Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar

Abstract

Abstract: Most maize disease are caused by fungi pathogens, such as northern leaf blight, southern leaf blight, ear and stalk rots, and so on. In normal years, this disease makes maize yield lost by 10% and this percentage may be increased by 30%-40% in its popular years. However, it is very difficult for conventional breeding to develop an elite maize material with the resistance to all the fungi disease. Therefore, we can use transgenic method to transfer some genes related to the resistance of these disease into maize in order to obtain resistance materials. Chitinase and β-1,3-glucanase are two important enzymes which play a key role in the hydrolysis reaction of fungal cell wall, so it is useful in the control of fungi disease, meanwhile, the herbicide resistant gene bar are usually used as a selectable marker in maize transformation.The purpose of this research is to introduce the fused gene of chitinase and β-1,3-glucanase and bar as well into elite maize inbred Zheng58 for improving its resistance to fungi disease. The wounded shoot apical meristem (SAM) of germinated seedlings were used as the material for Agrobacterium mediated transformation. We report here: 1) An optimized transformation system for Agrobacterium mediated transformation of shoot apical meristem, using the optimal concentration of bacterial culture (the value of OD₆₀₀ was 0.6) for infection, addition of 150 μmol/L acetosyringone (AS) in the bacterial suspension, the whole infection process was carried out in a vacuum desiccators with a negative pressure of 50 kPa for 12 minutes. 2) Identified by herbicide screening procedure and PCR detection, 13 transformed plants were obtained among the 32 herbicide-resistant plants, and the overall transformation was 2.6%.The preliminary evidences showed that the foreign genes had been introduced into the maize genome. This method circumvented the long period of tissue culture step and limitation of different seasons, in addition, many elite inbred lines which are recalcitrant in callus induction can be efficiently transformed by this method.

CLC Number:

FANG Yong-feng, LI Yong-sheng, PEN Yun-ling, WANG Fang, WANG Wei, MU Yan-zhao, WANG Han-ning. Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar[J]. Acta Prataculturae Sinica, 2012, 21(5): 69-76.

References

[1] 张世煌, 徐伟平, 李明顺, 等. 玉米育种面临的机遇和挑战[J]. 玉米科学, 2008, 16(6): 1-5.
[2] Grimsley N, Hohn T, Davies J W, et al. Agrobactcrium-mediated delivery of infectious maize streak virus into maize plants[J]. Nature, 1987, 325: 177-179.
[3] Ishida Y, Saito H, Ohta S, et al. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens[J]. Nature Biotechnology, 1996, 14(6): 745-750.
[4] 刘亚, 赵久然. 转基因玉米新种质创制若干技术环节的探讨[J]. 中国农业科技导报, 2011, 13(4): 46-52.
[5] Rafiqc M, Fatimaa T, Husnaina T, et al. Regeneration and transformation of an elite inbred line of maize (Zea mays L.), with a gene from Bacillus thuringiensis[J]. South African Journal of Botany, 2006, 72(3): 460-466.
[6] 周光宇, 翁坚, 龚蓁蓁, 等. 农业分子育种授粉后外源DNA导入植物的技术[J]. 中国农业科学, 1988, 21(3): 1-6.
[7] Wang J X, Sun Y, G Cui M, et al. Transgenic maize plants obtained by pollen-mediated transformation[J]. Acta Botanica Sinica, 2001, 43: 275-279.
[8] Sidorov V, Gilbertson L, Addae P, et al. Agrobacterium-mediated transformation of seedling-derived maize callus[J]. Plant Cell Reports, 2006, 25(4): 320-332.
[9] Wang J, Sun Y, Li Y. Maize genetic transformation by co-cultivating germinating seeds with Agrobacterium tumefaciens[J]. Biotechnology and Applied Biochemistry, 2007, 46(1): 51-55.
[10] 吕素莲, 尹小燕, 张举仁, 等. 农杆菌介导的棉花茎尖遗传转化及转betA植株的产生[J]. 高技术通讯, 2004, (11): 20-25.
[11] 梁欣欣, 刘录祥, 赵林姝, 等. 农杆菌介导法向小麦茎尖导入DREB1A基因的研究初报[J]. 麦类作物学报, 2007, 27(1): 16-19.
[12] 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[J]. 生物技术, 2007, 17(3): 2-5.
[13] 孙传波, 曲文利, 姜志磊, 等. 农杆菌介导向玉米茎尖导入HAL1基因的初步研究[J]. 玉米科学, 2009, 17(6): 32-34.
[14] 尹祥佳, 李永生, 王汉宁, 等. 抗双链RNA依赖性蛋白激酶PKR基因表达载体构建及在玉米中的遗传转化[J]. 华北农学报, 2011, 26(4): 15-22.
[15] Mauch F, Mauchman I B, Boll E R T. Antifungal hydrolases in pea tissue II. Inhibition of fungal growth by combination of chitinase and β-1, 3-glucanase[J]. Plant Physiology, 1988, 88: 936-942.
[16] 景岚, 康振生, 左豫虎. 植物抗真菌病害基因工程研究进展[J]. 干旱地区农业研究, 2004, 22(2): 205-210.
[17] 崔欣, 杨庆凯. 植物几丁质酶在抗真菌病害基因工程中的应用[J]. 植物保护, 2002, 28(1): 39-42.
[18] Broglie R, Roby D, Cressman R. Activation of a bean chitinase promoter in transgenic tobacco plants by phytopathogenic Fungi[J]. Journal of Cell Biochemistry, 1990, 14: 276-280.
[19] 程红梅, 简桂良, 倪万潮, 等. 转几丁质酶和β-1, 3-葡聚糖酶基因提高棉花对枯萎病和黄萎病的抗性[J]. 中国农业科学, 2005, 38(6): 1160-1166.
[20] 许明辉, 唐祚舜, 谭亚玲, 等. 几丁质酶-葡聚糖酶双价基因导入滇型杂交稻恢复系提高稻瘟病抗性的研究[J]. 遗传学报, 2003, 30(4): 330-334.
[21] 叶兴国, 程红梅, 徐惠君, 等. 转几丁质酶和β-1, 3-葡聚糖酶双价基因小麦的获得和鉴定[J]. 作物学报, 2005, 31(5): 583-586.
[22] 吴家和, 张献龙, 罗晓丽, 等. 转几丁质酶和葡聚糖酶基因棉花的获得及其对黄萎病的抗性[J]. 遗传学报, 2004, 31(2): 183-188.
[23] 黎定军, 赵开军, 周清明, 等. 转几丁质酶基因和β-1, 3-葡聚糖酶基因烟草的研究[J]. 湖南农业大学学报, 2002, 28(3): 211-213.
[24] Memelink J, Linthorst H J, Schilperoort R A, et al. Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns[J]. Plant Molecular Biology, 1990, 14(2): 119-126.
[25] 贾小霞, 张金文, 王汉宁, 等. Chi-Linker-Glu融合基因双价植物表达载体的构建及其相关鉴定[J].兰州大学学报(自然科学版), 2008, 12(6): 34-38.
[26] 贾小霞, 张金文, 王汉宁, 等. 抗真菌和抗除草剂基因多价植物表达载体构建及对烟草遗传转化的研究[J].草业学报, 2009, 18(1): 86-93.
[27] Hoisington D, Khairallah M, Gonzalez-de-leon D. Laboratory Protocols: CIMMYT Applied Molecular Genetics Laboratory[M]. Third Edition Mexico DF, CIMMYT, 2005.
[28] 萨姆布鲁克J, 拉塞尔D W. 分子克隆实验指南(第三版)[M]. 黄培堂, 译. 北京: 科学出版社, 2002: 597-598.
[29] 储俊, 许娜, 张强, 等. 农杆菌介导的药百合鳞片遗传转化体系的建立[J]. 草业学报, 2011, 20(6): 164-169.
[30] 蒋晓芳. 抗粗缩病RNA干扰载体的构建和玉米茎尖生长点原位转化[D]. 成都: 四川农业大学, 2010.
[31] 王琨, 胡银岗, 杨政伟, 等. 农杆菌介导TLPD34基因的玉米遗传转化及检测[J]. 西北农业学报, 2011, 20(5): 74-78.
[32] 张芳, 王舟, 宗俊勤, 等. 农杆菌介导的假俭草遗传转化体系的建立[J]. 草业学报, 2011, 20(2): 184-192.
[33] 徐开杰, 燕明献, 柴乖强, 等. 农杆菌不同菌液浓度对柳枝稷种子萌发及幼苗生长发育的影响[J]. 草业学报, 2012, 21(1): 206-212.
[34] Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant Journal, 1994, 6: 271-282.
[35] Zhao Z Y, Gu W, Tagliani L, et al. High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize[J]. Molecular Breeding, 2002, 8(4): 323-333.