AtGPX3和AtCHX23玉米转基因植株的获得
摘要
玉米是我国主要的粮食作物之一。玉米生产常受干旱和盐胁迫的影响,这些胁迫导致玉米大量减产,因此提高玉米的抗旱耐盐性对于保证我国的粮食安全至关重要。
随着基因工程技术的发展,利用转基因技术,可以有效改良玉米的抗逆性,获得抗旱和耐盐的新品种。根癌农杆菌介导转化玉米的方法具有转化机理清楚、外源基因低拷贝整合等优点,但该法转化玉米成功率低,其转化效率受到玉米基因型、外植体类型、外植体生长状态、培养基成分、激素成分和配比、农杆菌菌株类型、农杆菌侵染时的浓度、侵染液pH值以及农杆菌侵染后的培养条件等多种因素影响,因此建立遗传稳定性强、转化率高、易于再生的高效转化体系是玉米转基因成功的关键。
我们构建了抗旱基因AtGPX3和耐盐基因AtCHX23的转基因载体,以玉米杂交种Hi-Ⅱ和优良自交系郑58和齐319幼胚作为外植体诱导愈伤组织,利用根癌农杆菌介导的转化方法,将AtGPX3和AtCHX23转入愈伤组织中,通过继代、筛选、分化和植株再生等多种步骤,获得了转基因苗,进一步利用叶片涂抹抗生素PPT和PCR方法,鉴定得到了转基因阳性苗,已得到了Hi-Ⅱ转基因种子。同时,我们诱导获得了郑58和齐319等优良自交系的愈伤组织并转化AtGPX3和AtCHX23基因,获得了的转基因植物。对转基因体系进行优化,发现以D培养基为基础培养基,添加L-谷氨酰胺可有效提高郑58胚性愈伤组织的诱导率。
另外,农杆菌转化后,在后期筛选过程中,抑制农杆菌的繁殖,对抗性愈伤的再生具有积极作用,本实验优化了抑菌方法,即在不降低转化效率的前提下,使用较低的侵染液浓度(OD_(600)=0.3-0.4),较短的侵染时间(5 min左右)以及多次清洗愈伤组织和延长抗生素浸泡愈伤组织时间(60 min)等方法,可有效抑制农杆菌的繁殖,提高转基因效率。
Maize is one of the main grain crops in our country. Maize productivity is often adversely affected by drought and salt stresses, which can significantly decrease the yield of Zea may plants. Therefore, it is essential to improve drought and salt tolerance of maize plants for food security of our country.
With the development of genetic engineering techniques, people can use the transgenic technology to effectively improve stress tolerance of maize and obtain drought and salt resistant new maize varieties.The Agrobacterium-mediated transgenic method in maize has many advantages over other gene transformation methods, for example clear transformation mechanisms, a high frequency of single or low copy insertion events et al. However, the transformation efficiency using Agrobacterium in maize is relatively low. The efficiency is markedly influenced by many factors including maize genotype, explant type, explant growth state, medium composition, hormone composition and ratio, type of Agrobacterium strains, concentration and pH of infection solution, and the culture conditions after infection and so on. Hence, establishing an effective transgenic transformation system with stable heredity, high infection rate, easy and efficient maize regeneration method is a key step for obtaining transgenic maize plants.
We constructed two transformation vectors harbouring drought tolerance gene AtGPX3 and salt tolerance gene AtCHX23 and transformed these vectors into callus cells of immature embryos from hybrid Hi-Ⅱ, or inbred lines Zheng 58 and Qi 319 maize plants using Agrobacterium mediated transgenic method.
These calli were then subcultured, selected, differentiated, and regenerated. A large number of transgenic seedlingswere obtained. Furthermore, we identified some positive transgenic plants by smearing PPT on tender leaf tip or by using PCR method. Seeds of transgenic Hi-Ⅱplants have been obtained. At the same time, good calli of maize inbred lines Zheng 58 and Qi 319 were generated, and gene AtGPX3 and AtCHX23 were transferred into the callus cells. Transgenic plants from the cells were gained. Additionally, the Agrobacterium-mediated transformation protocols were reinvestigated. We found that the type D based medium with L-glutamine can help to improve the embryogenic callus inducing rate.
It is known that the inhibition of Agrobacterium propagation after Agrobacterium mediated transformation of maize calli is critical transgenic plant regeneration from callus cells.In this study, we optimized the methods of inhibiting reproduction of Agrobacterium by decreasing the concentration of the infection solution (OD600 = 0.3-0.4), shortening the infection time (about 5 min), washing the calli more times with sterile water and immersing the calli in Cef-contained water for a longer time (about 60 min), without reducing the transformation efficiency. The knowledge lays a solid foundation for the investigation of maize engineering in the future.
随着基因工程技术的发展,利用转基因技术,可以有效改良玉米的抗逆性,获得抗旱和耐盐的新品种。根癌农杆菌介导转化玉米的方法具有转化机理清楚、外源基因低拷贝整合等优点,但该法转化玉米成功率低,其转化效率受到玉米基因型、外植体类型、外植体生长状态、培养基成分、激素成分和配比、农杆菌菌株类型、农杆菌侵染时的浓度、侵染液pH值以及农杆菌侵染后的培养条件等多种因素影响,因此建立遗传稳定性强、转化率高、易于再生的高效转化体系是玉米转基因成功的关键。
我们构建了抗旱基因AtGPX3和耐盐基因AtCHX23的转基因载体,以玉米杂交种Hi-Ⅱ和优良自交系郑58和齐319幼胚作为外植体诱导愈伤组织,利用根癌农杆菌介导的转化方法,将AtGPX3和AtCHX23转入愈伤组织中,通过继代、筛选、分化和植株再生等多种步骤,获得了转基因苗,进一步利用叶片涂抹抗生素PPT和PCR方法,鉴定得到了转基因阳性苗,已得到了Hi-Ⅱ转基因种子。同时,我们诱导获得了郑58和齐319等优良自交系的愈伤组织并转化AtGPX3和AtCHX23基因,获得了的转基因植物。对转基因体系进行优化,发现以D培养基为基础培养基,添加L-谷氨酰胺可有效提高郑58胚性愈伤组织的诱导率。
另外,农杆菌转化后,在后期筛选过程中,抑制农杆菌的繁殖,对抗性愈伤的再生具有积极作用,本实验优化了抑菌方法,即在不降低转化效率的前提下,使用较低的侵染液浓度(OD_(600)=0.3-0.4),较短的侵染时间(5 min左右)以及多次清洗愈伤组织和延长抗生素浸泡愈伤组织时间(60 min)等方法,可有效抑制农杆菌的繁殖,提高转基因效率。
Maize is one of the main grain crops in our country. Maize productivity is often adversely affected by drought and salt stresses, which can significantly decrease the yield of Zea may plants. Therefore, it is essential to improve drought and salt tolerance of maize plants for food security of our country.
With the development of genetic engineering techniques, people can use the transgenic technology to effectively improve stress tolerance of maize and obtain drought and salt resistant new maize varieties.The Agrobacterium-mediated transgenic method in maize has many advantages over other gene transformation methods, for example clear transformation mechanisms, a high frequency of single or low copy insertion events et al. However, the transformation efficiency using Agrobacterium in maize is relatively low. The efficiency is markedly influenced by many factors including maize genotype, explant type, explant growth state, medium composition, hormone composition and ratio, type of Agrobacterium strains, concentration and pH of infection solution, and the culture conditions after infection and so on. Hence, establishing an effective transgenic transformation system with stable heredity, high infection rate, easy and efficient maize regeneration method is a key step for obtaining transgenic maize plants.
We constructed two transformation vectors harbouring drought tolerance gene AtGPX3 and salt tolerance gene AtCHX23 and transformed these vectors into callus cells of immature embryos from hybrid Hi-Ⅱ, or inbred lines Zheng 58 and Qi 319 maize plants using Agrobacterium mediated transgenic method.
These calli were then subcultured, selected, differentiated, and regenerated. A large number of transgenic seedlingswere obtained. Furthermore, we identified some positive transgenic plants by smearing PPT on tender leaf tip or by using PCR method. Seeds of transgenic Hi-Ⅱplants have been obtained. At the same time, good calli of maize inbred lines Zheng 58 and Qi 319 were generated, and gene AtGPX3 and AtCHX23 were transferred into the callus cells. Transgenic plants from the cells were gained. Additionally, the Agrobacterium-mediated transformation protocols were reinvestigated. We found that the type D based medium with L-glutamine can help to improve the embryogenic callus inducing rate.
It is known that the inhibition of Agrobacterium propagation after Agrobacterium mediated transformation of maize calli is critical transgenic plant regeneration from callus cells.In this study, we optimized the methods of inhibiting reproduction of Agrobacterium by decreasing the concentration of the infection solution (OD600 = 0.3-0.4), shortening the infection time (about 5 min), washing the calli more times with sterile water and immersing the calli in Cef-contained water for a longer time (about 60 min), without reducing the transformation efficiency. The knowledge lays a solid foundation for the investigation of maize engineering in the future.
引文
[1] Rhdes CA, Pierce DA, Mettles IJ, et al. Genetically transformed maize plants from protoplasts [J]. Science, 1988, 240: 204-207.
[2]王宏伟.抗草甘膦基因转入玉米自交系及农杆菌介导玉米遗传转化体系的优化[D].沈阳:沈阳农业大学,2006.
[3] Gould J, Devey M, Hasegawa O. Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex [J]. Plant Physiol, 1991(95): 426-434.
[4] Schlappi M, Hohn B. T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium [J]. Plant Journal, 1992, 10: 2355-2360.
[5]李世润,张举任,陈惠民.玉米胚性愈伤组织诱导和植株再生[J].山东大学学报, 1990, 25(1): 116-124.
[6]孙伟,张江丽,黄丛林,等.玉米遗传转化方法的研究进展[J].安徽农业科学, 2007, 35(25): 7772-7774.
[7]杨爱国.农杆菌介导玉米愈伤组织遗传转化体系优化及基因工程雄性不育系初步研究[D].雅安:四川农业大学, 2006.
[8] Potrytus, L?rz H, Wernicke W. Culture and plant regeneration of hyoscyamus protoplasts [J]. Thoer Appl Gent, 1979, 54: 209-214.
[9] Chourey PS, Zurawski DB. Callus formation from protoplasts of a maize cell culture. Thoer Appl Gent, 1981, 59: 341-344.
[10] Kamo KK, Hodges TK. Estabilishment and characterization of long-term embryogenic callus and cell suspension cultures [J]. Plant Science, 1986, 45: 111-117.
[11] Klein TM, Wolf ED, Wu, et al. High-velocity microporjectiles for delivering nucleic acid into living cells [J]. Nature, 1987, 327: 70-73.
[12] Klein TM, Arentzem R, Lewis PA, et al. Genetic transformation of maize cells by particle bombardment [J]. Plant Physiol, 1989, 91: 440-444.
[13]谢友菊,王国英,林爱星.遗传工程概论[M].北京:中国农业大学出版社, 2005: 235.
[14]张英,穆楠,朴红梅.转基因技术在玉米遗传育种中的应用[J].生物技术通报, 2009, (1): 64-68.
[15]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, 32(4): 381-389.
[16] Sanford JC, Klein TM, Wolf ED, et al.Delivery of substances into cells and tissues using a particle bombardment process [J]. Particulate Science and Technology, 1987, 6(1): 27-37.
[17]陈英,曹毅,蒋彦,等.转基因玉米的遗传转化方法研究[J].四川草原, 2000, (1): 51-55.
[18] Koziel MG, Beland GL, Bowman C, et al. Field performance of elite transgenic maize plant expressing an insecticidal protein derived form bacillus thuringiensis [J]. Biotechnology, 1993, (11): 194.
[19]王国英,张宏,丁群星,等.几种玉米基因转移技术的研究及转基因植株的获得[J].生物工程学报, 1996, 12(1):45-49.
[20]周光宇,龚纂纂,王自芬.远缘杂交的分子基础-DNA片段杂交假设的一个论证[J].遗传学报, 1979, 6(4):405-412.
[21]祁永红,李春秋,陈昌喜,等.外源DNA导入玉米自交系获得变异新品系[J].黑龙江农业科学, 1998, (5): 14.
[22]闫新甫.转基因植物[M].北京:科学出版社, 2002.
[23] Zhang W, Wu R. PEG-mediated transformation of rice protoplasts [J]. Theor Appl Genet, 1988, (76): 835-840.
[24] Golovkin MV, Abraham M, Morocz S, et al. Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts [J].Plant Sci, 1993, 90(1): 41-52.
[25] Fromm ME, Taylor LP, Malbot V. Inheritance and expression of chimeric genes in Progeny of transgenic Plants [J]. Plant Cell, 1990, (2): 603-618.
[26]张颖.玉米自交系转基因受体系统的建立及Barnase/Barstar基因的遗传转化[D].雅安:四川农业大学, 2004.
[27]王钰.农杆菌介导玉米自交系受体材料筛选及遗传转化体系的建立[D].雅安:四川农业大学. 2006.
[28] Grimsley N, Hohn T, Davies JW, et al. Agrobacterium mediated delivery of infectious maize streak virus into maize plants [J]. Nature, 1987, 325: 171-179.
[29] Ishida Y, Saito H, Hiei Y, et al. High efficiency transformation of Maize (Zea Mays) mediated by Agrobacterium tumfaciens [J]. Nature Biotechnol, 1996, 14: 745-750.
[30]黄璐,卫志明.不同基因型玉米的再生能力和胚性与非胚性愈伤组织DNA的差异[J].植物生理学报, 1999, 25(4): 332-338.
[31]陈敏.玉米Na+/H+反向转运器基因的克隆及耐盐转基因玉米的获得[D].北京:中国农业大学, 2003.
[32]程艳松.拟南芥抗旱耐盐基因在转基因玉米中的整合、表达及功能的研究[D].北京:中国农业大学, 2008.
[33]连云.人工改造的Cry1Ac、Cry1Ie基因在转基因烟草和玉米中的共表达[D].北京:中国农业大学, 2008.
[34]朱友银.玉米再生与遗传转化影响因素研究及转基因玉米的获得[D].贵阳:贵州大学, 2007.
[35]杨建波,许智宏,卫志明.几种禾谷类作物中诱导农杆菌Vir区基因活化的酚类物质分析[J].植物生理学报, 1993, 187(2): 179-184.
[36] Schlappi M, Hohn B. Competence of immature maize embryos for Agrobacterium-mediated gene transfer [J]. Plant Cell, 1992, 4(1): 7-16.
[37]黄雪清,卫志明.影响农杆菌介导玉米优良自交系遗传转化的因素[J].实验生物学报, 2004, 37(5): 398-407.
[38]张荣,王国英,张晓红,等.根癌农杆菌介导的玉米遗传转化体系的建立[J].农业生物技术学报, 2001, 9(1): 45-48.
[39] Huang X, Wei Z. Successful Agrobacterium-mediated genetic transformation of maize elited inbred lines [J].Plant Cell Tissue Organ Cult, 2005, (83): 187-200.
[40] Rainier DM. Agrobacterium-mediated transformation of rice (Oryza Satival) [J]. Bio/Technology, 1993, (8): 33-38.
[41]张艳贞,魏松红,胡汉桥,等.农杆菌介导的优良玉米自交系遗传转化体系的建立[J].沈阳农业大学学报, 2002, 33(3): 195-199.
[42]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[J].遗传, 2002, 24(1): 35-39.
[43]王景雪,孙毅,崔贵梅,等.花粉介导法获得玉米转基因植株[J].植物学报, 2001, 43(3): 275-279.
[44]王国英,张宏,谢友菊,等.玉米胚性愈伤组织转化及转Bt植株的抗虫性[J].农业生物技术学报, 1995, 3: 50-53.
[45]王罡,张艳贞,魏松红,等.花粉管通道法将Bt毒蛋白基因导入玉米优良自交系[J].吉林农业大学学报, 2002, 24(4): 40-44.
[46]张红伟,张红梅,郑祖平,等. Bt转基因玉米的获得及对玉米螟的抗性分析[J].西北植物学报, 2004, 24(7): 1266-1270.
[47]权瑞党,尚梅,王兆玉,等.农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系[J].西北植物学报, 2004, 24(5): 761-767.
[48] Murry LE, Elliott LG, Capitant SA, et a1. Transgenic com plants expressing MDMV strain B coat prote in are resistant to mixed infections of maize dwarf mosaic virus [J]. Bio-Technology, 1993, 11: 1559-1564.
[49]刘小红,张红伟,刘昕,等. MDMVCP基因的克隆及其转基因玉米的研究[J].生物工程学报, 2005, 21(1): 144-145.
[50]李陶圣,杨爱芳,张举仁.玉米愈伤组织的遗传转化及抗除草剂植株再生[J].科学通报, 2000, 45(20): 2181-2184.
[51]刘小红,张红梅,张红伟,等.抗除草剂转基因玉米研究[J].西南农业学报, 2007, 20(1): 53-57.
[52]何锶洁,董伟,李慧芬.转甜菜碱脱氢酶基因玉米及其耐盐性研究[J].高技术通讯, 1999, (2):50～52.
[53]刘岩,王国英,刘俊君,大肠杆菌gutD基因转入玉米及耐盐转基因植株的获得[J].中国科学(C辑), 1998, 28(6): 542-547.
[54] Torrent M, Alvarez I, Geli MI. Lyaine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms [J]. Plant Molecular Biology, 1997, 34: 139～149.
[55]张秀君,刘俊起,赵倩.用基因枪将高赖氨酸基因导入玉米及转基因植株的检测[J].农业生物技术学报, 1999, 7(4): 363～367.
[56] Sun XH, Ao GM, Yu JJ. Transfer of high lysine gene into maize inbred lines and the detection of transgenic plants [J]. Journal of Agricultural Biotechnology, 2001, 9(2): 156～158.
[57]吴玉峰.共转化方法获得无标记高赖氨酸转基因玉米[D].北京:中国农业大学, 2004.
[58]丁明忠,潘光堂,荣廷昭,等. 2l世纪玉米遗传育种展望[C].玉米遗传育种国际学术讨论会文集, 2000.
[59] Mariani TD, Halluin K, et at. The production and analysis of genetically engineered male sierile plants of maize [C]. In: Abstr 35th Ann Maize Genetics Conf. Fargo: University Press, 1993, 35: 40-45.
[60]刘大文,王守才,谢友菊,等.转Zm13-Barnase基因玉米的获得及其花粉育性研究[J].植物学报,2000, 42(6): 611-615.
[61] Miao YC, Lv D, Wang PC, et al. An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses [J]. The Plant Cell, 2006, 18: 2749-2766.
[62] Song CP, Guo Y, Qiu QS, et al. A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana [J]. PNAS, 2004, 101(27): 10211-10216.
[2]王宏伟.抗草甘膦基因转入玉米自交系及农杆菌介导玉米遗传转化体系的优化[D].沈阳:沈阳农业大学,2006.
[3] Gould J, Devey M, Hasegawa O. Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex [J]. Plant Physiol, 1991(95): 426-434.
[4] Schlappi M, Hohn B. T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium [J]. Plant Journal, 1992, 10: 2355-2360.
[5]李世润,张举任,陈惠民.玉米胚性愈伤组织诱导和植株再生[J].山东大学学报, 1990, 25(1): 116-124.
[6]孙伟,张江丽,黄丛林,等.玉米遗传转化方法的研究进展[J].安徽农业科学, 2007, 35(25): 7772-7774.
[7]杨爱国.农杆菌介导玉米愈伤组织遗传转化体系优化及基因工程雄性不育系初步研究[D].雅安:四川农业大学, 2006.
[8] Potrytus, L?rz H, Wernicke W. Culture and plant regeneration of hyoscyamus protoplasts [J]. Thoer Appl Gent, 1979, 54: 209-214.
[9] Chourey PS, Zurawski DB. Callus formation from protoplasts of a maize cell culture. Thoer Appl Gent, 1981, 59: 341-344.
[10] Kamo KK, Hodges TK. Estabilishment and characterization of long-term embryogenic callus and cell suspension cultures [J]. Plant Science, 1986, 45: 111-117.
[11] Klein TM, Wolf ED, Wu, et al. High-velocity microporjectiles for delivering nucleic acid into living cells [J]. Nature, 1987, 327: 70-73.
[12] Klein TM, Arentzem R, Lewis PA, et al. Genetic transformation of maize cells by particle bombardment [J]. Plant Physiol, 1989, 91: 440-444.
[13]谢友菊,王国英,林爱星.遗传工程概论[M].北京:中国农业大学出版社, 2005: 235.
[14]张英,穆楠,朴红梅.转基因技术在玉米遗传育种中的应用[J].生物技术通报, 2009, (1): 64-68.
[15]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, 32(4): 381-389.
[16] Sanford JC, Klein TM, Wolf ED, et al.Delivery of substances into cells and tissues using a particle bombardment process [J]. Particulate Science and Technology, 1987, 6(1): 27-37.
[17]陈英,曹毅,蒋彦,等.转基因玉米的遗传转化方法研究[J].四川草原, 2000, (1): 51-55.
[18] Koziel MG, Beland GL, Bowman C, et al. Field performance of elite transgenic maize plant expressing an insecticidal protein derived form bacillus thuringiensis [J]. Biotechnology, 1993, (11): 194.
[19]王国英,张宏,丁群星,等.几种玉米基因转移技术的研究及转基因植株的获得[J].生物工程学报, 1996, 12(1):45-49.
[20]周光宇,龚纂纂,王自芬.远缘杂交的分子基础-DNA片段杂交假设的一个论证[J].遗传学报, 1979, 6(4):405-412.
[21]祁永红,李春秋,陈昌喜,等.外源DNA导入玉米自交系获得变异新品系[J].黑龙江农业科学, 1998, (5): 14.
[22]闫新甫.转基因植物[M].北京:科学出版社, 2002.
[23] Zhang W, Wu R. PEG-mediated transformation of rice protoplasts [J]. Theor Appl Genet, 1988, (76): 835-840.
[24] Golovkin MV, Abraham M, Morocz S, et al. Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts [J].Plant Sci, 1993, 90(1): 41-52.
[25] Fromm ME, Taylor LP, Malbot V. Inheritance and expression of chimeric genes in Progeny of transgenic Plants [J]. Plant Cell, 1990, (2): 603-618.
[26]张颖.玉米自交系转基因受体系统的建立及Barnase/Barstar基因的遗传转化[D].雅安:四川农业大学, 2004.
[27]王钰.农杆菌介导玉米自交系受体材料筛选及遗传转化体系的建立[D].雅安:四川农业大学. 2006.
[28] Grimsley N, Hohn T, Davies JW, et al. Agrobacterium mediated delivery of infectious maize streak virus into maize plants [J]. Nature, 1987, 325: 171-179.
[29] Ishida Y, Saito H, Hiei Y, et al. High efficiency transformation of Maize (Zea Mays) mediated by Agrobacterium tumfaciens [J]. Nature Biotechnol, 1996, 14: 745-750.
[30]黄璐,卫志明.不同基因型玉米的再生能力和胚性与非胚性愈伤组织DNA的差异[J].植物生理学报, 1999, 25(4): 332-338.
[31]陈敏.玉米Na+/H+反向转运器基因的克隆及耐盐转基因玉米的获得[D].北京:中国农业大学, 2003.
[32]程艳松.拟南芥抗旱耐盐基因在转基因玉米中的整合、表达及功能的研究[D].北京:中国农业大学, 2008.
[33]连云.人工改造的Cry1Ac、Cry1Ie基因在转基因烟草和玉米中的共表达[D].北京:中国农业大学, 2008.
[34]朱友银.玉米再生与遗传转化影响因素研究及转基因玉米的获得[D].贵阳:贵州大学, 2007.
[35]杨建波,许智宏,卫志明.几种禾谷类作物中诱导农杆菌Vir区基因活化的酚类物质分析[J].植物生理学报, 1993, 187(2): 179-184.
[36] Schlappi M, Hohn B. Competence of immature maize embryos for Agrobacterium-mediated gene transfer [J]. Plant Cell, 1992, 4(1): 7-16.
[37]黄雪清,卫志明.影响农杆菌介导玉米优良自交系遗传转化的因素[J].实验生物学报, 2004, 37(5): 398-407.
[38]张荣,王国英,张晓红,等.根癌农杆菌介导的玉米遗传转化体系的建立[J].农业生物技术学报, 2001, 9(1): 45-48.
[39] Huang X, Wei Z. Successful Agrobacterium-mediated genetic transformation of maize elited inbred lines [J].Plant Cell Tissue Organ Cult, 2005, (83): 187-200.
[40] Rainier DM. Agrobacterium-mediated transformation of rice (Oryza Satival) [J]. Bio/Technology, 1993, (8): 33-38.
[41]张艳贞,魏松红,胡汉桥,等.农杆菌介导的优良玉米自交系遗传转化体系的建立[J].沈阳农业大学学报, 2002, 33(3): 195-199.
[42]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[J].遗传, 2002, 24(1): 35-39.
[43]王景雪,孙毅,崔贵梅,等.花粉介导法获得玉米转基因植株[J].植物学报, 2001, 43(3): 275-279.
[44]王国英,张宏,谢友菊,等.玉米胚性愈伤组织转化及转Bt植株的抗虫性[J].农业生物技术学报, 1995, 3: 50-53.
[45]王罡,张艳贞,魏松红,等.花粉管通道法将Bt毒蛋白基因导入玉米优良自交系[J].吉林农业大学学报, 2002, 24(4): 40-44.
[46]张红伟,张红梅,郑祖平,等. Bt转基因玉米的获得及对玉米螟的抗性分析[J].西北植物学报, 2004, 24(7): 1266-1270.
[47]权瑞党,尚梅,王兆玉,等.农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系[J].西北植物学报, 2004, 24(5): 761-767.
[48] Murry LE, Elliott LG, Capitant SA, et a1. Transgenic com plants expressing MDMV strain B coat prote in are resistant to mixed infections of maize dwarf mosaic virus [J]. Bio-Technology, 1993, 11: 1559-1564.
[49]刘小红,张红伟,刘昕,等. MDMVCP基因的克隆及其转基因玉米的研究[J].生物工程学报, 2005, 21(1): 144-145.
[50]李陶圣,杨爱芳,张举仁.玉米愈伤组织的遗传转化及抗除草剂植株再生[J].科学通报, 2000, 45(20): 2181-2184.
[51]刘小红,张红梅,张红伟,等.抗除草剂转基因玉米研究[J].西南农业学报, 2007, 20(1): 53-57.
[52]何锶洁,董伟,李慧芬.转甜菜碱脱氢酶基因玉米及其耐盐性研究[J].高技术通讯, 1999, (2):50～52.
[53]刘岩,王国英,刘俊君,大肠杆菌gutD基因转入玉米及耐盐转基因植株的获得[J].中国科学(C辑), 1998, 28(6): 542-547.
[54] Torrent M, Alvarez I, Geli MI. Lyaine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms [J]. Plant Molecular Biology, 1997, 34: 139～149.
[55]张秀君,刘俊起,赵倩.用基因枪将高赖氨酸基因导入玉米及转基因植株的检测[J].农业生物技术学报, 1999, 7(4): 363～367.
[56] Sun XH, Ao GM, Yu JJ. Transfer of high lysine gene into maize inbred lines and the detection of transgenic plants [J]. Journal of Agricultural Biotechnology, 2001, 9(2): 156～158.
[57]吴玉峰.共转化方法获得无标记高赖氨酸转基因玉米[D].北京:中国农业大学, 2004.
[58]丁明忠,潘光堂,荣廷昭,等. 2l世纪玉米遗传育种展望[C].玉米遗传育种国际学术讨论会文集, 2000.
[59] Mariani TD, Halluin K, et at. The production and analysis of genetically engineered male sierile plants of maize [C]. In: Abstr 35th Ann Maize Genetics Conf. Fargo: University Press, 1993, 35: 40-45.
[60]刘大文,王守才,谢友菊,等.转Zm13-Barnase基因玉米的获得及其花粉育性研究[J].植物学报,2000, 42(6): 611-615.
[61] Miao YC, Lv D, Wang PC, et al. An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses [J]. The Plant Cell, 2006, 18: 2749-2766.
[62] Song CP, Guo Y, Qiu QS, et al. A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana [J]. PNAS, 2004, 101(27): 10211-10216.