Engineering the K+ uptake regulatory pathway by MultiRound Gateway

In a previous study, we described improved versions of MultiRound Gateway vectors. Here, we engineered a calcineurin B-like (CBL) pathway for potassium (K+) nutrition to demonstrate their effectiveness. Using the two improved entry vectors pL12R34H-Ap and pL34R12-Cm, and through 2-4 rounds of Gateway recombination reactions, we generated five pMDC99-derived binary vectors [pK21 (CIPK23+CBL1), pK29 (CIPK23+CBL9), pK31 (CIPK23+CBL1+AKT1), pK39 (CIPK23+CBL9+AKT1), and pK4 (CIPK23+CBL1+AKT1+CBL9)], in which all four genes have the same pSuper promoter and tNos terminator. pK31, pK39 and pK4 were transformed into Arabidopsis. PCR analysis confirmed that all transgenes usually co-existed in the K31, K39 or K4 transgenic plants, and qRT-PCR analysis indicated that the transgenes were expressed at reasonably high levels. The eight overexpression lines, except K31-1, displayed significantly tolerant phenotypes to low-K+ and low-K+ combined with low-Ca2+ compared to the wild type. Significant differences between the K31, K39 and K4 lines were not observed. These results indicate that the improved MultiRound Gateway vectors efficiently assembled multiple transgenes, which were stably inherited and expressed in transformed plants, even with the same promoter and terminator.