Green fluorescence protein (GFP) reporter assay
The A. tumefaciens strain GV3101 containing one of the binary
vector (35s:5NBts-GFP or 35s:5NBmts-GFP ,35s: miR398b, 35s:miR157 ) was cultured in LB medium
overnight at 28°C. The A. tumefaciens samples with different
vectors were collected and mixed at the indicated optical density (O.D.)
using a DU800 spectrophotometer (Beckman Coulter). The A.
tumefaciens harboring different vectors were infiltrated into tobacco
leaves and the transfected cells were imaged by confocal laser scanning
microscopy (Olympus FV1200) 60 h after inoculation. For western blot
analysis, 100 mg of infiltrated leaf tissues was grounded into fine
powder in liquid nitrogen and homogenized in 400 μl of tobacco protein
extraction buffer (25 mM Tris-HCl, PH=7.5; 150 mM NaCl; 1 mM EDTA; 0.5%
Triton X-100; 5% glycerol; 1% protein inhibitor; 2 mM
phenylmethanesulfonyl fluoride). The same amount of extracted total
protein was separated on a 15% SDS-PAGE gel, and transferred to a
polyvinylidene fluoride membrane (Millipore). A 10,000-fold-diluted
Rabbit Anti-GFP sera (Ab290, Abcam) and Secondary-Goat Anti-Rabbit IgG
H&L (HRP) (Ab205718, Abcam) were used to detect GFP accumulation and
total protein was stained with Coomassie brilliant blue (R250) for a
loading control.