Ye G N, Hajdukiewicz P T, Broyles D, Rodriguez D, Xu C W, Nehra N, Staub J M
Monsanto Company, 700 Chesterfield Village Parkway North, St Louis, MO 63198, USA.
Plant J. 2001 Feb;25(3):261-70. doi: 10.1046/j.1365-313x.2001.00958.x.
Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.
质体转化（转质体）技术在生物技术应用方面具有若干潜在优势，包括使用未经修饰的原核基因进行工程改造、由于大多数作物植物的母系遗传而具有潜在的高水平基因表达和基因限制。然而，质体编码性状的功效可能会因质体数量和组织类型而改变。我们报告了一项在烟草质体中进行的可行性研究，以在营养组织和生殖器官中实现高水平的除草剂抗性。我们选择通过在质体中过表达5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）的耐受形式来测试草甘膦抗性。采用免疫学、酶学和全株测定方法来证明三种不同原核（无色杆菌、农杆菌和芽孢杆菌）EPSPS基因的功效。以农杆菌菌株CP4 EPSPS为模型，我们鉴定了指导蛋白质积累范围达10000倍（至叶片总可溶性蛋白的>10%）的翻译控制序列。质体表达的EPSPS可提供非常高水平 的草甘膦抗性，尽管营养组织和生殖组织中的抗性水平因EPSPS积累水平而异，并且与这些组织中的质体丰度相关。矛盾的是，与类似的核编码基因相比，质体表达的EPSPS蛋白积累似乎需要更高水平才能产生功效。尽管如此，利用转质体技术在营养器官和生殖器官中证明高水平的草甘膦耐受性为将该技术转移到其他作物物种提供了必要的一步。
