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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。
Based on the optimized Agrobacterium tumefaciens-mediated transformation system, the mature soybean cotyledon node 1 to 2 days after germination was used as explant. The bar gene was used as a marker marker for selection. Agrobacterium-mediated transformation of trypsin inhibitor gene Sporamin and Chitinase KDEL were transformed into soybean cultivar YC-2. T0 generation gave rooting 115, which using herbicide leaf smear method and PCR detection of the target gene identified 45 positive plants, of which selected 6 T0 plants in 4 independent strains of foreign genes in T1 generation genetic Two of the T1 lines showed a genetic segregation ratio of 3: 1, and the segregation ratios of the two T1 lines were greater than 3: 1. T0 and T1 generation were identified as positive plants by glufosinateate leaf smear method, and also showed positive by bar strip method and PCR method of target gene. Therefore, 135 mg.L-1 glufosinate leaf smear method Can be used for large scale screening of glufosinate-screened transgenic soybean plants.