Lysine racemase: a novel non-antibiotic selectable marker for plant transformation
Lysine racemase: a novel non-antibiotic selectable marker for plant transformation
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DOI：
10.1007/s11103-009-9558-y
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发表时间：
2010-01-01
影响因子：
5.1
通讯作者：
Hsu, Wen-Hwei
中科院分区：
文献类型：
--
作者：
Chen, I-Chieh;Thiruvengadam, Venkatesan;Hsu, Wen-Hwei
本研究以赖氨酸为选择剂，赖氨酸消旋酶（lyr）为选择标记基因，建立了一个非抗生素筛选体系。L-赖氨酸对植物有毒，并被Lyr转化为d-赖氨酸，其随后将被转基因植物用作氮源。转基因烟草和拟南芥植物在L-赖氨酸培养基上成功地回收，效率分别为23%和2.4%。转基因植物的表型特征清楚地揭示了正常的生长和发育特征的野生型植物的表达，这表明没有多效性与lyr基因的影响。在转基因烟草植物中选择L-赖氨酸的比活性范围为0.77至1.06 mU/mg蛋白质，而几乎没有活性在野生型植物中检测到。此外，游离氨基酸的组合物，除了天冬氨酸，不受影响的表达的lyr基因在转基因烟草植物表明非常有限的干扰内源氨基酸代谢。有趣的是，我们的研究结果还表明，植物天冬氨酸激酶可能具有区分赖氨酸对映体的反馈调节能力。据我们所知，这是第一个报告，证明赖氨酸消旋酶选择标记系统是新的，争议较少，比传统的选择系统便宜。
A non-antibiotic based selection system using l-lysine as selection agent and the lysine racemase (lyr) as selectable marker gene for plant transformation was established in this study. l-lysine was toxic to plants, and converted by Lyr into d-lysine which would subsequently be used by the transgenic plants as nitrogen source. Transgenic tobacco and Arabidopsis plants were successfully recovered on l-lysine medium at efficiencies of 23 and 2.4%, respectively. Phenotypic characterization of transgenic plants clearly revealed the expression of normal growth and developmental characteristics as that of wild-type plants, suggesting no pleiotropic effects associated with the lyr gene. The specific activity of Lyr in transgenic tobacco plants selected on l-lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in the wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of the lyr gene in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also suggested that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. To our knowledge, this is the first report to demonstrate that the lysine racemase selectable marker system is novel, less controversial and inexpensive than the traditional selection systems.