2.5 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
For expression pattern analysis of OsLHY , various tissues were collected from WT plants under natural LD conditions. For others, the plants were grown under natural LD conditions for 28 days (d), then were separately transferred to growth chambers with LD and SD conditions. After being entrained 14 d, leaves were separately harvested every 4 h during a 24 h period from WT and the oslhy mutant under LD and SD conditions. All collected samples with three replicates were frozen in liquid nitrogen and stored at -80 ℃. Total RNA was extracted using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. A total amount of 1 µg RNA per sample was used for reverse transcription. cDNAs were synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser following the manufacturer’s instructions (Code NO. RR047A Takara, Japan). The qRT-PCR was performed using SYBR Premix Ex Taq II (Code NO. RR820A/B Takara, Japan) with the Roche Real-Time PCR instrument (LightCycler 480). Sequence-specific primers were used for qRT-PCR (Supplemental Table 1) with ACTIN as an internal control. The qRT-PCR analysis was performed in three technical replicates for each sample. Relative gene expression levels were calculated using the 2-ΔΔCt method (Livak & Schmittgen, 2001).
2.6 Luciferase imaging and dual-luciferase assays
The promoters of flowering related genes were inserted into the pGreenII 0800-LUC vector to drive the firefly luciferase (LUC ) gene that used as the reporter. The pGreenII 0800-LUC vector also contains a renilla luciferase (REN ) gene driven by CaMV35S that served as positive control. While the effector vector carried the full coding sequence of OsLHY under the control of CaMV35S promoter. The 35S::GFP construct was used as a negative control. All constructs were introduced into the Agrobacterium strain EHA105-pSoup. The primers used for related vector construction are listed in Supplemental Table 1.
The tobacco (Nicotiana benthamiana ) plants were grown in the growth chamber at 25℃ under 16 h light/8 h dark photoperiod. Dual-luciferase assays were performed with approximately four-week-old tobacco leaves. The reporter and effector constructs were mixed in a ratio of 1:10 (v/v) and injected into tobacco leaves via needleless syringes. The negative control and tested pairs were injected into the same leaves but in different positions. At least 48 h after injection, the leaves were sampled for fluorescence signal observation and LUC activity detection. The luciferin (100 µM) was sprayed into the infiltrated tobacco leaves, and then were kept in a dark condition for 5 min before fluorescence observation. Tanon 5200 imaging system was used to capture the fluorescence signals images. The LUC and REN activities were assayed with dual-luciferase assay reagents (Promega) by using the GloMax 96 microplate luminometer (Promega), according to the manual provided by the manufacturer. The effect of OsLHY on the transcriptional activity of the tested flowering regulators was finally determined by the relative ratio of LUC/REN. At least five biological repeat were measured for each samples.