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Trimolecular Fluorescence Complementation (TriFC) Assay for Direct Visualization of RNA-Protein Interaction in planta
三分子荧光互补(TriFC)实验直接观察植物中RNA-蛋白质的相互作用   

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Abstract

RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-Protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) (Schonberger et al., 2012). Target RNA is tagged with 6xMS2 and MCP and RNA binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-Protein interaction in vivo is observed by confocal microscope.

Keywords: Long non-coding RNA(长非编码RNA), RNA-Protein interaction(RNA-蛋白相互作用), TriFC(TriFC), Tobacco transient expression(烟草瞬时表达)

Background

Recently, a variety of types of long-noncoding RNAs (lncRNAs) has been identified and shown to play important roles in transcriptional regulation and chromatin modification (St Laurent et al., 2015). So far, most of the molecular mechanisms for lncRNA-mediated functions are closely related with RNA-Protein interactions (St Laurent et al., 2015). Therefore, an experiment for RNA-Protein interaction is a key step in functional study of lncRNAs. In plants, molecular functions of lncRNAs are only beginning to be characterized, and the molecular basis of lncRNA-mediated gene regulation is still poorly understood. Though techniques for RNA visualization in plants have been well developed, visual assay for RNA-Protein interaction in plant is still poorly developed (Christensen et al., 2010). To develop the visual assay for RNA-Protein interaction in plants, we modified and combined MS2 system for RNA imaging technique with conventional BiFC system for protein-protein interaction (Schonberger et al., 2012) (Figure 1D). We generated binary Gateway vectors (pBA3130, 3132, 3134, and 3136) for transient BiFC assay (Seo et al., 2017) and got binary Gateway vectors (pBA-GW-6xMS2 and pBA-6xMS2-GW) for RNA tagging from Dr. Ulrich Z. Hammes (Schonberger et al., 2012). We tested and confirmed this TriFC assay was working well in plants with lncRNA, ELENA1, and MED19a protein (Seo et al., 2017). TriFC assay in plants will provide new insights in interaction between lncRNAs and proteins.

Materials and Reagents

  1. Pipette tips (Thermo Fisher Scientific, Fisher ScientificTM BasixTM Universal Tips)
  2. Fisherbrand sterile 100 x 15 mm polystyrene Petri dish (Fisher Scientific, catalog number: FB0875713 )
  3. 1 ml syringes (BD, catalog number: 302100 )
  4. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  5. Agrobacterium tumefaciens (strain GV3101)
  6. Nicotiana benthamiana (N. benthamiana) plants; 2-4 weeks old (6-10 leaves stage)
  7. Gateway entry clones for RNA, RNA binding protein (RNA-BP), and MCP
  8. TriFC Gateway destination vectors (pBA3130, 3132, 3134, 3136, pBA-GW-MS2, and pBA-MS2-GW)
  9. LR clonase II (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11791100 )
  10. LB medium powder (MP Biomedicals, catalog number: 113002082 )
  11. Spectinomycin (1,000x; 100 mg/ml) (Sigma-Aldrich, catalog number: S4014 )
  12. Gentamycin (1,000x; 50 mg/ml) (Sigma-Aldrich, catalog number: G1264 )
  13. Kanamycin (1,000x; 100 mg/ml) (Sigma-Aldrich, catalog number: K0200000 )
  14. Bacto agar (BD, BactoTM, catalog number: 214010 )
  15. Ethanol or DMSO
  16. MES (Sigma-Aldrich, catalog number: M8250 )
  17. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  18. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  19. LB media (see Recipes)
  20. LB agar media (see Recipes)
  21. 100 mM acetosyringone stock (see Recipes)
  22. LB-MES (pH 5.6) (see Recipes)
  23. Resuspension solution (see Recipes)

Equipment

  1. PIPETMAN ClassicTM Pipettes (Gilson, model: P1000, P100, P20, catalog number: F123602 , F123615 , F123600 )
  2. Centrifuge for 50 ml tubes (Beckman Coulter, model: Avanti® J-20XP )
  3. Spectrometer (Biochrom, model: Ultrospec 2100 pro )
  4. Confocal laser scanning microscope (ZEISS, model: LSM 780 )
  5. Autoclave (TOMY DIGITAL BIOLOGY, model: ES-215 )
  6. Laminar flow cabinet (NuAire, model: NU-440-400E )
  7. Incubator (MMM Medcenter Einrichtungen, model: INCUCELL 55 )
  8. Shaking incubator (Infors, model: Multitron Standard )

Software

  1. ZEN (Image analysis program for ZEISS confocal microscope)

Procedure

  1. Construct TriFC vectors by LR reactions (Gateway) with your entry clones (RNA, RNA-BP, and MCP) following the manufacturer’s instruction; LR reaction MCP and RNA-BP entry clones with Gateway BiFC vectors (pBA3130, 3132, 3134, and 3136; Figures 1A and 1B) (Seo et al., 2017). LR reaction RNA entry clones with Gateway MS2 tagging vectors (pBA-6xMS2-GW or pBA-GW-6xMS2; Figure 1C) (Schonberger et al., 2012). In this study, we used pENTR-ELENA1 for RNA entry clone, pENTR-MED19a for RNA-BP entry clone, and pENTR-MCP for MCP entry clone.


    Figure 1. Schematic representation of the vectors for TriFC system. A. Schematic diagram of expression vectors of RNA binding protein fused with YFP fragments; B. Schematic diagram of expression vectors of MCP fused with YFP fragments; C. Schematic diagram of MS2 tagged RNA expression vectors; D. Illustration of the TriFC system for RNA-Protein interaction.

  2. Transform 50 µl Agrobacterium competent cells (1010 cfu/ml) with each DNA construct (50 ng) (3 different vectors; Figures 1A, 1B, and 1C) by heat shock method (Höfgen and Willmitzer, 1988).
  3. Spread the cells on the LB/spectinomycin agar media (see Recipes) and grow for 2 days in an incubator at 28-30 °C.
  4. Inoculate one single colony of Agrobacterium in 5 ml LB with appropriate antibiotics (see Recipes). Grow overnight in a shaking incubator with 200 rpm at 28-30 °C.
  5. Use 250 μl of the overnight culture to inoculate 10 ml LB-MES (pH 5.6) (see Recipes) (with same antibiotics) plus 4 μl of 100 mM acetosyringone (see Recipes) and grow overnight in the shaking incubator with 200 rpm at 28-30 °C.
  6. Measure the A600 of overnight cultures (OD = 0.8-1.0).
  7. Collect bacterial cells (4,000 x g, 10 min) at room temperature and resuspend the pellet in resuspension solution (see Recipes). Adjust the final A600 to 0.4.
  8. Leave on a bench (room temperature) for over 4 h (or overnight) before infiltration.
  9. Transfer 1 ml of each Agrobacterium cell suspension into a 10 ml tube and mix well (Figure 2A).
  10. Perform the infiltration with a 1 ml syringe. Simple press the syringe (no needle) on the underside of a leaf (Note: Avoid cotyledons.), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading of ‘wetting’ area in the leaf and label the infiltrated region. (Figure 2B).
  11. Observe the YFP signals under a confocal laser scanning microscope 2-3 days after infiltration (Figure 2C). Excitation wavelength is 514 nm and detection rage of emission wavelength is 520-550 nm.


    Figure 2. Transient expression for TriFC assay in N. benthamiana. A. Mix three different Agrobacterium suspensions. 1 carries the RNA-BP construct (Figure 1A), 2 carries the MCP construct (Figure 1B), and 3 carries the MS2 tagged RNA construct (Figure 1C). B. Infiltration in tobacco leaves with a syringe; C. Observe the infiltrated region with a confocal microscope 2-3 days after infiltration.

Data analysis

  1. To avoid false positive signal, we checked MED19a and MCP interaction (Figure 3 [top]), and MED19a, MCP, and antisense ELENA1 combination (Figure 3 [middle]) as negative controls. We confirmed that there was no YFP signal in those negative controls (we tested three independent plants).
  2. We could observe YFP signal in MED19a, MCP, and sense ELENA1 combination (Figure 3 [bottom]). We tested three independent plants and observed YFP signals in all the plants.
  3. Detailed information about ELENA1 and MED19a is described in (Seo et al., 2017).


    Figure 3. Confocal microscopy images of TriFC assay. nYFP was fused to MED19a and cYFP was fused to MCP. 6xMS2 nucleotide sequences fused to sense or anti-sense ELENA1. Confocal images were taken 3 days after infiltration. Scale bars = 20 μm.

Notes

  1. An optimal combination of 6xMS2 tag, nYFP, and cYFP orientation is sometimes critical for the TriFC assay. Therefore, other combinations should be tried if the signal is not detected.
  2. Post-transcriptional gene silencing (PTGS) is a major cause for the lack of efficiency in transient expression experiments (Voinnet et al., 2003). Therefore, we usually co-infiltrate UBQ:p19, which suppresses PTGS, together with other constructs as it greatly improves the efficiency.

Recipes

  1. LB media
    LB media with appropriate antibiotics (kanamycin [50 µg/ml] for MS2 tagging vector [Figure 1C] and spectinomycin [50 µg/ml] for partial YFP fused vectors [Figures 1A and 1B])
  2. LB agar media
    LB media with 2% Bacto agar and appropriate antibiotics (kanamycin [50 µg/ml] for MS2 tagging vector [Figure 1C] and spectinomycin [50 µg/ml] for partial YFP fused vectors [Figures 1A and 1B])
  3. 100 mM acetosyringone stock
    100 mM acetosyringone stock in ethanol or DMSO, stored at -20 °C
  4. LB-MES (pH 5.6)
    LB with 10 mM MES (pH 5.6)
  5. Resuspension solution
    10 mM MgCl2
    10 mM MES-KOH (pH 5.6)
    100 μM acetosyringone
    Note: Added after autoclaving and immediately before using.

Acknowledgments

We thank Dr. Ulrich Z. Hammes for the Gateway 6xMS2 tagging vectors. This work was supported by Singapore NRF RSSS Grant (NRF-RSSS-002).

References

  1. Christensen, N. M., Oparka, K. J. and Tilsner, J. (2010). Advances in imaging RNA in plants. Trends Plant Sci 15(4): 196-203.
  2. Höfgen, R. and Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16(20): 9877.
  3. Schonberger, J., Hammes, U. Z. and Dresselhaus, T. (2012). In vivo visualization of RNA in plants cells using the λN22 system and a GATEWAY-compatible vector series for candidate RNAs. Plant J 71(1): 173-181.
  4. Seo, J. S., Sun, H. X., Park, B. S., Huang, C. H., Yeh, S. D., Jung, C. and Chua, N. H. (2017). ELF18-INDUCED LONG-NONCODING RNA associates with mediator to enhance expression of innate immune response genes in Arabidopsis. Plant Cell 29(5): 1024-1038.
  5. St Laurent, G., Wahlestedt, C. and Kapranov, P. (2015). The Landscape of long noncoding RNA classification. Trends Genet 31(5): 239-251
  6. Voinnet, O., Rivas, S., Mestre, P. and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5): 949-956.

简介

RNA-蛋白质相互作用在各种真核生物过程中起重要作用。 RNA /蛋白质复合物在植物中亚细胞定位的分子成像对于理解这些相互作用至关重要。然而,到目前为止,尚未开发在活植物中形成RNA-蛋白质相互作用的方法。最近,我们开发了一种三分子荧光互补(TriFC)系统,用于在烟草叶中瞬时表达的RNA-蛋白质相互作用的体内可视化。在这种方法中,我们将传统的双分子荧光互补(BiFC)系统与MS2系统(噬菌体MS2外壳蛋白[MCP]及其结合RNA序列[MS2序列])(Schonberger等人,2012)相结合, 。目标RNA用6xMS2标记,MCP和RNA结合蛋白与YFP片段融合。编码这种融合RNA和蛋白质的DNA构建体用土壤杆菌悬浮液渗入烟草叶中。通过共焦显微镜观察体内的RNA-蛋白质相互作用
【背景】近来,多种类型的长非编码RNA(lncRNA)已经被鉴定并显示出在转录调节和染色质修饰中起重要作用(St Laurent等人,2015)。到目前为止,lncRNA介导的功能的大多数分子机制与RNA-蛋白质相互作用密切相关(St Laurent等人,2015)。因此,RNA-蛋白质相互作用的实验是lncRNA功能研究的关键一步。在植物中,lncRNA的分子功能才开始被表征,而且lncRNA介导的基因调控的分子基础仍然很少被理解。虽然植物中RNA可视化技术已经发展良好,但是植物中RNA-蛋白质相互作用的视觉测定仍然很差(Christensen等,2010)。为了开发植物中RNA-蛋白质相互作用的视觉测定,我们将MS2系统用于RNA成像技术与传统的BiFC系统进行蛋白质 - 蛋白质相互作用(Schonberger等人,2012)(图1D) )。我们为瞬态BiFC测定(Seo等人,2017)生成二元网关向量(pBA3130,3132,3134和3136),并获得二进制网关向量(pBA-GW-6xMS2和pBA-6xMS2- GW)用于来自Ulrich Z.Hammes博士的RNA标记(Schonberger等人,2012)。我们测试并证实,该TriFC测定在具有lncRNA,ELENA1和MED19a蛋白质的植物中工作良好(Seo等人,2017)。植物中的TriFC测定将为lncRNA和蛋白质之间的相互作用提供新的见解。

关键字:长非编码RNA, RNA-蛋白相互作用, TriFC, 烟草瞬时表达

材料和试剂

  1. 移液器吸头(Thermo Fisher Scientific,Fisher Scientific TM Basix TM 通用提示)
  2. Fisherbrand无菌100 x 15 mm聚苯乙烯培养皿(Fisher Scientific,目录号:FB0875713)
  3. 1 ml注射器(BD,目录号:302100)
  4. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  5. 根癌土壤杆菌(GV3101)
  6. 本土烟草(Nicotiana benthamiana)( N。本哈米纳)植物; 2-4周龄(6-10叶阶段)
  7. 用于RNA,RNA结合蛋白(RNA-BP)和MCP的门户入口克隆
  8. TriFC网关目标向量(pBA3130,3132,3134,33136,pBA-GW-MS2和pBA-MS2-GW)
  9. LR克隆酶II(Thermo Fisher Scientific,Invitrogen TM,目录号:11791100)
  10. LB中等粉末(MP Biomedicals,目录号:113002082)
  11. 壮观霉素(1,000x; 100mg / ml)(Sigma-Aldrich,目录号:S4014)
  12. 庆大霉素(1,000x; 50mg / ml)(Sigma-Aldrich,目录号:G1264)
  13. 卡那霉素(1,000x; 100mg / ml)(Sigma-Aldrich,目录号:K0200000)
  14. Bacto琼脂(BD,Bacto TM ,目录号:214010)
  15. 乙醇或DMSO
  16. MES(Sigma-Aldrich,目录号:M8250)
  17. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  18. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  19. LB媒体(见食谱)
  20. LB琼脂培养基(见食谱)
  21. 100mM乙酰丁香酮(见配方)
  22. LB-MES(pH 5.6)(参见食谱)
  23. 再悬浮液(参见食谱)

设备

  1. PIPETMAN Classic TM 移液器(Gilson,型号:P1000,P100,P20,目录号:F123602,F123615,F123600)
  2. 离心机用于50ml管(Beckman Coulter,型号:Avanti J-20XP)
  3. 光谱仪(Biochrom,型号:Ultrospec 2100 pro)
  4. 共焦激光扫描显微镜(ZEISS,型号:LSM 780)
  5. 高压釜(TOMY DIGITAL BIOLOGY,型号:ES-215)
  6. 层流柜(NuAire,型号:NU-440-400E)
  7. 孵化器(MMM Medcenter Einrichtungen,型号:INCUCELL 55)
  8. 振动孵化器(Infors,型号:Multitron Standard)

软件

  1. ZEN(ZEISS共焦显微镜图像分析程序)

程序

  1. 按照制造商的说明,通过LR反应(Gateway)与您的进入克隆(RNA,RNA-BP和MCP)构建TriFC载体;具有Gateway BiFC载体(pBA3130,33132,3134和3136;图1A和1B)(Seo等人,2017)的LR反应MCP和RNA-BP进入克隆。具有Gateway MS2标记载体(pBA-6xMS2-GW或pBA-GW-6xMS2;图1C)的LR反应RNA进入克隆(Schonberger等人,2012)。在本研究中,我们使用pENTR-ELENA1进行RNA进入克隆,pENTR-MED19a用于RNA-BP进入克隆,pENTR-MCP用于MCP进入克隆。


    图1. TriFC系统载体的示意图。 A.与YFP片段融合的RNA结合蛋白的表达载体示意图; B.与YFP片段融合的MCP的表达载体示意图; MS2标记的RNA表达载体示意图; D.用于RNA-蛋白质相互作用的TriFC系统的图示
  2. 通过加热使每个DNA构建体(50ng)(3种不同载体;图1A,1B和1C)转化50μl感受态细胞(10μg/ ml cfu / ml)休克方法(Höfgenand Willmitzer,1988)
  3. 将细胞扩散到LB /壮观霉素琼脂培养基上(参见食谱),并在28-30℃的培养箱中培养2天。
  4. 在适当的抗生素的5ml LB中接种一个单一的土壤杆菌菌落(参见食谱)。在振荡的培养箱中,在28-30℃下以200rpm的速度生长过夜
  5. 使用250微升的过夜培养物接种10ml LB-MES(pH 5.6)(参见食谱)(含相同抗生素)加4μl100mM乙酰丁香酮(参见食谱),并在振荡培养箱中以200rpm在28℃下生长过夜-30°C。
  6. 测量过夜培养物的A 600(OD = 0.8-1.0)。
  7. 在室温下收集细菌细胞(4,000 x g,10分钟),并将沉淀重新悬浮在再悬浮溶液中(参见食谱)。将最终的A <600> 调整为0.4。
  8. 在长时间(室温)下放置超过4小时(或过夜),然后再渗透。
  9. 将1ml各种农杆菌细胞悬浮液转移到10ml管中并充分混合(图2A)。
  10. 用1 ml注射器进行渗透。简单地按下叶子下面的注射器(无针)(注意:避免子叶。),并用手指在另一侧施加反压力。经常观察到成功的渗透是叶片中“润湿”区域的扩散,并标记渗透区域。 (图2B)
  11. 在共聚焦激光扫描显微镜观察2-3天后观察YFP信号(图2C)。激发波长为514nm,发射波长的检测范围为520-550nm

    图2.NFC中TriFC测定的瞬时表达。本土汉堡。 A.混合三种不同的农杆菌悬浮液。 1携带RNA-BP构建体(图1A),2携带MCP构建体(图1B),3携带MS2标记的RNA构建体(图1C)。 B.用注射器在烟草叶中渗透; C.渗透后2-3天用共焦显微镜观察浸润区域

数据分析

  1. 为了避免假阳性信号,我们检查了MED19a和MCP相互作用(图3 [top])和MED19a,MCP和反义ELENA1组合(图3 [中])作为阴性对照。我们确认在这些阴性对照中没有YFP信号(我们测试了三个独立的植物)
  2. 我们可以在MED19a,MCP和感应ELENA1组合中观察到YFP信号(图3 [底部])。我们测试了三个独立的植物,并观察了所有植物中的YFP信号
  3. 有关ELENA1和MED19a的详细信息,请参见(Seo等人,2017)。


    图3. TriFC测定的共聚焦显微镜图像。将nYFP融合到MED19a,将cYFP融合到MCP。与感觉或反义ELENA1融合的6xMS2核苷酸序列。渗透后3天采集共聚焦图像。刻度棒=20μm。

笔记

  1. 对于TriFC测定,6xMS2标签,nYFP和cYFP方向的最佳组合有时是至关重要的。因此,如果未检测到信号,则应尝试其他组合。
  2. 转录后基因沉默(PTGS)是短暂表达实验中缺乏效率的主要原因(Voinnet等人,2003)。因此,我们通常会共同渗透UBQ:p19,抑制PTGS与其他结构,因为它大大提高了效率。

食谱

  1. LB媒体
    具有合适抗生素的LB培养基(用于MS2标记载体的卡那霉素[50μg/ ml]]和部分YFP融合载体的壮观霉素[50μg/ ml] [图1A和1B])
  2. LB琼脂培养基
    具有2%Bacto琼脂的LB培养基和用于部分YFP融合载体的适当抗生素(卡那霉素[50μg/ ml]用于MS2标记载体[图1C]和壮观霉素[50μg/ ml] [图1A和1B])
  3. 100毫克乙酰丁香酮股票
    100mM乙酰丁香酮在乙醇或DMSO中储存,储存于-20°C
  4. LB-MES(pH 5.6)
    具有10mM MES(pH 5.6)的LB /
  5. 重悬液
    10mM MgCl 2
    10mM MES-KOH(pH5.6)
    100μM乙酰丁香酮
    注意:在高压灭菌后立即使用之前添加。

致谢

我们感谢Ulrich Z.Hammes博士为Gateway 6xMS2标记向量。这项工作得到了新加坡NRF RSSS授权(NRF-RSSS-002)的支持。

参考

  1. Christensen,N.M.,Oparka,K.J。和Tilsner,J。(2010)。 植物中成像RNA的进展 趋势植物S ci 15(4):196-203。
  2. Höfgen,R。和Willmitzer,L。(1988)。 存放农杆菌转基因的感受态细胞 Nucleic Acids Res 16(20):9877.
  3. Schonberger,J.,Hammes,U.Z.and Dresselhaus,T。(2012)。 使用λN可植入植物细胞中的RNA, 22系统和候选RNA的GATEWAY兼容载体系列。植物J 71(1):173-181。
  4. Seo,J.S.,Sun,H.X。,Park,B.S.,Huang,C.H.,Yeh,S.D.,Jung,C.and Chua,N.H。(2017)。 ELF18诱导的长期非编码RNA与介体相关联,以增强先天免疫反应基因的表达>拟南芥。 植物细胞 29(5):1024-1038。
  5. St Laurent,G.,Wahlestedt,C.and Kapranov,P。(2015)。 长非编码RNA分类的风景趋势Genet < 31(5):239-251
  6. Voinnet,O.,Rivas,S.,Mestre,P。和Baulcombe,D。(2003)。 基于抑制番茄丛生特技的p19蛋白的基因沉默的植物中的增强的瞬时表达系统病毒。植物J 33(5):949-956。
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引用:Seo, J. and Chua, N. (2017). Trimolecular Fluorescence Complementation (TriFC) Assay for Direct Visualization of RNA-Protein Interaction in planta. Bio-protocol 7(20): e2579. DOI: 10.21769/BioProtoc.2579.
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