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To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.

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Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum
尖孢镰刀菌菌丝体的体内和体外葡萄糖的测定

生物化学 > 糖类 > 葡萄糖
作者: Carmen Ruiz-Roldan
Carmen Ruiz-RoldanAffiliation: Departamento de Genetica, Universidad de Cordoba, Campus de Excelencia Agroalimentario (ceiA3), Cordoba, Spain
For correspondence: ge2rurom@uco.es
Bio-protocol author page: a3318
 and M. Isabel G. Roncero
M. Isabel G. RonceroAffiliation: Departamento de Genetica, Universidad de Cordoba, Campus de Excelencia Agroalimentario (ceiA3), Cordoba, Spain
Bio-protocol author page: a3319
Vol 6, Iss 14, 7/20/2016, 1399 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1868

[Abstract] To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.
Keywords: Glucose determination(血糖测定), Fusarium(镰刀菌), Glucose utilization(葡萄糖的利用)

[Abstract]

Materials and Reagents

  1. Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15 )
  2. 2 ml Eppendorf tubes
  3. Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
  4. 5 mm-diameter glass beads (Sigma-Aldrich, catalog number: 18406-500G )
  5. Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
  6. Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and Δcon7-1 mutant strains
  7. Sterile dH2O
  8. Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483 )
  9. Glycerol (Merck, catalog number: 104092 )
  10. Glucose (Coger SAS, catalog number: 24379.363 )
  11. Na2CO3 (Sigma-Aldrich, catalog number: S2127 )
  12. Glucose (GO) Assay Kit (Sigma-Aldrich, catalog number: GAGO-20 )
    GO Assay includes:
    1. Glucose Oxidase/Peroxidase Reagent (Sigma-Aldrich, catalog number: G3660 )
    2. o-Dianisidine Reagent (Sigma-Aldrich, catalog number: D2679 )
    3. Glucose Standard Solution (Sigma-Aldrich, catalog number: G3285 )
  13. H2SO4, ACS reagent (Merck, catalog number: 108131.1000 )
  14. MgSO4·7H2O (Merck, catalog number: 1058865.5000 )
  15. KH2PO4 (Merck, catalog number: 104873.1000 )
  16. KCl (Merck, catalog number: 104933.0500 )
  17. NH4NO3 (Merck, catalog number: 101187.1000 )
  18. FeSO4 (Merck, catalog number: 103965.0500 )
  19. ZnSO4·7H2O (Merck, catalog number: 108883 )
  20. MnSO4·H2O (Merck, catalog number: 105941 )
  21. 0.1% benzoic acid
  22. Extraction buffer (see Recipes)
  23. Synthetic medium (SM) (see Recipes)
  24. H2SO4 (12 N) (see Recipes)

Equipment

  1. Mini-BeadBeater -16 homogenizer (BioSpec Products)
  2. Orbital incubator (Infors, Multitron Pro)
  3. Sterile spatula (Fisher Scientific, catalog number: S50821 )
  4. Microcentrifuge (Fisher Scientific, EppendorfTM MiniSpin plusTM)
  5. Hemocytometer (Thoma) (Marienfeld, catalog number: 06 407 10 )
  6. Fluorimeter (TECAN, SpectraFluorPlus, model: F129005 )
  7. Freeze-dryer (Virtis, model: BT4KZL-105 )
  8. Water bath (Selecta, model: 6000138 )
  9. Vortex (IKA, model: MS2 Mini shaker )

Procedure

  1. F. oxysporum f.sp. lycopersici wild type strain 4287 (race 2) was obtained from J. Tello, Universidad de Almeria, Spain. Δcon7-1 mutant, lacking a transcription factor essential for morphogenesis and virulence, was described previously (Ruiz-Roldan et al., 2015). Strains are stored at -80 °C with 30% glycerol as microconidial suspension.
  2. For fresh microconidia production, aliquots of frozen microconidial stocks are inoculated into 100 ml of potato dextrose broth (PDB) and incubated for 3 days at 150 rpm and 28 °C in an orbital incubator. Cultures are then filtered through Monodur nylon filters placed on funnels to separate mycelia (Figure 1A), and centrifuged at 3,020 x g for 5 min to collect microconidia. Finally, pellets containing fresh microconidia are resuspended into 1 ml sterile dH2O and counted using a hemocytometer.


    Figure 1. Filtration and mycelia harvesting procedure. A. Filtration of a fungal culture through a Monodur nylon membrane placed on a funnel. B. Subsequent harvesting of mycelia by scraping a nylon membrane using a spatula.

  3. Aliquots containing 4 x 108 freshly obtained microconidia are inoculated into 100 ml of SM containing either 0.05 or 2.5% glucose and grown at 170 rpm and 28 °C for 24 h.
  4. After harvesting by Monodur filtration, mycelia are washed twice with sterile dH2O, separated from the filter by scraping using a spatula (Figure 1B) and finally lyophilized.
  5. Mycelia dry-weight is determined using a precision weighing scale.
    Note: Normally, the amount of dry mycelia obtained from 100 ml cultures varies between 5 and 10 µg.
  6. After dry-weight determination, samples are introduced into 2 ml-Eppendorf tubes containing a 5 mm-diameter glass bead and disrupted by 3 cycles of 30 sec agitation each using a Mini-BeadBeater homogenizer. Samples are then resuspended into 250 μl of 0.25 M Na2CO3 and incubated at 95 °C on a water bath for 4 h.
  7. The supernatant is harvested by centrifugation at 5,000 x g for 3 min.
  8. The amount of glucose remaining in culture supernatants from the different strains is also determined.
  9. Glucose levels are calculated from glucose standard curves and referred to mycelium dry-weight in each strain. Aliquots (40 μl each) are used for quantification of intracellular glucose levels using the glucose determination kit GAGO (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/gago20bul.pdf) following these instructions of the manufacturer with some modifications (indicated in underlines).
    Note: The assay was repeated three times with independent biological samples.
    1. Reagent Preparation
      1. Glucose Oxidase/Peroxidase Reagent. Store the unopened kit reagent at 2-8 °C. Each capsule contains 500 units of glucose oxidase (Aspergillus niger), 100 purpurogallin units of peroxidase (horseradish) and buffer salts. Dissolve the contents of the capsule in an amber bottle with 39.2 ml of deionized water. The solution is stable up to one month at 2-8 °C and for at least 6 months frozen at -20 °C. Discard if turbidity develops.
      2. o-Dianisidine Reagent. Store the unopened kit reagent at 2-8 °C. Minimize exposure to light. The preweighed vial contains 5 mg of o-dianisidine dihydrochloride. Reconstitute the contents of the o-dianisidine vial with 1.0 ml of deionized water. Invert the vial several times to dissolve. Avoid exposing the reagent to light. Solution is stable for 3 months at 2-8 °C.
      3. Prepare the Assay Reagent by adding 0.8 ml of the o-Dianisidine Reagent to the amber bottle containing the 39.2 ml of Glucose Oxidase/Peroxidase Reagent. Invert bottle several times to mix. Minimize exposure to light. Solution is stable up to 1 month at 2-8 °C. Discard if turbidity develops or color forms.
      4. Glucose Standard Solution. D-Glucose, 1.0 mg/ml in 0.1% benzoic acid. This standard is traceable to an NIST standard and is supplied ready-to-use. It is stable at 2-8 °C for at least six months. Discard if turbidity develops.
    2. Standard curve
      1. Prepare low-concentration D-Glucose standard solutions by diluting the 1.0 mg/ml stock solution in dH2O to final concentrations ranging from 0 (reagent blank) to 80 μg/ml.
      2. Pipette 40 µl aliquots of diluted D-Glucose standard solutions into a microtiter plate.
      3. Start the reaction by adding 80 µl of Assay Reagent to each well and mixing.
      4. Incubate exactly 30 min at 37 °C. Stop the reaction by adding 80 µl of 12 N H2SO4 into each well. Mix thoroughly by pipetting several times.
      5. Measure the absorbance against thereagent blank at 540 nm.
      6. Plot Absorbance at 540 nm (Y axis) vs µg of glucose (X axis) (Figure 2). If the standard curve is not linear, results will be inaccurate. R-values of around 0.99 are normally obtained with this kit.
    3. Sample analysis
      1. Follow the directions given in Standard Curve, substituting D-Glucose standard solutions for 40 µl fungal experimental samples diluted 250 times.
        Note: Depending on the sample, it might be necessary to optimize the dilution factor.
      2. Calculate the amount of D-glucose in the experimental samples from the standard curve (Figure 2).
      3. Multiply the mg glucose determined above by the dilution factor made in sample preparation (x 250).
      4. Express mg of glucose in µg and divide it by the amount of dry mycelium in each case expressed in µg.

Representative data

  1. Figure 2 shows a representative standard curve for calculation of glucose concentration.


    Figure 2. A representative standard curve for calculation of glucose concentration. A. Representative Absorbance values at 540 nm of the diluted D-Glucose standard solutions. B. Graphical representation of Absorbance values (Y axis) vs. µg of glucose of the standards (X axis).

    In this example, an Absorbance value at 540 nm of 0.35 indicates that our test sample contains:

  2. Figure 3 shows a representative example of data obtained following this protocol.


    Figure 3. Extracellular glucose in culture supernatants of different fungal strains after 24 h growth in synthetic medium containing 2.5% glucose calculated using the Glucose determination kit GAGO. Bars represent standard errors calculated from three independent experiments with three replicates each.

Recipes

  1. Extraction buffer
    Dissolve 0.25 M Na2CO3 in deionized water.
  2. Synthetic medium (SM)
    0.2 g/L MgSO4·7H2O
    0.2 g/L KH2PO4
    0.2 g/L KCl
    1 g/L NH4NO3
    0.01 g/L FeSO4
    0.01 g/L ZnSO4·7H2O
    0.01 g/L MnSO4·H2O
    10 g/L glucose
    Dissolve in deionized water an sterilize by autoclaving
  3. H2SO4 (12 N)
    Add 16.7 ml of 36 N stock solution to 33.3 ml deionized water

Acknowledgments

This research was supported by Junta de Andalucia (Proyecto de Excelencia CVI-7319) and the Spanish Ministerio de Economia y Competitividad (grant BIO2013-47870 and the Ramon y Cajal Program). This protocol was adapted from Ruiz-Roldan et al., 2015.

References

  1. Bergmeyer, H. U. and Bernt, E. (1974). Methods of enzymatic analysis. 2nd edition. New York, NY: 1205-1212.
  2. Di Pietro, A. and Roncero, M. I. (1998). Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Mol Plant Microbe Interact 11(2): 91-98.
  3. Ruiz-Roldan, C., Pareja-Jaime, Y., Gonzalez-Reyes, J. A. and Roncero M. I. (2015). The Transcription factor Con7-1 Is a master regulator of morphogenesis and virulence in Fusarium oxysporum. Mol Plant Microbe Interact 28(1): 55-68.
  4. Xu, H. J., Xue, J., Lu, B., Zhang, X. C., Zhuo, J. C., He, S. F., Ma, X. F., Jiang, Y. Q., Fan, H. W., Xu, J. Y., Ye, Y. X., Pan, P. L., Li, Q., Bao, Y. Y., Nijhout, H. F. and Zhang, C. X. (2015). Two insulin receptors determine alternative wing morphs in planthoppers. Nature 519(7544): 464-467.

材料和试剂

  1. 直径15μm的Monodur尼龙过滤器(Filtravibracion S.L.,目录号:尼龙-15)
  2. 2 ml Eppendorf管
  3. 无菌塑料漏斗(直径80mm)(Tecnylab,目录号:45000150)
  4. 5mm直径的玻璃珠(Sigma-Aldrich,目录号:18406-500G)
  5. 微量滴定板(TermoFisher Scientific,目录号:2205)
  6. <尖孢镰刀菌 f.sp.来自野生型和Δ con 7-1 突变株的 lycopersici
  7. 无菌dH 2 O 2/b
  8. 马铃薯葡萄糖肉汤培养基(PDB)(Scharlau,目录号:01483)
  9. 甘油(Merck,目录号:104092)
  10. 葡萄糖(Coger SAS,目录号:24379.363)
  11. Na 2 CO 3(Sigma-Aldrich,目录号:S2127)
  12. 葡萄糖(GO)测定试剂盒(Sigma-Aldrich,目录号:GAGO-20) GO测定包括:
    1. 葡萄糖氧化酶/过氧化物酶试剂(Sigma-Aldrich,目录号:G3660)
    2. 邻联茴香胺试剂(Sigma-Aldrich,目录号:D2679)
    3. 葡萄糖标准溶液(Sigma-Aldrich,目录号:G3285)
  13. ACS试剂(Merck,目录号:108131.1000),H 2 SO 4 SO 4。
  14. MgSO 4·7H 2 O(Merck,目录号:1058865.5000)

  15. (Merck,目录号:104873.1000)</b>
  16. KCl(Merck,目录号:104933.0500)

  17. (Merck,目录号:101187.1000)

  18. (Merck,目录号:103965.0500)
  19. ZnSO 4·7H 2 O(Merck,目录号:108883)
  20. MnSO 4 H 2 O(Merck,目录号:105941)
  21. 0.1%苯甲酸
  22. 提取缓冲液(参见配方)
  23. 合成介质(SM)(参见配方)
  24. H sub 2 SO SO 4 sub(12 N)(参见配方)

设备

  1. Mini-BeadBeater-16匀浆器(BioSpec Products)
  2. 轨道孵化器(Infors,Multitron Pro)
  3. 无菌刮刀(Fisher Scientific,目录号:S50821)
  4. 微量离心机(Fisher Scientific,Eppendorf TM MiniSpin plus TM
  5. 血细胞计数器(Thoma)(Marienfeld,目录号:06 40710)
  6. 荧光计(TECAN,SpectraFluorPlus,型号:F129005)
  7. 冷冻干燥机(Virtis,型号:BT4KZL-105)
  8. 水浴(Selecta,型号:6000138)
  9. Vortex(IKA,型号:MS2 Mini摇动器)

程序

  1. F。尖孢镰孢野生型菌株4287(种族2)从J.Tello,Universidad de Almeria,Spain获得。 Δεcon7-1突变体缺乏对于形态发生和毒力必需的转录因子,如前所述(Ruiz-Roldan等人,2015)。将菌株在-80℃下与30%甘油一起储存为微小分枝悬浮液。
  2. 对于新鲜的小分生孢子生产,将冷冻的微小分生孢子的等分试样接种到100ml马铃薯葡萄糖肉汤(PDB)中,并在轨道培养箱中在150rpm和28℃下孵育3天。然后通过置于漏斗上的Monodur尼龙过滤器过滤培养物以分离菌丝体(图1A),并在3,020×g离心5分钟以收集微小分生孢子。最后,将含有新鲜微小分生孢子的沉淀重悬于1ml无菌dH 2 O中,并使用血细胞计数器计数。


    图1.过滤和菌丝体收获程序。A.通过置于漏斗上的Monodur尼龙膜过滤真菌培养物。 B.随后通过使用刮刀刮尼龙膜收获菌丝体
  3. 将含有4×10 8个新鲜获得的小分生孢子的等分试样接种到含有0.05或2.5%葡萄糖的100ml SM中,并在170rpm和28℃下生长24小时。
  4. 通过Monodur过滤收获后,用无菌dH 2 O 2洗涤菌丝体两次,使用刮刀通过刮擦从过滤器分离(图1B),并最后冻干。
  5. 菌丝体干重是使用精密称重秤测定的 注意:通常,从100ml培养物获得的干燥菌丝体的量在5和10μg之间变化。
  6. 在干重测定后,将样品引入含有5mm直径玻璃珠的2ml Eppendorf管中,并使用Mini-BeadBeater匀浆器分别通过3个循环的30个循环的30秒的搅拌进行破碎。然后将样品重悬于250μl的0.25M Na 2 CO 3中,并在95℃下在水浴上孵育4小时。
  7. 通过在5,000xg离心3分钟收获上清液。
  8. 还测定来自不同菌株的培养物上清液中残留的葡萄糖的量
  9. 从葡萄糖标准曲线计算葡萄糖水平,并参考每个菌株中的菌丝体干重。使用葡萄糖测定试剂盒GAGO( https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/gago20bul.pdf )这些说明的制造商有一些修改(下划线所示)。
    注意:用独立的生物样品重复测定三次。
    1. 试剂准备
      1. 葡萄糖氧化酶/过氧化物酶试剂。将未开封的试剂盒试剂保存在2-8°C。每个胶囊含有500单位的葡萄糖氧化酶(黑曲霉),100个紫红荧露单位的过氧化物酶(辣根)和缓冲盐。将该胶囊的内容物溶解在带有39.2ml去离子水的琥珀色瓶中。该溶液在2-8℃下至多一个月是稳定的,并且在-20℃下冷冻至少6个月。如果浑浊发展,则舍弃。
      2. - 联苯胺试剂。将未开封的试剂盒试剂保存在2-8°C。尽量减少暴露在光线下。预称重的小瓶含有5mg邻联茴香胺二盐酸盐。用1.0ml去离子水重构邻二氮甲啶小瓶的内容物。倒置小瓶几次溶解。避免将试剂暴露在光下。溶液在2-8℃下稳定3个月。
      3. 通过向含有39.2ml葡萄糖氧化酶/过氧化物酶试剂的琥珀色瓶中加入0.8ml的偶联物试剂来制备测定试剂。倒置瓶几次混合。尽量减少暴露在光线下。溶液在2-8℃下稳定至1个月。如果浑浊发展或出现颜色变化,请舍弃
      4. 葡萄糖标准溶液。 D-葡萄糖,1.0mg/ml在0.1%苯甲酸中。本标准可溯源至NIST标准,并提供即用型。它在2-8℃下稳定至少6个月。如果发生浑浊,请丢弃。
    2. 标准曲线
      1. 通过将dH 2 O中的1.0mg/ml储备液稀释至0(试剂空白)至80μg/ml的最终浓度来制备低浓度D-葡萄糖标准溶液。
      2. 移取40微升等分的稀释的D-葡萄糖标准溶液到微量滴定板中
      3. 通过向每个孔中加入80μl测定试剂并混合来开始反应
      4. 在37°C孵育恰好30分钟。通过向每个孔中加入80μl12N H 2 SO 4 4终止反应。用移液器充分混匀几次。
      5. 在540nm处测量针对试剂空白的吸光度
      6. 绘制540nm处的吸光度(Y轴)对葡萄糖的μg(X轴)(图2)。如果标准曲线不是线性的,结果将不准确。通常使用该试剂盒获得约0.99的R值。
    3. 样品分析
      1. 按照标准曲线中给出的指示,用D-葡萄糖标准溶液代替稀释250倍的40μl真菌实验样品。
        注意:根据样品,可能需要优化稀释因子。
      2. 从标准曲线计算实验样品中D-葡萄糖的量(图2)。
      3. 将上面测定的mg葡萄糖乘以样品制备中的稀释因子(×250)
      4. 以μg表示mg葡萄糖,在每种情况下以μg表示干燥菌丝体的量

代表数据

  1. 图2显示了用于计算葡萄糖浓度的代表性标准曲线

    图2.用于计算葡萄糖浓度的代表性标准曲线 A.稀释的D-葡萄糖标准溶液在540nm处的代表性吸光度值。 B.吸光度值(Y轴)对标准品葡萄糖的μg的图形表示(X轴)
    在本实施例中,540nm处的吸光度值为0.35表示我们的测试样品含有:

  2. 图3显示了遵循该协议获得的数据的代表性实例

    图3.在使用葡萄糖测定试剂盒GAGO计算的含有2.5%葡萄糖的合成培养基中生长24小时后不同真菌菌株的培养物上清液中的细胞外葡萄糖。条带代表从三个独立实验计算的标准误差,三次重复每个

食谱

  1. 提取缓冲区
    在去离子水中溶解0.25M Na 2 CO 3。
  2. 合成介质(SM)
    0.2g/L MgSO 4·7H 2 O·h/v 0.2g/L KH 2 PO 4 sub/
    0.2 g/L KCl
    1g/L NH 4 NO 3 sub/
    0.01g/L FeSO 4
    0.01g/L ZnSO 4 7H·7H 2 O 0.01g/L MnSO 4·H 2 O·n / 10g/L葡萄糖 溶解在去离子水中通过高压灭菌消毒

  3. 4 (12 N) 将16.7ml 36N储备溶液加到33.3ml去离子水中

致谢

这项研究由Junta de Andalucia(Proyecto de Excelencia CVI-7319)和西班牙经济部长竞争委员会(授予BIO2013-47870和Ramon y Cajal计划)支持。该协议改编自Ruiz-Roldan等人,2015年。

参考文献

  1. Bergmeyer,HU和Bernt,E。(1974)。  酶分析。 纽约,纽约:1205-1212
  2. Di Pietro,A。和Roncero,MI(1998)。  克隆,表达和在编码血管枯萎病原体尖孢镰刀菌的主要细胞外内聚半乳糖醛酸酶的pg1的致病性中的作用。 Mol Plant Microbe Interact 11(2) :91-98。
  3. Ruiz-Roldan,C.,Pareja-Jaime,Y.,Gonzalez-Reyes,JA和Roncero MI(2015)。  转录因子Con7-1是尖孢镰孢形态发生和毒力的主要调节剂 Mol Plant Microbe Interact 28(1):55-68。
  4. ,,Fan,HW,Xu,Ye,Y,Pan,Pan,,,Pan,,,,,Zhu, Li,Q.,Bao,YY,Nijhout,HF和Zhang,CX(2015)。  两种胰岛素受体决定在蚱蜢中的替代翼变体。 Nature 519(7544):464-467。
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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。

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How to cite this protocol: Ruiz-Roldan, C. and Roncero, M. I. (2016). Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum. Bio-protocol 6(14): e1868. DOI: 10.21769/BioProtoc.1868; Full Text



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