小麦花粉电穿孔转化因素优化和转基因植株获得及鉴定

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.

Factors Optimization of Pollen Electroporation Transformation and Identification of Transgenic Wheat

【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.