根癌农杆菌介导的大麦茎尖转化研究

Transformation of Shoot Apical Meristems of Elite Barley Cultivars via Agrobacterium-mediated Transformation

为了建立农杆菌介导的大麦茎尖遗传转化技术,以我国大麦主推品种鄂大麦9号、鄂大麦32122为材料,以农杆菌介导法转化大麦茎尖,经PPT筛选获得了转抗除草剂基因的大麦株系。PCR鉴定表明,以鄂大麦9号茎尖为受体,筛选获得4个转基因植株,阳性率为0.59%;以鄂大麦32122茎尖为受体,筛选获得10个阳性植株,阳性率为1.96%。转基因T1代Southern杂交分析证实,外源基因确已整合到大麦基因组中,已鉴定的转基因株系均含单个转基因拷贝,不同转基因株系整合的位点不同。因此大麦茎尖可作为农杆菌转化的受体。本研究结果为拓宽大麦遗传转化受体提供了技术和方法。

To develop a method for transformation of barley shoot apex through Agrobacterium-mediated strategy, two elite harley cultivars from China were used for transformation with their shoot apexes as explants. After selection on PPT media, transgenic lines resistant to herbicide were obtained. PCR identification showed that four transgenic lines derived from cultivar E-damai9, and ten transgenie lines from cultivar E-damai32122 contained expected transgene, with a transformation efficiency of 0.59% （E-Damai 9） and 1. 960//00 （E-Damai 32122）, respectively. Southern blot analysis of T1 transgenic lines demonstrated that foreign genes were indeed integrated into the barley genome. From the analyzed transgenic lines, it was clear that all transgenic lines contained one copy per genome, and different lines displayed varied patterns of the transgenic integration. Thus barley shoot apex can be used for genetic transformation via Agrobacterium tumefaciens, and the current results provided useful information for exploitation of barley shoot apex as explants for genetic transformation.