Yemets Alla, Radchuk Vladimir, Bayer Oleg, Bayer Galina, Pakhomov Alexey, Vance Baird W, Blume Yaroslav B
Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kiev, Ukraine.
Cell Biol Int. 2008 May;32(5):566-70. doi: 10.1016/j.cellbi.2007.11.012. Epub 2007 Nov 21.
A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.
利用一个经过修饰的微管蛋白基因作为选择标记，开发了一种植物转化和筛选系统。构建了携带来自牛筋草的赋予对二硝基苯胺除草剂抗性的突变α-微管蛋白基因的载体构建体，用于单子叶植物和双子叶植物的转化。这些构建体包含由泛素或花椰菜花叶病毒35S启动子驱动的β-和/或突变α-微管蛋白基因。这些构建体用于对龙爪稷和大豆进行基因枪转化，或用于对亚麻和烟草进行农杆菌介导的转化。二硝基苯胺除草剂的主要代表氟乐灵在实验中用作选择剂，以筛选转基因细胞、组织和幼苗。为每个物种估计的氟乐灵选择浓度如下：对龙爪稷、大豆、垂花烟草和林烟草为10微摩尔；对亚麻为3微摩尔。使用针对nptII、α-微管蛋白或β-微管蛋白基因的特异性探针，对转化株系进行PCR和Southern杂交分析，以确认再生植株的转基因性质。在转化的植株系中鉴定出了突变α-微管蛋白基因的特异性条带。结果证实了突变微管蛋白基因稳定整合到植物基因组中。本研究清楚地证明了植物突变微管蛋白作为植物转化选择基因的用途。
