Use of non-integrating Zm-Wus2 vectors to enhance maize transformation Non-integrating WUS2 enhances transformation
Use of non-integrating Zm-Wus2 vectors to enhance maize transformation Non-integrating WUS2 enhances transformation
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DOI：
10.1007/s11627-019-10042-2
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发表时间：
2020-01-02
影响因子：
2.6
通讯作者：
Gordon-Kamm, Bill
中科院分区：
文献类型：
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作者：
Hoerster, George;Wang, Ning;Gordon-Kamm, Bill
Baby Boom(BBM)和Wuschel2(Wus2)的使用使玉米在越来越广泛的自交系中的转化效率更高。然而，需要切除这些转化辅助成分以实现植株再生，这就带来了好处，这增加了T-DNA的大小和转化系统的复杂性。本文介绍了一种体积较小、操作简便的含T-DNA的可选择标记系统。首先，玉米Pltp启动子(Zm-Pltp(Pro))单独驱动Zm-Wus2的表达足以诱导玉米幼胚的盾片快速体细胞胚胎形成。研究还表明，用两株农杆菌共侵染，一株带有Wus2表达框，另一株同时带有可选标记和可视标记框，转化的T0植株只含有单一拷贝的可选择标记T-DNA，而没有整合Wus2。此外，通过改变两个农杆菌菌株的比例，并通过调节Wus2的表达来实现对不含Wus2的可选择标记T0植株的高频恢复，优化了这一过程。有几个因素可能是造成这一结果的原因。WUS2在定位细胞(S)中的表达似乎促进了邻近细胞的体细胞胚胎发生，包括那些整合了可选择标记的细胞。此外，在Wus2 T-DNA未整合但可选择标记T-DNA整合的细胞中，瞬时表达Wus2可刺激含有可选择标记的稳定T0植株的体细胞胚胎形成和再生。此外，用三种病毒增强子元件增强Pltp启动子以增加Wus2的表达，可刺激胚胎发生，但不能再生。这种现象现在被称为“利他主义转型”。
The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltp(pro)), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as "altruistic transformation."