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.