Expression and interaction proteins analysis of BjuFKF1 in stem mustard

Stem mustard (Brassica juncea var. tumida Tsen et Lee) is one of a unique vegetable in China and the tumor stem is its economic product. With the expansion planting area to high altitude in Fuling, early-bolting has become the most critical issue in its cultivation. Previous studies found that stem mustard early-bolting in high latitude closely related to short waves. FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) is a plant-specific LOV blue light receptor, mainly regulates photoperiodic flowering and cycle rhythm. We used BjuFKF1 as the bait protein and detected its interaction protein BjuPP2C52. Further, GST pull down assay confirmed the interaction in vitro. Yeast two hybrid assay showed that both the F-box domain and KELCH repeat domain of BjuFKF1 interacted with BjuPP2C52 in vivo. RT-PCR and qRT-PCR was used to analyze gene expression in different tissues and development stages, respectively. RT-PCR results indicated that BjuFKF1 was expressed in tumorous stem at an extremely high level, while BjuPP2C52 was highly expressed in both flowers and tumorous stem. qRT-PCR assays showed that both BjuFKF1 and BjuPP2C52 were up-regulated expressed in the reproductive growth stages. Sub-cellular assays showed that BjuFKF1 and BjuPP2C52 located in the nucleus and plasma membrane, respectively. Taken together, our results demonstrated that BjuFKF1 interacted with BjuPP2C52 in vitro and vivo, BjuFKF1 was expressed at an extremely high level in tumorous stem, BjuPP2C52 was highly expressed in flowers. BjuFKF1 and BjuPP2C52 were up-regulated expressed in reproductive growth stages. Sub-cellular assays showed that BjuFKF1 and BjuPP2C52 located in the nucleus and cell membrane, respectively. It was speculated that BjuFKF1 may through the pathway of “BjuFKF1-BjuPP2C52-BjuAGB1-BjuNH3-Bjuphot1” to regulate stem mustard response to blue light and eventually bolting and flowering time.