Tsai F-Y, Brotherton J E, Widholm J M
Edward R. Madigan Laboratory, Department of Crop Sciences, University of Illinois, 1201 W. Gregory, Urbana, IL 61801, USA.
Plant Cell Rep. 2005 Jan;23(8):548-56. doi: 10.1007/s00299-004-0849-0. Epub 2004 Sep 16.
A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS alpha-subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially alpha-methyltryptophan (alphaMT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on alphaMT, indicating that it might be possible to use ASA2 as a selectable marker gene and alphaMT as the selection agent.
采用根癌农杆菌-叶片片段共培养法，随后用卡那霉素筛选nptII基因，共培育出35株独立的转基因烟草植株。该载体还携带了对烟草反馈不敏感的邻氨基苯甲酸合酶基因（ASA2）。许多株系的ASA2 mRNA水平有所提高，但只有3株的游离色氨酸（Trp）含量增加，且许多株系的Trp含量低于未转化的对照。Trp水平最高的株系（是未转化对照的三倍）在叶片和源自该植株的细胞悬浮培养物中，邻氨基苯甲酸合酶活性（AS）均有所增加，而反馈不敏感性在悬浮培养物中最为明显。其他动力学数据还表明，ASA2编码的ASα亚基在组织培养中比在叶片中更为丰富。该株系的后代幼苗对某些有毒的Trp类似物具有抗性，尤其是α-甲基色氨酸（αMT），而对最常用的类似物5-甲基色氨酸的抗性则较弱。在αMT上，两个转基因株系的叶片比未转化对照的叶片更容易形成芽，这表明有可能将ASA2用作选择标记基因，将αMT用作选择剂。
