Inaba Yoshimi, Brotherton Jeffrey E, Ulanov Alexander, Widholm Jack M
Faculty of Medicine, Dentistry and Health Sciences, Department of Pediatrics, University of Melbourne, Melbourne, Australia.
Plant Cell Rep. 2007 Oct;26(10):1763-71. doi: 10.1007/s00299-007-0381-0. Epub 2007 Jun 14.
Soybean [Glycine max (L.) Merr.] embryogenic cultures were transformed by particle bombardment with the feedback-insensitive tobacco anthranilate synthase (AS) gene ASA2 driven by the CaMV 35S promoter and selected using hph as the selectable marker gene. Only one of eight regenerated lines that set seed and contained ASA2 expressed the gene highly and contained increased free tryptophan (Trp) levels in leaves, seeds and embryogenic cultures. Leaf extracts of the ASA2 expressing line contained about twice as much AS enzyme activity as the untransformed control and this activity was only slightly more feedback-insensitive. Amino acid analysis showed that both leaves and embryogenic tissue cultures of the ASA2 expressing line had four to five-times the normal levels of free Trp and slightly higher free tyrosine and phenylalanine. The seed total Trp content was only slightly increased. Metabolic profiling-analysis by GC-MS detected no other consistent differences. These studies show that the ASA2 gene can be expressed in soybean and that modest changes in Trp synthesis occurs.
利用由花椰菜花叶病毒35S启动子驱动的反馈不敏感型烟草邻氨基苯甲酸合酶（AS）基因ASA2，通过粒子轰击法对大豆[Glycine max (L.) Merr.]胚性培养物进行转化，并使用潮霉素磷酸转移酶（hph）作为选择标记基因进行筛选。在八个结籽且含有ASA2的再生株系中，只有一个株系高度表达该基因，并且其叶片、种子和胚性培养物中的游离色氨酸（Trp）水平有所增加。表达ASA2的株系的叶片提取物中AS酶活性约为未转化对照的两倍，并且这种活性仅略微更不敏感于反馈调节。氨基酸分析表明，表达ASA2的株系的叶片和胚性组织培养物中游离Trp的含量是正常水平的四到五倍，游离酪氨酸和苯丙氨酸的含量略高。种子中的总Trp含量仅略有增加。通过气相色谱-质谱联用（GC-MS）进行的代谢谱分析未检测到其他一致的差异。这些研究表明，ASA2基因可以在大豆中表达，并且色氨酸合成发生了适度变化。
