搜索

A Chemical Genetic Screening Procedure for Arabidopsis thaliana Seedlings
拟南芥幼苗的化学遗传筛选步骤   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Unbiased screening approaches are powerful tools enabling identification of novel players in biological processes. Chemical genetic screening refers to the technique of using a reporter response, such as expression of luciferase driven by a promoter of interest, to discover small molecules that affect a given process when applied to plants. These chemicals then act as tools for identification of regulatory components that could not otherwise be detected by forward genetic screens due to gene family redundancy or mutant lethality.
This protocol describes a chemical genetic screen using Arabidopsis thaliana seedlings, which has led to recognition of novel players in the plant general stress response.

Materials and Reagents

  1. Reporter line seeds (this protocol was developed using luciferase under control of a minimal promoter containing four copies of the rapid stress response element - 4xRSRE:LUCIFERASE)
  2. Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M0404 )
  3. Phytoagar (PlantMedia, catalog number: 40100072-2 )
  4. Sterile disposable reagent reservoir (Corning, catalog number: 07-200-128 )
  5. Bleach (Clorox concentrated, 8.25% sodium hypochlorite)
  6. Hydrochloric acid (HCl)
  7. Chemical library (source may vary)
  8. Potassium luciferin (Gold Biotechnology, model: LUCK-1G )
  9. ¼ strength MS media (see Recipes)
  10. 1 mM luciferin (see Recipes)

Equipment

  1. Micropore surgical tape (3M, model: 1530-0 )
  2. 1.5 ml tubes (SealRite, catalog number: 1615-5500 )
  3. 96 well plates with lid: Flat bottom, sterile but not tissue culture treated (SARSTEDT AG, catalog number: 82.1571.001 )
  4. Filter paper (here used Whatman 1440 125, pore size does not matter)
  5. Toothpicks
  6. Laminar flow hood or biosafety cabinet (here used SterilGARD ii, the Baker Company)
  7. Chemical fume hood (here used St. Charles)
  8. Vacuum chamber
  9. Multichannel pipette (2-20 and 20-200 µl) (Rainin)
  10. Temperature-controlled growth cabinet (here used Conviron, model: GR48 )
  11. Charge-Couple Device camera (CCD camera; Andor DU434-BV CCD)
  12. Dehesh lab Perl scripts (http://www-plb.ucdavis.edu/labs/dehesh/dehesh-lab-code.html)

Software

  1. ANDOR Solis analysis software (Andor technology, v15, http://www.andor.com/scientific-software)
  2. ImageJ image analysis software (http://imagej.nih.gov/ij/)

Procedure

  1. Preparation
    1. To synchronize germination and improve developmental consistency among seedlings, pre-select the larger and more uniformly sized seeds from selected Arabidopsis thaliana (Arabidopsis) reporter line by pouring seeds several times along a sheet of paper. The smallest seeds tend to stick to paper via electrostatic forces and can easily be discarded.
      Note: Using seeds harvested at the same time, from plants grown under the same conditions, will also improve seedling uniformity.
    2. Autoclave filter paper, toothpicks, and filter tips for 200 µl pipet. Surface-sterilize size-selected seeds:
      1. Aliquot approximately 50-100 µl size-selected seeds in 1.5 ml tube with chlorine-resistant label.
      2. In chemical fume hood, place open 1.5 ml tubes in rack in vacuum chamber with beaker containing ~100 ml bleach.
      3. Add 3 ml HCl to beaker and close vacuum chamber.
      4. Apply vacuum for 5-10 sec to seal chamber.
      5. After 2.5-3 h, open chamber (still in fume hood) and close lid of 1.5 ml tubes.
    3. Prepare solid ¼ strength MS media according to the recipe below.
      Note: This protocol is for seeds grown on solid media, but similar screens have been performed on plants grown in liquid media.

  2. Plating seeds
    1. “Pour” MS media into 96-well plates. Inside sterile hood, pour molten media into sterile reagent reservoir. Set multichannel pipet at desired volume + 10% (for 100 µl, set at 110). Draw media up from reservoir and eject into 96-well plate 1 row at a time.
      Notes:
      1. Bubbles in the wells make plating seeds difficult. To minimize the incidence of bubbles and mis-filled wells, (a) touch the pipet tips to the corner between bottom and side of well before ejecting and (b) do not eject the media fully, only to the first stop of the multichannel pipet. This results in some media remaining in pipet tips, but greatly reduces bubbles.
      2. If you do not work quickly enough, media may solidify in the reservoir or pipet tips. To avoid this we recommend starting with >65 °C media. If it becomes a problem, simply eject and replace tips or reservoir.
    2. Pour sterilized seeds onto filter paper. Using moistened tip of sterile toothpick, pick up and place one seed per well of the 96-well plate.
      Note: This allows a second opportunity for seed size selection. We observe significant differences between responses of border wells, at the edge of the plate, and internal wells. You should test for this difference and if necessary do not apply treatments or measure border wells. If border wells will not be measured then place any remaining smaller seeds in these, using uniformly larger seeds for internal wells.
    3. Tape edges of 96-well plates with micropore surgical tape to maintain sterility and humidity.

  3. Seedling growth
    1. Stratify seeds for 3-5 days in the dark at 4 °C.
    2. Grow seedlings under typical Arabidopsis plate growth conditions (22-24 °C under 16 h light (60-80 µE) /8 h dark).
    3. Six days after placing under light, seedlings should have expanded cotyledons and two visible true leaves, ready for treatment with luciferin (Figure 2A). To reduce background it is best to proceed with luciferin treatment 18-24 h before imaging.
      Note: We apply luciferin via spray, with detergent (see Recipes). To reduce false positives/negatives one must ensure uniform substrate availability by saturating all wells. In screening we achieved this uniformity by spraying twice from approximately 6 inches above the plate for each of left and right sides of the plate, followed by three sprays from approximately 9 inches above the plate tracking from left to right.

  4. Imaging
    1. At seven days post-stratification, move plates from growth chamber to the lab bench several hours prior to imaging.
      Note: In the case of 4xRSRE:LUCIFERASE-expressing seedlings, plates were moved at 8 am, four hours before imaging at noon. This was done to minimize modulation of physiological responses, and by extension luciferase activity, associated with changing plant environment. The time interval between moving and imaging may vary depending on the nature of the experiment, but beginning imaging at the same time throughout screen is advisable due to possible interaction with circadian-regulated processes.



      Figure 1. Organization of chemical template plate and corresponding treatment plates. In this example row 2 of the chemical template plate is used to treat rows 2 and 3 of both sample plates, row 3 of chemical template plate is used to treat rows 4 and 5 of both sample plates, and row 4 of the chemical template plate is used to treat rows 6 and 7 of both sample plates.

    2. Prepare chemicals 1: Dilution. It will likely be necessary to adjust the concentration of the chemical library by diluting in water or other appropriate solvent before treatment, based on the output observed. In our experiments we observed optimal working concentrations between 20 and 40 µM-few chemicals had any visible effect below 10 µM. Chemicals may be applied as a 10 µl drop at working concentration to treatment wells; dilute each chemical to produce a total volume sufficient for 12 µl per well to account for pipetting errors.
      Note: Some chemical solvents, such as dimethyl sulfoxide (DMSO), may affect the output signal. Prior to a large-scale screen, one should check for solvent effects at desired dilution level. We recommend a “diluted solvent” negative control, in addition to a no treatment control and where possible a positive control, on each plate.
    3. Prepare chemicals 2: Template plate. Prepare a chemical template plate on ice, with working concentration of chemicals in the same arrangement as they will be applied to treatment plate. We recommend applying each chemical to at least 8 replicate wells. This should yield at least 6 suitable seedlings to measure response. If replicate wells are placed in columns, one row on the chemical template plate may apply to multiple rows on the treatment plate (Figure 1).
      Notes:
      1. If border wells will not be assayed this must be reflected in the chemical template plate.
      2. The number of seedlings devoted to each chemical treatment should be determined by variability in the system of interest and practical constraints, i.e. the number of chemicals to be tested.
    4. Treat seedlings by transferring chemicals from template to treatment plate using multichannel pipette. It is important to not disturb the seedlings-if seedlings are wounded by a pipet tip it may change how chemicals are perceived and thus their response. We recommend touching the pipet tips to well sides away from seedlings, as close to agar as possible in order to minimize splashing or chemical adhesion to the side of the well (Figure 2B). Once all chemicals have been applied gently rock the plate for ~5 sec to ensure even chemical distribution in wells.
      Note: In this screening technique, it is not defined how the plant takes up the chemical. The chemical droplet may be taken up from the surface of the agar through the hypocotyl/cotyledon/leaves, in which case the working concentration is perceived. Alternatively, the chemical may be taken up through the roots after chemical diffusion into media. In this case, the media itself further dilutes the chemical and the perceived concentration may be as little as 1/10 the applied working concentration (10 µl chemical applied diluted into 100 µl media).


      Figure 2. Representative 96-well plates. (A) 7-day old seedlings. Note the set of four wells boxed in red, reflecting two of the potential issues in plating. This set of wells shows poor germination resulting in plants too small for reliable analysis, and two seedlings erroneously germinated in one well (marked with *), eliminating them for analysis. Panel (B) shows multichannel pipet chemical application.

    5. Imaging conditions will depend on reporter used, but we recommend imaging for up to triple the time of typical response, to allow for delayed response as chemical diffuses through media, etc. We used a 20 h time course of five minute exposures of a CCD camera within a light-tight box, controlled by the ANDOR Solis v15 image analysis software (http://www.andor.com/scientific-software).

  5. Analysis/re-screening
    1. Depending on the intensity and uniformity of response, an initial qualitative overview may be sufficient for first-round screening, to be followed by a quantitative analysis (Figure 3).
      1. For rapid quantification of combined information on what fraction of each seedling’s total area is luminescent and how bright that luminescence is, we used custom Perl scripts to interpret ImageJ analysis of images exported from ANDOR Solis v. 15. The commented scripts are available at http://www-plb.ucdavis.edu/labs/dehesh/. Briefly, this technique requires users to place a grid of round Regions Of Interest (ROI) in the ImageJ software using the plugin Microarray Profiler. ImageJ quantifies the “white” value for each pixel in these ROI, which can then be normalized to plant area. This technique allows for rapid screening of a large number of wells, but it does not distinguish between the pattern of luciferase activity (i.e. number of pixels with some non-zero “white” value) and the intensity of emitted light (i.e. magnitude of “white” value).
      2. For more defined quantification, small square ROI can be placed one-by-one on individual cotyledons using the ANDOR software. This eliminates the effect of area of luminescence while achieving greater precision in intensity. We found 4pixel-by-4pixel ROI to be an optimal size for seven-day-old Arabidopsis cotyledons. Commented Perl scripts for this analysis are also available at http://www-plb.ucdavis.edu/labs/dehesh/.


        Figure 3. Model data demonstrating two analysis methods. Shown are the same set of four wounded Arabidopsis seedlings at selected time points up to 6 h. In method (i), a grid of ROI (red circles) fits over each whole seedling. Luciferase actitivity is quantified as a fraction of theoretical maximum (picture at timepoint 0 with contrast enhanced). In method (ii), square ROI are placed on individual cotyledons, providing greater precision.

    2. Once putative chemical “hits” have been identified via first-round screening, they must be confirmed in a second and possibly third round. Second round screening should proceed similarly to first round, with the exception that we recommend at least doubling or preferably tripling the number of wells devoted to each chemical, followed by the more carefully defined quantification of response as described in step E1b.
      Note: In the first round of screening, we identified approximately 15% of screened chemicals as putatively affecting 4x RSRE: LUCIFERASE activity. Second round screening confirmed ~10% of these putative chemicals. These numbers should be expected to change depending on the output, sensitivity of reporter, and chemical library selected.
    3.  Confirmed chemical hits may lead to multiple avenues of experimentation, including concentration curves to determine active concentration, developmental time course of chemical effect, analysis of selected chemicals similar in structure or known function, and/or analysis of chemical effect on plant beyond screen output.

Notes

General screening notes

  1. The screen output described here is luminescence from luciferase driven by a minimal promoter containing four copies of the RSRE, but the screening technique may be applied to any reporter system, or visible effects on Arabidopsis seedlings. Simply substitute any pretreatment for luciferin application, necessary imaging equipment for the CCD camera/ANDOR software, and appropriate image analyses.
  2. This screen is designed to observe the immediate effect of chemical treatment, avoiding any secondary effects produced during prolonged exposure. However, if desired, the chemical treatment could be added to molten or solid media before placing seeds in the individual wells, or at any desired stage of Arabidopsis growth.
  3. If adding chemicals early, as well as during pretreatment with luciferin, take care to minimize contamination risks (i.e. use sterile water, keep containers closed as much as possible and if necessary work within sterile laminar flow hood). Contamination may affect the screening output, either directly or through effects on plant fitness.

Recipes

  1. ¼ strength MS media
    Dissolve 1.11 g MS salts in 1 L milli-Q pure H2O, and adjust pH to 5.7 with NaOH
    Measure out 8 g phytoagar into bottle, and pour in pH-adjusted MS solution
    Autoclave, liquid cycle, 30 min
    Note: Although ¼ strength MS media was used here, ½ MS or other plant growth media may work as well or better, and should be tested prior to screening.
  2. 1 mM luciferin
    Prepare 10x (31.824 mg/10 ml) or 100x (318.42 mg/10 ml) stock solution of potassium luciferin in milli-Q H2O
    Dilute down to 1 mM in sterile H2O, add Tween-20 to final concentration of 0.01%

Acknowledgments

This protocol is an expansion of that described in Bjornson et al. (2014). This work was supported by National Institute of Health (R01GM107311), and National Science Foundation (IOS-1036491and IOS-1352478), and Agricultural experimental station (CA-D-PLB-3510-H) grants awarded to KD.

References

  1. Benn, G., Wang, C. Q., Hicks, D. R., Stein, J., Guthrie, C. and Dehesh, K. (2014). A key general stress response motif is regulated non-uniformly by CAMTA transcription factors. Plant J 80(1): 82-92.
  2. Bjornson, M., Benn, G., Song, X., Comai, L., Franz, A. K., Dandekar, A. M., Drakakaki, G. and Dehesh, K. (2014). Distinct roles for mitogen-activated protein kinase signaling and CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 in regulating the peak time and amplitude of the plant general stress response. Plant Physiol 166(2): 988-996.
  3. Drakakaki, G., Robert, S., Szatmari, A. M., Brown, M. Q., Nagawa, S., Van Damme, D., Leonard, M., Yang, Z., Girke, T., Schmid, S. L., Russinova, E., Friml, J., Raikhel, N. V. and Hicks, G. R. (2011). Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proc Natl Acad Sci U S A 108(43): 17850-17855.
  4. Walley, J. W., Coughlan, S., Hudson, M. E., Covington, M. F., Kaspi, R., Banu, G., Harmer, S. L. and Dehesh, K. (2007). Mechanical stress induces biotic and abiotic stress responses via a novel cis-element. PLoS Genet 3(10): 1800-1812.

简介

无偏的筛选方法是能够识别生物过程中的新玩家的有力工具。 化学遗传筛选是指使用报告物应答(例如由感兴趣的启动子驱动的荧光素酶的表达)来发现当应用于植物时影响给定过程的小分子的技术。 这些化学物质因此作为用于鉴定由于基因家族冗余或突变致死性而不能通过正向遗传筛选检测的调节组分的工具。
此协议描述使用拟南芥幼苗的化学遗传屏幕,这导致在植物一般胁迫反应中的新玩家的识别。

材料和试剂

  1. 报告基因种子(该方案使用在含有四个快速应激反应元件--4xRSRE:LUCIFERASE的四个拷贝的最小启动子控制下的萤光素酶开发)
  2. Murashige和Skoog基础培养基(Sigma-Aldrich,目录号:M0404)
  3. Phytoagar(PlantMedia,目录号:40100072-2)
  4. 无菌一次性试剂槽(Corning,目录号:07-200-128)
  5. 漂白剂(Clorox浓缩,8.25%次氯酸钠)
  6. 盐酸(HCl)
  7. 化学图书馆(来源可能不同)
  8. 钾萤光素(Gold Biotechnology,型号:LUCK-1G)
  9. ¼强度MS介质(参见配方)
  10. 1mM荧光素(参见配方)

设备

  1. 微孔手术胶带(3M,型号:1530-0)
  2. 1.5ml管(SealRite,目录号:1615-5500)
  3. 带盖的96孔板:平底,无菌但不进行组织培养处理(SARSTEDT AG,目录号:82.1571.001)
  4. 滤纸(这里使用Whatman 1440 125,孔径无关紧要)
  5. 牙签
  6. 层流罩或生物安全柜(这里使用SterilGARD ii,贝克公司)
  7. 化学通风橱(这里使用圣查尔斯)
  8. 真空室
  9. 多通道移液器(2-20和20-200μl)(Rainin)
  10. 温控增长柜(这里使用Conviron,型号:GR48)
  11. Charge-Couple设备相机(CCD相机; Andor DU434-BV CCD)
  12. Dehesh lab Perl脚本( http://www-plb.ucdavis。 edu/labs/dehesh/dehesh-lab-code.html

软件

  1. ANDOR Solis分析软件(Andor技术,v15, http://www.andor.com/scientific-software
  2. ImageJ图像分析软件( http://imagej.nih.gov/ij/

程序

  1. 准备
    1. 同步萌发和提高发展一致性 幼苗,预选择更大和更均匀大小的种子 选择拟南芥( Arabidopsis )报道系 种子沿着一张纸几次。最小的种子往往 通过静电力粘到纸张上,可以很容易地丢弃 注意:使用同时收获的种子,从下生长的植物 相同的条件,也将提高幼苗的均匀性。
    2. 高压灭菌过滤纸,牙签和过滤嘴200μl移液器。表面消毒大小选择的种子:
      1. 等分大约50-100微升大小选择种子在1.5毫升管与耐氯标签
      2. 在化学通风橱中,在装有约100毫升漂白剂的烧杯的真空室中,将开放的1.5毫升管置于支架中
      3. 向烧杯中加入3 ml HCl,关闭真空室
      4. 施加真空5-10秒以密封腔室。
      5. 2.5-3小时后,打开室(仍然在通风橱)和关闭盖1.5ml管。
    3. 根据下面的配方准备固体1/4强度的MS培养基 注意:此协议是在固体培养基上生长的种子,但是对在液体培养基中生长的植物进行类似的筛选。

  2. 镀种子
    1. 将MS培养基倒入96孔板中。 内无菌罩,倒熔融 培养基进入无菌试剂池。 根据需要设置多通道移液器 体积+ 10%(对于100μl,设置为110)。 从水库中取出介质 一次排出96孔板1行 注意:
      1. 中的气泡 井使电镀种子困难。 最小化的发生率 气泡和填充不良的孔,(a)将移液管尖端触及角落 在底部和侧面之间排出之前和(b)不排出   媒体完全,只有第一站的多通道移液器。 这个 导致一些介质保留在移液管吸头中,但大大减少 气泡。
      2. 如果您的工作速度不够快,媒体可能会固化   储器或移液管尖端。 为了避免这种情况,建议从开始 65℃培养基。 如果它成为问题,只需弹出并更换提示 或水库。
    2. 将灭菌的种子倒在滤纸上。 使用 无菌牙签的润湿尖端,拾起并且每孔放置一个种子 的96孔板 注意:这允许第二次机会 种子大小选择。 我们观察到之间的显着差异 边缘孔,板边缘和内部孔的反应。   你应该测试这个差异,如果有必要不适用 处理或测量边界井。 如果边界井不被测量   然后将任何剩余的较小种子放在这些,使用均匀较大   种子用于内部井。
    3. 96孔板的带边缘用微孔外科胶带保持无菌和湿度。

  3. 幼苗生长
    1. 在4℃黑暗中将种子分层3-5天
    2. 在典型的拟南芥平板生长条件(22-24℃,16小时光照(60-80μE)/8小时黑暗)下生长幼苗。
    3. 放置后6天,苗木应该扩大 子叶和两个可看见的真实的叶子,准备好为治疗 荧光素(图2A)。 为了减少背景,最好继续 荧光素处理18-24小时后成像 注意:我们应用萤光素 通过喷雾,用洗涤剂(见配方)。 减少假 阳性/阴性一个必须确保均匀的底物可用性 饱和所有井。 在筛选中,我们实现了这种均匀性 从板上方约6英寸处喷射两次 左侧和右侧的板,然后三个喷雾 大约在板上方约9英寸从左到右跟踪。

  4. 成像
    1. 在分层后7天,在成像前数小时将板从生长室移至实验室。
      注意:在4xRSRE的情况下:表达LUCIFERASE的幼苗,平板 在中午8点前移动,在中午成像前4小时。 这样做了 最小化生理反应的调节,并通过延伸 荧光素酶活性,与变化的植物环境相关。 的 移动和成像之间的时间间隔可以根据不同而不同 实验的性质,但同时开始成像 整个屏幕是可取的,因为可能的交互 昼夜节律调节过程


      图1。 组织化学模板板及相应处理 板。 在此示例中,化学模板的第2行用于 处理两个样品板的行2和3,化学模板的行3 板用于处理两个样品板的行4和5,以及行4 化学模板用于处理两个样品的行6和7   板
    2. 准备化学品1:稀释。 它 可能需要调整化学品的浓度 库通过在水或其他适当的溶剂中稀释 治疗,基于观察到的输出。在我们的实验中我们观察 最佳工作浓度在20和40μM之间 - 几乎没有化学品 任何可见的效应低于10μM。化学品可以10μl液滴形式使用  在工作浓度到处理井;稀释每种化学品 产生足以满足每孔12μl的总体积 移液错误。
      注意:一些化学溶剂,如二甲基 亚砜(DMSO),可能会影响输出信号。之前大规模 屏幕,应该检查所需稀释水平的溶剂效果。  我们建议使用"稀释溶剂"阴性对照,除了没有 治疗控制,并在可能的情况下在每个板上进行阳性对照。
    3. 准备化学品2:模板。准备一个化学模板 板在冰上,与化学品的工作浓度相同 布置,因为它们将被应用于治疗板。我们推荐 将每种化学品应用于至少8个重复孔。这应该会产生 至少6个合适的幼苗来测量反应。如果重复井 被放置在列中,化学模板上的一行可以应用 到治疗板上的多排(图1) 注意:
      1. 如果不测试边界井,这必须反映在化学模板中。
      2. 用于每种化学处理的幼苗的数量应该是   由感兴趣的系统的可变性和实用性决定 约束,即要测试的化学品的数量。
    4. 对待 通过将化学物质从模板转移到处理板来培育幼苗 使用多通道移液器。 重要的是不要打扰 幼苗 - 如果幼苗受到移液管尖端的伤害,它可能会改变 化学品被感知,因此他们的反应。我们建议触摸 移液管尖端向远离幼苗的一侧,靠近琼脂 可能是为了使对侧面的飞溅或化学粘附最小化  的井(图2B)。一旦所有化学品已被轻轻应用 摇动板约5秒以确保孔中的化学分布均匀 注意:在这种筛选技术中,没有定义植物如何 吸收化学品。化学液滴可以从中吸收 表面的琼脂通过下胚轴/子叶/叶,其中 情况下感觉到工作集中。或者,化学品  可以在化学扩散到介质中之后通过根部被吸收。 在这种情况下,介质本身进一步稀释化学品 感知浓度可以小到施加的工作的1/10 浓度(10μl化学品稀释到100μl培养基中)。


      图2.代表性的96孔板。(A)7天龄的幼苗。注意 这套四口井装红,反映了两个潜力 电镀问题。这组孔显示差的萌发导致  植物太小,不能可靠分析,两个幼苗错误 在一个孔中发芽(用*标记),消除它们用于分析。 面板(B)显示多通道移液管化学应用
    5. 成像条件将取决于记者使用,但我们建议 成像时间达到典型响应的三倍,以允许 作为化学扩散通过介质的延迟响应,等。 我们使用了20小时   时间过程中的CCD相机的五分钟曝光 不透光盒,由ANDOR Solis v15图像分析控制 软件( http://www.andor.com/scientific-software )。

  5. 分析/重新筛选
    1. 根据响应的强度和均匀性,初始 定性概述可能足以用于第一轮筛查 然后进行定量分析(图3)
      1. 快速 量化组合信息中每个的百分比 幼苗的总面积是发光的,以及发光的亮度 是,我们使用自定义Perl脚本来解释图像的ImageJ分析 从ANDOR Solis v。15导出。注释脚本可在 http://www-plb.ucdavis.edu/labs/dehesh/。简而言之,这种技术 需要用户在圆形的感兴趣区域(ROI)中放置网格  ImageJ软件使用插件Microarray Profiler。 ImageJ量化  在这些ROI中的每个像素的"白"值,然后可以是 标准化为植物面积。这种技术允许快速筛选  大量的井,但它不区分模式 的荧光素酶活性(即具有一些非零"白"的像素数)  值)和发射光的强度(即"白"的大小) 值)。
      2. 对于更定义的量化,小方形ROI可以 使用ANDOR软件逐个放置在个体子叶上。 这在实现时消除了发光面积的影响 更高的强度精度。我们发现4像素乘4像素的ROI是一个 七日龄拟南芥子叶的最佳大小。评论Perl 此分析的脚本也可在 http://www-plb.ucdavis.edu/labs/dehesh/


        图3.模型数据 展示了两种分析方法。显示的是同一组四个 受伤的拟南芥幼苗在选择的时间点长达6小时。在 方法(i),ROI网格(红色圆圈)适合每个整个幼苗。 荧光素酶活性被定量为理论最大值的分数  (时间点0的图像,对比度增强)。在方法(ii)中,正方形  ROI放置在单独的子叶上,提供更高的精度。

    2. 一旦假定的化学"命中"已经通过第一轮确定 筛选,他们必须在第二轮和可能第三轮确认。 第二轮筛选应与第一轮类似,  例外,我们建议至少增加一倍或最好增加三倍 每个化学品的井数,其次是更多 仔细定义响应的量化,如步骤E1b所述 注意:在第一轮筛选中,我们发现约15% 的筛选化学品,推定影响4x RSRE:LUCIFERASE 活动。第二轮筛选证实这些推定的〜10% 化学品。这些数字应该根据不同而改变 输出,记者的灵敏度和选择的化学文库。
    3.  确认的化学品命中可能导致多种实验方法,  包括浓度曲线以确定活性浓度, 发展时间过程的化学效应,分析选择 化学结构或已知功能类似,和/或分析 化学效应对植物超越屏幕输出。

笔记

一般筛选记事

  1. 这里描述的屏幕输出是来自由含有四个拷贝的RSRE的最小启动子驱动的荧光素酶的发光,但是筛选技术可以应用于任何报道系统或对拟南芥幼苗的可见影响。 只需替换任何预处理的荧光素应用,CCD相机/ANDOR软件的必要成像设备,以及适当的图像分析。
  2. 此屏幕设计用于观察化学处理的即时效应,避免长时间暴露时产生的任何副作用。 然而,如果需要,可以将化学处理加入到熔融物中 固体培养基,然后将种子置于各个孔中,或在拟南芥生长的任何期望阶段。
  3. 如果早期添加化学品,以及在用荧光素预处理期间,注意最小化污染风险(即使用无菌水,尽可能保持容器关闭,并且如果必要在无菌层流罩内工作)。 污染可能直接或通过对植物健康的影响影响筛选输出

食谱

  1. ¼强度的MS媒体
    在1L milli-Q纯H 2 O中溶解1.11g MS盐,并用NaOH调节pH至5.7 测量8 g phytoagar到瓶中,并倾注pH调节的MS溶液
    高压灭菌,液体循环,30分钟
    注意:虽然这里使用了1/4强度的MS培养基,但是½MS或其他植物生长培养基可能工作得很好或更好,应在筛选前进行测试。
  2. 1mM荧光素
    准备10x(31.824mg/10ml)或100x(318.42mg/10ml)钾荧光素母液在milli-Q H 2 O中的量
    在无菌H 2 O中稀释至1mM,加入Tween-20至终浓度0.01%

致谢

该协议是Bjornson等人(2014)中描述的扩展。 这项工作得到国家卫生研究所(R01GM107311),国家科学基金会(IOS-1036491和IOS-1352478)和农业实验站(CA-D-PLB-3510-H)授予KD的支持。

参考文献

  1. Benn,G.,Wang,C.Q.,Hicks,D.R.,Stein,J.,Guthrie,C.and Dehesh,K。 关键的一般应激反应基序通过CAMTA 转录不一致地调节 因子。植物J 80(1):82-92
  2. Bjornson,M.,Benn,G.,Song,X.,Comai,L.,Franz,A.K.,Dandekar,A.M.,Drakakaki,G.and Dehesh,K。 促分裂原活化蛋白激酶信号和 CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3的不同作用调节植物一般胁迫应答的峰值时间和幅度。植物生理学166(2):988-996。
  3. Drakakaki,G.,Robert,S.,Szatmari,AM,Brown,MQ,Nagawa,S.,Van Damme,D.,Leonard,M.,Yang,Z.,Girke,T.,Schmid,SL,Russinova, E.,Friml,J.,Raikhel,NV和Hicks,GR(2011)。 生物活性化合物簇在体内靶向动态内膜网络。 Proc Natl Acad Sci USA 108(43):17850-17855。
  4. Walley,J.W.,Coughlan,S.,Hudson,M.E.,Covington,M.F.,Kaspi,R.,Banu,G.,Harmer,S.L.and Dehesh,K。 机械应激通过一种新颖的顺式元件诱导生物和非生物应激反应。 PLoS Genet 3(10):1800-1812。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bjornson, M., Song, X., Dandekar, A. M., Franz, A., Drakakaki, G. and Dehesh, K. (2015). A Chemical Genetic Screening Procedure for Arabidopsis thaliana Seedlings. Bio-protocol 5(13): e1519. DOI: 10.21769/BioProtoc.1519.
  2. Bjornson, M., Benn, G., Song, X., Comai, L., Franz, A. K., Dandekar, A. M., Drakakaki, G. and Dehesh, K. (2014). Distinct roles for mitogen-activated protein kinase signaling and CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 in regulating the peak time and amplitude of the plant general stress response. Plant Physiol 166(2): 988-996.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。