Cloning,subcellular location and rhythmic expression analysis of FaFKF1 gene in Festuca arundinacea

【Objective】 The blue light receptor family gene(FaFKF1) in Festuca arundinacea was cloned, its sequence characteristics and subcellular localization were analyzed, and its rhythmic expression characteristics under different light treatments were detected, so as to provide a theoretical basis for exploring its regulatory mechanism in adult flowers and the adaptive breeding of F. arundinacea optoperiood.【Method】 The RACE technology was used to clone the full length cDNA of FaFKF1 gene. Its bioinformatics analysis was conducted. pCAMBIA1300-FaFKF1-GFP fusion expression vector was constructed, infected tobacco epidermal cells by Agrobacterium mediated transfection to observe the fluorescent signal to determine protein subcellular localization.Meanwhile, the rhythmic expression characteristics of different light treatments and different growth stages of the FaFKF1 gene were detected using real-time fluorescence quantitative PCR.【Result】 The sequence analysis results showed that the full-length cDNA(2266 bp) was obtained with a 1881 bp open reading frame(ORF), which encoded a small molecular protein containing 626 amino acids, the relative molecular weight of FaFKF1 protein was about 68.89 kD and its theoretical isoelectric point(pI) was 5.76, and the fat coefficient 80.99, instability index 39.81, which was a stable hydrophilic protein. Subcellular localization results showed that FaFKF1 was located in the nucleus. In the secondary structure of FaFKF1 protein, 24.92% of alpha helix, 44.98% of random coil, 23.61% of extended strand and 7.03% of beta turn were observed. FaFKF1 protein had the high amino acid sequence similarity with FKF1 of Glycine max(NP_001235886.2), Hordeum vulgare(KAE8795993.1), and Brachypodium dilatatum (XP_003577479.1), reaching more than 85%. And the genetic distance with B. dilatatum was the shortest, followed by H. vulgare and wheat. Under long-day treatment, FaFKF1 gene was expressed in leaves during seedling, tillering, booting and heading stages, and the relative expression trend was similar, all peaked at ZT8(16:00 PM), showing a circadian rhythm of 24 h, but the relative expression was high in booting and heading stages. Under different light treatments based on long and short light exposure, the FaFKF1 gene could adapt to the environmental changes through autoregulation. Although the peak expression occurrence time was inconsistent, the overall circadian rhythm was maintained for 24 h.【Conclusion】 FaFKF1 gene may play an important role in the nucleus, and hasa rhythmic expression subcellular location under different lighting conditions, which is induced and regulated by the photoperiod.