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Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis
水果的土曲霉感染和高效液相色谱法(HPLC)定量测定土曲霉酮   

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Abstract

The opportunistic fungal human and plant pathogen Aspergillus terreus (A. terreus) can be isolated from sea water, soil or decaying organic matter such as rotting leaves and fruits. While growing on fruits A. terreus produces secondary metabolites such as terrein, which may ease its penetration into plant tissues. In addition, biological activities of terrein may support competition against other microorganisms. In summary, terrein is a small polyketide that reduces germination of seedlings, induces lesions on fruit surfaces but also shows moderate antifungal activity. With this manuscript we provide a fruit infection protocol with Aspergillus terreus with subsequent determination of terrein production rates on infected fruits using an HPLC-based quantification approach.

Keywords: Aspergillus terreus(土曲霉), Terrein(土曲霉酮), Secondary metabolite quantification(secondary metabolite量化), Ethyl acetate extraction(乙酸乙酯萃取), Banana(香蕉)

Materials and Reagents

  1. Petri dish with three cams (SARSTEDT AG & Co, catalog number: 82.1473 )
  2. T-shaped plastic spreaders (VWR International, catalog number: 30002-110 )
  3. 40 μm cell strainer (Corning, catalog number: 352340 )
  4. 50 ml screw cap tubes (Sigma-Aldrich, catalog number: T2318 )
  5. Glass beaker (1,000 ml)
  6. Scalpel, sterile (VWR International, catalog number: 95039-116 )
  7. 2 ml HPLC vials (VWR International, catalog number: 66020-950 )
  8. Plastic syringes with syringe filters (nylon, 0.45 μm) (Sigma-Aldrich, catalog number: Z260428 )
  9. Fluted cellulose filter MN 1672 (MACHEREY-NAGEL GmbH & Co. KG, catalog number: 57 20 11 )
  10. Aspergillus terreus conidia
    1. Strain SBUG844 (www.leibniz-hki.de/en/jena-microbial-resource-collection.html) [Jena Microbial Research Collection(JMRC)] or
    2. Strain FGSC A1156 (www.fgsc.net) [Fungal Genetics Stock Center(FGSC)]
  11. Organic fruits (preferable bananas, apples or peaches)
  12. KLEENEX® C-fold towels, Kimberly-Clark Professional® (cotton tissue) (VWR International, catalog number: 10815-990 )
  13. Deionised water, sterile
  14. Ethanol (Carl Roth GmbH + Co. KG, catalog number: 5054.4 )
  15. Ethyl acetate, acetic ester (Carl Roth GmbH + Co. KG, catalog number: 7336.1 )
  16. HPLC grade methanol (Carl Roth GmbH + Co. KG, catalog number: P717.1 )
  17. Terrein standard (Sigma-Aldrich, catalog number: T5705 )
  18. Formic acid (Sigma-Aldrich, catalog number: 56302 )
  19. D-Glucose (Carl Roth GmbH + Co. KG, catalog number: 6887.1 )
  20. NaOH (Carl Roth GmbH + Co. KG, catalog number: 6771.1 )
  21. Agar (Carl Roth GmbH + Co. KG, catalog number: 2266.1 )
  22. NaNO3 (Carl Roth GmbH + Co. KG, catalog number: A136.1 )
  23. KCl (Carl Roth GmbH + Co. KG, catalog number: 6781.1 )
  24. MgSO4·7H2O (Carl Roth GmbH + Co. KG, catalog number: P027.1 )
  25. KH2PO4 (Carl Roth GmbH + Co. KG, catalog number: 3904.1 )
  26. ZnSO4·7H2O (Carl Roth GmbH + Co. KG, catalog number: K301.1 )
  27. H3BO3 (Carl Roth GmbH + Co. KG, catalog number: 6943.1 )
  28. MnCl2·4H2O (Carl Roth GmbH + Co. KG, catalog number: T881.1 )
  29. FeSO4·7H2O (Carl Roth GmbH + Co. KG, catalog number: P015.1 )
  30. CoCl2·6H2O (Carl Roth GmbH + Co. KG, catalog number: T889.1 )
  31. CuSO4·5H2O (Carl Roth GmbH + Co. KG, catalog number: P024.1 )
  32. (NH4)6Mo7O24·4H2O (Sigma-Aldrich, catalog number: 09880-100G )
  33. Disodium ethylene diamine tetraacetate (Na2EDTA) (Sigma-Aldrich, catalog number: E9884-100G )
  34. KOH (Carl Roth GmbH + Co. KG, catalog number: 6751.1 )
  35. Aspergillus minimal medium (AMM) plates (see Recipes)
  36. 20x salt stock solution (see Recipes)
  37. 1,000x Hutner’s trace elements (see Recipes)
  38. HPLC solvent A (see Recipes)
  39. HPLC solvent B (see Recipes)

Equipment

  1. Porcelain pestles (VWR International, catalog number: 470149-080 )
  2. Counting cell chamber (Thoma chamber) (VWR International, catalog number: 101765-022 )
  3. Accuracy balance
  4. 500 ml round bottom flask (VWR International, catalog number: BOHLA158-09 )
  5. Magnetic stirrer
  6. Magnetic stirring bar (VWR International, catalog number: 58948-954 )
  7. Drying cabinet
  8. Rotary evaporator (e.g.,Carl Roth GmbH + Co. KG, catalog number: PE53.1 )
  9. High-performance liquid chromatography (HPLC) system equipped with a diode array detector (e.g., Agilent 1260 modular HPLC system) with bifunctional-phenylpropyl-modified C18 column (e.g., Sphinx RP, 4.0 x 3 x 250 mm; 5 mm) (MACHEREY-NAGEL GmbH & Co. KG, catalog number:760808.46) with a binary solvent system (solvents and running parameters see below)

Procedure

  1. Streak Aspergillus terreus conidia from a frozen glycerol stock (60%) onto Aspergillus Minimal Medium (AMM) plate (9.2 cm diameter, 25 ml medium solidified with 2% agar) and cultivate for 4 days at 37 °C in the dark (Figure 1A).


    Figure 1. Preparation of Aspergillus terreus conidia suspensions. A. AMM agar plate with Aspergillus terreus grown for 4 d at 37 °C. Note the cinnamon coloured conidia on top of the colonies. B. Aqueous suspension of conidia. C. Microscopic image of Aspergillus terreus conidia (arrows). Note the round shape of the conidia and an average size of 2.5-3.0 μm. Scale bar = 10 μm.

  2. Collect the conidia with a spreader by adding 10 ml distilled sterile water. Filter conidia over a 45 μm cell strainer to remove any clumps and hyphal fragments and collect in a 50 ml screw cap tube (Figure 1B).
  3. Centrifuge (10 min at 2,600 x g), wash the cells at least once with 10 ml distilled sterile water with subsequent second centrifugation (10 min, 2,600 x g) and suspend conidia in 5 ml distilled sterile water.
  4. Estimate the conidia concentration in suspension using a counting chamber (Thoma or Neubauer chamber) (Figure 1C). If necessary, dilute the crude suspension by factor 10 or 100 in sterile water prior to counting.
  5. Slightly wipe the surface of the fruits with 70% ethanol. Subsequently, wash with distilled water and dry the surface with a soft cotton tissue (Figure 2A).


    Figure 2. Preparation of fruits for fungal infection. A. Surface disinfection with 70% ethanol. B. Longitudinal sectioning with a sterile scalpel. C. Infection with conidia suspension in the longitudinal groove. D. Incubation in a sterile glass beaker (pre-weighed) for 5 to 7 days at room temperature.

  6. Cut the fruit longitudinally with a sterile scalpel (length around 10 cm; depth around 1.5 cm) (Figure 2B).
  7. Inoculate with 200 μl of a conidia suspension containing 2 x 108 conidia/ml. Disperse the volume evenly over the complete length of the groove (Figure 2C). For negative control use 200 μl of sterile water. Fruits used for infection and for the control must be taken from the same batch! At least three technical replicates (three fruits, each from the same batch or parental plant) and five biological (five different batches) replicates should be carried out.
  8. Weigh a sterile 1,000 ml glass beaker including a covering aluminium foil.
  9. Put the fruit into the sterile 1,000 ml beaker glass and cover it with aluminium foil (Figure 1D).
  10. Incubate the fruit for 5 to 7 days at room temperature (Figure 3).


    Figure 3. Cross-section of bananas after 7 days of incubation. A. Control (mock) infected banana. B. Banana infected with 200 μl of A. terreus conidia suspension (2 x 108 conidia/ml).

  11. Cut the fruit into pieces of approximately 2 x 2 cm size and smash the fruit in the glass beaker using a pestle.
  12. Add a magnetic stir bar and pour 150 ml ethyl acetate into the beaker.
  13. Extract the fruits while stirring on a magnetic stirrer at 150 rpm for 15 min.
  14. Decant the supernatant into a 500 ml round bottom flask and repeat the extraction procedure. Pool both extracts.
  15. For dry weight determination of bananas evaporate the fruit debris in the glass beaker under a fume hood. Subsequently dry it at 37-50 °C. Weigh the glass beaker including its cover and calculate the dry weight by subtracting the weight of the empty glass beaker as previously determined.
  16. Evaporate the ethyl acetate from the fruit extractions to dryness using a rotary evaporator.
  17. Solve the residue in 2 ml HPLC grade methanol. Dilute the extract in a ratio of 1:10, 1:20 and 1:50 in methanol. Filter the dilutions over a 0.45 μm nylon filter into a HPLC vial.
  18. Run a calibration curve by applying 10 μl serial dilutions of the terrein standard (2.5 to 500 μg/ml in methanol) to a bifunctional C18 column (e.g., Macherey-Nagel Sphinx RP, 4.0 x 3 x 250 mm; 5 mm) in a modular HPLC system equipped with DAD (e.g., Agilent 1260 modular HPLC system). The following gradient (solution A = water + 0.1% formic acid, solution B = methanol) with a flow rate of 0.8 ml/min should be applied: 0.5 min = 10% B, 0.5-20 min = 10%-70% B, 20-25 min = 70%-100% B, 25-28 min = 100% B, and 28-33 min = 100%-10% B. Terrein is detected at a wavelength of 254 nm.
  19. Generate a calibration curve from peak integrations at 254 nm.
  20. Apply 10 μl aliquots of the diluted fruit extracts to the column.
  21. Use the calibration curve to calculate the amount of terrein (μg) by peak integration from the UV profile. Note that the total volume of terrein extract in methanol was 2 ml.
  22. Determine the total terrein content as terrein (μg)/dry-weight of fruit (g).

Notes

  1. If the supernatant after the ethyl acetate extraction is not clear (contaminating fruit debris) the extract can be optionally filtered over a fluted cellulose filter.
  2. Typical terrein yields range from 10 to 450 μg terrein per (g) of fruit dry weight depending on the type of fruits and cultivation time. To increase the accuracy of measurements on a given type of fruit, at least 15 replicates (including technical and biological replicates) should be performed.
  3. Organic fruits should be used to avoid contaminating fungicides or pesticides, which could impede with fungal growth and terrein production. The amount of sugars in the fruits has an additional impact on terrein production and could cause experimental variations between different batches of fruits. Therefore, to quantify the terrein amount on fruits of different Aspergillus terreus strains a relative production rate (in %) can be calculated by simultaneous cultivation of a well-characterised producer such as strain SBUG844 (Zaehle et al., 2014).

Recipes

  1. Aspergillus minimal medium plates
    For 1 L:
    D-Glucose 10 g
    20x salt stock solution (see Recipe 2) 50 ml
    1,000x Hutner´s trace elements (see Recipe 3) 1 ml
    Water 900 ml
    Adjust to pH 6.5 with 10 M NaOH
    Agar 20 g
    Water make up to 1,000 ml
    Autoclave 20 min at 121 °C
  2. 20x salt stock solution
    For 1 L:
    NaNO3 120 g
    KCl 10.4 g
    MgSO4·7H2O 10.4 g
    KH2PO4 30.4 g
  3. 1,000x Hutner´s trace elements
    For 100 ml:
    ZnSO4·7H2O 2.2 g
    H3BO3 1.1 g
    MnCl2·4H2O 0.5 g
    FeSO4·7H2O 0.5 g
    CoCl2·6H2O 0.16 g
    CuSO4·5H2O 0.16 g
    (NH4)6Mo7O24·4H2O 0.11 g
    Na2EDTA 5.0 g
    Heat to boiling, cool to 60 °C, add KOH adjusting pH to 6.5-6.8. The colour of the solution should turn to deep purple after storing in the dark for several days.
  4. HPLC solvent A
    HPLC pure water + 0.1% formic acid
    Add 1 ml formic acid (98% purity) to 999 ml of water and filter
  5. HPLC solvent B
    Methanol (HPLC grade)

Acknowledgments

This protocol gives a detailed description of methods published previously in Gressler et al. (2015) and Zaehle et al. (2014). This work was financially supported by the German Science Foundation (DFG grant BR 2216/4-1) and internal funding from the Hans Knöll Institute, Jena (Germany).

References

  1. Gressler, M., Meyer, F., Heine, D., Hortschansky, P., Hertweck, C. and Brock, M. (2015). Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals. Elife 4.
  2. Zaehle, C., Gressler, M., Shelest, E., Geib, E., Hertweck, C. and Brock, M. (2014). Terrein biosynthesis in Aspergillus terreus and its impact on phytotoxicity.ChemBiol 21(6): 719-731.

简介

机体真菌人和植物病原菌土曲霉(A. terreus)可以从海水,土壤或腐烂的有机物质如腐烂的叶子和果实中分离出来。 当在水果上生长时,A. terreus产生次生代谢物如土曲杆菌,这可以缓解其渗透到植物组织中。 此外,土耳特蛋白的生物活性可能支持与其他微生物的竞争。 总而言之,土豆蛋白是一种减少幼苗发芽的小聚酮,诱发水果表面的病变,但也显示出中等的抗真菌活性。 使用这份手稿,我们提供了一种水果感染方案与土曲霉,随后使用基于HPLC的量化方法测定感染果实的土豆油生产率。

关键字:土曲霉, 土曲霉酮, secondary metabolite量化, 乙酸乙酯萃取, 香蕉

材料和试剂

  1. 带有三个凸轮的培养皿(SARSTEDT AG& Co,目录号:82.1473)
  2. T型塑料吊具(VWR International,目录号:30002-110)
  3. 40μm细胞过滤器(Corning,目录号:352340)
  4. 50ml螺旋盖管(Sigma-Aldrich,目录号:T2318)
  5. 玻璃烧杯(1000ml)
  6. 无菌手术刀(VWR International,目录号:95039-116)
  7. 2ml HPLC小瓶(VWR International,目录号:66020-950)
  8. 带注射器过滤器的塑料注射器(尼龙,0.45μm)(Sigma-Aldrich,目录号:Z260428)
  9. 槽纹纤维素过滤器MN 1672(MACHEREY-NAGEL GmbH& Co.KG,目录号:572011)
  10. Aspergillus terreus 分生孢子
    1. 菌株SBUG844( www.leibniz-hki.de/en /jena-microbial-resource-collection.html )[Jena Microbial Research Collection(JMRC)]或
    2. 菌株FGSC A1156( www.fgsc.net )[真菌遗传库存中心(FGSC)]
  11. 有机水果(优选香蕉,苹果或桃)
  12. KLEENEX C-fold毛巾,Kimberly-Clark Professional(棉花组织)(VWR International,目录号:10815-990)。
  13. 去离子水,无菌
  14. 乙醇(Carl Roth GmbH + Co.KG,目录号:5054.4)
  15. 乙酸乙酯,乙酸酯(Carl Roth GmbH + Co.KG,目录号:7336.1)
  16. HPLC级甲醇(Carl Roth GmbH + Co.KG,目录号:P717.1)
  17. Terrein标准品(Sigma-Aldrich,目录号:T5705)
  18. 甲酸(Sigma-Aldrich,目录号:56302)
  19. D-葡萄糖(Carl Roth GmbH + Co.KG,目录号:6887.1)
  20. NaOH(Carl Roth GmbH + Co.KG,目录号:6771.1)
  21. 琼脂(Carl Roth GmbH + Co.KG,目录号:2266.1)
  22. NaNO 3(Carl Roth GmbH + Co.KG,目录号:A136.1)
  23. KCl(Carl Roth GmbH + Co.KG,目录号:6781.1)
  24. MgSO 4·7H 2 O(Carl Roth GmbH + Co.KG,目录号:P027.1)
  25. KH <2> PO 4 (Carl Roth GmbH + Co.KG,目录号:3904.1 )
  26. ZnSO 4 ·7H 2 O(Carl Roth GmbH + Co.KG, :T881.1)
  27. FeSO 4 7H 2 O(Carl Roth GmbH + Co.KG, :P015.1)
  28. CoCl 2 ·6H 2 O(Carl Roth GmbH + Co. KG, :T889.1)
  29. CuSO 4 5H 2 O(Carl Roth GmbH + Co.KG, :P024.1)
  30. (NH 4)6 Mo 7 Mo 12 O 24(CH 4)2 (Sigma-Aldrich,目录号:09880-100G)
  31. 乙二胺四乙酸二钠(Na 2 EDTA)(Sigma-Aldrich,目录号:E9884-100G)
  32. KOH(Carl Roth GmbH + Co.KG,目录号:6751.1)
  33. 中等(AMM)板(请参阅食谱)
  34. 20x盐储备溶液(见配方)
  35. 1,000x Hutner的微量元素(参见配方)
  36. HPLC溶剂A(参见配方)
  37. HPLC溶剂B(参见配方)

设备

  1. 瓷杵(VWR International,目录号:470149-080)
  2. 计数细胞室(Thoma室)(VWR International,目录号:101765-022)
  3. 精度平衡
  4. 500ml圆底烧瓶(VWR International,目录号:BOHLA158-09)
  5. 磁力搅拌器
  6. 磁力搅拌棒(VWR International,目录号:58948-954)
  7. 干燥柜
  8. 旋转蒸发器(例如,Carl Roth GmbH + Co.KG,目录号:PE53.1)
  9. 装有二极管阵列检测器(例如,Agilent 1260模块HPLC系统)的双功能 - 苯丙基修饰的C18柱的高效液相色谱(HPLC)系统(例如,Sphinx RP,4.0×3×250mm; 5mm)(MACHEREY-NAGEL GmbH& Co.KG,目录号:760808.46),二元溶剂系统(溶剂和运行参数见下文)

程序

  1. (AMM)平板(直径为9.2cm,用2%琼脂固化的25ml培养基)将来自冷冻甘油储备液(60%)的条纹土曲霉欧文氏菌分生孢子 在37℃在黑暗中培养4天(图1A)

    图1.准备 曲霉菌 分生孢子悬浮液。 A. AMM琼脂平板用 Aspergillus terreus <在37℃下生长4天。注意在殖民地顶部的肉桂色分生孢子。 B.分生孢子的水性悬浮液。 C.土曲霉的分生孢子的显微镜图像(箭头)。注意分生孢子的圆形和平均尺寸为2.5-3.0μm。比例尺=10μm。

  2. 通过加入10ml蒸馏的无菌水用扩展器收集分生孢子。在45μm细胞过滤器上过滤分生孢子以除去任何团块和菌丝碎片,并收集在50 ml螺旋盖管中(图1B)。
  3. 离心(在2,600xg下10分钟),用10ml蒸馏的无菌水洗涤细胞至少一次,随后进行第二次离心(10分钟,2,600×g),并将分生孢子悬浮在5ml蒸馏无菌水
  4. 使用计数室(Thoma或Neubauer室)估计悬浮液中分生孢子浓度(图1C)。如有必要,在计数前将粗悬浮液稀释10倍或100倍,然后加入无菌水
  5. 用70%乙醇轻轻擦拭果实的表面。随后,用蒸馏水洗涤,并用软棉纸干燥表面(图2A)

    图2.用于真菌感染的水果的制备 A.用70%乙醇进行表面消毒。 B.用无菌手术刀纵向切片。 C.用在纵向槽中的分生孢子悬浮液感染。 D.在室温下在无菌玻璃烧杯(预称重)中孵育5至7天
  6. 用无菌手术刀(长度约10厘米,深度约1.5厘米)纵向切开果实(图2B)
  7. 用200μl含有2×10 8个分生孢子/ml的分生孢子悬浮液接种。在槽的整个长度上均匀地分散体积(图2C)。对于阴性对照使用200μl无菌水。用于感染和对照的水果必须取自同一批次!应该进行至少三次技术重复(三次果实,每次来自同一批次或亲本植物)和五次生物(五次不同批次)重复。
  8. 称量无菌的1000ml玻璃烧杯,包括覆盖铝箔。
  9. 将水果放入无菌的1000毫升烧杯中,并用铝箔覆盖(图1D)
  10. 在室温下孵育水果5至7天(图3)

    图3.在孵育7天后的香蕉的横截面。 A.对照(模拟)感染的香蕉。 B.用200μl的A感染的香蕉。 terreus 分生孢子悬浮液(2×10 8 分生孢子/ml)。
  11. 将果切成约2×2cm大小的碎片,并使用杵在玻璃烧杯中粉碎水果。
  12. 加入磁力搅拌棒,并将150ml乙酸乙酯倒入烧杯中
  13. 提取果实,同时在磁力搅拌器上以150rpm搅拌15分钟
  14. 将上清液倒入500ml圆底烧瓶中并重复提取程序。 池两个提取物。
  15. 对于香蕉的干重测定,在通风橱下将玻璃烧杯中的水果碎片蒸发。随后在37-50℃下干燥。称重玻璃烧杯,包括其盖子,并通过减去如前所确定的空玻璃烧杯的重量计算干重。
  16. 使用旋转蒸发仪将果实提取物中的乙酸乙酯蒸发至干
  17. 将残余物溶于2ml HPLC级甲醇中。在甲醇中以1:10,1:20和1:50的比例稀释提取物。将稀释物通过0.45μm尼龙过滤器过滤到HPLC小瓶中。
  18. 通过将10μl系列稀释的土链霉素标准品(2.5至500μg/ml,在甲醇中)应用于双功能C18柱(例如,Macherey-Nagel Sphinx RP,4.0×3×250 mm; 5mm)在装配有DAD(例如,Agilent 1260模块HPLC系统)的模块HPLC系统中。应当应用流速为0.8ml/min的以下梯度(溶液A =水+ 0.1%甲酸,溶液B =甲醇):0.5分钟= 10%B,0.5-20分钟= 10%-70%B ,20-25分钟= 70%-100%B,25-28分钟= 100%B,28-33分钟= 100%-10%B.在254nm的波长处检测到土曲霉毒素。
  19. 从峰值积分在254 nm处产生校准曲线。
  20. 将10μl等分的稀释水果提取物加入柱中。
  21. 使用校准曲线通过从UV谱的峰积分计算土曲霉素的量(μg)。 注意土曲霉提取物在甲醇中的总体积为2ml
  22. 确定总土壤蛋白含量为土粉(μg)/水果的干重(g)

笔记

  1. 如果乙酸乙酯萃取后的上清液不澄清(污染的水果碎片),则可以任选地在槽纹纤维素滤器上过滤萃取物。
  2. 根据水果的类型和培养时间,典型的土工产量范围为每(g)果干干重10至450μg土。 提高给定类型的测量的精度水果,应至少进行15次重复(包括技术和生物复制)
  3. 应使用有机果实,以避免污染杀真菌剂或农药,这可能阻碍真菌生长和土曲霉的生产。果实中的糖的量对土生土生产具有额外的影响,并且可能导致不同批次的果实之间的实验变化。因此,为了定量不同的土曲霉菌株的果实上的土壤杆菌素量,可以通过同时培养良好表征的生产菌株如菌株SBUG844(Zaehle等)来计算相对生产率(%), et al。,2014)。

食谱

  1. 曲霉基本培养基平板
    对于1 L:
    D-葡萄糖10g / 20x盐储备溶液(见配方2)50ml
    1,000x Hutner的微量元素(见配方3)1 ml
    水900毫升
    用10M NaOH调节至pH6.5 琼脂20克
    水补足1000毫升
    在121°C高压灭菌20分钟
  2. 20x盐储备液
    对于1 L:
    NaNO <3> 120克
    KCl 10.4 g
    MgSO 4·7H 2 O 10.4g·dm/s


  3. 1,000x Hutner的微量元素
    对于100 ml:
    ZnSO 4·7H 2 O 2.2g/g H sub 3 BO 3 1.1 g
    MnCl 2 2·4H 2 O 0.5g
    FeSO 4 7HH 2 O 0.5g
    CoCl 2 2·6H 2 O 0.16g/dm 2 CuSO 4·5H 2 O 0.16g/dm 2 (NH 4)6 Mo 7 SubO 24·4H 2 O O 0.11g
    Na 2 EDTA 5.0g
    加热至沸腾,冷却至60℃,加入KOH调节pH至6.5-6.8。 在黑暗中储存几天后,溶液的颜色应变成深紫色。
  4. HPLC溶剂A
    HPLC纯水+ 0.1%甲酸
    将1ml甲酸(98%纯度)加入到999ml水中并过滤
  5. HPLC溶剂B
    甲醇(HPLC级)

致谢

该协议详细描述了先前在Gressler等人(2015)和Zaehle等人(2014)中公开的方法。这项工作得到德国科学基金会(DFG赠款BR 2216/4-1)和来自德国汉娜·克诺尔研究所的内部资助的资助。

参考文献

  1. Gressler,M.,Meyer,F.,Heine,D.,Hortschansky,P.,Hertweck,C。和Brock,M.(2015)。植物毒素生产在  土曲霉是受独立环境信号的限制。 4.
  2. Zaehle,C.,Gressler,M.,Shelest,E.,Geib,E.,Hertweck,C。和Brock,M。(2014)。 土曲霉中的Terrein生物合成及其对植物毒性的影响。 ChemBiol 21(6):719-731。
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Copyright Gressler and Brock. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gressler, M. and Brock, M. (2016). Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis. Bio-protocol 6(12): e1845. DOI: 10.21769/BioProtoc.1845.
  2. Gressler, M., Meyer, F., Heine, D., Hortschansky, P., Hertweck, C. and Brock, M. (2015). Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals. Elife 4.07861.
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