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Isolation of Mitochondria from Potato Tubers
分离马铃薯块茎线粒体

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Abstract

One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as well as larger organelles. The crude mitochondria are then first purified by using a step Percoll gradient. The mitochondrial band from the step gradient is further purified by using a continuous Percoll gradient. The gradients remove contaminating amyloplasts and peroxisomes as well as ruptured mitochondria. The result is a highly purified, intact and functional mitochondrial preparation, which can be frozen and stored in liquid nitrogen in the presence of 5% (v/v) dimethylsulfoxide to preserve integrity and functionality for months.

Keywords: Mitochondria(线粒体), Potato(马铃薯), Percoll gradient(离心法), Subcellular localization(亚细胞定位)

Materials and Reagents

  1. Mannitol
  2. 3-morpholinopropane-1-sulfonic acid (MOPS)
  3. Potassium hydroxide (KOH)
  4. Bovine serum albumin (BSA)
  5. Ethylenediaminetetraacetic acid (EDTA)
  6. Cysteine
  7. Percoll
  8. Sucrose
  9. Dimethylsulfoxide (DMSO)
  10. Liquid nitrogen
  11. Extraction medium (see Recipes)
  12. Wash buffer (see Recipes)
  13. Gradient buffers (see Recipes)
  14. Percoll gradients (see Recipes)

Equipment

  1. Potato tubers
  2. 6 x 250 ml precooled angle rotor (e.g., Beckman Coulter, model: JA-14 or Thermo Fisher Scientific, model: SLA-1500 )
  3. 8 x 50 ml pre-cooled angle rotor (e.g., Beckman Coulter, model: JA-20 or Thermo Fisher Scientific, model: SS-34 )
  4. Juice extractor (kitchen appliance, e.g. Maginix Le duo XL)
  5. Centrifuge (for 50 and 250 ml tubes) (e.g. Beckman Coulter or SorvallTM)
  6. Paint brush (soft)
  7. Cotton cloth or nylon net (e.g., mesh 60-120 μm)
  8. Plastic Pasteur pipettes
  9. Beakers
  10. Measuring pipette
  11. pH meter
  12. Potato peeler
  13. Funnel

Procedure

Notes:

  1. All steps should be performed at 0-4 °C using detergent-free glass-ware or plastic!
  2. We will here describe the isolation of mitochondria from 1 kg of peeled tubers of the cultivar Folva. This will give a final yield of 8-12 mg mitochondrial proteins. Using other cultivars can give different yields. Total time from start of the homogenization (point 3) to freezing about 6 h.
  1. Pre-cool centrifuge rotor.
  2. Add cysteine and BSA to extraction buffer, adjust to pH 7.3. Cysteine is an antioxidant and BSA binds fatty acids and phenolics, which can interfere with mitochondrial function, BSA also works as a protease substrate to help protect the mitochondrial proteins from damage.
  3. Peel potatoes and homogenize the peeled potato using a juice extractor (1 kg gives approximately 500 ml juice) and let the juice run directly into about 1/2 volume extraction medium (250 ml here). Adjust to pH 7.2 using 2 M KOH immediately afterwards or, for larger preparations, after each kg homogenized.
  4. Leave homogenate (total volume about 750 ml) standing for 5 min allowing starch to sediment.
  5. Filter through two layers of cotton (or similar) using a funnel and transfer to centrifuge tubes.
  6. Transfer ~190 ml filtrate to each of four 250 ml centrifuge tubes. Balance tubes, and centrifuge at 3,000 x g for 5 min in a 6 x 250 ml precooled angle rotor.
  7. Pour supernatants carefully into fresh centrifuge tubes (avoid transferring the pellets), balance and centrifuge at 18,000 x g for 10 min.
  8. Discard the supernatants gently without disturbing the pellets and resuspend each pellet in 1 ml of 1x mannitol gradient buffer using a paint brush. Total volume of resuspended pellets 8-10 ml.
  9. Prepare two Percoll step gradients using plastic Pasteur pipettes. Avoid mixing the bands by gently layering first the 50%, then the 28% and finally the 20% Percoll (all in mannitol) on top of each other in 50 ml centrifuge tubes.
  10. Gently layer the crude fraction (4-5 ml maximum per gradient) on top of the two Percoll step gradients.
  11. Balance tubes and centrifuge at 40,000 x g for 30 min using an 8 x 50 ml pre-cooled angle rotor (see Figure 1A).
  12. Transfer the mitochondrial band from each tube using a Pasteur pipette to new 50 ml tubes, fill up to 40 ml with wash buffer and mix.
  13. Balance the two tubes against each other and centrifuge at 18,000 x g for 10 min.
  14. Carefully remove supernatant, resuspend the very loose pellets with wash buffer, fill up to 40 ml with wash buffer and mix.
  15. Balance and centrifuge at 18,000 x g for 10 min (see Figure 1B).
  16. Remove supernatant and resuspend the very loose pellets using a paint brush in 1 ml 1 x mannitol buffer and gently layer on top of two 28% Percoll sucrose gradients.
  17. Balance and centrifuge at 40,000 x g for 30 min using the 8 x 50 ml rotor (see Figure 1C).
  18. Transfer the mitochondrial bands to two fresh 50 ml centrifuge tubes, fill up to 40 ml with wash buffer and mix.
  19. Centrifuge at 18,000 x g for 10 min.
  20. Carefully remove supernatant and repeat wash (as steps 13-14) of pellets (see Figure 1D).
  21. Remove supernatant and resuspend each pellet in 500 µl wash buffer. Add 5% (v/v) DMSO for freezing of the intact organelles. The final volume from each pellet is around 800 µl containing 4-6 mg (total 8-12 mg) mitochondrial protein.
  22. Snap freeze and store aliquots of 100-200 µl in liquid nitrogen. In this way, the mitochondria maintain their intactness and respiratory function (see below) for months, if quickly thawed shortly before use.


    Figure 1. Percoll gradient purification of potato tuber mitochondria. A). Step gradient with a yellowish mitochondrial band at the 28% - 50% Percoll interface and a starch pellet (after step 11). B). The pellet obtained after removing the mitochondrial band from the step gradient and washing it (after step 15). The dark center consists of peroxisomes (Struglics et al., 1993). C). Continuous gradient with a whitish mitochondrial band (after step 17). D). The final pellet obtained after removing the mitochondrial band from the continuous gradient and washing it (after step 20).

Representative data

Properties of the isolated mitochondria

  1. The mitochondria isolated from potato tubers using this protocol have highly intact outer membranes as judged by >97% latency of cytochrome c oxidase and are highly purified as judged by the high specific activity of cytochrome c oxidase (3.5 µmol mg-1 min-1). The respiration rates in state 3 (presence of ADP) using single substrates (NADH, succinate and malate) are 150-350 nmol O2 mg-1 min-1, the respiratory control ratio of 2.5-3.5 and the ADP/O ratio 1.7-2.0 all depending on the substrate, the preparation and the cultivar. This is fully consistent with previous studies (Neuburger et al., 1982; Rasmusson and Møller, 1990; Struglics et al., 1993).
  2. The mitochondria were judged to be >95% pure by western blotting (Salvato et al., 2014), which is consistent with measurements of marker enzymes and marker compounds in previous studies (Neuburger et al., 1982; Struglics et al., 1993).
  3. Using one-dimensional polyacrylamide gel electrophoresis to separate the mitochondrial proteins according to size followed by tryptic digestion, separation of the tryptic peptides by liquid chromatography and identification by mass spectrometry a total of 1,060 different proteins were identified including about 500 proteins not previously identified in plant mitochondria (Salvato et al., 2014).
  4. Similar methods can be used to isolate mitochondria from other tissues and species. However, the homogenization methods will probably have to be changed as does the Percoll concentration used to collect the mitochondria and purify them further (28% here). See Meyer and Millar (2008) for a method for isolating mitochondria from Arabidopsis cell cultures. Isolating mitochondria from green leaves is more difficult because of the large amount of chloroplasts and thylakoid membranes released from the tissue, and Arabidopsis leaves are particularly difficult (Keech et al., 2005).

Recipes

  1. Extraction medium [600 (300) ml, pH 7.3]

    Chemical
    Concentration
    g/L
    Mannitol
    0.9 M
    163.95
    MOPS
    30 mM
    6.28
    EDTA
    3 mM
    0.87
    L-Cysteine
    25 mM
    3.03
    BSA
    0.3 % (w/v)
    3.00

    Dissolve mannitol, MOPS and EDTA in double-distilled water (ddH2O)
    Adjust pH to 7.2 and transfer 300 ml to new container to use as wash buffer
    On day of use add cysteine and BSA to the remaining 300 ml, adjust to pH 7.3 with KOH.
  2. Wash buffer (900 ml, pH 7.2)

    Chemical
    Concentration
    g/L
    Mannitol
    0.3 M
    54.65
    MOPS
    10 mM
    2.07
    EDTA
    1 mM
    0.29

    Use extraction medium (no cysteine and BSA) and dilute 3-fold by adding ddH2O
    Include 0.1% BSA if the mitochondria are to be used in a non-proteomics context
  3. Gradient buffers
    1. 2x mannitol buffer (150 ml, pH 7.2)

      Chemical
      Concentration
      g/L
      Mannitol
      0.6 M
      109.3
      MOPS
      20 mM
      4.14
      BSA
      0.2 % (w/v)
      2.0

      Use 35 ml for each of the two step gradients and dilute the remaining 115 ml 2-fold to use for resuspending pellets.
    2. 2x sucrose buffer (100 ml, pH 7.2)

      Chemical
      Concentration
      g/L
      Sucrose
      0.6 M
      205.4
      MOPS
      20 mM
      4.14
      BSA
      0.2 % (w/v)
      2.0

  4. Percoll gradients
    Total volume 35 ml without samples
    1. Step gradient

      Percoll, % (v/v)
      ddH2O, %
      2x mannitol buffer, %
      Volume per  gradient, ml
      20
      30
      50
      17.5
      28
      22
      50
      11.67
      50
      -
      50
      5.83

    2. Continuous gradient

      Percoll, % (v/v)
      ddH2O, %
      2x mannitol buffer, %
      Volume per gradient, ml
      28
      22
      50
      35

      Very carefully add the three layers of the step gradient one by one (highest first) holding the 50 ml tube at a 45 degree angle to avoid mixing

Acknowledgements

This work was supported by the Danish Council for Independent Research - Natural Sciences (to I.M.M.) and the OECD Cooperative Research Programme: Biological Resource Management for Sustainable Agricultural Systems (2012 sabbatical fellowship to J.J.T.).
The method was published in Neuburger et al. (1982) and it is an adaptation of the methods used by Neuburger et al. (1982), Struglics et al. (1993) and Considine et al. (2002).

References

  1. Considine, M. J., Goodman, M., Echtay, K. S., Laloi, M., Whelan, J., Brand, M. D. and Sweetlove, L. J. (2003). Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein. J Biol Chem 278(25): 22298-22302.
  2. Keech, O., Dizengremel, P. and Gardeström, P. (2005). Preparation of leaf mitochondria from Arabidopsis thaliana. Physiologia Plantarum 124(4): 403-409.
  3. Meyer, E. H. and Millar, A. H. (2008). Isolation of mitochondria from plant cell culture. In: Posch (ed). Methods Mol Biol. Humana Press. vol. 425: 2D PAGE: Sample Preparation and Fractionation, Volume 2A.
  4. Neuburger, M., Journet, E. P., Bligny, R., Carde, J. P. and Douce, R. (1982). Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll. Arch Biochem Biophys 217(1): 312-323.
  5. Rasmusson, A. G. and Møller, I. M. (1990). NADP-utilizing enzymes in the matrix of plant mitochondria. Plant Physiol 94(3): 1012-1018.
  6. Salvato, F., Havelund, J. F., Chen, M., Rao, R. S., Rogowska-Wrzesinska, A., Jensen, O. N., Gang, D. R., Thelen, J. J. and Møller, I. M. (2014). The potato tuber mitochondrial proteome. Plant Physiol 164(2): 637-653.
  7. Struglics, A., Fredlund, K. M., Rasmusson, A. G. and Møller, I. M. (1993). The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane. Physiologia Plantarum 88(1): 19-28.

简介

研究植物线粒体功能的一种方法是以未污染,完整和功能形式从植物组织中提取植物线粒体。还原性假设是这种制剂中存在的组分和体外可测量的功能或活性可靠地反映植物细胞内的细胞器的体内性质。在这里,我们描述了从相对均匀的植物组织,休眠马铃薯块茎分离线粒体的方法。使用榨汁机进行均质化,其是相对温和的均质化程序,其中线粒体仅暴露于强剪切力一次。在通过过滤除去淀粉和大的组织碎片后,使用差速离心以除去残余淀粉以及更大的细胞器。然后首先通过使用步骤Percoll梯度来纯化粗线粒体。通过使用连续的Percoll梯度进一步纯化来自阶梯梯度的线粒体条带。梯度去除污染的淀粉体和过氧化物酶体以及破裂的线粒体。结果是高度纯化的,完整和功能的线粒体制剂,其可以在5%(v/v)二甲基亚砜存在下在液氮中冷冻并保存,以保持数月的完整性和功能性。

关键字:线粒体, 马铃薯, 离心法, 亚细胞定位

材料和试剂

  1. 甘露醇
  2. 3-吗啉代丙烷-1-磺酸(MOPS)
  3. 氢氧化钾(KOH)
  4. 牛血清白蛋白(BSA)
  5. 乙二胺四乙酸(EDTA)
  6. 半胱氨酸
  7. Percoll
  8. 蔗糖
  9. 二甲基亚砜(DMSO)
  10. 液氮
  11. 提取介质(参见配方)
  12. 洗涤缓冲液(见配方)
  13. 渐变缓冲区(参见配方)
  14. Percoll渐变(参见配方)

设备

  1. 马铃薯块茎
  2. 6×250ml预冷角转子(例如,Beckman Coulter,型号:JA-14或Thermo Fisher Scientific,型号:SLA-1500)
  3. 8×50ml预冷角转子(例如,Beckman Coulter,型号:JA-20或Thermo Fisher Scientific,型号:SS-34)
  4. 榨汁机(厨房用具,如 Maginix Le duo XL)
  5. 离心机(对于50和250ml管)(例如Beckman Coulter或Sorvall TM
  6. 油漆刷(软)
  7. 棉布或尼龙网(如,网格60-120μm)
  8. 塑料巴斯德移液器
  9. 烧杯
  10. 测量移液器
  11. pH计
  12. 土豆削皮器
  13. 漏斗

程序

注意:

  1. 所有步骤应在0-4°C使用无洗涤剂的玻璃器皿或塑料!
  2. 我们将在这里描述从1kg的品种Folva的剥离的块茎中分离线粒体。 这将产生8-12mg线粒体蛋白的最终产量。 使用其他品种可以得到不同的产量。 从均质化开始(点3)至冷冻约6小时的总时间。
  1. 预冷离心机转子。
  2. 加入半胱氨酸和BSA至提取缓冲液,调至pH 7.3。 半胱氨酸是抗氧化剂,BSA结合脂肪酸和酚类物质,其可以干扰线粒体功能,BSA也作为蛋白酶底物 有助于保护线粒体蛋白免受损伤
  3. 剥皮土豆并使用榨汁机(1kg给出约500ml果汁)使剥皮的马铃薯均质化,并使汁直接进入约1/2体积的提取介质(此处为250ml)。 之后立即用2M KOH调节至pH7.2,或者对于较大的制剂,每kg均匀后调节至pH7.2
  4. 离开匀浆(总体积约750ml),静置5分钟,使淀粉沉淀
  5. 使用漏斗过滤两层棉花(或类似物),并转移到离心管中
  6. 将约190ml滤液转移到四个250ml离心管中的每一个中。 平衡管,并在6×250ml预冷角转子中以3,000xg离心5分钟。
  7. 将上清液小心地倒入新鲜离心管中(避免转移沉淀),平衡并以18,000×g离心10分钟。
  8. 轻轻丢弃上清液,不影响沉淀,并使用油漆刷将每个沉淀重悬于1ml 1x甘露醇梯度缓冲液中。 重悬浮颗粒的总体积为8-10ml。
  9. 使用塑料巴斯德移液器准备两个Percoll步骤梯度。 避免混合条带通过轻轻地分层50%,然后28%和最后20%Percoll(所有在甘露醇)在彼此顶部在50ml离心管中。
  10. 轻轻地将粗级分(每个梯度4-5ml)在两个Percoll阶梯梯度的顶部上
  11. 使用8×50ml预冷角转子(见图1A)将平衡管和离心机在40,000×g下离心30分钟。
  12. 使用巴斯德移液管将线粒体带从每个管转移到新的50ml管中,用洗涤缓冲液填充至40ml并混合。
  13. 将两个管彼此平衡,并以18,000×g离心10分钟
  14. 小心地除去上清液,用洗涤缓冲液重悬浮非常松散的颗粒,用洗涤缓冲液填充至40ml并混合。
  15. 平衡并以18,000×g离心10分钟(参见图1B)
  16. 除去上清液,并使用油漆刷在1ml 1×甘露醇缓冲液中重悬浮非常松散的颗粒,并轻轻地在两个28%Percoll蔗糖梯度的顶部上。
  17. 平衡并使用8×50ml转子在40,000×g离心30分钟(见图1C)。
  18. 转移线粒体带到两个新鲜的50毫升离心管,填充达40毫升与洗涤缓冲液和混合。
  19. 以18,000×g离心10分钟。
  20. 小心地除去上清液并重复洗涤(如步骤13-14)沉淀(见图1D)。
  21. 除去上清液,并将每个沉淀重悬在500μl洗涤缓冲液中。加入5%(v/v)DMSO冷冻完整的细胞器。每个沉淀的最终体积为约800μl,含有4-6mg(总8-12mg)线粒体蛋白
  22. 快速冷冻和存储100-200微升在液氮中的等分试样。这样,如果在使用前不久快速解冻,线粒体保持其完整性和呼吸功能(见下文)数月。


    图1.马铃薯块茎线粒体的Percoll梯度纯化。 A)。具有在28%-50%Percoll界面处的黄色线粒体条带和淀粉小球的步骤梯度(在步骤11之后)。 B)。从步骤梯度中除去线粒体带并洗涤(在步骤15之后)获得沉淀。暗中心由过氧化物酶体组成(Struglics等人,1993)。 C)。具有发白的线粒体带的连续梯度(在步骤17之后)。 D)。在从连续梯度中除去线粒体带并洗涤后(在步骤20之后)获得的最终沉淀物

代表数据

分离的线粒体的性质

  1. 使用该方案从马铃薯块茎分离的线粒体具有高度完整的外膜,如通过细胞色素c氧化酶的> 97%潜伏期所判断的,并且是高度纯化的,如通过细胞色素c氧化酶的高比活性(3.5μmolmg <-1> min -1 )。使用单一底物(NADH,琥珀酸盐和苹果酸盐)在状态3(存在ADP)下的呼吸速率为150-350nmol O 2 mg -1 sup -1 min- 1 ,呼吸控制比为2.5-3.5,ADP/O比为1.7-2.0都取决于底物,制剂和栽培种。这与以前的研究完全一致(Neuburger等人,1982; Rasmusson和Møller,1990; Struglics等人,1993)。
  2. 通过western印迹判断线粒体是纯度> 95%(Salvato et al。,2014),其与先前研究中的标记酶和标记化合物的测量一致(Neuburger等et al。,,1982; Struglics et al。,,1993)。
  3. 使用一维聚丙烯酰胺凝胶电泳根据大小分离线粒体蛋白质,随后胰蛋白酶消化,通过液相色谱法分离胰蛋白酶肽和通过质谱鉴定总共鉴定了1,060种不同的蛋白质,包括以前在植物中未鉴定的约500种蛋白质线粒体(Salvato et al。,2014)。
  4. 类似的方法可用于从其他组织和物种中分离线粒体。然而,均质化方法可能必须改变,用于收集线粒体并进一步纯化它们的Percoll浓度(在此为28%)。参见Meyer和Millar(2008)关于从拟南芥细胞培养物中分离线粒体的方法。从绿叶分离线粒体更困难,因为大量 叶绿体和类囊体膜从组织释放,并且拟南芥叶特别困难(Keech等人,2005)。

食谱

  1. 提取介质[600(300)ml,pH 7.3]

    化学
    集中
    g/L
    甘露醇
    0.9 M
    163.95
    MOPS
    30 mM
    6.28
    EDTA
    3 mM
    0.87
    L-半胱氨酸
    25 mM
    3.03
    BSA
    0.3%(w/v)
    3.00

    将甘露醇,MOPS和EDTA溶于双蒸水(ddH 2 O)中
    将pH调节至7.2,并将300ml转移至新容器以用作洗涤缓冲液
    使用当天,向剩余的300ml中加入半胱氨酸和BSA,用KOH调节至pH7.3
  2. 洗涤缓冲液(900ml,pH 7.2)

    化学
    集中
    g/L
    甘露醇
    0.3 M
    54.65
    MOPS
    10 mM
    2.07
    EDTA
    1 mM
    0.29

    使用提取介质(无半胱氨酸和BSA),并通过加入ddH 2 O稀释3倍
    如果线粒体用于非蛋白质组学环境,则包括0.1%BSA。
  3. 梯度缓冲液
    1. 2x甘露醇缓冲液(150ml,pH 7.2)
      化学
      集中
      g/L
      甘露醇
      0.6 M
      109.3
      MOPS
      20 mM
      4.14
      BSA
      0.2%(w/v)
      2.0

      每两步梯度使用35毫升,稀释剩余的115毫升2倍,用于重悬颗粒。
    2. 2x蔗糖缓冲液(100ml,pH 7.2)
      化学
      集中
      g/L
      蔗糖
      0.6 M
      205.4
      MOPS
      20 mM
      4.14
      BSA
      0.2%(w/v)
      2.0

  4. Percoll梯度
    总体积35 ml,无样品
    1. 渐变

      Percoll,%(v/v)
      ddH <2> O,%
      2x甘露醇缓冲液,%
      每张音量 梯度,ml
      20
      30
      50
      17.5
      28
      22
      50
      11.67
      50
      -
      50
      5.83

    2. 连续渐变

      Percoll,%(v/v)
      ddH <2> O,%
      2x甘露醇缓冲液,%
      每梯度体积,ml
      28
      22
      50
      35

      非常小心地将梯度梯度的三个层逐一(最高的第一个)以45度角保持50ml管,以避免混合

致谢

这项工作得到了丹麦自然科学研究自然科学委员会(至I.M.M.)和经合组织合作研究计划:可持续农业系统生物资源管理(2012年到J.J.T.的休会奖学金)的支持。
该方法公开在Neuburger等人(1982)中,并且是Neuburger等人(1982),Struglics等人所使用的方法的改编。(1993)和Considine等人(2002)。

参考文献

  1. Considine,M.J.,Goodman,M.,Echtay,K.S.,Laloi,M.,Whelan,J.,Brand,M.D.and Sweetlove,L.J。(2003)。 超氧化物刺激土豆线粒体中的质子泄漏,这与解偶联蛋白的活性有关。 J Biol Chem 278(25):22298-22302
  2. Keech,O.,Dizengremel,P。和Gardeström,P。(2005)。 从拟南芥中制备叶线粒体 Physiologia Plantarum 124(4):403-409。
  3. Meyer,E.H。和Millar,A.H。(2008)。从植物细胞培养物中分离线粒体。 In:Posch(ed)。 Methods Mol Biol。 Humana Press。 vol。 425:2D PAGE:Sample Preparation and Fractionation,第2A卷。
  4. Neuburger,M.,Journet,E.P.,Bligny,R.,Carde,J.P。和Douce,R。(1982)。 Percoll密度梯度中的等密度离心法纯化植物线粒体拱Biochem Biophys 217(1):312-323。
  5. Rasmusson,A.G。和Møller,I.M。(1990)。 NADP利用酶在植物线粒体基质中的应用植物生理学 94(3):1012-1018。
  6. Salvato,F.,Havelund,J.F.,Chen,M.,Rao,R.S.,Rogowska-Wrzesinska,A.,Jensen,O.N.,Gang,D.R.,Thelen,J.J.andMøller, 马铃薯块茎线粒体蛋白质组 植物生理学 164( 2):637-653。
  7. Struglics,A.,Fredlund,K.M.,Rasmusson,A.G.andMøller,I.M。(1993)。 完整马铃薯块茎过氧化物酶体膜中存在短的氧化还原链以及苹果酸脱氢酶与过氧化物酶体膜的结合。 > Physiologia Plantarum 88(1):19-28
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Havelund, J. F., Salvato, F., Chen, M., Rao, R., Rogowska-Wrzesinska, A., Jensen, O. N., Gang, D. R., Thelen, J. J. and Møller, I. M. (2014). Isolation of Mitochondria from Potato Tubers. Bio-protocol 4(17): e1226. DOI: 10.21769/BioProtoc.1226.
  2. Salvato, F., Havelund, J. F., Chen, M., Rao, R. S., Rogowska-Wrzesinska, A., Jensen, O. N., Gang, D. R., Thelen, J. J. and Møller, I. M. (2014). The potato tuber mitochondrial proteome. Plant Physiol 164(2): 637-653.
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