以bar基因为筛选标记转基因大豆的获得及鉴定

Acquisition and Identification of Transgenic Soybean using bar as a Selective Agent

在已优化的大豆农杆菌介导转化体系基础上,以发芽1～2 d的成熟大豆子叶节为外植体,以bar基因作为筛选标记基因,采用农杆菌介导法将胰蛋白酶抑制剂基因(Sporamin)和几丁质酶基因(Chitinase KDEL)转入大豆品种YC-2中。T0代得到生根苗115株,其中采用除草剂叶片涂抹法和目的基因PCR检测法鉴定出阳性植株45株,其中选取的6个T0代植株中有4个独立株系外源基因在T1代遗传,其中2个T1代株系呈3∶1的遗传分离比例,2个T1代株系分离比例大于3∶1。T0和T1代经草丁膦叶片涂抹法鉴定为阳性的植株,通过bar试纸条法和目的基因PCR检测法鉴定也均表现为阳性,因此,135 mg.L-1草丁膦叶片涂抹法可用于草丁膦筛选标记转基因大豆植株的大规模筛选。

On the basis of optimized soybean Agrobacterium-tumefaciens mediated transformation system,the trypsin inhibitor gene（sporamin）and chitinase gene（chitinase KDEL）were transformed into soybean cultivar YC-2 using germinated 1-2 days mature cotyledon node as explants and bar gene as a selectable agent in this paper.One hundred and fifteen putative transgenic soybean plants had been got and only 45 plants were positive transgenic plants identified by leaf painting 135 mg·L-1 Basta and polymerase chain reaction（PCR）methods.Six T0 positive plants were chosen,and four independent transgenic plants could be inherited into T1 progeny and the segregation rate of two T1 progenies were 3∶ 1,the other two T1 progenies were more than 3∶ 1.The results suggest that leaf painting 135 mg·L-1 Basta could be an effective and costless method to detect genetically modified soybeans progeny with bar gene as a selective agent.