利用子房滴注法获得无载体骨架序列和选择标记的转基因玉米

Generation of vector backbone-free and selectable marker-free transgenic maize (Zea mays L.) via ovary-drip method

转基因植物中的载体骨架序列和选择标记基因是引起生物安全性争论的根本原因,最直接、最有效的解决方法是在转化过程中不使用载体骨架序列和选择标记基因。本研究建立并优化了玉米子房滴注转化法,其操作要点是将DNA转化溶液直接滴加在完全去除花柱的子房上。利用子房滴注法将无载体骨架序列和选择标记的线性GFP基因表达框转化玉米。PCR结果表明:适合子房滴注法转化的玉米品种为9818,最佳转化时间为授粉后18～20 h,在此条件下得到最高的PCR阳性率,为3.01%;Southern blotting结果表明外源基因的整合方式简单(1～2条杂交带);RT-PCR结果表明转基因植株中GFP基因能够在RNA水平上正常表达;在转基因植株的根和幼胚中观察到GFP表达。

The presence of vector backbone sequences and selectable marker genes in transgenic plants has been the key concern for biosafety. A direct solution is to totally avoid the use of vector backbone sequences and selectable marker genes from the beginning of transgenic plant generation. In this study, the ovary-drip method was established and optimized. The key features of this method focused on the complete removal of the whole styles, and the subsequent application of a DNA solution directly to the ovaries. A vector backbone-free and selectable marker-free linear GFP cassette （Ubi-GFP -nos） was transformed into maize via the ovary-drip method. PCR analysis showed that suitable maize variety was 9818 and optimal transformation time was 18-20 h after pollination, which produced the highest PCR positive frequency （3.01%）. Southern blotting analysis showed that the transgenic plants had simple integration patterns （1-2 bands）. GFP transcription was detected by RT-PCR analysis. Green fluorescence was observed in roots and immature embryos of transgenic plants by a fluorescence microscopy.