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Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

In this experiment,

In this experiment, real-time quantitative PCR analysis was carried out using a BioRad iCycler IQ Real-Time PCR Detection System (Bio-Rad, USA) and a Primescript RT reagent (perfect real time) kit (TaKaRa, Japan). Each 25 µl reaction mixture contained 1 × SYBR Premix ExTaq, 0.5 µl cDNA template, and 200 nM gene-specific primers. Reactions were run in duplicate under the following conditions: 40 cycles of 95°C for 5 s, 61°C for 20 s, after an initial activation step at 95°C for 10 s, according the manufacturer's instructions. Melting curves and standard curves were calculated and analyzed for OsEATB and Actin. The relative mRNA expression levels were normalized against Actin gene expression levels. Yeast one hybrid assay The full open reading frame (ORF) of OsEATB was generated by PCR using the primers OsEATB-AD-S (5’-CATATGAACGACCTCAAGCACACTACTAG-3’) (forward) and OsEATB-AD-A (5’-GGATCCGCCTTCCAGTAATCTAATCCAAC-3’) (reverse). The ORF was then fused in-frame into pGAD424 to construct pGAD-OsEATB. To determine whether OsEATB interacted with the GCC-box, the construct was transformed into the yeast strain YM4271 (Clontech), which contained the reporter vector pHIS-4 and the GCC-box (5’-TAAGAGCCGCC-3’). The same yeast strain containing pHIS-4 and muGCC-box (5’-TAAGATCCTCC-3’), which had a 2bp substitution mutation at the center of the GCC-box, was used as the negative control. Electrophoretic mobility shift assays (EMSA) Nucleotide sequence of the double-stranded oligonucleotides of GCC-box element (2 × 5’-TAAGAGCCGCC-3’) was treated as the probe, and the double-stranded oligonucleotides of mGCC-box element (2 × 5’-TAAGATCCTCC-3’) was served as the competitor. Then, we used a DIG Gel Shift kit (Roche, Switzerland) to examine whether recombinant OsEATB protein could interact with the GCC-box element. The probe in a final volume of 20 μl DNA-protein complex was allowed to bind for 20 min at 20°C, and then proteins were separated on an 8% (w/v) polyacrylamide gel in 0.5 TBE. The gel was sandwiched and transferred to N+ nylon membrane (Millipore) in 0.5 TBE buffer at 380 22

mA at 4°C for 60 min. Bands were visualized by autoradiography. Subcellular localization assay The coding sequence of OsEATB was amplified with the cDNA clone as the template using the primers OsEATB-GFP-S (5’-GGATCCCGACCTCAAGCACACTACTAGC-3’) (forward) and OsEATB-GFP-A (5’-ACTAGTAAAACTAGACGCTGCACCGG -3’) (reverse). The PCR product was inserted into the pCAMBIA 1304 vector fused with GFP in-frame, under the control of the CaMV 35S promoter. The fused plasmid coprecipitated with tungsten particles and was introduced into onion epidermal cells by particle bombardment using the PDS-1000 system (Xinzhi, China) at 1,100 psi helium pressure. We observed GFP fluorescence under a fluorescence conversion microscope (Olympus, Japan) after incubation of transformed cells in the dark for 36 h. RNA Hybridization in situ Freshly collected plant tissues were fixed in 4% (w/v) formaldehyde at 4°C for 2–4 h and 10 µm microtome sections were mounted on RNase-free glass slides. The primers used to prepare the probe were 5’-GGGTAGTCATCAGGCTCCG-3’ (forward) and 5’- GCCGTTTCTTTGGCTGGG -3’ (reverse). The fragment was linked to pGEM-T (Promega, Madison, WI, USA) and the RNA probes were then produced by T7 and SP6 transcriptase labeled with digoxigenin (Roche, Switzerland). RNA hybridization in situ and immunological detection were carried out according to the instructions for the Enhanced Sensitive ISH Detection kit (POD, Boster Biotech, Wuhan, China). Stress and hormone treatments For stress and hormone treatments, 3-leaf-stage seedlings of wild-type and transgenic rice were incubated in Hoagland’s solution containing 200 mM NaCl, 50 μM ethephon, and 100 μM ABA at 28°C under a 14-h light/10-h dark photoperiod. For 200 mM NaCl treatment, the rate of seedlings without wilting and rolling was calculated every 6 h for 3 trials with 50 individuals for each trial. For 100 μM ABA treatment, the rate of seedlings without chlorosis was calculated every 6 h for 3 trials with 50 individuals for each trial. The 23

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