One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

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.

多基因转移技术的实现难度较高，这一现状正制约着植物研究领域的发展进程。本研究开发了一种适用于根癌农杆菌（Agrobacterium tumefaciens）介导转化的新型双元载体（binary vector）pHUGE-Red，并配套开发了一套可一次性转移最多9个基因的克隆方案。该载体借助Gateway克隆技术（Gateway technology）可实现大片段DNA的模块化克隆，并搭载DsRED1作为可视化筛选标记。此外，载体还引入了R/Rs诱导型重组系统（R/Rs inducible recombination system），可在后续步骤中移除新生成转基因植株内的筛选标记基因。我们通过将蒺藜苜蓿（Medicago truncatula）与根瘤菌建立共生关系所必需的8个基因，以一条74 kb的转移DNA（T-DNA）片段的形式，成功将其转移至草莓、杨树、番茄和烟草这4种非豆科植物中，验证了pHUGE-Red载体的实用性。研究结果证实，所有外源转基因均能在该类非豆科植物的根组织中正常表达。转化过程中的可视化筛选把控，配合后续标记基因的移除步骤，使pHUGE-Red载体成为实现多基因高效转移的优异工具。