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An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast
拟南芥防御素1型蛋白诱导酵母细胞中锌耐受性的能力的测试分析   

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Abstract

Heterologous expression of genes in budding yeast Saccharomyces cerevisiae (S. cerevisiae) is especially suitable to functionally study the corresponding encoded protein at the cellular level (Bonneaud et al., 1991). This is mainly because many strains defective in specific activities are available and could be complemented by homologous genes existing across the eukaryotic kingdom (http://www.yeastgenome.org/). However, the protocol we describe here is not a complementation but a “gain-of-function” assay. It is based on a drop-test assay that we have set up to assess the cellular zinc tolerance conferred by the expression of heterologous genes in the wild-type S. cerevisiae. Different dilutions of a yeast culture expressing the heterologous gene of interest are grown on a range of zinc-enriched plates, and are then compared to the control yeast expressing the empty vector. Working with different concentrations of both yeast and zinc are essential to succeed in describing zinc tolerance phenotype upon yeast transformation (Mirouze et al., 2006). This test has also proven to be valuable to differentiate among related members of gene families as exemplified for Arabidopsis Plant Defensin type1 (Shahzad et al., 2013).

Keywords: Plant defensin(植物防御素), Abiotic stress(非生物胁迫), Zinc tolerance(锌的耐受性), Yeast(酵母)

Materials and Reagents

  1. Plastic foil (Saran Wrap)
  2. Sterile square 12 cm x 12 cm Petri dishes (CML HealthCare, catalog number: BP 120SJ )
  3. Sterile-R Filtropur S (0.2 µm) (SARSTEDT AG, catalog number: 83.1826.001 )
  4. 20 ml sterile syringe without needle (Medline Scientific, catalog number: BS-20ES )
  5. 50 ml sterile tubes (SARSTEDT AG, catalog number: 62.547.254 )
  6. Autoclaved 1.5 ml Eppendorf tubes (SARSTEDT AG, catalog number: 72.690 )
  7. Sterile inoculating loops (Greiner Bio One International GmbH, catalog number: 731175 )
  8. Sterile pipet tips
  9. Parental BY4741 S. cerevisiae strain (Winston et al., 1995; Brachmann et al., 1998) having the genotypes MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) expressing: the pFL38H vector (Bonneaud et al., 1991), complementing the his3∆1 auxotrophy
  10. Transformed BY4741 yeast harbouring both: the pFL38H vector, complementing the his3∆1 auxotrophy and the pYX212 vector (Ingenius, R&D systems) complementing the ura3∆0 mutation and expressing the entire coding sequences of Arabidopsis PDF1s, i.e., including the signal peptide (Shahzad et al., 2013)
  11. Yeast nitrogen base (YNB) without amino acids and without ammonium sulfate (BD, Difco, catalog number: 233520 )
  12. Ammonium nitrate (NH4NO3) (Sigma-Aldrich, catalog number: A9642 )
  13. D(+)Glucose anhydrous (C6H12O6) (EUROMEDEX, catalog number: UG3050 )
  14. Succinic acid (C4H6O4) (Sigma-Aldrich, catalog number: S7501 )
  15. Potassium hydroxide (KOH) (Bio Basic Canada Inc., catalog number: PB0441 )
  16. Agarose type D-5 (EUROMEDEX, catalog number: D5 )
  17. Zinc sulphate heptahydrate (ZnSO4.7H2O) (Sigma-Aldrich, catalog number: Z4750 )
  18. L-leucine (C6H13NO2) (Sigma-Aldrich, catalog number: L8912 )
  19. L-methionine (C5H11NO2S) (Sigma-Aldrich, catalog number: M9625 )
  20. Sterile ultra-pure water
  21. 0.5 M zinc sulfate
  22. 10 N KOH (see Recipes)
  23. 0.075 M L-leucine (see Recipes)
  24. 0.1 M L-methionine (see Recipes)
  25. Sterile liquid and solid selective yeast nitrogen base (YNB) media (see Recipes)

Equipment

  1. Sterile 250 ml borosilicate glass Erlen flask
  2. 1,000 ml bottle
  3. Typical 20 µl pipet
  4. Autoclave
  5. Heating oven (Universal Memmert, model: 400 )
  6. New BrunswickTM Agitator (Eppendorf, model: Innova 44 )
  7. A normal cell density meter (Thermo Fisher Scientific, model: 40 )
  8. Laminar flow hood (ADS, model: VP120 )
  9. Centrifuge (Eppendorf, model: 5804R )
  10. Integral water purification system (Merck Millipore Corporation, model: Milli-Q+ )
  11. Scanner (Epson, model: 124OU )

Procedure

Note: For each yeast clone to be analyzed.
On day -1

  1. Use a sterile inoculating loop to inoculate each yeast clone to be tested in 20 ml liquid selective YNB media in sterile 250 ml glass Erlen flask.
    Note: At least three independent yeast clones are tested for each construct (harboring either recombinant or control plasmids).
  2. Culture yeast cells at 30 °C under 200 rpm agitation Over Night (O/N).

On day 0
Prepare Petri dish for drop assay.

  1. Prepare sterile selective YNB solid media containing the appropriate amino acids and appropriate zinc sulfate concentration to be tested.
  2. Under laminar flux hood, pour 50 ml of sterile selective YNB solid media in each square 12 cm x 12 cm Petri dish.
    Note: Tolerance to zinc excess is usually tested around the final concentrations of 23, 25, 27.5, 30 and 32.5 mM. The control condition is without zinc added (≈1.4 µM). The leucine and methionine auxotrophy are complemented by the addition of the corresponding amino acids to the final concentrations of 0.75 mM and 0.33 mM, respectively.
  3. Let media solidify, cover opened, for 15 min. Close and store square Petri dish under the hood.
    Prepare yeast for drop assay.
    Note: All subsequent manipulations with yeast are performed under sterile conditions in laminar flow hood.
  4. Transfer O/N yeast culture in sterile 50 ml Falcon tubes.
  5. Centrifuge for 5 min at 5,000 x g at room temperature.
  6. Discard supernatant.
  7. Suspend yeast culture with the same volume of sterile water (20 ml).
  8. Repeat steps 7 to 9.
  9. Measure optical density at 600 nm after suitable dilution (usually 1/10) to get an OD600nm of around 0.4.
  10. In a 1.5 ml sterile Eppendorf tube, make appropriate yeast culture dilution with sterile water in order to reach OD600nm of 1 in 500 µl volume and then make serial dilution in order to reach OD600nm of 10-1, 10-2, 10-3 in a maximum of 500 µl.
  11. Use a pipet and sterile filter tips to drop 10 µl of each yeast dilution onto selective YNB media supplemented with ZnSO4 at various final concentrations (see Figure 1).
    Notes:
    1. For steps 10 to 12: Yeast cells sediment particularly rapidly; hence make sure to fully agitate the yeast culture before taking a sample and / or before measuring optic density of a suspension and/or before drop assay.
    2. For drop assays, separate the center of each drop by ≈1 cm.
  12. Allow drops to dry under the laminar flux hood.
  13. Close Petri dish and wrap them plastic foil (type Saran Wrap).
  14. Incubate in 30 °C incubator upside down.
  15. Monitor yeast growth every day and record results by scanning the Petri dish under black screen at a 600 dots per inch (dpi) resolution (usually less than one minute per scan). For each zinc concentration, the yeast expressing the protein of interest is compared to the yeast harboring the empty pYX212.
    Note: Control plates, i.e., without zinc added, are usually read 2 days after incubation. This is necessary to verify that the expression of the protein of interest does not disturb yeast growth at very low zinc concentration. Plates for zinc tolerance assay (upon zinc addition) are usually read between 4 to 7 days after incubation.

Representative data


Figure 1. In vitro functional characterisation of amino-acid substitutions impacting zinc tolerance capability in AhPDF1.4 and AtPDF1.4. Serial dilutions of the S. cerevisiae BY4741 strain expressing the pYX212 empty vector (EV) or pYX212 harbouring AhPDF1.4, AtPDF1.4 or its mutated versions of were analysed. Replacement of either A28 to G (A28G) or S54 to R (S54R) or both (A28G- S54R) were generated. The various yeast transformants were spotted on medium supplemented with 1.4 µM (control), or 27.5 mM ZnSO4, as indicated above the panels. Each spot was made with 10 µl of a yeast culture diluted at the OD600nm mentioned below the drops. Pictures were taken at day 2 for control and day 11 for the Zn treatments; they are representative of the three experiments, which have been performed with three independent yeast transformants. Reproduced from (Shahzad et al., 2013; Supplementary Figure 4).

Notes

The BY4741 S. cerevisiae strain has auxotrophic markers, in particular for the production of the amino acid histidine (his3∆1). Free histidine is a potent metal chelator and could, in particular, chelate zinc if this metal is present in the culture medium. The histidine (his3∆1) auxotrophy must thus be complemented by the expression of the corresponding HIS gene (here expressed from the pFL38 plasmid) and not by the addition of free histidine, which would bias the zinc tolerance test.

Recipes

  1. 10 N KOH
    Filter sterilized using Sterile-R Filtropur S (0.2 µm)
    Keep at +4 °C
  2. 0.075 M L-leucine
    Filter sterilized using Sterile-R Filtropur S (0.2 µm)
    Keep at +4 °C
  3. 0.1 M L-methionine
    Filter sterilized using Sterile-R Filtropur S (0.2 µm)
    Keep at +4 °C
  4. Sterile liquid and solid selective YNB media (1 L)
    1. Mix in ultra-pure water
      YNB without amino acids without ammonium sulfate: 1.7 g
      NH4NO3: 6.4 g
      D-Glucose: 20 g
      Succinic acid: 5.9 g
    2. Adjust pH to 4.5 with 10 N KOH
    3. Adjust volume and dispatch 500 ml in 1,000 ml Bottle
    4. For solid media add:
      Agarose: 20 g/L
    5. Autoclave at 120 °C for 20 min
    6.  Upon cooling of solid media, add necessary sterile amino acid solutions complementing auxotrophy (here leucine and methionine) and sterile zinc in order to reach concentrations that will be tested.
      Note: Care should be taken to add necessary sterile amino acid solutions and sterile zinc when solid media has cooled down to ~ 40 to 45 °C. This will prevent both the degradation of added amino acids and the precipitation of the metal.

Acknowledgments

This work was supported by the University of Montpellier (LM), the French Ministère de l’Enseignement Supérieur et de la Recherche (PB), the Pakistani Higher Education Commission (ZS) and the Centre National de la Recherche Scientifique (FG).

References

  1. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G. Y., Labouesse, M., Minvielle-Sebastia, L. and Lacroute, F. (1991). A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7(6): 609-615.
  2. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. and Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2): 115-132.
  3. Mirouze, M., Sels, J., Richard, O., Czernic, P., Loubet, S., Jacquier, A., Francois, I. E., Cammue, B. P., Lebrun, M., Berthomieu, P. and Marquès, L. (2006). A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J 47(3): 329-342.
  4. Shahzad, Z., Ranwez, V., Fizames, C., Marquès, L., Le Martret, B., Alassimone, J., Gode, C., Lacombe, E., Castillo, T., Saumitou-Laprade, P., Berthomieu, P. and Gosti, F. (2013). Plant Defensin type 1 (PDF1): protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri. New Phytol 200(3): 820-833.
  5. Winston, F., Dollard, C. and Ricupero-Hovasse, S. L. (1995). Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11(1): 53-55.

简介

基因在出芽酵母酿酒酵母(酿酒酵母)中的异源表达特别适合于在细胞水平上功能性研究相应的编码蛋白质(Bonneaud等人, ,1991)。这主要是因为许多具有特异性活性缺陷的菌株是可获得的,并且可以通过真核生物界存在的同源基因来补充( http: /www.yeastgenome.org/)。然而,我们在这里描述的协议不是互补,而是"获得功能"测定。它是基于滴试验测定,我们已经设置以评估由异源基因在野生型S中的表达赋予的锌细胞耐受性。酿酒厂。将表达目的异源基因的酵母培养物的不同稀释液在一系列富锌平板上生长,然后与表达空载体的对照酵母进行比较。使用不同浓度的酵母和锌对于在酵母转化后成功描述锌耐受性表型是必要的(Mirouze等人,2006)。该试验也已经证明对于区分基因家族的相关成员是有价值的,如拟南芥属植物防御素类型1所例证的(Shahzad等人,2013)。

关键字:植物防御素, 非生物胁迫, 锌的耐受性, 酵母

材料和试剂

  1. 塑料箔(Saran Wrap)
  2. 无菌正方形12cm×12cm培养皿(CML HealthCare,目录号:BP 120SJ)
  3. Sterile-R Filtropur S(0.2μM)(SARSTEDT AG,目录号:83.1826.001)
  4. 20ml无针无菌注射器(Medline Scientific,目录号:BS-20ES)
  5. 50ml无菌管(SARSTEDT AG,目录号:62.547.254)
  6. 高压灭菌的1.5ml Eppendorf管(SARSTEDT AG,目录号:72.690)
  7. 无菌接种环(Greiner Bio One International GmbH,目录号:731175)
  8. 无菌移液器吸头
  9. 亲本BY4741酿酒酵母菌株(Winston等人,1995; Brachmann等人,1998)具有基因型MATa, his3Δ1,leu2Δ0,met15Δ0,ura3Δ0)表达:pFL38H载体(Bonneaud等,1991),补充his3Δ1/em>营养缺陷
  10. 含有补充his3Δ1营养缺陷型的pFL38H载体和补充ura3Δ0突变的pYX212载体(Ingenius,R& D systems)包括信号肽(Shahzad等人,2013)的拟南芥 ,即
  11. 不含氨基酸且不含硫酸铵的酵母氮碱(YNB)(BD,Difco,目录号:233520)
  12. 硝酸铵(NH 4 NO 3)(Sigma-Aldrich,目录号:A9642)
  13. D(+)无水葡萄糖(C 6 H 12 SO 6)(EUROMEDEX,目录号:UG3050)
  14. 琥珀酸(C 4 H 6 6 O 4)(Sigma-Aldrich,目录号:S7501)
  15. 氢氧化钾(KOH)(Bio Basic Canada Inc.,目录号:PB0441)
  16. 琼脂糖D-5型(EUROMEDEX,目录号:D5)
  17. 硫酸锌七水合物(ZnSO 4·7H 2 O 7H 2 O)(Sigma-Aldrich,目录号:Z4750)
  18. L-亮氨酸(C 6 H 13 NO 3 NO 2)(Sigma-Aldrich,目录号:L8912)
  19. L-甲硫氨酸(C 15 H 11 NO 2 S)(Sigma-Aldrich,目录号:M9625)
  20. 无菌超纯水
  21. 0.5 M硫酸锌
  22. 10 N KOH(见配方)
  23. 0.075 M L-亮氨酸(参见配方)
  24. 0.1 M L-甲硫氨酸(参见配方)
  25. 无菌液体和固体选择性酵母氮源(YNB)培养基(参见配方)

设备

  1. 无菌250ml硼硅酸盐玻璃锥瓶
  2. 1000 ml瓶
  3. 典型的20μl移液器
  4. 高压灭菌器
  5. 加热炉(Universal Memmert,型号:400)
  6. 新不伦瑞克 TM 搅拌器(Eppendorf,moldel:Innova 44)
  7. 正常细胞密度计(Thermo Fisher Scientific,型号:40)
  8. 层流罩(ADS,型号:VP120)
  9. 离心机(Eppendorf,型号:5804R)
  10. 整体式水净化系统(Merck Millipore Corporation,型号:Milli-Q +)
  11. 扫描仪(爱普生,型号:124OU)

程序

注意:对于每个要分析的酵母克隆。
第-1天

  1. 使用无菌接种环接种待测试的每个酵母克隆 在20ml无菌250ml玻璃瓶中的液体选择性YNB培养基中 注意:对每个构建体(含有重组质粒或对照质粒)测试至少三个独立的酵母克隆。
  2. 在30℃下在200rpm搅拌下培养酵母细胞过夜(O/N)。

第0天
准备培养皿滴试验。

  1. 制备无菌选择性YNB固体培养基,含有适当的 氨基酸和适当的硫酸锌浓度进行测试
  2. 在层流通量罩下,在每个正方形12cm×12cm培养皿中倒入50ml无菌选择性YNB固体培养基。
    注意:对锌过量的耐受性通常在最后测试 浓度为23,25,27.5,30和32.5mM。控制条件为 ?不含锌(≈1.4μM)。亮氨酸和甲硫氨酸营养缺陷型是 ?通过向其中添加相应的氨基酸来补充 最终浓度分别为0.75mM和0.33mM。
  3. 让介质固化,盖子打开,在15分钟。关闭和存放方形的培养皿在敞篷下。
    准备酵母进行液滴测定。
    注意:所有随后用酵母的操作在无菌条件下在层流罩中进行。
  4. 在无菌的50ml Falcon管中转移O/N酵母培养物
  5. 在室温下以5,000xg离心5分钟。
  6. 弃去上清液。
  7. 用相同体积的无菌水(20ml)悬浮酵母培养物
  8. 重复步骤7到9.
  9. 在合适的稀释度(通常为1/10)后测量600nm处的光密度,以获得约0.4的OD <600> nm 。
  10. 在1.5ml无菌Eppendorf管中,进行适当的酵母培养 用无菌水稀释,以便在500μl中达到OD 600nm 1分之一 体积,然后进行连续稀释,以便达到10μM-1的OD 600nm sub。 ?10 -2 ,10 -3
  11. 使用移液器和无菌 过滤嘴将每种酵母稀释液10μl滴到选择性YNB上 补充有各种最终浓度的ZnSO 4的培养基 ?1)。
    注意:
    1. 对于步骤10至12:酵母细胞沉积 特别快;因此确保充分搅拌酵母菌培养物 ?在采集样品之前和/或在测量a的光密度之前 悬浮和/或滴液测定之前。
    2. 对于液滴测定,将每个液滴的中心分开≈1cm。
  12. 允许滴在层流通量罩下干燥
  13. 关闭培养皿,并包装他们的塑料箔(类型Saran Wrap)。
  14. 在30°C孵育箱中倒置孵育
  15. 每天监测酵母生长,并通过扫描记录结果 培养皿在黑色屏幕下以每英寸600点(dpi)分辨率 (通常每次扫描少于一分钟)。对于每个锌浓度, 将表达目的蛋白质的酵母与酵母进行比较 拥有空的pYX212 注意:对照板,即不含锌 添加,通常在孵育后2天读取。这是必要的 验证目标蛋白的表达不会干扰 酵母在非常低的锌浓度下生长。镀锌板 测定(在加入锌时)通常在4至7天后读取 孵化。

代表数据


图1. 体外 氨基酸取代影响AhPDF1.4和AtPDF1.4锌耐受能力的功能特征。 系列稀释的 酿酒酵母BY4741菌株,其表达携带AhPDF1.4,AtPDF1.4或其突变形式的pYX212空载体(EV)或pYX212。产生A28至G(A28G)或S54至R(S54R)或两者(A28G-S54R)的替换。将各种酵母转化体点样在补充有1.4μM(对照)或27.5mM ZnSO 4的培养基上,如上图所示。每个点用10μl在下面提到的OD 600nm处稀释的酵母培养物制备。在第2天拍照用于对照,第11天用于Zn处理;它们是用三个独立的酵母转化体进行的三个实验的代表。转载自(Shahzad等人,2013;补充图4)。

笔记

BY4741 S。酿酒酵母菌株具有几种营养缺陷型,特别是用于产生氨基酸组氨酸(his3Δ1)。游离组氨酸是有效的金属螯合剂,并且如果该金属存在于培养基中,则可以特别地螯合锌。因此,组氨酸(ε3Δ1)营养缺陷型必须通过相应的HIS 基因(这里从pFL38质粒表达)的表达互补,而不是通过加入游离组氨酸,这将偏差锌耐受性试验。

食谱

  1. 10 N KOH
    使用Sterile-R Filtropur S(0.2μM)过滤灭菌 保持在+4°C
  2. 0.075μML-亮氨酸 使用Sterile-R Filtropur S(0.2μM)过滤灭菌 保持在+4°C
  3. 0.1M L-甲硫氨酸 使用Sterile-R Filtropur S(0.2μM)过滤灭菌 保持在+4°C
  4. 无菌液体和固体选择性YNB培养基(1 L)
    1. 混合在超纯水中
      不含氨基酸而不含硫酸铵的YNB:1.7g
      NH 4 NO 3:6.4g
      D-葡萄糖:20g
      琥珀酸:5.9g
    2. 用10 N KOH将pH调节至4.5
    3. 调整体积并在1000 ml瓶中分装500 ml
    4. 对于固体媒体添加:
      琼脂糖:20g/L
    5. 在120℃下高压灭菌20分钟
    6.  冷却固体培养基后,加入必要的无菌氨基酸 补充营养缺陷型(这里是亮氨酸和甲硫氨酸)的溶液 无菌锌,以达到将要测试的浓度 注意:应注意加入必要的无菌氨基酸溶液 ?和固体培养基冷却至约40至45℃时的无菌锌。 这将防止加入的氨基酸的降解和 金属沉淀。

致谢

这项工作得到了蒙彼利埃大学(LM),法国巴黎高等教育委员会(PB),巴基斯坦高等教育委员会(ZS)和国家科学研究中心(FG)的支持。

参考文献

  1. Bonneaud,N.,Ozier-Kalogeropoulos,O.,Li,G.Y.,Labouesse,M.,Minvielle-Sebastia,L。和Lacroute,F。(1991)。 低和高拷贝复制,整合和单链的家族。酿酒酵母。大肠杆菌穿梭载体。 酵母 7(6):609-615。
  2. Brachmann,C.B.,Davies,A.,Cost,G.J.,Caputo,E.,Li,J.,Hieter,P.and Boeke,J.D。(1998)。 来源于酿酒酵母的设计者缺失菌株S288C:一组有用的菌株 酵母 14(2):115-132。
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引用:Shahzad, Z., Marquès, L., Berthomieu, P. and Gosti, F. (2015). An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast. Bio-protocol 5(22): e1653. DOI: 10.21769/BioProtoc.1653.
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