搜索

Subchromoplast Fractionation Protocol for Different Solanaceae Fruit Species
不同茄科果实物种有色体亚群的分级分离方法   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Macromolecules, proteins, lipids, and other small molecules, such as carotenoids can be studied within different tissues and organelles using an array of in vitro and in vivo methodologies. In the case of tomato and other fleshy fruit the predominant organelle in ripe fruit is the chromoplast. The characteristic feature of this organelle is the presence of pigments, carotenoids at high levels. In order to fully understand the underlying biological mechanisms that operate within the chromoplast, it is necessary to perform studies at the subchromoplast level. This protocol allows the separation of plastoglobules (lipoprotein particles, which are coupled to thylakoid membranes in the chloroplasts) and membranes (thylakoid, envelope-like) of chromoplasts through a sucrose gradient. The subchromoplast compartments can then be analysed independently. Comparisons between mutant/transgenic genotypes and their backgrounds can be performed accurately with simultaneous processing during the same fractionation run. The procedure was initially developed for ripening tomato fruit but translation to sweet and hot pepper has been shown.

Materials and Reagents

  1. 50 ml Falcon tubes (Sigma-Aldrich, catalog number: Greiner227261 )
  2. Muslin (MacCulloch & Wallis, catalog number: 4470 )
  3. Plants
    1. Tomato (90 to 150 g of breaker +3 to 5 days fruit per condition)
    2. Sweet bell pepper (30 to 120 g of ripe fruit per condition; depending on fruit pigment content)
    3. Hot chilli pepper (~30 g of ripe fruit per condition)
    Note: Breaker implies the first visual appearance of yellow/orange colour on the fruit. It is usually difficult to get ~10 tomato fruits (for 90 to 150 g total) at the same ripening stage on the same day, so that is why we harvest fruit from breaker + 3 days to breaker + 5 days. Of course, if you can have enough fruit at the same ripening stage, it is even better.
  4. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  5. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  6. Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503 )
  7. EDTA (Sigma-Aldrich, catalog number: E5134 )
  8. Tricine (Sigma-Aldrich, catalog number: T0377 )
  9. Sodium bisulphite (sodium metabisulfite) (Sigma-Aldrich, catalog number: S9000 )
  10. Extraction buffer (see Recipes)
  11. Gradient buffers (see Recipes)

Equipment

  1. Cold room (4 °C)
  2. -20 °C room
  3. Centrifuge Sorvall RC-5C (Thermo Scientific)
  4. Fixed angle rotor Sorvall GSA-3 (purple colour) (Thermo Scientific)
  5. Fixed angle rotor Sorvall GSA-5 (green colour) (Thermo Scientific)
  6. Ultracentrifuge Beckman L7 (Beckman Coulter)
  7. Swing rotor SW28 (Beckman Coulter)
  8. 500 ml centrifuge bottles (Thermo Fisher Scientific, NalgeneTM, model: 3141-0500 )
  9. 50 ml centrifuge tubes (Thick-wall UltraTubes) (Thermo Fisher Scientific, NalgeneTM, model: 3110-0500 )
  10. 38.5 ml centrifuge tubes (Beckman Coulter, Ultra-ClearTM, model: 344058 )
  11. Glass flask conical narrow neck 5 L (Fischer Scientific, FisherbrandTM, model: 11597422 )
    Note: This product has been withdrawn by the manufacturer. [Replacement item: Borosilicate glass narrow neck Erlenmeyer flasks (Fischer Scientific, FisherbrandTM, model: 15479103 )]
  12. Waring blender (small laboratory blender 8010ES) (Scientific Labs, model: MIX1126 )
  13. Potter-Elvehjem tissue grinder (VWR International, model: 14231-372 )
  14. Fraction collector (Gilson, model: 203B )
  15. Suitable ice containers
  16. Funnel (glass or plastic, to fit 5 L conical flasks)

Procedure

  1. Rapid homogenisation and isolation of the crude chromoplast fraction
    Day 0:
    1. Harvest 90 to 150 g of tomatoes at the ripening stage of breaker + 3 to + 5 days per condition (for instance, wild type and transgenic tomatoes). The fruits need to be firm. Ensure an equal weight is used for both conditions (WT and transgenic tomatoes). For pepper fruits, harvest 30 to 120 g of fruit depending on the type of pepper and the colour of the fruit. This step varies enormously depending on the fruit, whether they contain high pigment (carotenoids) levels or not. Care must be taken at this step to avoid the use of too much fruit, as this will result in overloaded sucrose gradients and consequently, a clear separation will not be achieved. On the contrary, if there is not enough fruit, no enriched material within the gradient will be observed. It is advisable to perform several trials to optimise the correct amount of fruit needed for your experiment and laboratory conditions.
      Note: Red bell peppers contain higher levels of pigments compared to orange or yellow bell peppers. So, we recommend to use 30 g of red bell peppers and 120 g of orange or yellow bell peppers. We also use 30 g for red chili peppers. However, if you are studying a high pigment or low pigment line, these weights might need to be optimised. It is only by doing the fractionation that you will see if your gradient is overloaded (same colour throughout, no clear separation). If it is the case, you will have to use less fruits. On the contrary, if you don’t see a lot of colour or no crystal, that may indicate that you did not use enough fruits to start with and you need to increase the initial fruit weight.
    2. Wash the fruit with tap water or distilled water, deseed them.
    3. Cut the pericarp into pieces (± 1 cm2) (Figure 1).


      Figure 1. Examples of Bell and Chilli pepper material prepared ready for homogenisation. A, B, C, and 1, 2, and 3 indicate the different accessions of pepper studied.

    4. Place them in a plastic tray, covered with foil, overnight at 4 °C (in order to reduce the amount of starch in the fruit). This is particularly important when carrying out the procedure with green chloroplast containing fruit. It is also important to note that some mutants have altered ripening and thus carbohydrate levels and texture will vary.
    5. Prepare the extraction buffer (2 L) and the gradient buffers (without the DTT) and place them overnight in the cold room.
    6. In the -20 °C room, store the plastic containers (for the ice), eight centrifuge tubes (50 ml), four centrifuge bottles (500 ml), and two glass flasks (5 L). The plastic ware can be pre-washed with distilled water.
    7. Place the centrifuge rotors in the fridge overnight.

      Day 1:
    8. Start promptly in the morning (8.00 am), switch on the centrifuges and cool them to 4 °C.
    9. Place the rotor GSA-3 in the centrifuge RC-5C.
    10. Place the glass flasks and the centrifuge bottles (500 ml) on ice (in the ice containers) and place the experimental materials in the cold room.
      Note: Work in the cold room in the following steps (steps 4-19 of Day 1), with everything on ice where possible.
    11. Place the pieces of fruits in the blender (Clean the blender vessel with distilled water before re-use it for the next condition).
    12. Add extraction buffer (do not forget to add the DTT from the stock solution) to cover the fruits (1:3 ratio, the fruit pieces need to be adequately covered) (Figure 2).


      Figure 2. Pepper pericarp in extraction buffer before homogenisation

    13. Leave the buffer to infiltrate into the tissues for 5 min.
    14. Use rapid blast for 3 sec (5 sec for pepper fruit) from the Waring blender, repeated twice at high speed. This should break the cell walls but keep the chromoplasts intact, due to the sucrose maintaining a constant osmotic pressure (Figure 3).
      Note: It is important that the tissues are not over homogenised, as this will result in broken plastids and the formation of gelatinous carbohydrate particulate material.


      Figure 3. The extract produced following rapid homogenisation and prior to filtration

    15. Use a glass flask, a funnel and muslin to filter the slurry through 2 to 4 layers of muslin. Pass the slurry through the muslin by squeezing gently (Figure 4).
      Note: It is important not to apply too much force to avoid the extrusion of polysaccharide material that will affect the pelleting of the plastid material. 


      Figure 4. Representation of the extract being filtered through two to four layers of muslin to create an extract free of debris

    16. Divide the tomato juice (filtered extract) into two centrifuge bottles (500 ml). Add extraction buffer to the suspension until it reaches 2/3 of the bottle. As a result, two 500 ml centrifuge bottles per condition are generated (Figure 5).


      Figure 5. Examples of the extracts ready for centrifugation in suitable centrifuge pots capable of pelleting the crude plastid fraction

      Video 1. Obtaining the filtered pepper extract (protocol Day 1, step 7 to 9)

    17. Balance the centrifuge bottles carefully using a top loading balance. Tare the first one, then the second should be equivalent to ± 1 g.
    18. Spin the centrifuge bottles in the centrifuge (RC-5C) with the GSA-3 rotor (purple, pre-cooled) at 5,000 x g for 10 min at 4 °C. This step is to remove the cell debris (supernatant) (Figure 6).
      Note: To operate the centrifuge RC-5C, check that the rotor is well attached, and close the lid by first turning the bigger cog and then the smaller one. To open it, first turn the small cog and then the big one.


      Figure 6. Pepper extracts before centrifugation

    19. Discard the supernatant carefully by pouring it from the opposite side of the pellet.
    20. Leave some volume (5 ml) of supernatant in the pot in order to resuspend the pellet (Figure 7).


      Figure 7. Pellets representing the crude plastid fractions obtained from the pepper extracts after centrifugation

    21. Resuspend the pellet by swirling the pots or using a glass rod with a rubber teat attached, alternatively for strong residue aggregates a pipette with a cut end tip can be used.
    22. Transfer the content of the centrifuge bottle in two centrifuge tubes (50 ml; this step can vary depending on the thickness and constituency of the pellet. The content of one centrifuge bottle could be transferred in only one centrifuge tube, if needed.) and then add extraction buffer in order to fill up to ¾ of the volume of the tube. At this point there are four centrifuge tubes (50 ml) per condition (Figure 8).


      Figure 8. Pepper pellets resuspended in extraction buffer ready for the second centrifugation

    23. Balance the centrifuge tubes carefully.
    24. Spin the tubes with the centrifuge RC-5C using the GSA-5 rotor (green) at 9, 000 x g for 10 min at 4 °C to pellet the plastid (Potentially, there can be some nuclear and mitochondrial contamination at this point.).
    25. Discard the supernatant carefully by pouring it from the opposite side of the pellet.
    26. This time, discard all the supernatant. The pellet should be “silky” packed in a manner that does not allow slippage (Figure 9).


      Figure 9. Pepper pellet obtained after the second centrifugation

  2. Breaking the chromoplasts
    1. Resuspend the pellet of each tube with 3 ml of the 45% sucrose gradient buffer. The tubes can be vortexed. Pour and pool the chromoplast juice from the 50 ml centrifuge tubes of the same condition into the potter-Elvehjem tissue grinder, which is on ice. Wash the tubes with 0.5 ml (or more if needed) of 45% sucrose buffer and pour it into the potter-Elvehjem as well.
    2. Homogenise the chromoplast juice using the potter-Elvehjem (approximately 10 times) to break the chromoplasts (Video 2). Keep it vertical to avoid the glass potter from breaking (Figure 10).

      Video 2. Homegenisation of the pepper chromoplast juice using a potter-Elvehjem tissue grinder


      Figure 10. Homogenisation of the pepper chromoplast juice using a potter-Elvehjem tissue grinder

  3. Separating the subchromoplast compartments using a sucrose gradient
    1. Pour everything (now the subchromoplast compartment suspension) into a labelled Falcon tube (50 ml). Keep on ice. To wash the potter-Elvehjem, use 5 ml of 45% buffer twice and add it into the Falcon tube.
    2. Prepare the sucrose gradient in three 38.5 ml Ultra-ClearTM centrifuge tubes per condition (if not enough of the suspension is available, prepare only two tubes per condition).
      1. First add 8 ml of the subchromoplast compartment juice (already in the 45% sucrose buffer) to the bottom of the tube
      2. Then 6 ml of 38% sucrose buffer
      3. Then 6 ml of 20% sucrose buffer
      4. Then 4 ml of 15% sucrose buffer
      5. And finally, 8 ml of 5% sucrose buffer to the top of the gradient.
      6. Add each layer drop by drop on the inner wall of the tube following a circular movement, a Pasteur pipette or a Gilson pipette can be used to perform this task (Video 3).

        Video 3. Preparation of the discontinuous sucrose gradient


        Figure 11. Ultra-ClearTM centrifuge tube containing a tomato subchromoplast compartment suspension in sucrose gradient before centrifugation

    3. Place the 38.5 ml centrifuge tubes in their metal container ready for centrifugation. Balance them (including the lid of the metal tube) using 5% sucrose buffer so they weigh exactly the same amount. Use the Beckman support to transport the centrifuge tubes to the Beckman L7 ultracentrifuge.
    4. Place the tubes in the SW28 rotor (tubes are hanging). Check their position by slightly twisting them and pulling them vertically and then towards the exterior of the centrifuge.
    5. Spin at 24,000 rpm (100,000 x g) at 4 °C for 19 h. Choose hold “on”, vacuum “on” and press start. If there is a problem with the balance, the centrifuge will stop at around 600 rpm. If there is a problem with the vacuum, the centrifuge will stop at around 2,000 rpm.

      Day 2:
    6. Stop the centrifuge: Press the stop button and when the pressure is low switch off the vacuum. The centrifuge lid can then be opened.
    7. Be very careful whilst removing the tubes from the rotor.
    8. Put them on ice.
    9. Take pictures of the different gradients (Figure 12).


      Figure 12. Separation of the pepper subchromoplast compartments in sucrose gradient for the different accessions of pepper (A, B, C, and 1, 2, 3)

    10. Collect the fractions with the Gilson 203B fraction collector & pump system (Figure 13). Set the speed to 24.5 on the pump system (or -22.5 with anticlockwise) in order to collect 1 ml in each 1.5 ml tube. Click on the clock arrow. Keep the needle in the blank (water) for the first minute, then put it in your tube and press start on the fraction collector. Always keep the needle at the surface (just under the meniscus). Do not collect the pellet, leave some millilitres at the end (the fractions that are mixed with the pellet). When all fractions are collected, remove the needle from the tube and place in the blank (1 ml). Thus, the solution in the tubing can be retrieved. Click on the “stop” button to stop the pump system and click on the “end” button to stop the fraction collector. Collect the last millilitres with a pipette in another 1.5 ml tube.


      Figure 13. Chilli pepper gradient collected. Numbers indicate the order of the fractions collected (from the top of the Ultra-ClearTM centrifuge tube).

    11. Fractions can now be analysed individually.
    12. Keep them at -20 °C or at -80 °C (if enzyme assays are carried out).

Representative data


Figure 14. Subchromoplast compartments separation on the 5th step of sucrose gradient at Day 2 (Transgenic CrtB+I Ailsa Craig tomato, breaker + 3 to 5 days, 150 g).
Each ml collected is called a fraction (see Day 2, point 5). The first faction corresponds to the first ml collected from the top of the tube.


Figure 15. Example of data from Nogueira et al. (2013). A. Fractionation of subplastidial components of chromoplasts from AC (the wild type) and CrtB+I lines. Chromoplasts were extracted from 90 g of a mix of breaker + 3 to + 5 d tomatoes and then broken with a handheld potter and separated in a discontinuous gradient of 5, 15, 20, 38, and 45% sucrose (weight per volume). Fractions of 1 ml were collected for further analysis. Typically, a total of 30 fractions were collected per centrifuge tube. Fractions from six replicates were used to achieve all the experiments shown in Figure 6B. Validation of subplastidial components using antibodies to biomarker proteins and analysis of lipid species. B. (i) Protein profile. Proteins, extracted from each fraction, were separated and visualized using SDS-PAGE followed by silver staining. Selected proteins were identified by nano-LC-MS-MS: 1, Plastoglobulin-1; 2, plastid lipid-associated protein CHRC; 3, ATP synthase subunit b; 4, photosystem I reaction center subunit II; 5, photosystem II 22-kD protein; 6, oxygen evolving enhancer protein 1; 7, oxygen evolving enhancer protein 2; 8, heat shock cognate 70-kD protein-1; 9, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit binding protein subunit b. Details of the identification of these proteins are shown in Supplemental Figure 6 online. (ii) Immunoblot (I. blot). Immunolocalization of biomarker proteins in the fractions were determined by immunoblotting: plastoglobulin (PGL, 35 kD), photosystem II protein D1 (PSBA, 28 kD), TIC (45 kD), TOC (75 kD), and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL, 52 kD). (iii) Lipid profile. Lipids derived from the fractions were separated in a thin layer chromatography silica plate with a mixture of acetone:toluene:water (91:30:7). Standards for lipid species were used for identification: a, triglycerides; b, monogalactodiacylglycerol; c, digalactodiacylglycerol; d, phosphatidylethanolamine; e, phosphatidylserine/phosphatidylcholine; asterisk, contaminant. C. Ultrastructure component of isolated fractions. After collection, the fractions were dialyzed against phosphate buffer and then fixed in osmium tetroxide. D. Carotenoids and a-tocopherol contents of the isolated fractions. Metabolites were extracted from each fraction and separated by liquid chromatography using a ultrahigh performance liquid chromatograph. The carotenoids and a-tocopherol were identified and quantified using calibration curves of standards. Contents are given as a percentage in a fraction compared with the total content in the tube. E. Localization of the heterologous phytoene synthase (CRTB, 38 kD) and phytoene desaturase (CRTI, 56 kD) enzymes and the endogenous phytoene synthase (PSY-1, 35 kD) enzyme within the subplastidial component of the AC and CrtB+I chromoplasts. Specific antibodies were used to immunodetect these enzymes in each collected fraction. Experiments were performed with the hemizygous CrtB+I line and a concurrent control.

Notes

The ripening stage of the fruit and the amount of fruit used are critical for this work. Here, the best combination (Breaker + 3 to 5 days & 90 to 150 g) for Ailsa Craig tomatoes is provided. However, fruits from different backgrounds can have different characteristics (firmness, quantity of pigments, etc.). Consequently, it is possible that several trials are needed before obtaining the best separation.

Recipes

  1. Extraction buffer
    0.4 M sucrose
    50 mM Trizma base
    1 mM DTT
    1 mM EDTA
    pH = 7.8
    Add the DTT (from a 1 M stock solution) just before to use the buffer
  2. Gradient buffers
    45% w/v or 38% or 20% or 15% or 5% sucrose
    50 mM Tricine
    2 mM EDTA
    2 mM DTT
    5 mM sodium bisulphite
    pH = 7.9
    Add the DTT (from a 1 M stock solution) just before to use the buffer

Acknowledgments

This work has been funded through the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No244348 METAPRO and No613513 DISCO.
The initial preparation of the crude plastids has been reported in Fraser et al. (1994).

References

  1. Fraser, P. D., Truesdale, M. R., Bird, C. R., Schuch, W. and Bramley, P. M. (1994). Carotenoid biosynthesis during tomato fruit development (evidence for tissue-specific gene expression). Plant Physiol 105(1): 405-413.
  2. Nogueira, M., Mora, L., Enfissi, E. M., Bramley, P. M. and Fraser, P. D. (2013). Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations. Plant Cell 25(11): 4560-4579.

简介

可以使用体外和体内方法的阵列在不同的组织和细胞器中研究大分子,蛋白质,脂质和其它小分子,例如类胡萝卜素。在番茄和其他肉果的情况下,成熟果实中的主要细胞器是色质体。这种细胞器的特征是存在高水平的颜料,类胡萝卜素。为了完全理解在chromoplast内操作的基本生物机制,有必要在subchromoplast水平进行研究。该方案允许通过蔗糖梯度分离塑料球(脂蛋白颗粒,其与叶绿体中的类囊体膜偶联)和膜(类囊体,包膜样)的色原体。然后可以独立地分析亚氯化银色室。突变体/转基因基因型与其背景之间的比较可以在同一分馏运行期间同时进行精确进行。该程序最初是为了成熟番茄果实开发的,但已经显示翻译成甜和辣椒。

材料和试剂

  1. 50ml Falcon管(Sigma-Aldrich,目录号:Greiner227261)
  2. Muslin(MacCulloch& Wallis,目录号:4470)
  3. 植物
    1. 番茄(90至150克破碎剂+3至5天水果每种条件)
    2. 甜椒(每种条件30至120g成熟果实;取决于水果色素含量)
    3. 辣椒辣椒(约30克成熟果实/条件)
    注意:断路器意味着水果上第一个黄色/橙色的视觉外观。在同一天在相同的成熟阶段通常难以获得?10个番茄果实(总共90至150g),因此这就是为什么我们从破碎剂+ 3天到破碎剂+ 5天收获果实。当然,如果你在同一个成熟阶段有足够的水果,它会更好。
  4. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  5. 蔗糖(Sigma-Aldrich,目录号:SO389)
  6. Trizma碱(Tris)(Sigma-Aldrich,目录号:T1503)
  7. EDTA(Sigma-Aldrich,目录号:E5134)
  8. Tricine(Sigma-Aldrich,目录号:T0377)
  9. 亚硫酸氢钠(焦亚硫酸钠)(Sigma-Aldrich,目录号:S9000)
  10. 提取缓冲液(参见配方)
  11. 渐变缓冲区(参见配方)

设备

  1. 冷室(4℃)
  2. -20°C房间
  3. 离心Sorvall RC-5C(Thermo Scientific)
  4. 定角转子Sorvall GSA-3(紫色)(Thermo Scientific)
  5. 定角转子Sorvall GSA-5(绿色)(Thermo Scientific)
  6. 超速离心机Beckman L7(Beckman Coulter)
  7. 摆动转子SW28(Beckman Coulter)
  8. 500ml离心瓶(Thermo Fisher Scientific,Nalgene TM ,型号:3141-0500)
  9. 50ml离心管(厚壁UltraTubes)(Thermo Fisher Scientific,Nalgene TM ,型号:3110-0500)
  10. 38.5ml离心管(Beckman Coulter,Ultra-Clear TM ,型号:344058)
  11. 玻璃烧瓶锥形窄颈5L(Fischer Scientific,Fisherbrand TM ,型号:11597422)
    注意:此产品已由制造商撤销。 [替代物品:硼硅酸盐玻璃窄颈锥瓶(Fischer Scientific,Fisherbrand TM ,型号:15479103)]
  12. Waring混合器(小实验室混合器8010ES)(Scientific Labs,型号:MIX1126)
  13. Potter-Elvehjem组织研磨机(VWR International,型号:14231-372)
  14. 馏分收集器(Gilson,型号:203B)
  15. 合适的冰箱
  16. 漏斗(玻璃或塑料,适合5 L锥形瓶)

程序

  1. 快速匀化和分离粗的色原体级分
    第0天:
    1. 在断路器的成熟阶段每个条件(例如,野生型和转基因番茄)收获90至150g番茄,+ 3至+ 5天。水果需要坚定。确保两种条件(WT和转基因番茄)使用相等的重量。对于胡椒果实,根据胡椒的类型和果实的颜色收获30至120g的水果。这个步骤根据水果而不同,它们是否含有高色素(类胡萝卜素)水平。在该步骤中必须小心以避免使用太多的水果,因为这将导致过量的蔗糖梯度,并且因此将不能实现清楚的分离。相反,如果没有足够的水果,则不会观察到梯度内的富集材料。建议进行多次试验,以优化实验和实验室条件所需的正确量的水果。
      注意:与橙色或黄色甜椒相比,红灯笼椒含有更高水平的颜料。所以,我们建议使用30克红椒和120克橙色或黄色甜椒。我们也使用30克红辣椒。然而,如果你正在研究高颜料或低颜料线,这些重量可能需要优化。只有通过分馏,你才会看到你的梯度是否重载(相同的颜色,没有明确的分离)。如果是这样,你将不得不使用更少的水果。相反,如果你没有看到很多颜色或没有水晶,这可能表明你没有使用足够的水果开始,你需要增加初始水果重量。
    2. 用自来水或蒸馏水洗涤水果,然后进行
    3. 将果皮切成片(±1cm 2 )(图1)

      A,B,C和1,2和3表示研究的胡椒的不同种质。

    4. 将它们放在塑料托盘中,用箔覆盖,在4℃过夜(以减少果实中淀粉的量)。当用含有绿色叶绿体的水果进行该程序时,这是特别重要的。还必须注意,一些突变体具有改变的成熟,因此碳水化合物水平和质地将不同。
    5. 准备提取缓冲液(2 L)和梯度缓冲液(无DTT),并将其在冷室中过夜。
    6. 在-20℃的室中,储存塑料容器(用于冰),八个离心管(50ml),四个离心瓶(500ml)和两个玻璃烧瓶(5L)。塑料制品可以用蒸馏水预先清洗。
    7. 将离心机转子放在冰箱中过夜。

      第1天:
    8. 在早晨(8.00 am)立即开始,打开离心机并将其冷却至4℃
    9. 将转子GSA-3放在离心机RC-5C中。
    10. 将玻璃烧瓶和离心瓶(500 ml)放在冰上(在冰容器中),将实验材料放在冷室中。
      注意:在寒冷的房间在以下步骤(第1天的步骤4-19)工作,尽可能在冰上一切。
    11. 将水果放在搅拌机中(用蒸馏水清洗搅拌容器,然后重新用于下一个条件)。
    12. 加入提取缓冲液(不要忘记从原液中加入DTT)以覆盖水果(1:3比例,水果片需要充分覆盖)(图2)。


      图2.均质前的提取缓冲液中的胡椒果皮

    13. 让缓冲液渗入组织5分钟。
    14. 使用快速爆破3秒(胡椒水果5秒)从Waring搅拌器,重复两次高速。这应该打破细胞壁,但保持完整的染色质,因为蔗糖保持恒定的渗透压(图3)。
      注意:重要的是,组织不会过度匀浆,因为这将导致破碎的质体和形成凝胶状碳水化合物颗粒物质。


      图3.快速匀浆后和过滤前产生的提取物

    15. 使用玻璃烧瓶,漏斗和细棉布通过2至4层棉布过滤浆料。通过轻轻挤压使浆液通过细棉布(图4)。
      注意:重要的是不要施加太大的力以避免多糖材料的挤出,这将影响质体材料的造粒。 


      图4.通过两到四层棉布过滤的提取物的表示,以创建没有碎片的提取物

    16. 将番茄汁(过滤的提取物)分成两个离心瓶(500毫升)。向悬浮液中加入萃取缓冲液,直至达到瓶的2/3。结果,每种条件下产生两个500ml离心瓶(图5)

      图5.准备在能够沉淀粗质体级分的合适离心机中离心的提取物的实例

    17. 使用顶部装载平衡小心地平衡离心机瓶。去皮第一个,第二个相当于±1克
    18. 使用GSA-3转子(紫色,预冷却)在离心机(RC-5C)中以5000xg在4℃下旋转离心瓶10分钟。这一步是去除细胞碎片(上清液)(图6) 注意:要操作离心机RC-5C,请检查转子是否连接良好,首先转动较大的齿轮,然后转动较小的齿轮,关闭盖子。要打开它,首先转动小齿轮,然后转动大齿轮。


      图6.离心前的胡椒提取物

    19. 通过从颗粒的相对侧倾倒掉上清液,小心地弃去上清液
    20. 将一定体积(5ml)的上清液留在罐中以便重悬沉淀(图7)

      图7.表示离心后从胡椒提取物获得的粗质体部分的颗粒

    21. 通过旋转锅或使用附有橡胶奶嘴的玻璃棒重悬颗粒,或者对于强残余聚集体,可以使用具有切割末端的移液管。
    22. 将离心瓶中的内容物转移到两个离心管(50 ml;此步骤可以根据颗粒的厚度和选区而变化,一个离心瓶的内容可以在一个离心管中转移,如果需要),然后加入提取缓冲液以填充管的体积的3/4。此时,每种条件下有四个离心管(50ml)(图8)

      图8. Pepper丸粒重新悬浮在提取缓冲液中,准备进行第二次离心

    23. 小心平衡离心管。
    24. 使用GSA-5转子(绿色)在9000×g下在4℃下用离心机RC-5C旋转管子10分钟以沉淀质体(潜在地,可以有一些核和线粒体污染)。
    25. 通过从颗粒的相对侧倾倒掉上清液,小心地弃去上清液
    26. 这一次,丢弃所有的上清液。颗粒应该是"丝质"包装,不允许滑脱(图9)。


      图9.第二次离心后获得的胡椒丸

  2. 打破色原体
    1. 用3 ml的45%蔗糖梯度缓冲液重悬每个管的沉淀。可以涡旋管。倾倒并将来自相同条件的50ml离心管的染色质液汁倒入在冰上的potter-Elvehjem组织研磨机中。用0.5ml(或更多,如果需要)45%蔗糖缓冲液洗涤试管,并将其倒入陶器-Elvehjem中。
    2. 使用potter-Elvehjem(约10次)均质化染色质汁以破坏色原体(视频2)。保持垂直,避免玻璃壶破裂(图10)。


      图10.使用potter-Elvehjem组织研磨机均匀化胡椒色素浆汁

  3. 使用蔗糖梯度分离亚溴化物隔室
    1. 将一切(现在的次溴化物室悬浮液)倒入标记的Falcon管(50ml)中。保持在冰上。要洗涤陶器-Elvehjem,使用5毫升45%的缓冲液两次,并将其添加到Falcon管。
    2. 在每个条件下在三个38.5ml Ultra-Clear TM离心管中制备蔗糖梯度(如果没有足够的悬浮液,则每种条件只准备两个管)。
      1. 首先向管底部加入8ml的次氯化物隔室汁(已经在45%蔗糖缓冲液中)
      2. 然后加入6ml 38%蔗糖缓冲液
      3. 然后加入6ml 20%蔗糖缓冲液
      4. 然后加入4ml 15%蔗糖缓冲液
      5. 最后,将8ml 5%蔗糖缓冲液加到梯度顶部。
      6. 在圆形运动后,将每层逐滴添加到管的内壁上,可以使用巴斯德吸管或Gilson吸管来执行此任务(视频3)。


        图11.超离心 TM 离心管在离心前在蔗糖梯度中含有番茄亚质谱支架悬浮液

    3. 将38.5 ml离心管放置在其金属容器中以备离心。使用5%蔗糖缓冲液平衡它们(包括金属管的盖子),因此它们称重量完全相同。使用Beckman支架将离心管运输到Beckman L7超速离心机。
    4. 将管放在SW28转子(管悬挂)。通过稍微扭转并垂直拉动,然后朝离心机外部检查其位置。
    5. 在24℃以24,000rpm(100,000xg)旋转19小时。选择保持"开",真空"开",然后按开始。如果天平出现问题,离心机将在600 rpm左右停止。如果真空有问题,离心机将在约2,000 rpm停止
      第2天:
    6. 停止离心机:按下停止按钮,当压力低时关闭真空。然后可以打开离心机盖。
    7. 从转子上取下管子时要非常小心
    8. 把它们放在冰上。
    9. 拍摄不同梯度的照片(图12)。


      图12.对于胡椒的不同种质(A,B,C和1,2,3),在蔗糖梯度中分离胡椒亚质谱板区段

    10. 用Gilson 203B级分收集器收集级分,泵系统(图13)。在泵系统上设置速度为24.5(或逆时针方向为-22.5),以在每个1.5ml管中收集1ml。点击时钟箭头。将针头留在空白(水)中第一分钟,然后将其放入您的管中,并在馏分收集器上开始。始终保持针在表面(刚好在半月板下面)。不要收集沉淀,在最后留下一些毫升(与沉淀混合的馏分)。当收集所有级分时,从管中取出针并置于空白(1ml)中。因此,可以取回管中的溶液。点击"停止"按钮停止泵系统,点击"结束"按钮停止馏分收集器。用移液管在另一个1.5 ml管中收集最后一毫升。


      图13.收集辣椒梯度。数字表示收集的级分的顺序(从Ultra-Clear TM 离心管的顶部)。

    11. 现在可以单独分析分数。
    12. 将其保存在-20°C或-80°C(如果进行酶测定)

代表数据


图14.在第2天(转基因Crt B + I Ailsa Craig番茄,破碎剂+ 3至5天,150g)的蔗糖梯度的步骤5上的次溴化物区室分离 。 收集的每毫升称为分数(见第2天,第5点)。第一个阵营对应于从管顶部收集的第一个ml。


(2013)。 的来自AC(野生型)和CrtB + I品系的色质体的亚质粒组分。从90g破碎剂+ 3至+ 5d番茄的混合物中提取色原体,然后用手持式搅拌器破碎,并以5,15,20,38和45%蔗糖(重量/体积)的不连续梯度分离。收集1ml的级分用于进一步分析。通常,每个离心管收集总共30个级分。使用来自六个重复的级分来实现图6B中所示的所有实验。使用抗体生物标记蛋白质和脂质种类的分析验证亚质粒组分。 (i)蛋白质谱。分离从每个级分提取的蛋白质,并使用SDS-PAGE,然后银染色显现。所选蛋白质通过纳米LC-MS-MS鉴定:1,Plastoglobulin-1; 2,质体脂相关蛋白CHRC; 3,ATP合酶亚基b; 4,光系统I反应中心亚基II; 5,光系统II 22-kD蛋白; 6,氧放出增强蛋白1; 7,氧放出增强蛋白2; 8,热激同源70-kD蛋白-1; 9,核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基结合蛋白亚基b。这些蛋白质的鉴定的细节在附图6中在线示出。 (ii)免疫印迹(I.印迹)。通过免疫印迹测定级分中生物标志物蛋白的免疫定位:通过免疫印迹测定:平滑肌蛋白(PGL,35kD),光系统II蛋白D1(PSBA,28kD),TIC(45kD),TOC(75kD)和核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基(RBCL,52kD)。 (iii)脂质谱。将来自级分的脂质在薄层色谱法二氧化硅板中用丙酮:甲苯:水(91:30:7)的混合物分离。脂质标准品用于鉴定:a,甘油三酯; b,一缩二甘油甘油; c,二半乳糖二甘油; d,磷脂酰乙醇胺; e,磷脂酰丝氨酸/磷脂酰胆碱;星号,污染物。 C.分离级分的超基因组分。收集后,将级分对磷酸盐缓冲液透析,然后固定在四氧化锇中。 D.分离级分的类胡萝卜素和α-生育酚含量。从每个级分中提取代谢物,并使用超高效液相色谱通过液相色谱法分离。使用标准品的校准曲线鉴定和定量类胡萝卜素和α-生育酚。内容以与管中总含量相比的分数的百分比给出。 E.在AC和CrtB + I染色质的亚质体组分内的异源八氢番茄红素合酶(CRTB,38kD)和八氢番茄红素去饱和酶(CRTI,56kD)酶和内源八氢番茄红素合酶(PSY-1,35kD) 。使用特异性抗体在每个收集的级分中免疫检测这些酶。用半合子CrtB + I品系和同时对照进行实验。

笔记

水果的成熟阶段和使用的水果量对于这项工作至关重要。这里,提供了用于Ailsa Craig番茄的最佳组合(破碎器+ 3至5天和90至150g)。然而,来自不同背景的水果可以具有不同的特性(坚实度,颜料量等)。因此,有可能在获得最佳分离之前需要进行几次试验。

食谱

  1. 提取缓冲区
    0.4 M蔗糖 50 mM Trizma碱基
    1 mM DTT
    1mM EDTA
    pH = 7.8
    在使用缓冲区
    之前添加DTT(来自1 M储备溶液)
  2. 渐变缓冲区
    45%w/v或38%或20%或15%或5%蔗糖 50 mM Tricine
    2mM EDTA 2 mM DTT
    5mM亚硫酸氢钠 pH = 7.9
    在使用缓冲液之前添加DTT(来自1 M储备溶液)

致谢

这项工作是通过欧盟第七框架计划资助的,研究,技术开发和示范在授权协议No244348 METAPRO和No613513 DISCO。
原始质体的初始制备已在Fraser等人(1994)中报道。

参考文献

  1. Fraser,PD,Truesdale,MR,Bird,CR,Schuch,W.and Bramley,PM(1994)。  在番茄果实发育期间的类胡萝卜素生物合成(组织特异性基因表达的证据)植物生理学105(1):405-413。
  2. Nogueira,M.,Mora,L.,Enfissi,EM,Bramley,PM and Fraser,PD(2013)。 
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nogueira, M., Berry, H., Nohl, R., Klompmaker, M., Holden, A. and Fraser, P. D. (2016). Subchromoplast Fractionation Protocol for Different Solanaceae Fruit Species. Bio-protocol 6(13): e1861. DOI: 10.21769/BioProtoc.1861.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。