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Indole Derivative Feeding Test and Detection of TRP and Indole derivatives by Thin Layer Chromatography
吲哚衍生物的喂养试验及薄层色谱法检测TRP和吲哚衍生物   

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参见作者原研究论文

本实验方案简略版
New Phytologist
Oct 2012

Abstract

The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits could be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica (Hilbert et al., 2012).

Materials and Reagents

  1. Indole derivative:
    TRP (Sigma-Aldrich, catalog number: T0254-500g )
    IAD (indole-3-acetaldehyde) (Sigma-Aldrich, catalog number: I1000-100mg )
    IAA (Sigma-Aldrich, catalog number: I5148-2g )
  2. Standard microscope coverslips
  3. Standard microscope slides
  4. 0.002% (v/v) Tween water 20
  5. 0.9% NaCl
  6. FeCl3
  7. HClO4
  8. NaNO3
  9. MgSO4.7H2O
  10. KH2PO4
  11. Orthophosphoric acid
  12. Glucose
  13. Peptone
  14. Yeast extract
  15. Casamine acids
  16. Microelements
  17. Agar
  18. Tryptophan (Sigma-Aldrich, catalog number: T0254-500g)
  19. Ethyl acetate
  20. Aluminum foil
  21. p-dimethylaminobenzaldehyde (Sigma-Aldrich)
  22. Van Urk reagent (see Recipes)
  23. Salkowski test reagents (see Recipes)
  24. 10 mM orthophosphoric acid (see Recipes)
  25. Complete medium (see Recipes)
  26. 20x salt solution (see Recipes)
  27. Microelements (see Recipes)

Equipment

  1. Erlenmeyer flasks (100 ml)
  2. Neubauer improved counting chamber (Marienfeld-Superior, Lauda, Königshofen, Germany)
  3. Scalpel (sterile)
  4. Whatman paper
  5. Miracloth filter 15 cm x 15 cm (Merck KGaA, catalog number: 475855 )
  6. Drigalski spatula (sterile)
  7. Disposable cuvettes
  8. Preval sprayer
  9. Thin layer chromatography (TLC) chamber
  10. Petri dishes (12 mm in diameter)
  11. 15 ml sterile Falcon tubes
  12. 2 ml Eppendorf tubes
  13. Glass test tubes
  14. Incubator e.g. Certomat BS-1 (B. Braun Biotech International)
  15. Speedvac sc110 (Savant, Thermo Fisher Scientific)
  16. Biofuge Primo R (Heraeus) (Rotor, catalog number: 7590 )
  17. Heraeus Pico 17 centrifuge (Heraeus)
  18. Spectrophotometer e.g. Ultrospec 3,000 proUV/Visible (GE Healthcare)
  19. Clean bench Hera Safe (Heraeus)
  20. Hybridization oven (Hybaid Shake n’ Stack, Thermo Fisher Scientific)

Procedure

  1. Collect spores from 3 to 4 weeks old Piriformospora indica CM agar plate cultures grown at 28 °C (see Figure 1).


    Figure 1. Four-week-old P. indica agar plate

    1. Pour approximately 5 ml sterile 0.002% Tween water 20 on 3-4 weeks old P. indica plate under sterile condition at room temperature (RT).
    2. Scratch plate with Drigalski spatula and/or scalpel and mix.
    3. Pour spore solution through miracloth filter and collect flow through in 50 ml falcon tube.
    4. Centrifuge for 7 min at 3,500 rpm, discard supernatant.
    5. Wash pellet with 5-10 ml 0.002% Tween water 20.
    6. Centrifuge for 7 min at 3,500 rpm, discard supernatant.
    7. Wash pellet with 5-10 ml 0.002% Tween water 20.
    8. Centrifuge for 7 min at 3,500 rpm, discard supernatant.
    9. Resuspend spore pellet in 10 ml 0.002% Tween water 20, count spores with counting chamber (e.g. Neubauer improved) and dilute to requested spore concentration (e.g. 500,000 spores ml-1).
  2. Inoculate 50 ml “complete medium” CM liquid medium (Pham et al., 2004) with 400 μl of 500,000 spores/ml spore solution in 100 ml Erlenmeyer flask.
  3. Let spores germinate for 1 week at 28 °C and 130 rpm.
  4. Inoculate with 2.5 mM TRP (prepare the stock 1 day before, dissolve slowly; work in darkness auxin is light sensitive).
  5. Incubate cultures in darkness (alternatively wrap flasks with aluminium foil) for 3 days.
  6. Collect 11 ml supernatant through miracloth filter (check the mass of each filter) into 15 ml falcon tube:
    1. Wash mycelium with 0.9% NaCl and let the whole miracloth filter with fungal biomass dry overnight in oven (85 °C).
  7. 15 μl aliquot of the culture supernatant can be used for the determination of TRP and indole derivatives by LC-MS/MS (see Hilbert et al., 2013") From the falcon tube take 1 ml into glass test tube for Salkowski test.
  8. Salkowski test: Mix 1 ml supernatant with 2 ml Salkowski reagent and 50 μl of 10 mM orthophosphoric acid, incubate for 25 min at RT, measure OD530.
  9. Add 5 ml ethyl acetate to the remaining 10 ml of supernatant and incubate for 3 to 4 h at 200 rpm in darkness (supernatant can be frozen at -20 °C overnight).
  10. For good phase separation centrifuge 5 min at 4 °C at 3,500 rpm (if needed leave falcons overnight at 4 °C in darkness).
  11. Transfer 2 ml of organic phase (upper phase) into a 2 ml eppendorf tube and evaporate in SpeedVac concentrator for 30 min with medium heating.
  12. Add 2 ml of the remaining organic phase to the pellet and repeat the SpeedVac process.
  13. Resuspend pellet in 60 μl ethyl acetate (Storage in -20 °C).
  14. Calculate the dry biomass.
  15. Fill the TLC chamber with Whatman paper.
  16. Saturate TLC chamber for approximately 1 h with approximately 200 ml of running buffer chloroform: ethanol: water (84:14:1).
  17. Load extracted samples onto the TLC plate in a volume, that each spot represents the amount of indole derivatives obtained from the samples of equal biomass (use not more than 6 μl for loading on TLC plate).
  18. Use commercially available indole derivatives as marker control (e.g. TRP, IAD and IAA).
  19. Let TLC run for 1 to 1.5 h in darkness.
  20. Dry TLC plate for 5 min at RT for approximately 2 to 5 min.
  21. Develop TLC plate by spraying on it a mixture of van Urk and Salkowski reagents in the proportion of 1:3 (Ehmann, 1977) and incubate in an incubator at 90 °C up to 10 min.
  22. Calculate retention factor (Rf) for each spot (the distance travelled by the compound divided by the distance travelled by the solvent) and compare it with Rf calculated for each indole derivative control.

Recipes

  1. Van Urk reagent
    1 g p-dimethylaminobenzaldehyde dissolved in 50 ml concentrated HCl and 50 ml ethanol. This reagent is stable for several months at room temperature when stored in a brown/light protected glass bottle.
  2. Salkowski test reagents
    Prepare stock solution of 0.5 M FeCl3 (1.35 g in 10 ml water)
    Use 1 ml of this stock to mix with 49 ml of 35 % HClO4
  3. 10 mM orthophosphoric acid
    115 μl (of 85 %) in 100 ml water
  4. Complete medium (CM) 1 L
    50 ml   20x salt solution
    20 g    glucose
    2 g      peptone
    1 g      yeast extract
    1 g      casamine acids
    1 ml    microelements
    15 g    agar
  5. 20x salt solution 1 L
    120 g    NaNO3
    10.4 g   KCl
    10.4 g   MgSO4.7H2O
    30.4 g   KH2PO4
  6. Microelements 1 L
    6 g          MnCl2.4H2O
    1.5 g       H3BO3
    2.65 g     ZnSO4.7H2O
    750 mg    KI
    2.4 mg    Na2MO4.2H2O
    130 mg   CuSO4.5H2O

Acknowledgments

This protocol is adapted from Hilbert et al. (2012).

References

  1. Hilbert, M., Voll, L. M., Ding, Y., Hofmann, J., Sharma, M. and Zuccaro, A. (2012). Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots. New Phytol 196(2): 520-534.
  2. Hilbert, M., Voll, L., Hofmann, J. and Zuccaro, A. (2013). Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS. Bio-protocol 3(12): e800. 
  3. Ehmann, A. (1977). The van urk-Salkowski reagent--a sensitive and specific chromogenic reagent for silica gel thin-layer chromatographic detection and identification of indole derivatives. J Chromatogr 132(2): 267-276.

简介

共生根内生真菌(Pyriformospora indica)殖民广泛的植物,并且这种真菌对根细胞的定植通常与对其宿主的有益效果相关,例如生长促进和增加的生物和非生物胁迫耐受性 。 这些性状可以基于许多不同植物物种共有的一般机制和信号途径。 一种这样的机制可以是通过P募集植物激素途径。 indica 。 已知许多共生微生物能够在与其宿主植物相互作用期间合成和分泌植物激素。 该方案已经成功地用于通过P分析吲哚-3-乙酸(IAA)及其吲哚衍生物的色氨酸(TRP)依赖性生物合成。 疟疾(Hilbert et al 。,2012)。

材料和试剂

  1. 吲哚衍生物:
    TRP(Sigma-Aldrich,目录号:T0254-500g) IAD(吲哚-3-乙醛)(Sigma-Aldrich,目录号:I1000-100mg) IAA(Sigma-Aldrich,目录号:I5148-2g)
  2. 标准显微镜盖玻片
  3. 标准显微镜载玻片
  4. 0.002%(v/v)吐温水20
  5. 0.9%NaCl
  6. FeCl <3>
  7. HClO 4
  8. NaNO 3
  9. MgSO 4。 。 O
  10. KH 2 PO 4
  11. 正磷酸
  12. 葡萄糖
  13. 蛋白胨
  14. 酵母提取物
  15. 酪蛋白酸
  16. 微元件
  17. Agar
  18. 色氨酸(Sigma-Aldrich,目录号:T0254-500g)
  19. 乙酸乙酯
  20. 铝箔
  21. 对二甲基氨基苯甲醛(Sigma-Aldrich)
  22. Van Urk试剂(参见配方)
  23. Salkowski试剂(见配方)
  24. 10 mM正磷酸(见配方)
  25. 完整介质(见配方)
  26. 20x盐溶液(见配方)
  27. 微量元素(参见配方)

设备

  1. 锥形烧瓶(100ml)
  2. Neubauer改进了计数室(Marienfeld-Superior,Lauda,Königshofen,德国)
  3. 手术刀(无菌)
  4. Whatman纸
  5. Miracloth过滤器15cm×15cm(Merck KGaA,目录号:475855)
  6. Drigalski铲(无菌)
  7. 一次性比色杯
  8. 前吹式喷雾器
  9. 薄层色谱(TLC)室
  10. 培养皿(直径12mm)
  11. 15 ml无菌Falcon管
  12. 2 ml Eppendorf管
  13. 玻璃试管
  14. 孵化器 Certomat BS-1(B. Braun Biotech International)
  15. Speedvac sc110(Savant,Thermo Fisher Scientific)
  16. Biofuge Primo R(Heraeus)(Rotor,目录号:7590)
  17. Heraeus Pico 17离心机(Heraeus)
  18. 分光光度计例如 Ultrospec 3000 proUV/Visible(GE Healthcare)
  19. 清洁台Hera Safe(Heraeus)
  20. 杂交炉(Hybaid Shake n'Stack,Thermo Fisher Scientific)

程序

  1. 收集3至4周龄生长于28℃的印度梨孢囊线虫CM琼脂平板培养物的孢子(参见图1)。


    图1.四周龄的 p。 楝子琼脂板

    1. 在3-4周龄的P上倒约5ml无菌的0.002%Tween水20。 在无菌条件下在室温(RT)下培养。
    2. 刮板用Drigalski铲和/或解剖刀并混合
    3. 通过miracloth过滤器倒入芽孢溶液,收集流过50毫升falcon管
    4. 以3500rpm离心7分钟,弃去上清液
    5. 用5-10ml 0.002%吐温水20洗涤沉淀。
    6. 以3500rpm离心7分钟,弃去上清液
    7. 用5-10ml 0.002%吐温水20洗涤沉淀。
    8. 以3500rpm离心7分钟,弃去上清液
    9. 在10ml 0.002%吐温水中重悬孢子沉淀20,用计数室计数孢子(例如,Neubauer改善)并稀释至所需的孢子浓度(例如 500,000孢子ml < -1 )。
  2. 用400μl500,000孢子/ml孢子溶液在100ml锥形瓶中接种50ml"完全培养基"CM液体培养基(Pham等人,2004)。
  3. 让孢子在28℃和130rpm下发芽1周
  4. 接种2.5毫米TRP(准备股票1天前,缓慢溶解;在黑暗中工作生长素是光敏感的)。
  5. 在黑暗中孵育培养物(或者用铝箔包裹烧瓶)3天
  6. 通过miracloth过滤器收集11ml上清液(检查每个过滤器的质量)到15ml falcon管:
    1. 用0.9%NaCl洗涤菌丝体,并使整个miracloth过滤器与真菌生物质在烘箱(85℃)中干燥过夜。
  7. 15μl的培养上清液等分试样可用于通过LC-MS/MS测定TRP和吲哚衍生物(参见Hilbert等人,2013")。从falcon管中取1ml进入玻璃用于Salkowski试验的试管
  8. Salkowski试验:将1ml上清液与2ml Salkowski试剂和50μl10mM正磷酸混合,在室温下温育25分钟,测量OD 530。
  9. 向剩余的10ml上清液中加入5ml乙酸乙酯,在黑暗中以200rpm孵育3至4小时(上清液可在-20℃下冷冻过夜)。
  10. 对于良好的相分离离心机在4℃下以3,500rpm离心5分钟(如果需要,在4℃下在黑暗中离开隼类过夜)。
  11. 将2毫升有机相(上相)转移到2毫升的eppendorf管中,在SpeedVac浓缩器中用介质加热蒸发30分钟。
  12. 将2ml剩余的有机相加入到沉淀中并重复SpeedVac方法。
  13. 将沉淀重悬在60μl乙酸乙酯中(-20℃保存)
  14. 计算干生物量。
  15. 用Whatman纸填充TLC室。
  16. 饱和TLC室用约200ml运行缓冲液氯仿:乙醇:水(84:14:1)洗涤约1小时。
  17. 将提取的样品以一定体积装载到TLC板上,每个点代表从等量生物质的样品获得的吲哚衍生物的量(在TLC板上使用不超过6μl)。
  18. 使用市售的吲哚衍生物作为标记物对照(例如TRP,IAD和IAA)。
  19. 让TLC在黑暗中运行1至1.5小时
  20. 干燥TLC板在RT下5分钟约2至5分钟
  21. 通过在其上喷洒比例为1:3(Ehmann,1977)的van Urk和Salkowski试剂的混合物并在90℃的培养箱中孵育至多10分钟来开发TLC板。
  22. 计算每个点的保留因子(Rf)(化合物移动的距离除以溶剂行进的距离),并将其与对于每个吲哚衍生物对照计算的Rf进行比较。

食谱

  1. Van Urk试剂
    1g对二甲基氨基苯甲醛溶解在50ml浓HCl和50ml乙醇中。 当储存在棕色/光保护的玻璃瓶中时,该试剂在室温下稳定几个月
  2. Salkowski试剂
    制备0.5M FeCl 3(1.35g,在10ml水中)的储备溶液 使用1ml该原料与49ml 35%HClO 4
    混合
  3. 10mM正磷酸 115μl(85%)在100 ml水中
  4. 完成介质(CM)1 L
    50 ml  20x盐溶液
    20克   葡萄糖
    2 g     蛋白胨
    1 g     酵母提取物
    1 g     酪蛋白酸
    1 ml   微型元素
    15克    agar
  5. 20x盐溶液1 L
    120克   NaNO 3
    10.4 g  KCl
    10.4 g  MgSO 4。 。 O
    30.4克 KH 2 PO 4
  6. 微量元素1 L
    6 g          MnCl <2> 4H <2> O
    1.5 g      H 3 BO 3
    2.65克   ZnSO 4 。 7H O
    750 mg    KI
    2.4 mg    Na 2 MO 4 2H 2 O
    130毫克 CuSO 4 5H sub 2 O

致谢

该协议改编自Hilbert等人(2012)。

参考文献

  1. Hilbert,M.,Voll,L.M.,Ding,Y.,Hofmann,J.,Sharma,M。和Zuccaro,A。(2012)。 由根内生真菌生成的吲哚衍生物 Piriformospora indica 不是生长所必需的 促进但是用于大麦根的生物营养定殖。新植物 196(2):520-534。
  2. Hilbert,M.,Voll,L.,Hofmann,J。和Zuccaro,A。(2013)。 通过LC-MS测定培养上清液中的海藻孢子虫中的TRP和吲哚衍生物的生长测定和检测 MS/MS。 生物协议 3(12):e800。
  3. Ehmann,A。(1977)。 Van urk-Salkowski试剂 - 一种用于硅胶薄层色谱的灵敏和特异性显色试剂 检测和鉴定吲哚衍生物。 132(2):267-276。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hilbert, M., Voll, L. M., Hofmann, J. and Zuccaro, A. (2013). Indole Derivative Feeding Test and Detection of TRP and Indole derivatives by Thin Layer Chromatography. Bio-protocol 3(12): e801. DOI: 10.21769/BioProtoc.801.
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