GFP绿色荧光法辅助筛选玉米转基因植株.docx

、 GFP绿色荧光法辅助筛选玉米转基因植株 段学清1，郑莉茹1，刘文波1，侯绪刚1，郭玉玉1，谢芳蕊1，王文倩1，安海龙1* 1山东农业大学生命科学学院，作物生物学国家重点实验室，泰安271018。 摘要 在玉米（Zea mays L.）遗传转化研究工作中，人们通常使用未成熟胚或其胚性愈伤组织作为转化受体，抗生素或除草剂筛选产生抗性愈伤组织和抗性芽。玉米的抗性愈伤组织和抗性芽中往往会存在比例不等的假阳性现象，这显著地增加了组织培养和后期分子鉴定工作的工作量。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，基因枪法轰击了玉米自交系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3周，轰击后10天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，获得了一批玉米转基因植株，转化频率0.3-0.5%。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。这一工作揭示了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。 关键词：玉米，转基因植株，GFP，假阳性 基金项目：国家自然科学基金（No.和转基因生物新品种培育重大专项（No. 2011ZX08009-002） *通讯作者。E-mail:  HYPERLINK mailto:hlan@ hlan@ Production of Transgenic Maize Plants via the GFP Green Fluorescence-assisted Selection Xueqing Duan1, Liru Zheng1, Wenbo Liu1, Xugang Hou1, Yuyu Guo1, Fangrui Xie1, Wenqian Wang1, Hailong An1* 1State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian 271018, China. Abstract During the genetic transformation of maize (Zea mays L.), the immature embryos or calli from them were used frequently as recipients for transformation and endured the prolonged selection by antibiotics or herbicides, to produce resistant calli and shoots. Due to the fact that escapes are quite common from the selection by antibiotics or herbicides in maize transformation, additional jobs are required to deal with this. Using a plasmid containing GFP (Green Fluorescence Protein) gene as a visual marker, the calli of immature embryos of maize inbreds A188 and Qi319 were bombarded. In a transient expression experiment, the GFP fluorescence was observed within 20 days and kept at a high level in 10 days after bombardment. Under the selection of hygromycin and by the visual check of GFP fluorescence, a few transgenic maize plants were produced with a frequency of 0.3-0.5%. The identity of the transgenics was confirmed by the molecular analysis assays and?genetic analysis experiments of offspring later. This work suggests that GFP i