转AM79-EPSPS基因耐草甘膦大豆的获得及功能验证

Acquisition and functional validation of transgenic AM79-EPSPS glyphosate-resistant soybean(Glycine max L.)

以天隆一号大豆为受体材料,通过农杆菌介导的遗传转化方法,从消毒时间、乙酰丁香酮(acetosyringone,AS)添加浓度、草甘膦添加浓度和生根阶段吲哚丁酸(indole-3-butyric acid,IBA)浸没时间等因素出发,探究并建立高效的转AM79-EPSPS基因大豆转化体系,将具有我国自主知识产权的新基因AM79-EPSPS导入大豆,并验证它在转基因大豆中的功能。结果表明:当种子消毒时间为6~8 h,AS添加浓度为200μmol/L,草甘膦添加浓度为100μmol/L,IBA浸没时间为30 s时,转化效果最好。通过该转化体系获得了6株转AM79-EPSPS基因耐草甘膦大豆苗,并对其T0、T1、T2代植株进行了蛋白检测及草甘膦喷施实验。结果表明:在各世代转基因植株中均能检测出AM79-EPSPS蛋白,说明AM79-EPSPS基因能够在大豆中正常表达;喷施草甘膦后,非转基因大豆植株全部枯萎,而转AM79-EPSPS基因大豆植株均能正常生长,表现出明显的草甘膦耐受性。

In order to introduce a new gene AM79-EPSPS with independent intellectual property rights into soybean and verify its function in transgenic soybean,an efficient transformation system of AM79-EPSPS gene was established through Agrobacterium-mediated genetic transformation from disinfection time,acetosyringone(AS)concentration,glyphosate concentration and indole-3-butyric acid(IBA)immersion time at the rooting stage with the receptor material of Tianlong No.1 soybean.It was clear that the transformation effect was the best under the following optimal condition:the disinfection time of 6-8 h,the AS concentration of 200μmol/L,the glyphosate concentration of 100μmol/L,and the IBA immersion time of 30 s.Six transgenic AM79-EPSPS glyphosate-resistant soybeans were obtained,and the protein detection and glyphosate spraying experiments were carried out on the T0,T1 and T2 generation plants.The results showed that AM79-EPSPS protein could be detected in all generation plants containing AM79-EPSPS gene,which indicated that AM79-EPSPS gene could be expressed normally in the soybean.After spraying glyphosate,all non-transgenic soybean plants withered,while the transgenic AM79-EPSPS gene soybean plants could normally grow and showed obvious glyphosate tolerance.