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Determination of Luciferase Activity in Arabidopsis seedling
拟南芥幼苗中荧光素酶活性的测定   

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Abstract

There are different direct and indirect methods available to study gene expression in plant systems. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the control of phosphate transporter PHT1;1 promoter. This assay was originally optimized for analyzing the repression of PHT1;1 promoter in response to arsenate As (V) which can be adapted to study the kinetics of transcriptional regulation of any gene in response to biotic or environmental stimuli measuring Luciferase activity in Arabidopsis thaliana.

Keywords: Luciferase(荧光素酶), Gene expression(基因的表达), Arabidopsis(拟南芥)

Materials and Reagents

  1. Transgenic Arabidopsis seedlings expressing Luciferase fused to promoter of interest (i.e. PHT1;1:Luc in pGWB435 binary vector)
    Note: Vector information can be find at: http://shimane-u.org/nakagawa/pgwb-tables/2.htm.
  2. KH2PO4 (Sigma-Aldrich, catalog number: P5655 )
  3. Sodium arsenate dibasic heptahydrate (Sigma-Aldrich, catalog number: A6756 )
  4. 30 mM arsenate (see Recipes)
  5. 0.36 mM D-Luciferin (Sigma-Aldrich, catalog number: L9504 ) (see Recipes)
  6. Johnson media (see Recipes)

Equipment

  1. Growth chamber [16-h-light/8-h-dark regime (24 °C/21 °C)]
  2. White 96-well plates (Greiner Bio-One GmbH, catalog number: 655074 )
  3. MicroAmp optical adhesive film (Life Technologies, Applied Biosystem®, catalog number: 4311971 )
  4. Luminometer (centro LB 960) (Berthold Technologies)

Software

  1. Mikro Win 2000 software

Procedure

  1. Sow the transgenic seeds minimum (24 plants for each treatment) in Petri dish containing Johnson media (0.6% agar) with 1 mM phosphate and incubate in growth chamber with 16-h-light/8-h-dark regime (24 °C/21 °C) for 7 days.
  2. Transfer the seedlings to Johnson media (0.6% agar) lacking phosphate and leave for 3 days.
  3. Add 200 µl of Johnson liquid media (without agar) to White 96-well plate and then transfer single seedlings to each well by placing upside down.
    Note: Since PHT1;1 is prominently expressing in root we submerged the seedlings upside down.
  4. Add D-Luciferin substrate (50 µM final concentration) to each well and incubate for 1 h.
  5. Measure the initial luminescence i.e. Luminescence (0).
  6. Add arsenate to each well (30 µM final concentration) and cover the plate with MicroAmp optical adhesive Film (refer to Reference 1 to understand the behavior of PHT1;1 in response to Arsenic).
  7. Measure the luminescence of the plate in every 10 min interval with the luminometer using Mikron Win 2000 software (counting time for each well is two seconds).
  8. Calculate the percentage of luciferase activity (%LUC activity) as follows:
    %LUC activity = [Luminescence(n) – Luminescence(0)]/Luminescence(0)*100
    Luminescence (0) = Luminescence at time zero
    Luminescence (n) = Luminescence at 2nd and consecutive readings
    Once we calculate the %LUC activity for each time point, we have to calculate the average Luminescence of all the independent plants at each time point.
  9. Luciferase activity can be also determined in Nicotiana benthamiana (N. benthamiana) using 1 cm discs of agro infiltrated leaves transferred to a microplate and proceed as described above.

Recipes

  1. 30 mM arsenate
    Dissolve 468 mg of sodium arsenate dibasic heptahydrate in 50 ml water
    Filter sterilize
  2. 0.36 mM D-Luciferin
    Mix 1 mg of D-Luciferin with 8 ml dH2O
    Warm at 80-90 °C for 30 to 40 sec to dissolve it
    Add dH2O to 10 ml
    Filter sterilize (0.25 μm)
    Stored at -20 °C
  3. Johnson media

    Macronutrientes
    Stock
    ml stock/L
    KNO3 (4 °C)
    100 g/L
    6.06 ml
    Ca(NO3).4H2O (4 °C)
    100 g/L
    9.446 ml
    MgSO4.7H2O (4 °C)
    100 g/L
    2.46 ml

    Micronutrientes
    g for 100 ml stock 1,000x
    ml stock/L
    KCl (-20 °C)
    0.378 g
    1 ml
    H3BO3 (-20 °C)
    0.15457 g
    MnSO4.H2O (-20 °C)
    0.0845 g
    ZnSO4.7H2O (-20 °C)
    0.0575 g
    CuSO4. 5H2O (-20 °C)
    0.01248 g
    H2MoO4 (-20 °C)
    0.00161 g


    g for 200 ml stock 500x
    ml stock/L
    FeSO4.7H2O (4 °C)
    (ajustar pH a 3.5 con H2SO4 diluido)
    Proteger de la luz
    0.1112 g
    2 ml


    Stock
    stock/L
    Mio-inositol (4 °C)
    50 g/L
    2 ml

    MES
    0,5 g/l
    1% sacarosa
    10 g/L
    0.5% sacarosa
    5 g/L

    Make volume to 1 L using Milli Q water
    Adjust the pH to 5.7 with KOH
    0.6% Agar
    After Autoclave add phosphate KH2PO4
    1 M KH2PO4: 6.8 g/50 ml (filter sterilize)
    Add 1 ml/L to get final concentration of 1 mM

Acknowledgments

This protocol is adapted from the original paper by Castrillo et al., 2013. This work was supported by fellowships from the Spanish Ministry of Science and Innovation (G.C) and from La Caixa/CNB International PhD program (to M.T.C.) as well as research grants (BIO2004-03759, BIO2007-66104, BIO2010-16687, AGL2007-61705, AGL2010-15151, CSD2007-00057, EUI2009-03993, and BIO2011-29085) from the Spanish Ministry of Science and Innovation.

References

  1. Castrillo, G., Sanchez-Bermejo, E., de Lorenzo, L., Crevillen, P., Fraile-Escanciano, A., Tc, M., Mouriz, A., Catarecha, P., Sobrino-Plata, J., Olsson, S., Leo Del Puerto, Y., Mateos, I., Rojo, E., Hernandez, L. E., Jarillo, J. A., Pineiro, M., Paz-Ares, J. and Leyva, A. (2013). WRKY6 transcription factor restricts arsenate uptake and transposon activation in Arabidopsis. Plant Cell 25(8): 2944-2957.

简介

有不同的直接和间接方法可用于研究植物系统中的基因表达。 在该协议中,我们描述了使用转基因植物表达在磷酸转运蛋白PHT1; 1启动子控制下的荧光素酶基因的修饰表达测定。 该测定最初被优化用于分析响应于砷酸盐As(V)的PHT1; 1启动子的抑制,其可以适于研究响应于测量荧光素酶活性的生物或环境刺激的任何基因的转录调节的动力学。 拟南芥(Arabidopsis thaliana)。

关键字:荧光素酶, 基因的表达, 拟南芥

材料和试剂

  1. 表达与感兴趣的启动子融合的荧光素酶(pIGWB435二元载体中的

    PHT1; 1:Luc)的转基因拟南芥 注意:可以在以下网址找到矢量信息: http://shimane-u .org/nakagawa/pgwb-tables/2.htm
  2. KH sub 2 PO 4(Sigma-Aldrich,目录号:P5655)
  3. 砷酸氢钠七水合物(Sigma-Aldrich,目录号:A6756)
  4. 30 mM砷酸盐(参见配方)
  5. 0.36mM D-荧光素(Sigma-Aldrich,目录号:L9504)(参见Recipes)
  6. 约翰逊媒体(见配方)

设备

  1. 生长室[16小时光/8小时黑暗区(24℃/21℃)]
  2. 白色96孔板(Greiner Bio-One GmbH,目录号:655074)
  3. MicroAmp光学粘合膜(Life Technologies,Applied Biosystem ,目录号:4311971)
  4. 发光计(centro LB 960)(Berthold Technologies)

软件

  1. Mikro Win 2000软件

程序

  1. 在含有具有1mM磷酸盐的约翰逊培养基(0.6%琼脂)的培养皿中播种最小的转基因种子(每次处理24株植物),并在具有16小时光/8小时黑暗区域(24℃/21℃)7天
  2. 将幼苗转移到缺乏磷酸盐的约翰逊培养基(0.6%琼脂),并放置3天
  3. 向白色96孔板中加入200μl约翰逊液体培养基(无琼脂),然后通过倒置将单个幼苗转移到每个孔中。
    注意:由于PHT1; 1在根中显着地表达,我们将幼苗颠倒。
  4. 向每个孔中加入D-荧光素底物(50μM终浓度),并孵育1小时
  5. 测量初始发光,即发光(0)。
  6. 向每个孔中加入砷酸盐(30μM终浓度),并用MicroAmp光学粘合剂膜覆盖板(参考参考文献1以了解PHT1的行为; 1响应于砷)。
  7. 使用Mikron Win 2000软件(每个孔的计数时间为2秒),使用光度计测量板每隔10分钟的发光。
  8. 计算荧光素酶活性百分比(%LUC活性)如下:
    %LUC活性= [发光(n) - 发光(0)] /发光(0)* 100
    发光(0)=在零时刻的发光率
    发光(n)=在2个和连续读数下的发光
    一旦我们计算每个时间点的%LUC活性,我们必须计算每个时间点的所有独立植物的平均发光。
  9. 也可以使用转移到微量培养板上的农杆渗入叶的1cm圆片在本氏烟草(<本生烟草)中测定萤光素酶活性,并如上所述进行。

食谱

  1. 30mM砷酸盐 将468mg七水合砷酸氢钠溶于50ml水中
    过滤灭菌
  2. 0.36mM D-荧光素 将1mg D-荧光素与8ml dH 2 O混合 在80-90℃温热30〜40秒使其溶解
    将dH 2加到10ml ml/h 过滤灭菌(0.25μm)
    储存于-20°C
  3. 约翰逊媒体

    Macronutrientes
    库存
    ml stock/L
    KNO <3>(4℃)
    100 g/L
    6.06 ml
    (4℃)的水溶液中的溶解性。
    100 g/L
    9.446 ml
    MgSO 4 .7H 2 O(4℃)。
    100 g/L
    2.46 ml

    Micronutrientes
    g for 100 ml stock 1,000x
    ml stock/L
    KCl(-20℃)
    0.378克
    1 ml
    (-20℃)。
    0.15457克
    (20℃)。
    0.0845克
    (-20℃)。
    0.0575克
    CuSO 4 5H 2 O(-20℃)
    0.01248克

    MoO 4(-20℃)
    0.00161克


    g for 200 ml stock 500x
    ml stock/L
    (4℃)的FeSO 4
    (ajustar pH a 3.5 con H 2 SO 4>稀释剂)
    Proteger de la luz
    0.1112克
    2 ml


    库存
    库存/L
    肌醇(4℃)
    50克/升
    2 ml

    MES
    0.5 g/l
    1%sacarosa
    10 g/L
    0.5%sacarosa
    5 g/L

    使用Milli Q水使体积达到1升
    用KOH调节pH至5.7 0.6%琼脂
    在高压灭菌后,加入磷酸盐KH 2 PO 4 4/
    1M KH 2 PO 4:6.8g/50ml(过滤灭菌)
    加入1ml/L,使终浓度为1mM

致谢

该方案由Castrillo等人于2013年的原始文章改编。这项工作得到了来自西班牙科学和创新部(GC)和La Caixa/CNB国际博士课程的研究金的支持到MTC)以及来自西班牙科学和创新部的研究授权(BIO2004-03759,BIO2007-66104,BIO2010-16687,AGL2007-61705,AGL2010-15151,CSD2007-00057,EUI2009-03993和BIO2011-29085) 。

参考文献

  1. Castrillo,G.,Sanchez-Bermejo,E.,de Lorenzo,L.,Crevillen,P.,Fraile-Escanciano,A.,Tc,M.,Mouriz,A.,Catarecha,P.,Sobrino-Plata,J 。,Olsson,S.,Leo Del Puerto,Y.,Mateos,I.,Rojo,E.,Hernandez,LE,Jarillo,JA,Pineiro,M.,Paz-Ares,J.and Leyva, )。 WRKY6转录因子限制拟南芥中的砷酸盐摄取和转座子活化。 a> Plant Cell 25(8):2944-2957。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:TC, M., Castrillo, G. and Leyva, A. (2014). Determination of Luciferase Activity in Arabidopsis seedling. Bio-protocol 4(12): e1160. DOI: 10.21769/BioProtoc.1160.
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