新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定

草甘膦是世界上应用最广泛的广谱性除草剂,目前我国还没有自主知识产权的抗草甘膦油菜品种。本研究利用农杆菌介导的油菜下胚轴遗传转化方法,将新型抗草甘膦基因I.variabilis EPSPS转入甘蓝型油菜品系J9707中,获得了126株阳性转化株,阳性率为97.0%。这些转化单株中的T-DNA插入以单拷贝为主(占44.8%)。通过反向PCR确定了EPS-2、EPS-6和EPS-7等油菜转化体中T-DNA插入位置,并设计转化体特异性引物对它们的T0~T3代材料进行检测,证明了它们的T-DNA在基因组水平上整合的稳定性。RNA和蛋白水平的表达分析证实,I.variabilis EPSPS转基因及其蛋白产物在各转化株系不同世代能够稳定表达。苗期进行不同剂量的除草剂喷施处理发现,EPS-1、EPS-2、EPS-5、EPS-6和EPS-7等株系可耐受4倍田间推荐使用剂量的草甘膦。本研究所创建的新型抗草甘膦油菜种质资源将为我国抗除草剂油菜品种培育奠定了重要基础。

Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance

Glyphosate is the most widely used broad-spectrum herbicide in the world.However,at present there is no glyphosate-tolerant rapeseed variety with independent intellectual property rights in China.In the study,a novel glyphosate-resistant genes I.variabilis EPSPS was transferred to the Brassica napus pure line J9707 via the Agrobacterium tumefaciens-mediated hypocotyl method,and 126 T0-positive transgenic plants with 97.0%positive rate were generated.The T-DNA insertion with a single copy(44.8%)is dominant.The insertion locations of T-DNA in the lines of EPS-2,EPS-6,and EPS-7 were identified by inverse PCR method.The stability of the T-DNA insertion in these lines were further confirmed by insertion-specific PCR in their T0 to T3 plants.The gene expression analysis revealed that the I.variabilis EPSPS gene and its protein was stably expressed in different generations of transgenic lines in RNA and protein levels.Treatments with different doses of glyphosate indicated that the lines of EPS-1,EPS-2,EPS-5,EPS-6,and EPS-7 could tolerate four times of the recommended dose of glyphosate in production.Thus,the novel glyphosate-tolerant rapeseed lines generated in the present study will lay the foundation for the herbicidetolerance rapeseed breeding in China.