以草甘膦抗性基因Epsps为标记基因, 在原核Kanr基因两侧引入Cre(环化重组酶)基因识别的Lox-P位点, 同时以编码花青素合成转录因子的Bi和Cl基因为可视化选择报告基因, 构建了Bt杀虫蛋白基因Cry1Ab/c的可视化跟踪表达载体pBAC9017。用PDS1000/He基因枪转化玉米(Zea mays)自交系501的幼胚和胚性愈伤组织, 获得147个草甘膦抗性的玉米再生植株。其中106棵植株获得了结实种子, 16棵植株的结实种子有紫红色花青素基因的表达。经PCR检测表明, 外源Cry1Ab/c基因已经整合到玉米的基因组中。转基因植株种子蛋白粗提物用BT-Cry1Ab/1Ac金标免疫检测试纸条和ELISA检测, 结果表明, Cry1Ab/c在部分转基因植株后代中表达。
Bi and Cl genes, encoding anthocyanin synthesis of transcription factor, were used as visual selection reporter genes, and the glyphosate-resistance gene EPSPS as a selective marker, to generate a visualizing track vector pBAC9017. We used this vector to transform Cry1Ab/c driven by an enhanced CaMV 35S (E35S) promotor, into immature embryos and embryonic callus of maize inbred line 501 by microprojectile bombardment. We obtained 147 transformed, and among 106 plants with harvested T0 ears, 16 ears were decorated with mosaic purple seeds. PCR analysis showed that the Cry1Ab/c gene was integrated into the genome of maize. Extracts of seed protein were tested by BT-Cry1Ab/1Ac immune-strip and ELISA, showing that Bt-Cry1Ab/c gene was expressed in transgenic lines.
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