Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

Expression of Cre recombinase by AtRps5a_pro or AtDD45_pro 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_pro and the ZsGreen coding sequence in F₁ progeny upon genotyping but no ZsGreen expression in F₁ seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3_pro. Strong ZsGreen expression in F₁ seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45_pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F₁ 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 AtRps5a_proCre lines pollinated by the AtUbq3_prolox line supported the early activation mode.