抗草铵膦除草剂转基因油菜新种质的创制及鉴定

Development and Identification of Transgenic Rapeseed Resistant to Herbicide Glufosinate

草铵膦是一种在世界范围内广泛使用的内吸型除草剂。目前,我国还未研发出具有自主知识产权的抗草铵膦除草剂油菜品种。本研究通过油菜下胚轴遗传转化方法成功将1个新型草铵膦抗性基因Syn1-RePAT转化到甘蓝型油菜中,通过筛选获得了27株阳性单株。利用Southern blot技术筛选出其中的单拷贝T-DNA插入株系,并通过后代分离比检测对重要株系进行了拷贝数验证。利用Inverse-PCR技术对OV40-7、OV40-15、OV40-16和OV40-17等转化体进行T-DNA侧翼序列分析与比对,分离获得了各转基因株系的插入位点信息。对不同转化体株系设计特异性引物,通过PCR技术检测外源基因序列在T1~T3代材料的整合稳定性。RT-PCR和qRT-PCR表达分析证实,Syn1-RePAT转基因在多个转化株系的不同世代稳定超量表达。对T1代转化体进行功能鉴定,各转化体对草铵膦具有良好的抗性,至少可耐受6倍田间推荐施用剂量的草铵膦。本研究所创建的新型抗草铵膦油菜种质资源极具应用前景,为我国抗草铵膦良种培育工作提供了有力支持。

Glufosinate is a non-selective broad-spectrum herbicide applicable in weeds control worldwide.However,in China there is no glufosinate-tolerant rapeseed variety with independent intellectual property rights to date. In this study,a glufosinate-resistant gene Syn1-RePAT was successfully transformed to the Brassica napus L. via Agrobacterium tumefaciens-mediated hypocotyl method,and 27 transgenic plants were obtained.The transgenic lines with a single copy of T-DNA insertion were identified by Southern blot analysis,and some of them on T-DNA copy number variation were analyzed by progeny segregation assay. The T-DNA insertion sites in the lines of OV40-7,OV40-15,OV40-16 and OV40-17 were identified by inverse PCR method. The stability of the T-DNA insertion was further confirmed by insertion-specific PCR in their T1 to T3 generations. RTPCR and qRT-PCR expression analysis revealed that the Syn1-RePAT gene was stably overexpressed in different generations of transgenic lines. Treatments with different doses of glufosinate indicated that lines OV40-6, OV40-7 and OV40-16 were tolerant to at least six times of the recommended dose of glufosinate in production.Thus,the novel glufosinate-tolerant rapeseed lines generated in the present study will lay the foundation for the herbicide-tolerance rapeseed breeding in China.