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In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts
拟南芥粗提物中硝酸还原酶体外活性测定   

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参见作者原研究论文

本实验方案简略版
Nature Communications
Jul 2011

Abstract

Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.

Keywords: Nitrate reductase (硝酸盐还原酶), in vitro NR activity assay (体外硝酸盐还原酶活性测定), Nitrite concentration (亚硝酸盐浓度)

Background

Nitrogen is crucial macronutrient required by plants and is mainly absorbed in the form of nitrate. Nitrate reductase is the first enzyme of the nitrogen assimilation in higher plants. Homodimers of plant nitrate reductase catalyze the NAD(P)H-dependent reduction of nitrate to nitrite as follows:

NO3- + NADH + H+ → NO2- + NAD+ + H2O

Methods to measure NR activity may be a powerful tool to investigate biological factors influencing NR activity (Park et al., 2011). Nitrogen assimilation affects the contents of amino acid in plant, thus regulating NR activity could be used for increasing quality of some crop (Croy and Hageman, 1970; Dalling and Loyn, 1977; Ruan et al., 1998).

In this protocol, increased nitrite concentrations during limited time in optimized buffer condition are acquired as comparable values. Nitrite concentration is measured by its absorbance at 540 nm through Griess assay. Briefly, nitrite forms a diazonium salt with sulfanilic acid, then N-(1-naphthyl) ethylenediamine dihydrochloride is formed colored azo compound. It is possible to compare the values to determine how samples have different NR activity. Furthermore, the values could be converted to exact increased nitrite concentration through a simple process.

Materials and Reagents

  1. 3MTM MicroporeTM surgical tape (3M, MicroporeTM, catalog number: 1530-1 )
  2. Reaction tube, 1.5 ml (Greiner Bio One International, catalog number: 616201 )
  3. 150 x 25 mm (d x h) plastic Petri dish (SPL Life Sciences, catalog number: 10151 )
  4. Cuvette (Ratiolab, catalog number: 2712120 )
  5. Arabidopsis seeds
  6. Ethanol (Merck, EMSURE®, catalog number: 1009831011 )
  7. Liquid nitrogen
  8. Potassium nitrite (Sigma-Aldrich, catalog number: P8394 )
  9. Murashige & Skoog medium including vitamins (Duchefa Biochemie, catalog number: M0222 )
  10. MES monohydrate (Duchefa Biochemie, catalog number: M1503 )
  11. Potassium hydroxide (Merck, catalog number: 814353 )
  12. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  13. Plant agar (Duchefa Biochemie, catalog number: P1001 )
  14. Tris-HCl (Duchefa Biochemie, catalog number: T1513 )
  15. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
  16. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1003 )
  17. Flavin adenine dinucleotide disodium salt hydrate (FAD-Na2) (Sigma-Aldrich, catalog number: F8384 )
  18. Dithiothreitol (DTT) (Duchefa Biochemie, catalog number: D1309 )
  19. Bovine serum albumin (BSA) (Merck, Probumin®, catalog number: 821006 )
  20. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  21. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 78830 )
  22. Sodium nitrate (NaNO3) (Sigma-Aldrich, catalog number: S5506 )
  23. Sodium phosphate dibasic (Na2HPO4) (Bio Basic, catalog number: S0404 )
  24. Sodium phosphate monobasic, anhydrous (NaH2PO4) (Bio Basic, catalog number: SB0878 )
  25. Nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich, catalog number: 43420 )
  26. Hydrochloric acid (HCl) (DAEJUNG CHEMICAL & METALS, catalog number: 4090-4405 )
  27. Sulfanilamide (Sigma-Aldrich, catalog number: S9251 )
  28. N-(1-naphthyl) ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488 )
  29. MS agar media (see Recipes)
  30. Extraction buffer (see Recipes)
  31. Reaction buffer (see Recipes)
  32. 1% sulfanilamide solution (see Recipes)
  33. 0.05% N-(1-naptyl) ethylenediamine hydrochloride (see Recipes)

Equipment

  1. Pipette kit (Gilson, model: PIPETMAN® Classic, catalog number: F167300 )
  2. 2 L flask (DWK Life Sciences, Duran®, catalog number: 21 216 63 )
  3. Arabidopsis growth chamber (Hanbaek, model: HB-301L-3 )
  4. Stainless steel tweezers
  5. Mortar & pestle (Silico & Chemico Porcelain Works, catalog number: J-753 )
  6. Centrifuge (Thermo Fisher scientific, model: SorvallTM LegendTM 17 , catalog number: 75002431)
  7. Spectrophotometer (Biochrom, model: Libra S22 )
  8. Magnetic stirrer (Vision Scientific, model: VS-130SH )
  9. Stirring bar
  10. pH meter (Fisher Scientific, model: accumetTM AB15 )
  11. Autoclave (Hanbaek, model: HB-506-6 )

Procedure

  1. Plant preparation
    1. Sterilize Arabidopsis seeds with 70% EtOH three times.
    2. Plate the seeds on Murashige and Skoog (MS) medium (see Recipes) containing 0.75% agar.
    3. Wrap 3M micropore tape around the circumference of each MS plate and store the plates in the dark at 4 °C for 2 days.
    4. After 2 days, move the plates to fully automated growth chambers under long day conditions (16 h, 22 °C under light/8 h, 20 °C under dark).

  2. Tissue extraction
    1. After 15 days, take tissue of interest (0.5 g fresh weight) using stainless steel tweezer.
    2. Freeze the samples in liquid nitrogen.
    3. Grind samples in liquid nitrogen with chilled mortar and pestle.
    4. Add 750 μl of chilled extraction buffer (see Recipes) to ground sample to homogenize.
    5. Collect the homogenate into new 1.5 ml tubes through a pipette.
    6. Centrifuge at 17,000 x g for 5 min.
    7. Collect supernatant into new 1.5 ml tubes and discard pellet.

  3. Nitrate reductase enzymatic assay
    1. Add 150 μl of supernatant to 850 μl of reaction buffer (see Recipes) in a 1.5 ml tube.
      Note: The reaction buffer contains substrate (nitrate) and NADH to initiate reaction.
    2. Incubate at room temperature for 2 h.
      Note: To make blank, skip incubation procedure and do next step.
    3. Add 200 μl of 1% sulfanilamide and 200 μl of 0.05% N-(1-naphthyl) ethylenediamine hydrochloride by pipetting to stop the reaction.
    4. Incubate at room temperature for 15 min.

  4. Measure absorbance
    1. Transfer 1 ml of the reaction mixture to a cuvette.
    2. Measure the absorbance of the reaction mixture at 540 nm.

  5. Preparation of nitrite standard curve
    1. Prepare a nitrite dilution series using potassium nitrite (0, 5, 10, 20, and 40 μM) in 1,250 μl of 0.6x diluted extraction buffer (add 500 μl of ddH2O to 750 μl of extraction buffer).
      Note: The dilution ratio of extracted buffer for standards is determined based on the assumption that 1 mg fresh weight has a volume of about 1 µl.
    2. Add 150 μl of dilution series to 850 μl of reaction buffer (see Recipes) in a 1.5 ml tube.
    3. Add 200 μl of 1% sulfanilamide and 200 μl of 0.05% N-(1-naptyl) ethylenediamine hydrochloride by pipetting.
    4. Incubate at room temperature for 15 min.
    5. Transfer 1 ml of the reaction mixture to a cuvette.
    6. Measure the absorbance of the reaction mixture at 540 nm.
    7. Generate standard curve for known concentrations of nitrite.

  6. Waste disposal
    1. All waste must be disposed of in accordance with federal, state and local environmental control regulations.

Data analysis

Values acquired from this protocol are quantity of nitrite molecules. Thus, calculate the NR activity as follows:
ΔA = Asample - Ablank
Each sample is assayed in triplicate and ΔA value is blank subtracted from the average of triplicates.
ΔA value means the increased concentration of nitrite which is produced during 2 h incubation time (see Step C2) and it could be used as a comparable scale of NR activity. For someone who wants to acquire the exact increased nitrite concentration, calculate the concentration change using the standard curve. The ratio of increasing nitrite content of the sample (nmol NO2- g-1 FW h-1) using the formula as follows:
[change of nitrite concentration (μM)] x [extracted volume (ml)]/[fresh weight (g)]/[reaction time (h)]

Recipes

  1. MS agar media
    4.4 g Murashige Skoog basal salt mixture and 0.5 g MES monohydrate in 1 L of deionized water
    Adjust pH to 5.8 with 5 M KOH
    Add 10 g sucrose
    Add 7.5 g plant agar
    Autoclave at 121 °C for 15 min
    Store the medium at room temperature
    Pour media to 150 x 25 mm (d x h) plastic Petri dish
    Poured plated can be stored at 4 °C
  2. Extraction buffer
    250 mM Tris-HCl (pH 8.0)
    1 mM EDTA
    1 μM Na2MoO4
    5 μM flavin adenine dinucleotide(FAD)
    3 mM dithiothreitol
    1% BSA
    12 mM 2-mercaptoethanol
    250 μM PMSF
    Note: Extraction buffer should be prepared fresh, used immediately and stored on ice.
  3. Reaction buffer
    40 mM NaNO3
    80 mM Na2HPO4
    20 mM NaH2PO4 (pH 7.5)
    0.2 mM NADH
    Note: Reaction buffer without NADH can be stored at 4 °C for several months. Since NADH is added, the buffer should be used immediately.
  4. 1% sulfanilamide solution
    Dissolved in 3 M HCl
    Note: This solution is stable for several months.
  5. 0.05% N-(1-naphthyl) ethylenediamine hydrochloride
    Dissolved in distilled water. Store the solution in a dark bottle
    Note: This solution is stable for 1 month.

Acknowledgments

This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ01327601), Rural Development Administration, Republic of Korea.
This protocol was adapted and modified as described previously (Samuelson and Larsson, 1993; Yu et al., 1998, Hachiya and Okamoto, 2016). The authors declare that they have no conflict of interest.

References

  1. Croy, L. I. and Hageman, R. H. (1970). Relationship of nitrate reductase activity to grain protein production in wheat. Crop Science 10(3): 280-285.
  2. Dalling, M. and Loyn, R. (1977). Level of activity of nitrate reductase at the seedling stage as a predictor of grain nitrogen yield in wheat (Triticum aestivum L.). Aust J Agr Res 28(1):1-4.
  3. Hachiya, T. and Okamoto, Y. (2016). Simple spectroscopic determination of nitrate, nitrite, and ammonium in Arabidopsis thaliana. Bio-protocol 7(10):e2280.
  4. Park, B. S., Song, J. T. and Seo, H. S. (2011). Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1. Nat Commun 2: 400.
  5. Ruan, J., Wu, X., Ye, Y. and Härdter, R. (1998). Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensis L). J Sci Food Agr 76(3):389-396.
  6. Samuelson, M. E. and Larsson, C. M. (1993). Nitrate regulation of zeation riboside levels in barley roots: effects of inhibitors of N assimilation and comparison with ammonium. Plant Science 93(1-2):77-84.
  7. Yu, X., Sukumaran, S. and Mrton, L. (1998). Differential expression of the Arabidopsis Nia1 and Nia2 genes. cytokinin-induced nitrate reductase activity is correlated with increased nia1 transcription and mrna levels. Plant Physiol 116(3): 1091-1096.

简介

硝酸还原酶(NR)将主要的植物氮源NO 3 N-2还原为NO 2 - 2。 NR活性可以通过其最终产物,亚硝酸盐在最佳条件下通过其吸光度来测量。 在这里,我们提供了一个详细的协议,用于测量来自拟南芥粗提物的NR的相对酶活性。 该协议提供简单的程序和数据分析来比较多个样品的NR活性。

【背景】氮是植物所需的主要营养素,主要以硝酸盐的形式吸收。硝酸还原酶是高等植物中首次同化氮的酶。植物硝酸还原酶的同型二聚体如下催化硝酸根的NAD(P)H依赖性还原为亚硝酸根:

NO <3> + NADH + H +→NO <2> - + NAD < +> + H <2>

测量NR活性的方法可能是研究影响NR活性的生物因素的有力工具(Park等人,2011)。氮同化影响植物中氨基酸的含量,因此调节NR活性可用于提高某些作物的质量(Croy和Hageman,1970; Dalling和Loyn,1977; Ruan等人,1998) )。

在该协议中,在优化的缓冲液条件下限制时间内亚硝酸盐浓度增加作为可比值获得。亚硫酸盐浓度通过Griess测定法通过其在540nm处的吸光度来测量。简言之,亚硝酸盐与磺胺酸形成重氮盐,然后N-(1-萘基)乙二胺二盐酸盐形成有色偶氮化合物。可以比较这些值以确定样品如何具有不同的NR活性。此外,通过简单的过程可以将这些数值转换为精确增加的亚硝酸盐浓度。

关键字:硝酸盐还原酶, 体外硝酸盐还原酶活性测定, 亚硝酸盐浓度

材料和试剂

  1. 1. 3M TM Micropore TM医用胶带(3M,Micropore TM TM,目录号:1530-1)
  2. 反应管,1.5毫升(Greiner Bio One International,目录号:616201)

  3. 150 x 25毫米(d x h)塑料培养皿(SPL Life Sciences,目录号:10151)
  4. 比色杯(Ratiolab,产品目录号:2712120)
  5. 拟南芥种子
  6. 乙醇(Merck,EMSURE ®,目录号:1009831011)
  7. 液氮
  8. 亚硝酸钾(Sigma-Aldrich,目录号:P8394)
  9. Murashige&amp; Skoog培养基包括维生素(Duchefa Biochemie,目录号:M0222)
  10. MES一水合物(Duchefa Biochemie,目录号:M1503)
  11. 氢氧化钾(Merck,目录号:814353)
  12. 蔗糖(Duchefa Biochemie,目录号:S0809)
  13. 植物琼脂(Duchefa Biochemie,目录号:P1001)
  14. Tris-HCl(Duchefa Biochemie,目录号:T1513)
  15. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
  16. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(Sigma-Aldrich,目录号:M1003)
  17. 黄素腺嘌呤二核苷酸二钠盐水合物(FAD-Na 2)(Sigma-Aldrich,目录号:F8384)
  18. 二硫苏糖醇(DTT)(Duchefa Biochemie,目录号:D1309)
  19. 牛血清白蛋白(BSA)(Merck,Probumin,目录号:821006)
  20. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  21. 苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:78830)
  22. 硝酸钠(NaNO 3)(Sigma-Aldrich,目录号:S5506)
  23. 磷酸二氢钠(Na 2 HPO 4)(Bio Basic,目录号:S0404)
  24. 磷酸二氢钠无水(NaH 2 PO 4)(Bio Basic,目录号:SB0878)
  25. 烟酰胺腺嘌呤二核苷酸(NADH)(Sigma-Aldrich,目录号:43420)
  26. 盐酸(HCl)(DAEJUNG CHEMICAL&amp; METALS,目录号:4090-4405)
  27. 磺胺(Sigma-Aldrich,目录号:S9251)
  28. N-(1-萘基)乙二胺二盐酸盐(Sigma-Aldrich,目录号:222488)
  29. MS琼脂培养基(见食谱)
  30. 提取缓冲液(见食谱)
  31. 反应缓冲液(见食谱)
  32. 1%磺胺溶液(见食谱)
  33. 0.05%N-(1-萘基)乙二胺盐酸盐(见配方)

设备

  1. 移液器试剂盒(Gilson,型号:PIPETMAN Classic Classic,产品目录号:F167300)
  2. 2L烧瓶(DWK Life Sciences,Duran,目录号:21 216 63)
  3. 拟南芥生长室(Hanbaek,型号:HB-301L-3)
  4. 不锈钢镊子
  5. 砂浆&amp;杵(Silico&amp; Chemico Porcelain Works,目录号:J-753)
  6. 离心机(Thermo Fisher科学公司,型号:Sorvall TM Legend TM 17,目录号:75002431)
  7. 分光光度计(Biochrom,型号:Libra S22)
  8. 磁力搅拌器(Vision Scientific,型号:VS-130SH)
  9. 搅拌棒
  10. pH计(Fisher Scientific,型号:accumet TM TM AB15)
  11. 高压灭菌器(Hanbaek,型号:HB-506-6)

程序

  1. 植物准备

    1. 用70%乙醇消毒拟南芥种子三次。
    2. 将种子铺在含有0.75%琼脂的Murashige和Skoog(MS)培养基(见食谱)上。
    3. 将3M微孔胶带缠绕在每块MS板的周围,并将板在黑暗中4℃保存2天。
    4. 2天后,在长日照条件下(16小时,22℃光照/ 8小时,20℃黑暗条件下),将平板移至全自动生长室。

  2. 组织提取
    1. 15天后,使用不锈钢镊子取出感兴趣的组织(0.5克鲜重)。
    2. 将样品冷冻在液氮中。
    3. 使用冷冻研钵和研杵在液氮中研磨样品。

    4. 加入750μl冷冻提取缓冲液(见配方)到研磨样品中均匀化

    5. 通过移液管将匀浆收集到新的1.5 ml管中。

    6. 在17,000×g的条件下离心5分钟。
    7. 收集上清到新的1.5毫升管和丢弃颗粒。

  3. 硝酸还原酶酶试验

    1. 在150 ml反应缓冲液中加入150μl上清液(见配方) 注意:反应缓冲液含有底物(硝酸盐)和NADH以引发反应。

    2. 在室温下孵育2小时 注意:要空白,跳过孵化程序并执行下一步。

    3. 加入200μl1%对氨基苯磺酰胺和200μl0.05%N-(1-萘基)乙二胺盐酸盐,通过移液停止反应。
    4. 在室温下孵育15分钟。

  4. 测量吸光度
    1. 将1毫升反应混合物转移到比色杯中。
    2. 测量540 nm处反应混合物的吸光度。

  5. 亚硝酸盐标准曲线的制备
    1. 使用亚硝酸钾(0,5,10,20和40μM)在1,250μl0.6x稀释的提取缓冲液(加入500μlddH 2 O至750μl提取物中)中制备亚硝酸盐稀释系列缓冲区)。
      注:标准提取缓冲液的稀释比例是基于1mg鲜重具有约1μl体积的假设来确定的。

    2. 在150 ml反应缓冲液中加入150μl稀释系列(见配方)

    3. 加入200μl1%磺胺和200μl0.05%N-(1-萘基)乙二胺盐酸盐。
    4. 在室温下孵育15分钟。
    5. 将1毫升反应混合物转移到比色杯中。
    6. 测量540 nm处反应混合物的吸光度。
    7. 为已知的亚硝酸盐浓度生成标准曲线。

  6. 废物处理
    1. 所有的废物必须按照联邦,州和地方的环境控制法规进行处理。

数据分析

从这个协议获得的价值是亚硝酸盐分子的数量。因此,计算NR活动如下:
ΔA= A sample - blank
每个样品一式三份,ΔA值从三份平均值中减去空白。
ΔA值表示在2小时培养时间内产生的亚硝酸盐浓度增加(参见步骤C2),并且其可以用作NR活性的可比较尺度。对于想要获得准确增加的亚硝酸盐浓度的人,请使用标准曲线计算浓度变化。样品中亚硝酸盐含量增加的比例(nmol NO 2 - g -1 -1 FW h -1)使用公式如下:
[亚硝酸盐浓度(μM)的变化]×[提取体积(ml)] / [鲜重(g)] / [反应时间(h)]

食谱

  1. MS琼脂培养基
    4.4克Murashige Skoog基础盐混合物和0.5克MES一水合物在1升去离子水中
    用5 M KOH调节pH至5.8 加10克蔗糖
    添加7.5克植物琼脂

    在121°C高压灭菌15分钟 将介质存放在室温下
    将介质倒入150 x 25毫米(d x h)的塑料培养皿中。
    浇注电镀可以在4°C储存
  2. 提取缓冲区
    250 mM Tris-HCl(pH 8.0)
    1 mM EDTA
    1μMNa 2 MoO 4 4/2 5μM黄素腺嘌呤二核苷酸(FAD)
    3毫米二硫苏糖醇
    1%BSA
    12 mM 2-巯基乙醇
    250μMPMSF
    注意:提取缓冲液应该准备新鲜,立即使用并储存在冰上。
  3. 反应缓冲液
    40mM NaNO 3 /
    80mM Na 2 HPO 4 4/2 20mM NaH 2 PO 4(pH 7.5)
    0.2 mM NADH
    注意:不含NADH的反应缓冲液可以在4°C下保存数月。由于添加了NADH,缓冲区应立即使用。
  4. 1%磺胺溶液
    溶于3M HCl
    注意:此解决方案在几个月内保持稳定。
  5. 0.05%N-(1-萘基)乙二胺盐酸盐
    溶于蒸馏水。将溶液储存在黑瓶中
    注意:此解决方案在1个月内保持稳定。

致谢

这项工作得到了大韩民国农村发展管理局下一代BioGreen 21项目(植物分子育种中心PJ01327601)的资助。
如前所述(Samuelson和Larsson,1993; Yu等人,1998,Hachiya和Okamoto,2016)对该协议进行了修改和修改。作者声明他们没有利益冲突。

参考

  1. Croy,L.I。和Hageman,R.H。(1970)。 硝酸还原酶活性与籽粒蛋白质产量的关系在小麦中。 <作物科学 10(3):280-285。
  2. Dalling,M.和Loyn,R。(1977)。 苗期硝酸还原酶活性水平可作为小麦籽粒氮产量的预测指标( Triticum aestivum L.) Aust J Agr Res 28(1):1-4。
  3. Hachiya,T.和Okamoto,Y。(2016)。 简单的光谱测定拟南芥中的硝酸盐,亚硝酸盐和铵。 Bio-protocol 7(10):e2280。
  4. Park,B.S。,Song,J.T。和Seo,H.S.(2011)。 拟南芥硝酸还原酶活性受E3 SUMO连接酶AtSIZ1刺激。 / a> Nat Commun 2:400.
  5. Ruan,J.,Wu,X.,Ye,Y.和Härdter,R。(1998)。 Sci Food Agr 76(3):389-396。
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引用:Kim, J. Y. and Seo, H. S. (2018). In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts. Bio-protocol 8(7): e2785. DOI: 10.21769/BioProtoc.2785.
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