Experiments

Culture of the Tobacco plant (strain Yun yan 87)

1. Sterilize the seeds
• Put 50-100 seeds in a 2ml sterilized EP tube
• Wash the seed with 1ml 70% ethanol for 30s, gently flip the EP tube
• Wash the seed with 1ml sterilized water for 2 time
• Wash the seeds with 1ml 3% Sodium hypochlorite solution for 5min, gently flip the ep tube
• Resine the seeds with 1ml sterilized water for 5 time
2. Use a pipet tip to plant seed on 1/2 N6 medium(3g/L N6 salt, 10g/L sucrose, 7g/L agar; autoclave 121℃ 20min) in a Petri dish, use a sterilized filter paper to soak excess water on the dish surface.
3. Place the Petri dish in 4℃ for 48h
4. Place the Petri dish in 21℃, 16h light 8h dark cycle light incubator for 14 days
5. Transfer the tobacco plant into a hydroponic cell for further experiment

Low phosphate treatment of tobacco plants

1. Prepare the following solutions:
2. Prepare the following solutions:
   • Culture solution A:
   • 2mMCa(NO3)2
   • 0.65mMMgSO4
   • 25μM Fe·EDTA
   • 5μM MnSO4
   • 2μM ZnSO4
   • 0.5μM CuSO4
   • 0.005μM (NH4)6Mo24·4H2O
   • 25μM H3BO4
   • 50μM KCl
   • Normal culture solution: 1 mM KH2PO4 + Culture solution A
   • Low phosphate culture solution: 10 μM KH2PO4, 0.99 μM KCl+ Culture solution A
3. Grow the control group in normal culture solution and the low phosphate group in low phosphate culture solution, in a light incubator with 21℃, 16h light 8h dark cycle.

Electrophoretic mobility shift assay (EMSA) essay

1. Express the candidate protein in Ecoli and purify with His-tag
2. The Probe was synthesis by Tskingke Biotech by two single strain oligos with 5' Biotin modify
3. Mix the two strains with a 1:1 mole ratio in TE buffer, put them in a 95℃ water bath, turn off the water bath, and allow it to cool down to room temperature. This will anneal the two strains into one single strain.
4. Perform the Emsa experiment and transfer to a nylon membrane
5. Follow the instruction and use DIG DNA Labeling and Detection Kit (Roche, 11093657910) to detect the DNA probe on the membrane.
6. Use a CCD chemiluminescence image system to visualize the result.

Extraction of plant tissue protein

1. Weight 0.1g plant tissue sample and placed in a 2ml EP tube
2. Frozen with liquid nitrogen and grind into powder
3. Add 0.3ml RIPA buffer and place on ice for 30min
4. Centrifuge at 4℃ 12,000g for 10min and keep the supernatant

Detection of protein through western blot

1. The protein has been transformed from an SDS-PAGE gel to PVDF membrane (millipore, R1NB740V8) (soak in Methanol 3min before use) at 300V for 45min.(put the device in ice to cool down)
2. The membrane was blocked in 5% non-fat milk(dissolve in TBS buffer) for 1h at room temperature.
3. The membrane was washed in TSBT buffer for 10min
4. The membrane was incubated with anti-HA tag antibody(1:10000 in block buffer) and antiβ-actin antibody(internal reference) at 4℃ overnight.
5. The membrane was washed in TBST buffer for 15min * 4 times
6. The membrane was incubated with Rabbit Anti-Mouse IgG H&L (HRP)(Abcam, ab6728) (1:5000in block buffer) for 1h at room temperature.
7. The membrane was washed in TBST buffer for 15min * 4 times
8. The buffer A and buffer B of the ECL detection reagent(Beyotime,P0018S) was mixed and dropped on the membrane, signal was detected by the Tanon 5200 chemiluminescence system.

Transformation of tobacco leaf using carbon nanodots

1. Ligate the vector with carbon nanodots by mixing CDP 10ul, Plasmid vector (final concentration 10ng/ul), 50X MES buffer 10ul, 10% glycerol 25ul, ddH2O to total volume 500ul
2. Incubate the mixture in 37℃ for 30min
3. Brush on the leaf at 3:00pm every day for 3 or more days.

Other minor protocols - Click to open PDF