Department of Horticulture and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 2356 Rainwater Rd, Tifton, GA, 31793, USA.
Plant Cell Rep. 2022 Jan;41(1):119-138. doi: 10.1007/s00299-021-02789-z. Epub 2021 Sep 30.
Expression of Cre recombinase by AtRps5a or AtDD45 enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3 and the ZsGreen coding sequence in F progeny upon genotyping but no ZsGreen expression in F seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3. Strong ZsGreen expression in F seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45 to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aCre lines pollinated by the AtUbq3lox line supported the early activation mode.
AtRps5a 或 AtDD45 表达 Cre 重组酶可在杂交时的早期胚胎发育阶段通过 Cre/lox 介导的重组激活杂种豇豆和烟草中的转基因。基因工程理想情况下可实现转基因表达的精确时空控制。为了在受精时仅在杂种中激活转基因，我们评估了一种 Cre/lox 介导的基因激活系统，该系统使用在卵细胞和早期胚胎中具有活性的 AtRps5a 或 AtDD45 启动子表达 Cre 重组酶。在 Cre 重组酶系与携带lox 切除报告盒的转基因系杂交后，用 AtUbq3 启动子驱动 ZsGreen，在 Cre/lox 介导的重组后，我们在 F 代中通过基因分型观察到lox 侧翼的间隔 DNA 序列在 AtUbq3 和 ZsGreen 编码序列之间完全切除，但在 F 代种子或幼苗中没有 ZsGreen 表达。无法观察到 ZsGreen 荧光归因于 AtUbq3 的活性。当 ZsGreen 由 AtUbq10 启动子驱动时，在重组后观察到 F 代种子中强烈的 ZsGreen 表达。使用 AtDD45 表达 Cre 导致转基因系之间和杂交中的重组频率变化更大。无论用于调节 Cre 的启动子如何，重组的嵌合体 F 代都很少，这表明在早期胚胎发育阶段激活了基因。用 AtUbq3lox 系授粉的 AtRps5aCre 系杂交的球形期 ZsGreen 表达的烟草胚胎的观察结果支持了早期激活模式。
