): 108-13.
[29]赵军营,常永义,朱建兰.杏树受嗜果刀孢菌侵染后叶片内苯丙氨酸解氨酶活性的变化[J].果树科学, 2000, 17(4): 277-281.
[30] Petig N.Changes in POD and PPO associated with natural and induced resistance of tomato to F.wilt. Physiol[J].Plant Patho1, 1974, 4: 14-50.
[31] Park W M. Peroxidase activity in leaftissue of rice infected by Pyricularia oryzae[J]. Korean J. Plant Patho1. 1985, 1(3): 178-183.
[32]王雅平,吴兆苏,刘伊强.小麦抗赤霉病性的生化研究及其机制的探讨[J].作物学报, 1994, 20(3): 327-33.
[33]庄炳昌,王玉民,谢雪菊.抗性不同大豆品种感染灰斑病后若干生化反应[J].1993, 19 (6): 567-570
[34]李靖,利容千,袁文静.黄瓜感染霜霉病菌叶片中一些酶活性的变化[J].植物病理学报, 1991, 21(4): 277-283.
[35]杨莉,徐昌杰,陈昆松.果树转基因研究进展与产业化展望[J].果树学报, 2003, 20(6): 331-337.
[36] James DJ, Passey AE,Barbara DJ, el al. Cenelic transformation of apple (Malus pumila mill.) using a disarmed Ti-binary veclar [J]. Plant Cell Rep, 1989, 7: 658-661.
[37] Liu Q, Ingersall J, Owens L, el al. Response of transgenic Royal Gala apple (Malus domestica Barh h) schools carrying a modified cecropin MB39 gene, Lo Erwinia amylovara[J]. Plant Cell Rep, 2001, 20: 306-312.
[38]刘庆忠,赵红军,刘鹏,等.抗菌肽MB39基因导入皇家嘎拉苹果及其四倍体植株的培育[J].园艺学报, 2001, 28(5): 392-398.
[39] Norelli JL, Barejsza-Wysacha E, Reynaird JP, el al. Transgenic Royal Gala apple expressing allacin E has increased field resistance la Erwinia amylovara(fire blight)[J]. Acla Hort, 2000, 538: 631-633.
[40] Norelli JL, Bolar JP, Harman CE, el al. Transgennic apple plants expressing chilinases from Trichoderma have increased resistance lacab(Luria inacq ualis)[J]. Acla Hort , 2000, 538: 617-618.
[41] Bolar JP, Norelli JL, Wang KW, et al.Expression of endacilinase from Trichoderma harsianum in transgenic apple increase resistance la apple scab and reduces vigor [J]. Phylopalhol, 2000, 90: 72-77.
[42] Kikkert J R, Hebert Sorle D. Transgenic plantlets of‘Chancellor’grapevine (Vitis sp.) from ballistic transformation of embryogenic cell suspensions. Plant Cell Rep., 1996, 15: 311-16.
[43] Reyniord JP,Mourgurs F, Norelli J, et al. First evident for difference in fire blight resistance among transgenic pear clones expression attacin E gene from Hyalophora cecropia[J]. Plant Sci, 1999, 149: 23-31.
[44] Mourgues F, Brisset MN, Chevreau E, et al. Activily of different antibacterial peptides on Eruina amylovora growth, and evaluation of the phyloloxicily and slahilily of cecropins[J]. Plant Sci, 1998, 139: 83-91.
[45] Pulerka CJ, Bocchetti C, Dang P, et al. Pear transformed with a lytic peptide gene for disease control affects nontargel organism,pear psylla(Homoptera , Psyllidae)[J], J Entomol, 2002, 95: 797-802.
[46] Moffal AS, Crop engineering goes south [J]. Science, 1999, 285: 370-371.
[47]方宏筠,王关林,王火旭,等.抗菌肽基因转化樱桃矮化砧木获得抗根瘤病的转基因植株[J].植物学报, 1999, 41(11): 1192-1198.
[48] Fitch MMM, Manshardt RM,Consalves D, et al. Stable transformation of papaya via microprojectile, bombardment[J]Plant Cell Rep, 1990, 9: 189-194.
[49] Fitch MMM,Manshardt RM,Consalves D, D, et al. Virus resistant papaya plants derived from tissues bombarsed with the coat protein gene of papaya ringspot virus[J], Bio/Technol, 1992, 10: 1466-1472.
[50]李亚新.首例商品化的转基因果树-番木瓜[J].园艺学报, 2000, 28(1) :51.
[51]赵志英,周鹏,曾宪松,等.核酶基因转化番木瓜的研究[J].热带作物学报, 1998, 19(2): 20-25.
[52]汤浩茹,Wallbraun M,任正隆,等.通过农杆菌介导法将哈兹木酶几丁质酶ThEn-42基因导入核桃[J].园艺学报, 2001, 28(1): 12-18.
[53] Finslad K, Marlin RR, Transformation of strawberry for virus ressitance [J].Acta Hort, 1995, 385: 86-90.
[54]张丽丽,等.农杆菌介导β-1,3-葡聚糖酶基因转入嘎拉苹果的研究[D].保定:河北农业大学,2007
[55]王贵章,等.抗真菌病害基因转化苹果(Malus domestica)的研究[D].西安:西北农林科技大学,2006.
[56] Cheng M, Hu T C, Layton J, et al. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat [J]. In Vitro Cellular & Developmental Biology-Plant, 2003, 39: 595-604.
[57]丁莉萍,陈泠,李圣纯,等.根癌农杆菌介导小麦成熟胚遗传转化影响因素的研究[J].麦类作物学报,2007, 27(5): 761-766.
[58]黄培堂,等译.分子克隆实验指南[M].北京:科学出版社, 2002, 27.
[59] Neill S J, Desikan R, Hancock J T. Nitric oxide signaling in plants. Neu Phytologist, 3003, 1: 11-12.
[60]赵晓刚,徐张红,何奕昆,等.NO在植物中的调控作用.植物学报[J], 2004, 21(1): 44-51.
[61]李际红,邢世岩,焦振,等.窄冠白杨组培快繁与再生体系的建立[J].农业生物技术科学, 2008, 24(7): (60-64).
[62]韩小娇,杨洪强,由淑贞,等.平邑甜茶叶片不定芽再生及NO的效应[J],园艺学报, 2008, 35(3): 419-422.
[63]Tun N N, Holk A, Scherer F E. Rapid increase of NO release in plant cell culture induced by cytokinin . FEBS.
[64] ?tv?s K, Pasternak T P, et al.Nitric oxide is required for , and promotes auxin-mediated activation of , cell division and em-bryogenic cel formation but dose not influence cell cycle progression in alfafa cell cultures. The Plant Journal, 2005, 43: 849-860.
[65]师校欣,王斌,杜国强等.根癌农杆菌介导豇豆胰蛋白酶抑制剂基因转入苹果主栽品种[J].园艺学报, 2000, 27(3): 282-284.
[66]施利利,王松文,蔡宝立等.影响农杆菌介导的水稻遗传转化效率的因素[J].天津农学院学报, 2003, 10(4): 53-57.
[67]李达旭,张杰,赵建等.根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织[J].植物生理与分子生物学报, 2006, 32 (1): 45-51.
[68]毕瑞明,贾海燕,封德顺等.农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析[J].生物工程学报, 2006, 22 (3): 431-437.
[69]余叔文.植物生理与分子生物学[M].北京:北京科学出版社,1998.
[70]万钧,覃筱婷,周俊初.三叶草素基因在快生型大豆根瘤菌中的表达[J].高技术通讯, 1996, 11: 50-52.
[71]郭丽红,陈善娜,龚明.玉米幼苗热激诱导抗冷性过程中钙的效应[J].云南大学学报,2003, 25(02): 148-152.
[72]张志宏,景士西,王关林.果树基因工程研究进展[J].果树科学, 1995, 12(3): 188~193.
[73] Jamea DJ.Passey AJ, Barbara DJ, Genetic transformation of apple (Malus punila Mill.) using a disarmed Ti-binary vector [J]. Plant Cell Reports, 1989, 7: 658~661.
[74] Danderkar AM, Urastu SL, Matsuta N. Factors influencing virulence in Agrobacterium-mediated transformation of apple[J]. Acta Hort, 1990, 280: 483~494.
[75] Lambert C, Tepfer D. Use of agrobacterium rhizogens to creat transgenic apple trees having at altered organogenic response to hormones[J]. Theor Apple Genet, 1992, 85: 105~109.
[76]程家胜,鄂超苏,田颖川等.转Bt抗虫基因苹果植株再生[J].中国果树, 1994(4): 14~15.
[77]程家胜, Dandekar AM, Urastu SL.苹果基因转移技术初报[J].园艺学报, 1992, 19(2): 101~104.
[78]潘增光,邓秀新.苹果组织培养再生技术研究进展[J].果树科学, 1998, 15(3): 261~266.
[79]刘北东,等.农杆菌介导几丁质酶基因转化大豆子叶主要影响因素的研究[D].哈尔滨:东北农业大学,2001.
[80]张振霞,刘萍,杜雪玲等.农杆菌介导GUS基因对多年生黑麦草转化的研究[J].广西植物.2002, 27(1): 121-126.
[81]易自力,曹守云,王力,等.提高农杆菌转化水稻频率的研究[J] .遗传学报,2001, 28( 4) : 352- 358.
[82] Ming C, Tianci H,Jeanne L, Chong N L, Oyce E F.Desiccation of plant tissues post-Agrobacterium infection enhancesT-DNA delivery and increases stable transformation efficiency inwheat. Vitro Cell Dev Biol,2003, 39: 595-604.
[83]王艳丽,叶兴国,杜丽璞等.农杆菌敏感小麦基因型的筛选研究[J].麦类作物学报.2005, 25(6): 6-10.
[84]石云鹭,丁在松,张彬等.影响农杆菌介导旱稻转化效率主要因素的研究[J].作物学报,2007, 33(7): 1135-1140.
[85]梁业红,叶兴国,张世煌.适用4种玉米基因型的农杆菌转化方法的探讨[J].作物学报.2007, 33(5): 771-775.
[86]丁莉萍,陈泠,李圣纯等.跟癌农杆菌介导小麦成熟胚遗传转化影响因素的研究[J].麦类作物学报.2007, 27(5): 761-766.
[87]马慧,赵开军,徐正进.农杆菌介导的水稻遗传转化现状及展望[J].中国生物工程杂志,2003, 23(4): 23~31.
[88] Rshid H, Yokoi S, Toriyama K. Transgenic plant production mediated by Agrobacterium in Indian rice [J]. Plant Cell Rep, 1996, 15: 727~730.
[89]洪玉梅,李美娜,乔壮,等.不同培养条件对苹果轮纹烂果病病菌的影响[J].中国果树,2001(2):29-30.
[90]刘晓琳,马丽亚,王勇,等.苹果轮纹病致病菌的生物学特性初探[J].天津农业科学,1998, 4(2): 10-12.
[91]吴桂本,刘传德,王继秋,等.苹果轮纹病和炭疽病病原菌生物学研究[J].中国果树, 2001, (1): 7-11.
[92]薛莲,檀根甲.采后苹果果实轮纹病的研究进展[J].安徽农业科学,2004, 32(6): 1227-1230, 1266.
[93]刘菊花.龙牙百合几丁质酶和β-1,3-葡聚糖酶基因的遗传转化研究[D].长沙:湖南农业大学,2003.
[94] Marjori A M et al. 1995. Plant Physiol, 107: 679-685.
[95]吴刚等.生物技术. 2000. 10(2): 27-32.
[96] Matzke M A et al. 1995. Trends, inGenetic. 11: 1-3.
[97]王关林,方宏筠.植物基因工程(第二版). 2002: 640-646.