PEG介導(dǎo)擬南芥葉片原生質(zhì)體瞬時(shí)表達(dá)方法.doc

PEG介導(dǎo)擬南芥葉片原生質(zhì)體瞬時(shí)表達(dá)方法 1. B5培養(yǎng)基上萌發(fā)擬南芥種子，待根長(zhǎng)至1-3厘米時(shí)即可移栽到土里，溫室培養(yǎng)，光照12h/12h，150E。 2. 準(zhǔn)備好一個(gè)90mm培養(yǎng)皿，稱1.82克D甘露醇于20ml雙蒸水中。培養(yǎng)皿的蓋子用來(lái)切葉片。 3. 取4周后未抽臺(tái)前的葉片，約90片。切成1mm寬的長(zhǎng)條，置于甘露醇溶液中?？梢砸贿吳幸贿厪闹仓晟先?。 4. 配酶解液，100ml三角瓶，15ml酶解體系。 5. 將步驟3中細(xì)條撈出，置于酶解液中。黑暗，23，40-50rpm酶解3小時(shí)。 6. 酶解液過(guò)100-200目的篩子，將過(guò)濾后的綠色混合物置于15ml離心管（直徑約1cm）中，均分為兩管。4，60 g，15min，brake 設(shè)為4-5。 7. 棄上清，沉淀用冰冷的W5溶液輕柔洗滌，每管4ml。4，100 g，1min，brake 設(shè)為4-5。 8. 棄上清，沉淀用冰冷的W5溶液輕柔洗滌，每管4ml。冰上放置30min。 9. 23，100 g，1min，brake 設(shè)為4-5。棄上清，每管沉淀用0.5ml MaMg重懸。（本步驟及以下操作均在23。） 10. 取約10-20ug 質(zhì)粒于1.5ml EP管中，加100ul 步驟9中的原生質(zhì)體。用200ul 槍頭（剪去前端）輕柔混勻。 11. 加入110ul PEG/Ca 溶液，輕柔混勻。放置20-30min。 12. 加入0.44ml W5 溶液，來(lái)回顛倒混勻。23，100 g，1min，brake 設(shè)為4-5。 13. 棄上清，加100ul W5，混勻。加900ul W5，混勻。 14. 上述混合液體置于六孔板內(nèi)，23，弱光，孵育6-18小時(shí)。 15. 熒光觀察，觀察之前100g，常溫，離心2分鐘，終體積控制在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 生長(zhǎng)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.較短的光照時(shí)間(10-13小時(shí)光照 23/11-14小時(shí)黑暗 20) 40-65%的相對(duì)濕度. 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:經(jīng)過(guò)我們實(shí)驗(yàn)室的反復(fù)驗(yàn)證,總的來(lái)說(shuō)擬南芥對(duì)各種環(huán)境變化非常敏感(如干旱,水淹,極端溫度和不停地機(jī)械性干擾)。所以盡量為植株提供一個(gè)持續(xù)穩(wěn)定的生長(zhǎng)環(huán)境。Protoplast isolation _ TIMING 45 h原生質(zhì)體分離耗時(shí)4-5小時(shí)2| Choose well-expanded leaves from 34-week-old plants (usually true leaf numbers five to seven) before flowering。選擇3-4周完全伸展為抽苔植株的葉片（一般第5到第7片真葉最好）見(jiàn)圖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.選擇合適生長(zhǎng)階段的健康的葉片是原生質(zhì)體試驗(yàn)非常重要的因素。從經(jīng)過(guò)逆境脅迫(如干旱,水淹,極端溫度或持續(xù)的機(jī)械性干擾)又恢復(fù)過(guò)來(lái)的葉片中獲得的原生質(zhì)體看起來(lái)可能與正常葉片一樣,但是我們經(jīng)常遇到這樣的原生質(zhì)體轉(zhuǎn)化效率很低.高壓脅迫誘導(dǎo)的細(xì)胞狀態(tài)改變?cè)谘芯棵{迫、防御和激素信號(hào)轉(zhuǎn)導(dǎo)通路時(shí)也會(huì)造成麻煩。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). 用一個(gè)新的鋒利的刀片切取葉片的中部成0.5-1mm寬的細(xì)條，注意切條時(shí)盡量減少葉片擦傷。一次高效的制備能夠獲得107個(gè)原生質(zhì)體/克鮮重(大約40-60ml酶液消化100-150片葉)，一般試驗(yàn)中，用5-10ml酶液消化10-20片葉就能夠獲得0.5-1 x 106個(gè)原生質(zhì)體，足夠25-100個(gè)樣品用(1-2 x104 個(gè)原生質(zhì)體每個(gè)樣品)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片葉換一個(gè)刀片，我們經(jīng)常在一個(gè)固定的干凈的試驗(yàn)臺(tái)上鋪一張白紙，在白紙上切葉片，這樣能夠提供一個(gè)好的支持并且能夠檢測(cè)傷害和擠壓（一般會(huì)出現(xiàn)暗綠色）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.把切好的葉片細(xì)條小心迅速地轉(zhuǎn)移到酶液中(10-20片葉to5-10ml)，用一個(gè)平頭鑷子把葉條完全浸入到酶液中。 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：快速地把葉片浸沒(méi)到酶液中非常重要，在紙上切葉片時(shí)，葉片干了會(huì)影響酶液對(duì)葉片的消化，原生質(zhì)體產(chǎn)量會(huì)嚴(yán)重下降。5| Vacuum infiltrate leaf strips for 30 min in the dark using a desiccator.暗中抽真空30分鐘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.室溫條件下，暗中繼續(xù)消化3小時(shí)以上，不用搖動(dòng)。之后輕搖酶液會(huì)變綠，這說(shuō)明原生質(zhì)體已經(jīng)釋放出來(lái)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：消化時(shí)間的長(zhǎng)短取決于實(shí)驗(yàn)?zāi)康?，往往需要?jīng)驗(yàn)來(lái)優(yōu)化。一般情況下，經(jīng)過(guò)3小時(shí)的酶解消化，野生型的擬南芥葉片都能釋放出大多數(shù)的原生質(zhì)體。當(dāng)然，不同的生態(tài)型和遺傳背景的材料，原生質(zhì)體分離的效率也會(huì)有所不同。延長(zhǎng)暗中消化時(shí)間可以消除葉肉細(xì)胞的生理響應(yīng)并且很有效的。但是這種stress在其他原生質(zhì)體方法中對(duì)去分化和再生過(guò)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.顯微鏡下檢測(cè)酶液中原生質(zhì)體釋放程度，擬南芥葉肉原生質(zhì)體大小約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.在過(guò)濾除去沒(méi)有消化的葉片組織前用等體積的W5溶液稀釋原生質(zhì)體酶液。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尼龍膜以去除酒精 (尼龍膜一般保存在95%的酒精中)，過(guò)濾前去除尼龍膜上多余的水，再用W5溶液濕潤(rùn)尼龍膜，過(guò)濾含有原生質(zhì)體的酶液。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 圓底管100g離心1-2分鐘，以使原生質(zhì)體呈球狀。盡可能地去除上清，輕轉(zhuǎn)以重懸原生質(zhì)體小球。 CRITICAL STEP A higher speed (200g) of centrifugation may help to increase protoplast recovery.稍高的離心力（200g）能夠幫助原生質(zhì)體恢復(fù)。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.通過(guò)顯微鏡觀察計(jì)算過(guò)原生質(zhì)體數(shù)目后，用W5溶液重懸至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.如果想要研究冷凍效應(yīng)，就把原生質(zhì)體放置室溫。盡管原生質(zhì)體能夠在冰上放置至少24小時(shí)，但在研究基因表達(dá)調(diào)控，信號(hào)傳導(dǎo)和蛋白轉(zhuǎn)運(yùn)、加工和定位時(shí)盡量使用新鮮制備的原生質(zhì)體。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.重懸之后原生質(zhì)體在重力的作用下15分鐘以后會(huì)沉到試管底部。去除上清的W5溶液而避免碰到原生質(zhì)體小球。再用MMG溶液重懸至2105 ml1，室溫放置。DNA-PEGcalcium transfection _ TIMING Less than 1 h for 20 samplesPEG-鈣離子介導(dǎo)的轉(zhuǎn)染 20個(gè)樣品耗時(shí)少于1小時(shí)13| Add 10 l DNA (1020 g of plasmid DNA of 510 kb in size) to a 2-ml microfuge tube.加10ul質(zhì)粒DNA（10-20ug 5-10kb大小的質(zhì)粒DNA）到一2ml離心管中。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的原生質(zhì)體（2104個(gè)），輕輕混勻。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，輕彈試管以充分混勻（每次轉(zhuǎn)染一般轉(zhuǎn)染6-10個(gè)樣品）16| Incubate the transfection mixture at room temperature for up to 15 min (5 min is sufficient). 室溫孵育轉(zhuǎn)染細(xì)胞15分鐘以內(nèi)（5分鐘就可以了） CRITICAL STEP Transfection time should be optimized empirically. 具體的最佳轉(zhuǎn)染時(shí)間要靠試驗(yàn)來(lái)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.室溫400-440ulW5溶液稀釋轉(zhuǎn)染細(xì)胞，輕微振蕩或顛倒混勻以停止轉(zhuǎn)染反應(yīng)。18| Centrifuge at 100g for 2 min at room temperature using a bench-top centrifuge and remove supernatant.用臺(tái)式離心機(jī)室溫100g離心2分鐘，棄上清。 19| Re-suspend protoplasts gently with 1 ml WI in each well of a 6-well tissue culture plate. 用1ml WI 溶液重懸原生質(zhì)體。 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.室溫（20-25）孵育原生質(zhì)體，時(shí)間視要求而定。（2-16個(gè)小時(shí)） 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.重懸原生質(zhì)體，室溫100g離心2分鐘收集。22| Remove the supernatant and freeze samples on dry ice.棄上清，冰上放置。 PAUSE POINT Samples can be stored at 80 until further analysis.這時(shí)候可以-80凍存以備進(jìn)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