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SGR-based Reporter to Assay Plant Transcription Factor-promoter Interactions
基于拟南芥滞绿报告基因(SGR)分析植物转录因子与启动子之间的相互作用   

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Abstract

We developed an in vivo method to assay plant transcription factor (TF)–promoter interactions using the transient expression system in Nicotiana benthamiana (N. benthamiana) plants. The system uses the Arabidopsis stay green (SGR) gene as a reporter. Induction of SGR expression in N. benthamiana causes chlorophyll degradation and causes leaves to turn yellow.

Materials and Reagents

  1. Plant material
    1. 4 to 5 week old healthy N. benthamiana plants

  2. Vectors and bacteria strains
    1. pDONR221 or other gateway DONR vector (Life Technologies, InvitrogenTM, catalog number: 12536-017 )
    2. Escherichia coli (E. coli) DH10B or similar cells for molecular cloning
    3. SGR reporter destination vector, SPDK 2388
      Note: This vector contains the reporter gene SGR without promoter.
    4. Binary vector for TFs over-expression (e.g. LIC6 from Arabidopsis Biological Resource Center)
    5. Binary vector for expression of negative control protein [e.g. Actin7 (At5g09810) in LIC6]
    6. Agrobacterium tumefaciens (A. tumefaciens) strain GV2260

  3. Other materials
    1. BP and LR clonase enzyme Kit (Life Technologies, InvitrogenTM, catalog numbers: 11789-013 and 11791-043 )
    2. LB media
    3. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
    4. Infiltration medium (see Recipes)

Equipment

  1. Incubator (42 °C)
  2. 1 ml Tuberculin syringes without needle (Tyco, catalog number: 8881501400 )
  3. Controlled environment plant growth chamber (12 h of light per day, 21 °C)

Procedure

  1. Cloning of transcription factor of interest into SGR reporter vector
    1. Amplify promoter fragments (1-2 kb) from plant genomic DNA by high-fidelity PCR, and clone them into Gateway entry vector (pDONR221 or pDONR207) using BP reaction according to manufacturer’s instruction. This will generate the entry vector containing the promoter fragments.
    2. Transfer the promoter fragments into the destination vector SPDK2388 via LR reaction. The LR reaction should be set up as below:
      100 ng   entry vector containing promoters
      100 ng   destination vector SPDK2388
      2 µl    LR enzyme buffer
      2 µl    LR enzyme
      ddH2O to 10 µl
      Incubate at 25 °C overnight. Add 1 µl Proteinase K, incubate at 37 °C for 10 min. Transform the reaction mix into chemical competent cells of E. coli DH10B or similar cells, and select the transformants on 50 µg/ml spectinomycin (spectinomycin50) containing LB plates.

  2. Prepare plants and Agrobacterium strains
    1. Grow N. benthamiana plants with 12 h day light, 22 °C in the growth chamber. At 4 to 5 weeks, the plants should have three to four large healthy leaves suitable for infiltration.
    2. Transform separately the destination vector SPDK2388 with the TF of interest and the control into A. tumefaciens GV2260 by using chemical or electro competent cells. Typically, 200-500 ng plasmids and 50 µl competent cells are used for the transformation. Plate all the transformed cells and select the transformants on spectinomycin100, streptomycin100, Rifampicin25, and Carbenicillin50 (SSRC) containing LB plates. More than 100 transformants should be expected.

  3. Evaluate background expression of the promoter::SGR reporter constructs in N. benthamiana leaves
    1. Grow Agrobacterium GV2260 strains harboring the promoter::SGR construct overnight in ~20 ml LB media with SSRC.
    2. Spin down the Agrobacterium culture at 3,000 x g for 10 min, discard the supernatant, and suspend in infiltration media. Adjust the concentration of the culture to a series of OD600. For example, 0.05, 0.1, 0.3, and 0.6 OD. Incubate the culture at the room temperature for 3 h.
    3. Infiltrate N. benthamiana leaves with different concentration of Agrobacterium culture prepared in step C2 above. Make a small cut on the abaxial side of the leaves, use 1 ml syringes without needle to infiltrate Agrobacterium culture into the leaves. The culture should cover a spot of ~1 cm in diameter (see Figure 1). Infiltrate 1-2 spots for each OD concentration and infiltrate all samples with different ODs onto a single leaf. Repeat the infiltration onto a second leaf within the same plant.
    4. Keep infiltrated plants for 48 or 72 h in the same growth chamber and check the spots for signs of yellowing. Choose the highest OD concentration that shows no or slight yellowing for the following experiment. 


      Figure 1. SGR reporter-based transcription factors and promoter interaction assay in Nicotiana benthamiana. The SUR1 promoter::SGR reporter construct was co-infiltrated with different MYB TFs (spots 1 to 7) or with a negative control Actin (spot 0). Only two MYBs (spot 1 and 2) showed severe yellowing signs indicating that these two MYBs activate SUR1 promoter.

  4. Assay for TF-promoter interaction
    1. Grow separately Agrobacterium GV2260 strains harboring the promoter::SGR construct, TF construct, and actin control overnight in LB liquid media with SSRC.
      Note: We have constructed an ATEC library in LIC6 binary vector for over-expression of 15,000 Arabidopsis genes, which include more than 700 transcription factors. All these clones are deposited to and can be ordered from ABRC. In ABRC, the ATEC clones are names as “DKL” plus AGI numbers. See Reference 3 for more details.
    2. Spin down the cultures min and re-suspend them in infiltration media. Adjust the OD of the reporter culture to the concentration determined in step C4. Adjust the OD of TF and actin control to 1.0.
    3. Mix Agrobacterium culture with the reporter and the TFs or control with equal volume (~ 1 ml each). Incubate at room temperature for 3 h.
    4. Infiltrate N. benthamiana leaves with the mixed cultures from above onto 1-2 spots of ~1 cm onto the same leaf. Control and treatment samples should be present on the same leaf to overcome leaf-to-leaf variability.
    5. Keep infiltrated plants for 48 to 96 h in the growth chamber. Record signs of yellowing of each spot at 24, 48, 72, and 96 h post infiltrations. The severity of yellowing indicates the strength of reporter gene expression by the corresponding TF. The spots containing TFs should be compared to the control spots to assess specificity of TF-promoter interactions. See example shown in Figure 1. Photograph the leaves to document the results.
    6. Further validation of the observed results could be performed using luciferase-based reporter assay as described in Ma et al. (2013).

Recipes

  1. Infiltration medium
    Note: Prepare fresh media every time; sterilization is not required.
    10 mM MgCl2
    10 mM MES
    200 µM acetosyringone

Acknowledgments

This protocol is adapted from the following paper: Ma et al. (2013). This work is supported by National Science Foundation grants DBI-0723722 and DBI-1042344, and UC Davis funds to SPDK.

References

  1. Hortensteiner, S. (2009). Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends Plant Sci 14(3): 155-162.
  2. Ma, S., Shah, S., Bohnert, H. J., Snyder, M. and Dinesh-Kumar, S. P. (2013). Incorporating motif analysis into gene co-expression networks reveals novel modular expression pattern and new signaling pathways. PLoS Genet 9(10): e1003840.
  3. Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Seay, M., Gerstein, M., Snyder, M. and Dinesh-Kumar, S. P. (2007). Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc Natl Acad Sci U S A 104(11): 4730-4735.

简介

我们开发了一种体内方法,使用本氏烟草中的瞬时表达系统测定植物转录因子(TF) - 启动子相互作用(< )植物。 系统使用拟南芥绿色( SGR )基因作为报道基因。 在 N中感应 SGR 表达。 本生烟雾导致叶绿素降解并导致叶变黄。

材料和试剂

  1. 植物材料
    1. 4至5周龄健康的N。 本生植物

  2. 载体和细菌菌株
    1. pDONR221或其它网关DONR载体(Life Technologies,Invitrogen TM ,目录号:12536-017)
    2. 大肠杆菌(大肠杆菌)DH10B或类似细胞用于分子克隆
    3. SGR报道目的载体,SPDK 2388
      注意:该载体含有没有启动子的报告基因SGR。
    4. TFs过表达的二元载体(例如来自拟南芥生物资源中心的 LIC6)
    5. 用于表达阴性对照蛋白的双元载体[例如LIC6中的肌动蛋白7(At5g09810)]
    6. 根瘤土壤杆菌(根瘤土壤杆菌)菌株GV2260

  3. 其他材料
    1. BP和LR克隆酶Kit(Life Technologies,Invitrogen TM ,目录号:11789-013和11791-043)
    2. LB媒体
    3. Acetosyringone(Sigma-Aldrich,目录号:D134406)
    4. 渗透介质(参见配方)

设备

  1. 培养箱(42℃)
  2. 1ml无针结核菌素注射器(Tyco,目录号:8881501400)
  3. 受控环境植物生长室(每天12小时,21℃)

程序

  1. 将感兴趣的转录因子克隆到SGR 报告载体中
    1. 通过高保真PCR扩增来自植物基因组DNA的启动子片段(1-2kb),并根据制造商的说明使用BP反应将它们克隆到Gateway进入载体(pDONR221或pDONR207)中。这将产生含有启动子片段的入门载体
    2. 通过LR反应将启动子片段转移到目的载体SPDK2388中。 LR反应应设置如下:
      100 ng  含有启动子的入门载体
      100 ng  目标向量SPDK2388
      2μl     LR酶缓冲液
      2μl     LR酶
      ddH 2 O至10μl
      在25℃孵育过夜。加入1μl蛋白酶K,37°C孵育10分钟。将反应混合物转化为E的化学感受态细胞。大肠杆菌DH10B或类似细胞,并在含有LB平板的50μg/ml壮观霉素(壮观霉素 50 )上选择转化体。

  2. 制备植物和土壤杆菌菌株
    1. 成长N。本生烟草具有12小时日光,22℃在生长室中。在4至5周时,植物应该有三到四个大的健康叶适合渗透
    2. 分别将目标向量SPDK2388与感兴趣的TF和控件转换为 A。 tumefaciens GV2260通过使用化学或电感受态细胞。通常,200-500ng质粒和50μl感受态细胞用于转化。平板所有转化的细胞并选择在壮观霉素100,链霉素100,Rifampicin 25和Carbenicillin 50上的转化子。 (SSRC)。应该预计有100多个转化体

  3. 评估启动子:: SGR报道构建体在N中的背景表达。本生植物叶
    1. 使含有启动子:: SGR构建体的土壤杆菌属GV2260菌株在〜20mL具有SSRC的LB培养基中生长过夜。
    2. 在3,000×g下将土壤杆菌培养物旋转10分钟,弃去上清液,并悬浮于浸润培养基中。将培养物的浓度调节至一系列OD 600。例如,0.05,0.1,0.3和0.6OD。在室温下孵育培养物3小时
    3. 渗透 N。本生烟草叶用不同浓度的上述步骤C2中制备的土壤杆菌培养物培养。在叶的背面侧做一个小切口,使用1ml无针头的注射器将土壤杆菌培养物浸润到叶子中。培养物应覆盖直径约1cm的斑点(参见图1)。每个OD浓度渗透1-2个点,并将具有不同OD的所有样品渗透到单叶上。重复渗透到同一植物中的第二片叶子上。
    4. 在同一生长室中保持浸润的植物48或72小时,并检查斑点的黄化迹象。选择最高的OD浓度,显示没有或轻微泛黄的以下实验。


      图1.本塞姆氏烟草中基于SGR报告基因的转录因子和启动子相互作用测定 .SUR1启动子:: SGR报道构建体与不同的MYB TFs共浸润(点1至7)或用阴性对照肌动蛋白(斑点0)。只有两个MYB(斑点1和2)显示严重的黄化迹象,表明这两个MYB激活SUR1启动子。

  4. 测定TF-启动子相互作用
    1. 在具有SSRC的LB液体培养基中分别生长含有启动子:: SGR构建体,TF构建体和肌动蛋白对照的土壤杆菌属GV2260菌株过夜。
      注意:我们已经在LIC6二元载体中构建了ATEC文库,用于过表达15,000个拟南芥基因,其包括超过700个转录因子。所有这些克隆被保藏并可从ABRC订购。在ABRC中,ATEC克隆名称为"DKL"加上AGI编号。有关详细信息,请参阅参考文献3。
    2. 旋转文化min和重新悬浮在浸润介质中。将报告培养物的OD调节至步骤C4中测定的浓度。将TF和肌动蛋白控制的OD调整为1.0
    3. 将农杆菌培养物与报告物和TF或对照物等体积(每个约1ml)混合。在室温下孵育3小时
    4. 渗透 N。本氏烟叶用来自上面的混合培养物到〜1cm的1-2个斑点到同一叶上。对照和处理样品应存在于同一叶上以克服叶对叶的变异性。
    5. 在生长室中保持浸润的植物48至96小时。 在浸润后24,48,72和96小时记录每个斑点黄化的迹象。 黄化的严重性表示通过相应的TF的报告基因表达的强度。 含有TF的斑点应当与对照斑点进行比较以评估TF-启动子相互作用的特异性。 参见图1所示的示例。拍摄叶子以记录结果。
    6. 观察结果的进一步验证可以使用基于荧光素酶的报告基因测定法进行,如Ma等人所述。 (2013年)。

食谱

  1. 渗透介质
    注意:每次准备新鲜培养基; 不需要灭菌。
    10mM MgCl 2/
    10 mM MES
    200μM乙酰丁香酮

致谢

该协议改编自以下论文:Ma et al。(2013)。 这项工作是由国家科学基金会拨款DBI-0723722和DBI-1042344和加州大学戴维斯基金SPDK支持。

参考文献

  1. Hortensteiner,S。(2009)。 Stay-green调节叶绿素和叶绿素结合蛋白在衰老过程中的降解。 Trends Plant Sci 14(3):155-162。
  2. Ma,S.,Shah,S.,Bohnert,H. J.,Snyder,M。和Dinesh-Kumar,S.P。 将基序分析并入基因共表达 表达网络揭示了新的模块表达模式和新的信号通路。

    9(10):e1003840。
  3. Popescu,S.C.,Popescu,G.V.,Bachan,S.,Zhang,Z.,Seay,M.,Gerstein,M.,Snyder,M.and Dinesh-Kumar,S.P。 钙调蛋白相关蛋白与其靶标的差异结合通过高密度拟南芥 em>蛋白质微阵列。 Proc Natl Acad Sci USA 104(11):4730-4735。
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Copyright: © 2017 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. Ma, S. and Dinesh-Kumar, S. (2014). SGR-based Reporter to Assay Plant Transcription Factor-promoter Interactions. Bio-protocol 4(16): e1214. DOI: 10.21769/BioProtoc.1214.
  2. Ma, S., Shah, S., Bohnert, H. J., Snyder, M. and Dinesh-Kumar, S. P. (2013). Incorporating motif analysis into gene co-expression networks reveals novel modular expression pattern and new signaling pathways. PLoS Genet 9(10): e1003840.
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