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利用GFP(Green Fluorescence Protein)绿色荧光蛋白基因作为辅助性筛选标记基因,基因枪法轰击玉米自交系A188和Qi319未成熟胚的胚性愈伤组织。瞬间表达实验结果表明,轰击后GFP表达可持续近20 d,轰击后10 d左右表达量仍保持在较高的水平。采用潮霉素筛选、GFP荧光法鉴定,获得一批玉米转基因植株,转化频率为0.33%~0.47%。分子鉴定和后代分析的结果表明,外源基因已经整合到了玉米基因组中,并能够稳定地表达和遗传。GFP作为辅助性筛选标记基因可以直观、快速和有效地消除玉米遗传转化工作中的假阳性现象。
Green fluorescent protein (GFP) green fluorescent protein gene was used as a secondary screening marker gene to bombard embryogenic callus of maize inbred line A188 and Qi319 immature embryos by particle bombardment. The results of transient expression showed that the expression of GFP lasted nearly 20 days after bombardment and remained at a high level about 10 days after bombardment. A series of maize transgenic plants were obtained by hygromycin selection and GFP fluorescence assay. The transformation frequency was 0.33% -0.47%. The results of molecular identification and progeny analysis showed that foreign genes have been integrated into the maize genome and expressed stably and genetically. GFP as a helper marker gene can eliminate false positives quickly and efficiently in maize genetic transformation.