以草甘膦为筛选标记的大豆转基因体系的建立及抗除草剂转基因大豆的培育

Establishment of an Efficient Transformation Protocol for Soybean Using Glyphosate As Selective Agent and the Development of Glyphosate-Tolerant Transgenic Soybean Lines

抗草甘膦转基因大豆能显著提高大豆生产效率,具有重大的应用前景.本实验室前期研究建立了农杆菌介导、草胺膦为筛选剂的大豆转基因体系,转化效率在4%以上.在此基础上,利用G6-EPSPS和G10-EPSPS 2个具有自主知识产权的草甘膦抗性基因,通过优化转化体系,成功建立了以草甘膦为筛选剂的大豆遗传转化体系,转化效率达1%以上.浓度梯度实验发现,当草甘膦的筛选浓度为100 mg/L时,虽然丛生芽的再生率下降了50%～60%,但最终转化效率不受影响.进一步通过基因表达分析、Western blot、Southern blot和除草剂抗性鉴定等方法对转基因大豆进行了分子检测和验证,最后获得了分子特征明确、对草甘膦抗性稳定的抗草甘膦转基因大豆后代.结果对国内抗草甘膦转基因大豆转基因方法研究及抗除草剂新品种选育具有意义.

Herbicide-tolerant soybean can significantly reduce the weed control cost, thus has great potential applications in agriculture. In a previous study, we described an efficient soybean transformation protocol using glufosinate as selective agent. The transformation efficiency was up to 4%. In this study, we attmpted to establish a transformation system using the newly isolated EPSPS genes, G6-EPSPS and GIO-EPSPS as the selective marker gene. By optimization of the factors affecting each step of the transformation process, we were able to achieve a stable transformation system with efficiency higher than 1%. While the high glyphosate concentration （100 mg/L） signficantly reduced the numbers of proliferated shootlets in the cotyledonary nodes of soybean explants, the final transformation efficiency was not affected. Molecular analyses using Southern blot, reverse transcription PCR （RT-PCR）, and Western blot verfied the insertions of the transgene in the transgenic soybean lines. The transgenic soybean plants were able to tolerant to spray of Roundup~ up to 6 kg/hectares, which is 4.9 folds of the recommended field application rate. These results are valuable for soybean trangenic research and development of transgenic soybean for field application.