Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, Wageningen, The Netherlands.
PLoS One. 2012;7(10):e47885. doi: 10.1371/journal.pone.0047885. Epub 2012 Oct 24.
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
很难实现多个基因的转移。在这里，我们提出了一种新的用于根癌农杆菌介导转化的二元载体 pHUGE-Red，以及一种适合一次转移多达九个基因的克隆策略。该载体通过使用 Gateway 技术实现了大 DNA 片段的模块化克隆，并包含 DsRED1 作为可视选择标记。此外，还包含了一个 R/Rs 诱导的重组系统，允许在新生成的转基因植物中随后去除选择标记。我们通过将 8 个对苜蓿建立共生关系至关重要的基因转移到 pHUGE-Red 中，展示了其成功的应用，将一个 74kb 的 T-DNA 作为一个整体转化到四个非豆科物种中；草莓、杨树、番茄和烟草。我们提供的证据表明，所有的转基因都在非豆科植物的根组织中表达。转化过程中的可视化控制和随后的标记基因去除使得 pHUGE-Red 载体成为高效转移多个基因的优秀工具。
