PEG介导拟南芥叶片原生质体瞬时表达方法.doc

PEG介导拟南芥叶片原生质体瞬时表达方法 1. B5培养基上萌发拟南芥种子，待根长至1-3厘米时即可移栽到土里，温室培养，光照12h/12h，150E。 2. 准备好一个90mm培养皿，称1.82克D甘露醇于20ml双蒸水中。培养皿的盖子用来切叶片。 3. 取4周后未抽台前的叶片，约90片。切成1mm宽的长条，置于甘露醇溶液中。可以一边切一边从植株上取。 4. 配酶解液，100ml三角瓶，15ml酶解体系。 5. 将步骤3中细条捞出，置于酶解液中。黑暗，23，40-50rpm酶解3小时。 6. 酶解液过100-200目的筛子，将过滤后的绿色混合物置于15ml离心管（直径约1cm）中，均分为两管。4，60 g，15min，brake 设为4-5。 7. 弃上清，沉淀用冰冷的W5溶液轻柔洗涤，每管4ml。4，100 g，1min，brake 设为4-5。 8. 弃上清，沉淀用冰冷的W5溶液轻柔洗涤，每管4ml。冰上放置30min。 9. 23，100 g，1min，brake 设为4-5。弃上清，每管沉淀用0.5ml MaMg重悬。（本步骤及以下操作均在23。） 10. 取约10-20ug 质粒于1.5ml EP管中，加100ul 步骤9中的原生质体。用200ul 枪头（剪去前端）轻柔混匀。 11. 加入110ul PEG/Ca 溶液，轻柔混匀。放置20-30min。 12. 加入0.44ml W5 溶液，来回颠倒混匀。23，100 g，1min，brake 设为4-5。 13. 弃上清，加100ul W5，混匀。加900ul W5，混匀。 14. 上述混合液体置于六孔板内，23，弱光，孵育6-18小时。 15. 荧光观察，观察之前100g，常温，离心2分钟，终体积控制在50ul左右。 Solution Recipes Enzyme solution 1ml 15cellulase R10 (RS is too strong) 1ml 4.5macerozyme R10 (Yakult Honsha, Tokyo, Japan) 1.09 g mannitol 1ml 0.3M KCl 1ml 0.3M MES, pH 5.7 Heat the enzyme solution at 55oC for 10 min (to inactivate proteases and enhance enzymesolubility) and cool it to room temperature before adding 1ml 0.15M CaCl2 1ml 0.75mM -mercaptoethanol 1ml 1.5% BSA PEG solution (40%, v/v) 1 g PEG4000 (Fluka, #81240) *Very Important! 0.75 ml H2O 0.625 ml 0.8 M mannitol 0.25 ml 1M CaCl2 W 5 1000 ml 154 mM NaCl 9.0 g 125 mM CaCl2 18.4 g 5 mM KCl 0.37 g 5 mM glucose 0.9 g 0.03% MES 0.3 g pH to 5.8 with KOH autoclave 20 minutes in 125 bottles MaMg solution 100 ml 15 mM MgCl2 1.5 ml 1M MgCl2 0.1% MES 0.1 g 0.4 M mannitol 7.3 g pH to 5.6 with KOH autoclave 20 minutes in 125 ml bottles References 1. Sheen, J. 2002, A transient expression assay using Arabidopsis mesophyll protoplasts. /sheenweb/ 2. Doelling & Pikaard. 1993, Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures. Plant Cell Reports 12: 241-244Enzyme solution Prepare 20 mM MES (pH 5.7) containing 1.5% (wt/vol) cellulase R10, 0.4% (wt/vol) macerozyme R10, 0.4Mmannitol and 20mMKCl.Warm the solution at 55 for 10 min to inactivate DNAse and proteases and enhance enzyme solubility. Cool it to room temperature (25 ) and add 10mM CaCl2, 15 mM -mercaptoethanol (ptional) and 0.1% BSA. CRITICAL :Addition of 15 mM -mercaptoethanol is optional, and its use should be determined according to the experimental purpose. CRITICAL Before the enzyme powder is added, the MES solution is preheated at 70 for 35 min. The final enzyme solution should be clear light brown. Filter the final enzyme solution through a 0.45msyringe filter device into a Petri dish (100x 25mm2 for 10 ml enzyme solution). CRITICAL The enzyme solution should be prepared fresh.WI solution Prepare 4 mM MES (pH 5.7) containing 0.5 M mannitol and 20 mM KCl. The prepared WI solution can be stored at room temperature (2225 ).W5 solution Prepare 2mM MES (pH 5.7) containing 154mM NaCl, 125mM CaCl2 and 5 mM KCl. The prepared W5 solution can be stored at room temperature.MMG solution Prepare 4 mM MES (pH 5.7) containing 0.4 M mannitol and 15mMMgCl2. The preparedMMG solution can be stored at room temperature. PEGcalcium transfection solution Prepare 2040% (wt/vol) PEG4000 in ddH2O containing 0.2 M mannitol and 100 mM CaCl2. CRITICAL Prepare PEG solution at least 1 h before transfection to completely dissolve PEG. The PEG solution can be stored at room temperature and used within 5 d. However, freshly prepared PEG solution gives relatively better protoplast transfection efficiency. Do not autoclave PEG solution.Protoplast lysis buffer Prepare 2.5 mM Trisphosphate (pH 7.8) containing 1 mM DTT, 2 mM DACTAA, 10% (vol/vol) glycerol and 1% (vol/vol) Triton X-100. The lysis buffer should be prepared fresh.MUG substrate mix for GUS assay Prepare 10 mM TrisHCl (pH 8) containing 1 mM MUG and 2 mM MgCl2. The prepared GUS assay substrate can be stored at 20 PROCEDUREPlant growth _ Timing 34 weeks 生长3-4周1| Grow Arabidopsis plants on either Metro-Mix 360 or Jiffy7 soil in a greenhouse or an environment-controlled chamber with a relatively short photoperiod (1013 h light at 23 /1114 h dark at 20) under low light (5075 E m2 s1) and 4065% relative humidity.较短的光照时间(10-13小时光照 23/11-14小时黑暗 20) 40-65%的相对湿度. CRITICAL STEP Col-0 and Ler have been extensively tested in our lab. In general, Arabidopsis plants are very sensitive to all kinds of environmental changes (e.g., drought, flooding, extreme temperature and constant mechanical perturbation). Try to maintain aconstant environment as much as possible.注意:经过我们实验室的反复验证,总的来说拟南芥对各种环境变化非常敏感(如干旱,水淹,极端温度和不停地机械性干扰)。所以尽量为植株提供一个持续稳定的生长环境。Protoplast isolation _ TIMING 45 h原生质体分离耗时4-5小时2| Choose well-expanded leaves from 34-week-old plants (usually true leaf numbers five to seven) before flowering。选择3-4周完全伸展为抽苔植株的叶片（一般第5到第7片真叶最好）见图1(see Fig. 1a). CRITICAL STEP The selection of healthy leaves at the proper developmental stage is a very important factor in protoplast experiments. Protoplasts prepared from leaves recovered from stress conditions (e.g., drought, flooding, extreme temperature and constant mechanical perturbation) may look similar to those from healthy leaves. However, we have often experienced low transfection efficiency with the protoplasts from stressed leaves. High stressinduced cellular status can also be a problem in the study of stress, defense and hormonal signaling pathways.选择合适生长阶段的健康的叶片是原生质体试验非常重要的因素。从经过逆境胁迫(如干旱,水淹,极端温度或持续的机械性干扰)又恢复过来的叶片中获得的原生质体看起来可能与正常叶片一样,但是我们经常遇到这样的原生质体转化效率很低.高压胁迫诱导的细胞状态改变在研究胁迫、防御和激素信号转导通路时也会造成麻烦。3| Cut 0.51-mm leaf strips from the middle part of a leaf using a fresh sharp razor blade without tissue crushing at the cutting site. A good preparation yields approximately 107 protoplasts per gram fresh weight (approximately 100150 leaves digested in 4060 ml of enzyme solution). For routine experiments, 1020 leaves digested in 510 ml enzyme solution will give 0.51 x 106 protoplasts, enough for more than 25100 samples (12 x104 protoplasts per sample). 用一个新的锋利的刀片切取叶片的中部成0.5-1mm宽的细条，注意切条时尽量减少叶片擦伤。一次高效的制备能够获得107个原生质体/克鲜重(大约40-60ml酶液消化100-150片叶)，一般试验中，用5-10ml酶液消化10-20片叶就能够获得0.5-1 x 106个原生质体，足够25-100个样品用(1-2 x104 个原生质体每个样品)CRITICAL STEP Change the blade after cutting four to five leaves. We routinely cut leaves on a piece of clean white paper(8_ 11) on top of the solid and clean laboratory bench, which provides for good support and easy inspection of wounded/crushed tissue (juicy and dark green stain).切4到5片叶换一个刀片，我们经常在一个固定的干净的试验台上铺一张白纸，在白纸上切叶片，这样能够提供一个好的支持并且能够检测伤害和挤压（一般会出现暗绿色）4| Transfer leaf strips quickly and gently into the prepared enzyme solution (1020 leaves in 510 ml) by dipping both sides of the strips (completely submerged) using a pair of flat-tip forceps.把切好的叶片细条小心迅速地转移到酶液中(10-20片叶to5-10ml)，用一个平头镊子把叶条完全浸入到酶液中。 CRITICAL STEP Immediate dipping and submerging of leaf strips is very critical for protoplast yield. When leaf strips are dried out on the paper during cutting, the enzyme solution cannot penetrate and protoplast yield is decreased significantly.注意：快速地把叶片浸没到酶液中非常重要，在纸上切叶片时，叶片干了会影响酶液对叶片的消化，原生质体产量会严重下降。5| Vacuum infiltrate leaf strips for 30 min in the dark using a desiccator.暗中抽真空30分钟6| Continue the digestion, without shaking, in the dark for at least 3 h at room temperature. The enzyme solution should turn green after a gentle swirling motion, which indicates the release of protoplasts.室温条件下，暗中继续消化3小时以上，不用摇动。之后轻摇酶液会变绿，这说明原生质体已经释放出来了。 CRITICAL STEP Digestion time depends on the experimental goals, desirable responses and materials used, and it needs to be optimized empirically. After 3 h digestion, most protoplasts are released from leaf strips in case of Col-0. However, the protoplast isolation efficiency differs significantly for different ecotypes and genotypes. The digesting time has to be optimized for eachecotype and genotype. Prolonged incubation of leaves (1618 h) in the dark is stressful and might eliminate physiological responses of leaf cells. However, the stress might be important for the dedifferentiation and regeneration processes recommended in other protoplast protocols.注意：消化时间的长短取决于实验目的，往往需要经验来优化。一般情况下，经过3小时的酶解消化，野生型的拟南芥叶片都能释放出大多数的原生质体。当然，不同的生态型和遗传背景的材料，原生质体分离的效率也会有所不同。延长暗中消化时间可以消除叶肉细胞的生理响应并且很有效的。但是这种stress在其他原生质体方法中对去分化和再生过程有重要作用。7| Check for the release of protoplasts in the solution under the microscope; the size of Arabidopsis mesophyll protoplasts is approximately 3050 m.显微镜下检测酶液中原生质体释放程度，拟南芥叶肉原生质体大小约30-50m CRITICAL STEP It is not necessary to release all the protoplasts from leaf strips. Be gentle with protoplasts, but you can handle them with regular pipettes and pipette tips.8| Dilute the enzyme/protoplast solution with an equal volume of W5 solution before filtration to remove undigested leaf tissues.在过滤除去没有消化的叶片组织前用等体积的W5溶液稀释原生质体酶液。9| Wash a clean 75-m nylon mesh with water to remove ethanol (the mesh is normally kept in 95% ethanol) then remove excess water before protoplast filtration. Filter the enzyme solution containing protoplasts after wetting the 75-mm nylon mesh with W5 solution.用水清洗75um尼龙膜以去除酒精 (尼龙膜一般保存在95%的酒精中)，过滤前去除尼龙膜上多余的水，再用W5溶液湿润尼龙膜，过滤含有原生质体的酶液。10| Centrifuge the flow-through at 100g to pellet the protoplasts in a 30-ml round-bottomed tube for 12 min. Remove as much supernatant as possible and re-suspend the protoplast pellet by gentle swirling. 用30ml 圆底管100g离心1-2分钟，以使原生质体呈球状。尽可能地去除上清，轻转以重悬原生质体小球。 CRITICAL STEP A higher speed (200g) of centrifugation may help to increase protoplast recovery.稍高的离心力（200g）能够帮助原生质体恢复。11| Re-suspend protoplasts at 2105 ml1 in W5 solution after counting cells under the microscope (x100) using a hemacytometer. Rest the protoplasts by keeping on ice for 30 min.通过显微镜观察计算过原生质体数目后，用W5溶液重悬至2105 ml1，冰上放置30mins。 CRITICAL STEP If cold response is desired, keep protoplasts at room temperature. Although the protoplasts can be kept on ice for at least 24 h, freshly prepared protoplasts should be used for the study of gene expression regulation, signal transduction and protein trafficking, processing and localization.如果想要研究冷冻效应，就把原生质体放置室温。尽管原生质体能够在冰上放置至少24小时，但在研究基因表达调控，信号传导和蛋白转运、加工和定位时尽量使用新鲜制备的原生质体。12| Protoplasts should begin to settle at the bottom of the tube by gravity after 15 min. Remove the W5 solution as much as possible without touching the protoplast pellet. Re-suspend protoplasts at 2105 ml1 in MMG solution kept at room temperature.重悬之后原生质体在重力的作用下15分钟以后会沉到试管底部。去除上清的W5溶液而避免碰到原生质体小球。再用MMG溶液重悬至2105 ml1，室温放置。DNA-PEGcalcium transfection _ TIMING Less than 1 h for 20 samplesPEG-钙离子介导的转染 20个样品耗时少于1小时13| Add 10 l DNA (1020 g of plasmid DNA of 510 kb in size) to a 2-ml microfuge tube.加10ul质粒DNA（10-20ug 5-10kb大小的质粒DNA）到一2ml离心管中。CRITICAL STEP To study the role of a candidate regulator, three plasmids expressing a regulatory effector, a specific reporter and a transfection control reporter are used at the ratio of 5:4:1. However, it is highly recommended to optimize the plasmid ratio depending on each reporters sensitivity. For example, a highly sensitive reporter needs only half the amount. Whenever the plasmid amount is changed, the same total plasmid DNA amount is compensated with a control plasmid that does not express any effector or reporter. The plasmid DNA has to be purified appropriately to support high transfection efficiency. Additional information on the preparation of plasmid DNA using the economical CsCl gradient is provided on the Sheen lab website (/sheenweb/protocols.html).14| Add 100 ml protoplasts (2104 protoplasts) and mix gently.加入100ul的原生质体（2104个），轻轻混匀。15| Add 110 ul of PEG solution, and then mix completely by gently tapping the tube (handle six to ten samples for each transfection).加入110ul的PEG溶液，轻弹试管以充分混匀（每次转染一般转染6-10个样品）16| Incubate the transfection mixture at room temperature for up to 15 min (5 min is sufficient). 室温孵育转染细胞15分钟以内（5分钟就可以了） CRITICAL STEP Transfection time should be optimized empirically. 具体的最佳转染时间要靠试验来摸索。17| Dilute the transfection mixture with 400440 ul W5 solution at room temperature and mix well by gently rocking or inverting the tube to stop the transfection process.室温400-440ulW5溶液稀释转染细胞，轻微振荡或颠倒混匀以停止转染反应。18| Centrifuge at 100g for 2 min at room temperature using a bench-top centrifuge and remove supernatant.用台式离心机室温100g离心2分钟，弃上清。 19| Re-suspend protoplasts gently with 1 ml WI in each well of a 6-well tissue culture plate. 用1ml WI 溶液重悬原生质体。 CRITICAL STEP We also use 12-well (0.5 ml WI) or 24-well (0.25 ml WI) plates for larger-scale experiments (50100 samples). The culture plate is normally coated with 5% (vol/vol) sterile calf serum for a short time (12 s) to prevent sticking of the protoplasts to the plastic surface. The depth of the WI solution is approximately 0.1 mm to prevent hypoxia stress during protoplast incubation. CRITICAL STEP The use of carrier DNA is unnecessary for this type of DNA delivery.CRITICAL STEP High transfection efficiency can be achieved using 1020% PEG final concentration. The optimal PEG concentration should be determined empirically for each experimental purpose. Visual reporters such as GFP can be used to determine optimal DNA transfection conditions (Fig. 1d, Table 1). If protoplasts are derived from healthy leaf materials, most protoplasts should remain intact throughout the isolation, transfection, culture and harvesting procedures.CRITICAL STEP Protoplast transfection can be scaled up or down depending on the experimental needs. Approximately 1034 protoplasts are sufficient for reporter enzyme assays, 1045 protoplasts are required for protein labeling and immunoprecipitation or western blot analysis and 106 protoplasts for RNA extraction and microarray analysis. If the protoplast transfection efficiency is low (less than 20%), the quality of plasmid DNA or the ratio of plasmid DNA and protoplast number should be systematically examined to find the optimal condition.? TROUBLESHOOTINGProtoplast culture and harvest _ TIMING 224 h20| Incubate protoplasts at room temperature (2025 ) for the desired period of time.室温（20-25）孵育原生质体，时间视要求而定。（2-16个小时） CRITICAL STEP A stimulus can be applied during the incubation according to the experimental purposes. For example, the synthetic flg22 peptide derived from bacteria can be added for 10 min or 1 h for the analysis of protein kinase activity or early transcription responses, respectively. For the inducible gene expression system (Fig. 2c,d), DEX can be added to the protoplasts 1530 min after DNA transfection.CRITICAL STEP In general, the protoplast incubation time is 26 h for RNA analysis and 216 h for enzyme activity analysis and protein labeling. The TEAMP system is most suitable for the study of early events in signal transduction pathways, gene regulation and cell death. CRITICAL STEP The protoplast incubation temperature has to be optimized according to experimental needs. CRITICAL STEP It is not necessary to perform experiments under sterile conditions in general. Ampicillin (50 ug ml1) can be added during the protoplast incubation after DNA transfection if bacterial growth is a concern.21| Re-suspend and harvest protoplasts by centrifugation at 100g for 2 min.重悬原生质体，室温100g离心2分钟收集。22| Remove the supernatant and freeze samples on dry ice.弃上清，冰上放置。 PAUSE POINT Samples can be stored at 80 until further analysis.这时候可以-80冻存以备进一步分析。Reporter assays _ TIMING 12 h23| Several assays can be carried out using option A (LUC assay), B (GUS assay) or C (GFP assay).(A) LUC assay(i) Add 100 ml of protoplast lysis buffer to the frozen protoplasts and mix vigorously by vortexing for 2 s to rupture the protoplasts.(ii) After 5 min incubation on ice, centrifuge at 1,000g