小麦花粉电穿孔转化因素优化和转基因植株获得及鉴定

doi:10.3864/j.issn.0578-1752.2013.12.001

摘要/Abstract

摘要： 【目的】通过优化电穿孔转化技术主要参数，得到候选转基因植株，探索普通小麦（Triticum aestivum L.）电穿孔遗传转化体系。【方法】以冬小麦品种济麦19为受体，GUS为外源基因，将扬花期成熟花粉以最适电场强度和操作温度进行电穿孔处理后，人工授粉济麦19，最终收获种子；利用PCR技术、Southern技术、化学染色方法对T1、T2植株进行鉴定。【结果】电穿孔转化以电场强度6 kV•cm-1、冰上处理花粉，花粉密度为5×106个/mL，以及去雄后第5天授粉为最佳参数；Southern杂交检测结果表明，T2阳性植株的2个株系检测到杂交信号，同时其幼根、叶片等组织经GUS表达活性的组织化学染色检测，呈现蓝色反应。【结论】利用小麦花粉电穿孔转化法可以成功将外源基因整合到小麦基因组中并稳定遗传至T2，且外源基因GUS能够得到表达。

关键词: 小麦 , 花粉 , 花粉电穿孔转化 , 参数优化

Abstract: 【Objective】The objective of this study was to establish a pollen electroporation transformation system for common wheat (Triticum aestivum L.) by optimizing the main parameters of transformation for obtaining transgenic wheat plants.【Method】The pollens of wheat were collected in the pollen germination buffer mixed with GUS gene and treated with electroporation, and then pollinated Jimai19 to obtain transgenic plants. The putative transformed plants were confirmed by PCR, Southern blot，histochemical staining of GUS positive reaction.【Result】The optimization parameters of pollen electroporation transformation were summarized: pulse field strength 6 kV•cm-1, operating on ice, pollen density 5×106 pollens/mL, and pollinating on the 5th day after castration. Southern blot proved that two T2 transgenic wheat lines showed positive signals of GUS gene. Meanwhile, histochemical staining showed that the blue color was detected in young roots, leaves of the transgenic plants.【Conclusion】Exogenous gene was integrated into the genome of common wheat by the pollen electroporation transformation method and stably expressed in progeny of transgenic plant.

Key words: wheat , pollen , pollen electroporation transformation , optimization parameters

张小红, 赵雪晶, 李波, 黎飞飞, 刘沛璇, 闵东红. 小麦花粉电穿孔转化因素优化和转基因植株获得及鉴定[J]. 中国农业科学, 2013, 46(12): 2403-2411.

ZHANG Xiao-Hong, ZHAO Xue-Jing, LI Bo, LI Fei-Fei, LIU Pei-Xuan, MIN Dong-Hong. Factors Optimization of Pollen Electroporation Transformation and Identification of Transgenic Wheat[J]. Scientia Agricultura Sinica, 2013, 46(12): 2403-2411.

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参考文献

[1]黎裕, 王建康, 邱丽娟, 马有志, 李新海, 万建民. 中国作物分子育种现状与发展前景. 作物学报, 2010, 36(9): 1425-1430.

Li Y, Wang J K, Qiu L J, Ma Y Z, Li X H, Wan J M. Crop molecular breeding in China: Current status and perspectives. Acta Agronomica Sinica, 2010, 36(9): 1425-1430. (in Chinese)

[2]Moose S P, Mumm R H. Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiology, 2008, 147: 969-977.

[3]王维, 郎明林, 杨学举. 小麦转基因技术及转化功能基因研究进展. 中国农业通报, 2012, 28(18): 1-6.

Wang W, Lang M L, Yang X J. Advances of wheat transgenic techniques and its transferred functional genes. Chinese Agricultural Science Bulletin, 2012, 28(18): 1-6. (in Chinese)

[4]叶兴国, 陈明, 杜丽璞, 徐惠君. 小麦转基因方法及其评述. 遗传, 2011, 33(5): 422-430.

Ye X G, Chen M, Du L P, Xu H J. Description and evaluation of transformation approaches used in wheat. Hereditas, 2011, 33(5): 422-430. (in Chinese)

[5]周岩, 薛晓锋, 魏琦超, 张洁, 何男男, 游建. 小麦转基因技术研究进展. 生物技术通报, 2012(2): 53-58.

Zhou Y, Xue X F, Wei Q C, Zhang J, He N N, You J. Advances in wheat transgenic technology research. Biotechnology Bulletin, 2012(2): 53-58. (in Chinese)

[6]喻修道, 徐兆师, 陈明, 李连城, 马有志. 小麦转基因技术研究及其应用. 中国农业科学, 2010, 43(8): 1539-1553.

Yu X D, Xu Z S, Chen M, Li L C, Ma Y Z. The progress and application of wheat transformation technology. Scientia Agricultura Sinica, 2010, 43(8): 1539-1553. (in Chinese)

[7]Zaharoff D A, Henshaw J W, Yuan F, Mossop B, Yuan F. Mechanistic analysis of electroporation-induced cellular uptake of macromolecule. Experimental Biology and Medicine, 2008, 233(1): 94-105.

[8]Zhu T, Luo C X, Huang J Y, Xiong C Y, Ouyang Q, Fang J. Electroporation based on hydrodynamic focusing of microfluidics with low dcvoltage. Biomedical Microdevices, 2010, 12(1): 35-40.

[9]Lee Y K, Deng P G. Review of micro/nano technologies and theories for electroporation of biological cells. Science China-Physics, Mechanics and Astronomy, 2012, 55(6): 996-1003.

[10]Van Wert S L, Saunders J A. Electrofusion and electroporation of plants. Plant Science, 1992, 99: 365-367.

[11]张有明. 第七章电穿孔转殖技术//叶锡东, 陈良筑. 植物基因转殖之原理与应用.台中: 植物生物技术教学资源中心, 2004.

Zhang Y M. The transgenic technology of electroporation//Ye X D, Chen L Z. The Principle and Application of Plant Transgenic. Taizhong: Plant Biotechnology Teaching Resource Center, 2004. (in Chinese)

[12]Saunders J A, Lin C H, Hou B H, Cheng J, Tsengwa N, Lin J J, Smith C R, Mclntosh M S, Wert S V. Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen. Journal of Molecular Microbiology and Biotechnology, 1995(3): 181-190.

[13]Van Wert S L, Saunders J A. Reduction of nuclease activity release from germinating pollen under conditions used for pollen electrotransformation. Plant Science, 1992, 84: 11-16.

[14]Li S T, Wang S H, Yang H Y. Transformation of plant young proembryos by electroporation. Chinese Science Bulletin, 2000, 45(13): 1202-1206.

[15]柯遐义, 黄粤, 石和平, 李宝健. 利用电激法转化小麦幼胚的研究. 武汉植物学研究. 1997, 15(2): 103-107.

Ke X Y, Huang Y, Shi H P, Li B J. A study on gene transformation of immature wheat embryos by electroporation. Journal of Wuhan Botanical Research, 1997, 15(2): 103-107. (in Chinese)

[16]Ren Y J, Zhao J. Optimization of electroporation parameters for immature embryos of indica rice. Rice Science, 2008, 15(1): 43-50.

[17]Abdul-Baki A A, Saunders J A, Matthews B F, Pittarelli G W. DNA uptake during electroporation of germinating pollen grains. Plant Science, 1990, 70: 181-190.

[18]Saunders J A, Matthews B F, Van Wert S L. Pollen electrotransformation in tobacco. Molecular Biology Reports, 1992, 55: 81-88.

[19]王清岚. 以花粉做为受体之玉米转殖方法[D]. 嘉义: 国立嘉义大学农业生物技术研究所, 2006.

Wang Q L. A simple and quick method for efficient production of transgenic maize using pollen as a carrier of transgenes[D]. Jiayi: Graduate Institute of Agricultural Biotechnology, National Chiayi University, 2006. (in Chinese)

[20]谌丽斌, 梁文艳, 曲久辉, 解明曙, 雷鹏举, 刘会娟. FDA-P I双色荧光法检测蓝藻细胞活性的研究. 环境化学, 2005, 24(5): 554-557.

Chen L B, Liang W Y, Qu J H, Xie M S, Lei P J, Liu H J. The viability determination of Cyano-Bacteria by double staining with fluorescein diacetate and Propidium Iodide. Environmental Chemistry, 2005, 24(5): 554-557. (in Chinese)

[21]崔群香, 王倩, 李英, 侯喜林. 不结球白菜小孢子胚胎发生过程及发育途径研究. 南京农业大学学报, 2012, 35(2): 21-26.

Cui Q X, Wang Q, Li Y, Hou X L. Studies on microspore-derived embryogenesis and developmental pathway in non-heading Chinese cabbage. Journal of Nanjing Agricultural University, 2012, 35(2): 21-26. (in Chinese)

[22]闵东红, 何莎, 张彦, 夏兰琴. 基因枪转化小麦主要轰击参数的优化. 作物学报, 2013, 39(1): 60-67.

Min D H, He S, Zhang Y, Xia L Q. Optimization of key bombardment parameters in biolistic mediated transformation in wheat. Acta Agronomica Sinica, 2013, 39(1): 60-67. (in Chinese)

[23]Saunders J A, Matthews B F. Pollen electrotransformation for gene transfer in plant//Guide to Electroporation and Electrofusion. America, 1992: 227-247.

[24]Ingrain H M, Power J B, Lowe K C, Davey M R. Optimisation of procedures for microprojectile bombardment of microspore- derived embryos in wheat. Plant Cell Tissue and Organ Culture, 1999, 57: 207-210.

[25]李永春, 王潇, 陈雷, 尹钧. 中国小麦转基因研究的现状及前景. 中国农业通报, 2008, 24(5): 90-94.

Li Y C, Wang X, Chen L, Yin J. The progress and prospect of wheat transgenic research in China. Chinese Agricultural Science Bulletin, 2008, 24(5): 90-94. (in Chinese)

[26]张立, 王建峰, 王晓杰, 康振生, 韩德俊. 花粉管通道法介导小麦抗病相关基因的转化和抗锈性鉴定. 麦类作物学报, 2013, 33(1): 1-5.

Zhang L, Wang J F, Wang X J, Kang Z S, Han D J. Wheat transformation of resistant genes to diseases using oollen-tube pathway. Journal of Triticeae Crop, 2013, 33(1): 1-5. (in Chinese)

[27]Wang J X, Sun Y, Cui G M, Hu J J. Transgenic maize plants obtained by pollen- mediated transforrrration. Acta Botanica Sinica, 2001, 43(3): 275-279.

[28]杜春芳, 刘惠民, 李朋波, 孙毅, 李润植. 花粉介导法获得油菜转基因植株研究. 作物学报, 2006, 32(5): 749-754.

Du C F, Liu H M, Li P B, Sun Y, Li R Z. Pollen-mediated transformation of rape. Acta Agronomica Sinica, 2006, 32(5): 749-754. (in Chinese)

[29]Wang J Y, Li Y H, Liang C. Recovery of transgenic plants by pollen-mediated transformation in Brassica juncea. Transgenic Research, 2008, 17: 417-424.

[30]张小娟, 张荣光. 影响电穿孔法转化效率的因素. 医学综述, 2011, 17(5): 666-668.

Zhang X J, Zhang R G. Factors which influence the efficiency of electroporation. Medical Recapitulate, 2011, 17(5): 666-668. (in Chinese)

[31]Bates G W. Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana. Planta, 1985, 165: 217-224.

[32]徐乃瑜, 王许莲, 李泳洲, 黄玉凤, 高汉娟. 小麦不同恢复系间花药大小和花粉数量的比较及其与恢复力强弱关系的探讨. 武汉大学学报: 自然科学版, 1980, 2: 92-96.

Xu N Y, Wang X L, Li Y Z, Huang Y F, Gao H J. The comparison of pollen size and number and the relationship with restorability during different restorers in wheat. Journal of Wuhan University: Natural Science Edition, 1980, 2: 92-96. (in Chinese)

[33]陈曦, 周忠泽, 曹景林. 不同品种烟草花粉电子显微镜观察. 西北植物学报, 2004, 24(1): 43-49.

Chen X, Zhou Z Z, Cao J L. Comparative study of tobacco pollen morphology with scanning electronic microscope. Acta Botanica Boreali-Occidentalia Sinica, 2004, 24(1): 43-49. (in Chinese)

[34]黄洪云, 那日. 电激法介导作物种子基因转移的研究与进展. 种子, 2007, 26(2): 52-55.

Huang H Y, Na R. Progress of gene transfer technology of crop seed mediated by electroporation method. Seed, 2007, 26(2): 52-55. (in Chinese)

[35]崔贵梅, 孙毅, 郝耀山, 杜建中, 王亦学. 玉米花粉体外萌发方法改进及其对花粉介导转基因的作用. 植物学报, 2012, 47(2): 155-161.

Cui G M, Sun Y, Hao Y S, Du J Z,Wang Y X. The improvement of maize pollen in vitro germination method and its role in pollen- mediated plant genetic transformation. Chinese Bulletin of Botany, 2012, 47(2): 155-161. (in Chinese)

[36]申家恒, 申业, 王艳杰. 小麦花粉管生长途径及受精过程经历时间的研究. 作物学报, 2006, 32(4): 522-526.

Shen J H, Shen Y, Wang Y J. Pathway of pollen tube growth and the duration of different phases of fertilization process in wheat. Acta Agronomica Sinica, 2006, 32(4): 522-526. (in Chinese)

[37]胡茂兴, 田晓梅. 小麦开花习性观察初报. 上海农学院学报, 1988, 6(1): 95-100.

Hu M X, Tian X M. The initial report about observed on blossom characteristic of wheat. Journal of Shanghai Agricultural College, 1988, 6(1): 95-100. (in Chinese)

[38]Shi H Z, Xu B F, Yang H Y, Zhou C. Transient transformation of pollen protoplasts via electroporation and temporal expression of Zm13-260-GUS-NOS chimeric gene in Brassica campestris var. purpurea. Chinese Science Bulletin, 1996, 41(4): 339-342.