原生质体制备.doc

本氏烟草原生质体制备Schweiger R and Schwenkert S. 2014. Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves. Journal of Visualized Experiments. 85( 51327): 1- 8. 1 Protoplast PreparationProtoplast preparation of tobacco leaves was adapted from Koop et al.16 and slightly modified.1.1 Buffer preparation1. Prepare F-PCN medium: Macro-salts KNO3 (1012 g/ml), CaCl22H2O (440 g/ml), MgSO47H2O (370 g/ml), KH2PO4 (170 g/ml),NH4-succinate (20 mM; prepare a 2 M stock solution (succinate (236 g/ml) and NH4Cl (106 g/ml), adjust to pH 5.8 to dissolve),Micro-salts EDTA-Fe(III) x Na-salt (40 g/ml), KJ (0.75 g/ml), H3BO3 (3 g/ml), MnSO4H2O (10 g/ml), ZnSO47H2O (2 g/ml), Na2MoO47H2O (0.25 g/ml), CuSO45H2O (0.025 g/ml), CoCl26H2O (0.025 g/ml), MES (390 g/ml), glucose (approximately 80 g/ml) osmolarity 550 mOsm, pH 5.8 (KOH). Store in aliquots at -20 C.2. Prepare F-PIN medium: All ingredients as F-PCN but instead of glucose use sucrose (approximately 110 g/ml), osmolarity 550mOsm, pH 5.8 (KOH). Store in aliquots at -20 C.3. Prepare W5 medium: 150 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM M ES, osmolarity 550-580 mOsm, pH 5.7 (KOH). Store at 4 C (sterile filter through a 0.2 m filter to prevent bacterial growth during longer storage).4. Prepare fresh enzyme solution for protoplast isolation (0.1 g cellulase, 0.03 g macerozym in 10 ml F-PIN). Incubate the solution at 55C for 10 min and cool to RT. Add 100 l of 10% BSA to 10 ml solution.1.2 Isolation of protoplasts1. Place one infiltrated leaf into a Petri dish and add the fresh enzyme solution. Use a new razorblade to cut the leaf into approximately 0.5 cm2 sized pieces. Transfer the leaf-pieces with the enzyme solution into a vacuum-infiltration flask and vacuuminfiltrate for approximately 20 sec until air bubbles emerge from the leaves (release vacuum very carefully).2. Shake the flask for 90 min at 40 rpm in darkness.3. Release protoplasts by shaking for 1 min at 90 rpm. Filter the solution through gauze (100 M) into a 15 ml centrifugation tube (round bottom).4. Overlay the protoplast solution with 2 ml F-PCN buffer and centrifuge for 10 min at 70 x g (slow acceleration and deceleration) at RT.5. Intact protoplasts accumulate at the interface of enzyme solution and F-PCN. Take a wide orifice 1 ml pipette tip to transfer the intact protoplasts into a fresh centrifuge tube and fill up with W5 buffer. Centrifuge for 2 min at 100 x g (slow acceleration and deceleration) to pellet the protoplasts.6. Remove the supernatant carefully by using a pipette and resuspend pellet in approximately 200 l W5 buffer, depending on the amount of protoplasts.7. Always use wide orifice tips to prevent rupturing of intact protoplasts.用W5液补满重悬浮移液器吸弃上清，视原生质体产量，约保留200微升W5液以广口1ml移液器吸取酶液与F-PCN界面间的原生质体，移入新离心管加入2 ml F-PCN图1. 本氏烟草原生质体制备流程拟南芥原生质体准备Zhai Z, Jung H,and Vatamaniuk O K. Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana. Journal of Visualized Experiments, 2009: 1-3.1 Reagents preparationTVL:0.3 M sorbitol; 50 mM CaCL2 Filter-sterilize and store at -20 C.Enzyme Solution: 0.5 M sucrose, 10 mM MES-KOH pH 5.7, 20 mM CaCl2, 40 mM KCl, 1% Cellulase (Onozuka R-10), 1% Macerozyme (R10). Filter-sterilize and freshly use.W5 Solution:0.1% (w/v) glucose, 0.08% (w/v) KCl, 0.9% (w/v) NaCl, 1.84% (w/v) CaCl2, 2 mM MES-KOH pH 5.7. Filter-sterilize and store at room temperature.2 Isolation of protoplasts from Arabidopsis seedlings.1. Slice 2 g of 14-day-old seedlings with a fresh razor blade in 15 ml of filter-sterilized TVL Solution. We chop plant material in sterile disposable Petri dishes.2. Transfer chopped tissues into 200 ml beaker, add 20 ml of filter-sterilized Enzyme Solution, swirl the beaker to let tissues mix with Enzyme Solution, cover with parafilm and aluminum foil.3. Shake plant tissues at 35 rpm in the dark at room temperature for 16-18 h.4. Collect the released protoplasts into 50 ml Falcon tube by sieving through 8 layers of the cheese cloth, pre-wet in W5 Solution.5. Sieve protoplasts from the cheese-cloth one more time by washing the cloth with 15 ml of W5 Solution.6. Carefully overlay protoplasts with 10 ml of W5 Solution, do not disturb the sugar gradient; centrifuge for 7 min at 100 g.7. Collect 10 ml of protoplasts at the interface of Enzyme Solution and W5 Solution (Fig. 1B) and transfer to a new 50 ml Falcon tube.8. Add 15 ml W5 Solution, centrifuge for 5 min at 60 g. Remove the supernatant.9. Wash protoplasts free from Enzyme Solution by resuspending protoplasts in 15ml of W5 Solution, centrifuge for 5min at 60 g10. Remove the supernatant, resuspend pelleted protoplasts in 1-3ml W5 Solution.11. Evaluate protoplast yield by cell counting with a hemocytometer.Protoplast IsolationSheen, J. 2002, A transient expression assay using Arabidopsis mesophyll protoplasts.Enzyme solution1-1.5 % cellulase R10 (RS is too strong)0.2-0.4% macerozyme R10 (Yakult Honsha, Tokyo, Japan)0.4 M mannitol20 mM KCl20 mM MES, pH 5.7Heat the enzyme solution at 55oC for 10 min (to inactivate proteases and enhance enzymesolubility) and cool it to room temperature before adding10 mM CaCl25 mM -mercaptoethanol (optional)0.1% BSA (Sigma A-6793)The enzyme solution is light brown but clear (passed through a 0.45 m filter).Washing and incubation solution (WI)0.5 M mannitol,4 mM MES, pH 5.720 mM KClW5 solution154 mM NaCl125 mM CaCl25 mM KCl2 mM MES (pH 5.7) (no glucose since we use glucose as a signal)Plant MaterialsBE plants grown on the B5 mediumGreenhouse-grown BE, Col, Ler and C24 plants are fine Use well expanded leaves from 3-4 weeks old plants (the second and/or third/fourth pair, 1-2 cm) before flowering. Shorter photoperiod (12-13 h light or less, 50-150 E) is recommendedfor Col and Ler that flower earlier under long-day condition.Protoplast Isolation ProcedureCut 0.5-1 mm leaf strips with fresh razor blades without wounding. This is perhaps the most tedious part for most people. However, I consider it easier and more efficient than peeling the lower epidermis of the leaves one by one. It takes some practice to do a good job in cutting leaves. A very good preparation yields around 107 protoplasts/g fresh weight (about 100 to 150 leaves digested in 40-60 ml of enzyme solution). For a practice or for most small scale experiments, 10-20 leaves digested in 5-10 ml cellulase/macerozyme solution will give 0.5 -1 x 106 protoplasts that are enough for more than 50-100 samples (1-2 x 104 protoplasts per sample). Note that it is not necessary to use 106 protoplasts per sample for gene expressionanalysis as commonly recommended in other protoplast protocols. The experiments can be easily scaled up or down as long as the recommended DNA/protoplast ratio is followed (see below).Use a flask (125 ml flask for 10 ml enzyme solution) with a side arm for leaf digestion and apply vacuum infiltration for 5-30 min or just put leaf strips in a Petri dish with enzyme solution and put it into to a vacuum desiccator. Continue the digestion for about 3 h without shaking in the dark (digestion time is depending on the experimental goals and desirable responses). This step needs to be tested empirically for your own assay. The usual prolonged incubation of leaves for 16-18 h in the dark for protoplast isolation is stressful and might eliminate physiological responses of leaf cells. However, the stress might be potentially important for the dedifferentiation and regeneration processes. The enzyme solution should turn green after a gentle swirling motion, which indicates the release of round protoplasts (check under microscope, the size of Arabidopsis mesophyll protoplasts is around 30 to 50 m). We do not intend to release protoplasts 100%. Be gentle with protoplasts but you can handle them with regular pipets and pipet tips. Filter the enzyme solution containing protoplasts with a 35-75 m nylon mesh.Spin at 100 x g to pellet the protoplasts in a round-bottomed tube for 1-2 min (speed 3 with an IEC clinical centrifuge). Higher speed or the addition of CaCl2 (50 mM) may be used if the protoplast recovery is poor. The pelleted protoplasts should be resuspended easily by gentle shaking. Wash protoplasts once in cold washing/incubation (WI) solution for electroporation or W5