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Dissecting the gene regulatory networks (GRNs) underlying developmental processes is a central goal in biology. The characterization of the GRNs underlying flower development has received considerable attention, however, novel approaches are required to reveal temporal and spatial aspects of these GRNs. Here, we provide an overview of the options available to perform dynamic gene perturbations to identify downstream response genes at specific stages of development in the flowers of Arabidopsis thaliana.

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Strategies for Performing Dynamic Gene Perturbation Experiments in Flowers
在花朵中进行动态基因扰动的实验策略

植物科学 > 植物发育生物学 > 综合
作者: Emmanuelle Graciet
Emmanuelle GracietAffiliation: Department of Biology, National University of Ireland, Maynooth, Ireland
Bio-protocol author page: a3025
Diarmuid S. Ó’Maoiléidigh
Diarmuid S. Ó’MaoiléidighAffiliation 1: Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland
Affiliation 2: Max Planck Institute for Plant Breeding Research, Cologne, Germany
For correspondence: omaoil@mpipz.mpg.de
Bio-protocol author page: a3026
 and Frank Wellmer
Frank WellmerAffiliation: Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland
Bio-protocol author page: a3027
Vol 6, Iss 7, 4/5/2016, 1532 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1774

[Abstract] Dissecting the gene regulatory networks (GRNs) underlying developmental processes is a central goal in biology. The characterization of the GRNs underlying flower development has received considerable attention, however, novel approaches are required to reveal temporal and spatial aspects of these GRNs. Here, we provide an overview of the options available to perform dynamic gene perturbations to identify downstream response genes at specific stages of development in the flowers of Arabidopsis thaliana.
Keywords: Arabidopsis thaliana(拟南芥), Flower Development(花的发育), Inducible promoters(诱导型启动子), Genomics(基因组学), Metabolomics(代谢组学)

[Abstract]

[Introduction] Gene activity perturbation followed by expression analysis of downstream targets represents a powerful method to understand gene function and dissect regulatory hierarchies. The transcriptomes of “static” mutant lines (e.g. caused by point mutations, transfer-DNA insertions, or constitutive expression) are often compared with those of wild type counterparts, which can confound the biological interpretation of the expression data. For example, a null mutant (noted ag-1) of the transcription factor AGAMOUS (AG) lacks stamens and carpels, which are replaced by sepals and petals (Figure 1A and 1B) (Bowman et al., 1989). A comparison of the transcriptomes of ag-1 flowers with wild-type flowers may lead to the conclusion that AG regulates the expression of genes controlling the specification of mature tissues in the anther and gynoecium. This interpretation is inappropriate, as AG is required for the specification of these organ-types (Bowman et al., 1989; Yanofsky et al., 1990). Therefore, the cause of the expression differences observed may be a result of tissue biases (Wellmer et al., 2004). Dynamic perturbations circumvent these and other issues: i) the tissues harvested will be morphologically identical, as tissue from “mock-treated” and inducer-treated plants would normally be harvested within a 24 h time-frame, ii) the response of downstream targets can be measured in the range of minutes and hours, which can be useful to infer direct and indirect interactions, iii) the development-dependent functions of the regulators of interest can be dissected (O'Maoileidigh et al., 2015). Flowers of the model plant Arabidopsis thaliana are minute, particularly at early stages of development. Furthermore, they are initiated in a sequential manner such that no two flowers are at the exact same stage of development on any given inflorescence (Smyth et al., 1990). Until recently, these traits have inhibited investigations into the molecular mechanisms underlying the earliest stages of flower development. There are many strategies available to isolate tissue from young flower buds including laser capture microdissection and fluorescent activated cell sorting (Birnbaum et al., 2003; Mantegazza et al., 2014; Wuest and Grossniklaus, 2014). We prefer to use a floral induction system (FIS) to isolate flowers at specific stages of development, as it is a user friendly, low-cost approach (O'Maoileidigh et al., 2015; Wellmer et al., 2006; O'Maoileidigh and Wellmer, 2014). This system is based on the reintroduction of APETALA1 (AP1) activity into the apetala1-1 cauliflower-1 (ap1-1 cal-1) background via either a rat glucocorticoid (Wellmer et al., 2006; O'Maoileidigh and Wellmer, 2014; O’Maoileidigh et al., 2013) or a mouse androgen (O'Maoileidigh et al., 2015) receptor ligand binding domain (noted GR and AR, respectively) fusion with AP1. Once the FIS ap1-1 cal-1 inflorescences are treated with dexamethasone or dihydrogentestosterone (DHT, 5α-androstan-17β-ol-3-one), respectively, AP1 translocates to the nucleus to regulate transcription, which leads to the formation of many flowers on a single inflorescence that are at similar stages of development (Wellmer et al., 2006; O'Maoileidigh and Wellmer, 2014). We have shown that stage-specific flowers can be reliably harvested from early to late stages of development (Wellmer et al., 2006; Ryan et al., 2015). We have also demonstrated that these FISs can be combined with two-component inducible systems that facilitate dynamic perturbations (O'Maoileidigh et al., 2015; O’Maoileidigh et al., 2013; Wuest et al., 2012). We have generated FISs that are responsive to dexamethasone and DHT, which we have combined with the ethanol-responsive AlcApro/AlcR and the OPpro/GR-LhG4 dexamethasone-responsive two-component systems, respectively, to express artificial microRNAs (amiRNAs) that perturb gene activity. We have successfully used these lines to assess the stage-specific functions of genes of interest (O'Maoileidigh et al., 2015; O’Maoileidigh et al., 2013; Wuest et al., 2012). Notably, we also observed that these two-component inducible systems can influence the expression of the A. thaliana genome independently of the intended amiRNA-mediated perturbation (O'Maoileidigh et al., 2015). Therefore, we developed and described several control measures that must be taken in order to account for these experimental artifacts (O'Maoileidigh et al., 2015). In the following protocol, we provide technical advice for implementing dynamic perturbation strategies during flower development and the establishment of the experimental design. As a guide, we briefly discuss the perturbation strategies implemented to understand the functions of the homeotic gene AG, including our published and unpublished results, as well as data from the literature.

Materials and Reagents

  1. 20 cm x 32 cm x 50 cm trays with plastic lids [Romberg & Sohn, catalog number: 51221K (trays) and 74051K (lids)]
  2. 50 ml centrifuge tubes (Sigma-Aldrich, catalog number: CLS430829-500E )
  3. Disposable pasteur pipettes (Thermo Fisher Scientific, catalog number: 12837625 )
  4. Mouse AR coding sequence [obtained from the vector pBJ36-mAR (O'Maoileidigh et al., 2015)]
    Note: Only the ligand-binding domain was fused to AP1.
  5. Rat GR coding sequence [obtained from the vector pBJ36-GR (O’Maoileidigh et al., 2013)]
    Note: Only the ligand-binding domain was fused to AP1.
  6. SRDX domain fusions [produced using the vector p35SSRDXG (Mitsuda et al., 2011)]
    Note: The SRDX amino acid sequence is LDLELRLGFA (Ikeda and Ohme-Takagi, 2009).
  7. The WUSB coding sequence [obtained from the vector p35SWUSB (Ikeda et al., 2009)]
    Note: The WUSB amino acid sequence is HRRTLPLFPMHGED (Ikeda et al., 2009).
  8. VP16 domain fusions [produced using the vector p35SVP16 (Mitsuda et al., 2011)]
    Note: Genbank accession KM486811 for VP16 activation domain sequence in a cloning vector.
  9. AlcA sequence [obtained from AlcApro-pBJ36 (Leibfried et al., 2005)]
  10.  AlcR coding sequence [obtained from 35Spro:AlcR-pML-BART (Leibfried et al., 2005)]
  11. GR-LhG4 coding sequence [obtained from 35Spro:GR-LhG4-pML-BART (Wuest et al., 2012)]
  12. OPpro sequence [obtained from 6xOPpro-pBJ36 (Wuest et al., 2012)]
  13. Floral induction system (FIS) [Various versions of the FISs are available from Dr. Frank Wellmer (O'Maoileidigh et al., 2015; Wellmer et al., 2006; O'Maoileidigh and Wellmer, 2014)]
  14. CONTROL-amiRNA sequence [obtained from pBJ36-AlcApro:CTRL-amiRNA (O'Maoileidigh et al., 2015)]
  15. GR antibody [obtained from Santa Cruz ( sc-1002 ) (Kaufmann et al., 2010)]
  16. Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
  17. α-Androstan-17β-ol-3-one (DHT) (Sigma-Aldrich, catalog number: A8380 )
  18. General reagents for molecular cloning and expression analysis
  19. Liquid nitrogen
  20. 10 mM dexamethasone stock solution (see Recipes)
  21. 100 mM DHT stock solutionpowder (see Recipes)
  22. GR activation solution (see Recipes)
  23. AR activation solution (see Recipes)

Equipment

  1. Sharp Forceps (Sigma-Aldrich, catalog number: T5790 )
  2. Dissection microscope
  3. General equipment for molecular cloning and expression analysis

Procedure

  1. Gain-of-function-mediated dynamic perturbation
    The use of inducible gain-of-function strategies to understand regulatory hierarchies can be extremely informative. For example, Gomez-Mena et al. (2005) overexpressed an AG-GR fusion under the control of the Cauliflower Mosaic Virus (CMV) 35S promoter (Benfey and Chua, 1990), to identify targets of AG (Gomez-Mena et al., 2005). Many of the genes they identified as differentially expressed in response to AG activation were shown to be direct targets (O’Maoileidigh et al., 2013; Gomez-Mena et al., 2005). Ito et al. (2004) also used a 35Spro:AG-GR line to perform perturbation experiments in the presence of the protein biosynthesis inhibitor cycloheximide, which was used to infer the direct regulation of the SPOROCYTELESS gene by AG. In addition, Ito et al. (2007) introduced the 35Spro:AG-GR transgene into the ag-1 background and determined the temporal requirements for AG activity, particularly during stamen development, using an AG dosage-dependent rescue assay (Ito et al., 2004). Although these gain-of-function-mediated perturbation strategies are extremely useful, they can also produce artifactual results, as the activity of the protein of interest may behave differently due to its altered spatio-temporal pattern of expression (Smith et al., 2011).
    To perform a gain-of-function-mediated dynamic perturbation experiment, the protein of interest can simply be expressed from a promoter of interest in combination with the glucocorticoid (GR) or androgen (AR) receptor fusion technique (O'Maoileidigh et al., 2015; Yamaguchi et al., 2015; Sun et al., 2009). Alternatively, the chemically inducible two-component promoter systems OPpro/GR-LhG4 or AlcApro/AlcR can be employed (Roslan et al., 2001; Deveaux et al., 2003; Craft et al., 2005). Use of these promoter systems would not require modification of the protein of interest, which simplifies the design process relative to the GR/AR fusion techniques and eliminates the possibility that the GR/AR domain will interfere with protein function.
    1. Fuse the coding region of your gene of interest to the coding region of the GR/AR ligand binding domain (Notes 1-2). Be sure to preserve the open reading frames of the gene of interest and the GR/AR domain. Place the gene fusion downstream of a promoter of choice (Note 3) in a binary vector. Alternatively, utilize a two-component inducible system (see above and Note 4).
    2. Transform wild-type or mutant plants (Note 5) with the inducible transgene by floral dip and identify transformants using the appropriate selection technique (Clough and Bent, 1998).
    3. Discard first generation (T1) plants displaying phenotypes in the absence of the inducing agent.
    4. Treat the inflorescences of the remaining first generation plants with the appropriate inducer (Note 6).
    5. Phenotype the flowers at least every second day after treatment. A gradient of phenotypes would be expected, depending on the stage of a given flower when it was treated (Note 7).
    6. Alternatively, the plants can also be characterized on a molecular level to select candidates for further analysis (Notes 8-9). However, the phenotype produced after activating the transgene should be carefully examined.
    7. Select several independent lines and, if using the GR/AR fusion technique, perform Western blotting analysis, using an anti-GR or anti-AR antibody, in the second generation to determine the level of expression of the fusion protein.
    8. Select lines that produce full-length proteins, desirable phenotypes and/or desirable mRNA levels. Isolate lines that are homozygous for the transgene.
    9. At this point, an inducible perturbation experiment can be initiated. However, it may be difficult to dissect the stage-specific functions of the factor of interest on a molecular level. Therefore, we recommend crossing the transgenic lines with the appropriate floral induction system.


      Figure 1. Perturbation of AG activity. A. A wild-type Landsberg erecta flower; B. An ag-1 flower; C. A flower from a plant containing a 35Spro:AG-SRDX transgene (unpublished); D. A flower from a plant containing a 35Spro:AG-WUSB transgene (unpublished). Inset: An inflorescence from an independent line containing the 35Spro:AG-WUSB transgene (unpublished); E. A flower from a plant containing a 35Spro:RNAi-AG transgene (unpublished); F. A flower from a plant containing a 35Spro:AG-4-amiRNA transgene (O’Maoileidigh et al., 2013).

  2. Transcriptional effector domain-mediated dynamic perturbation
    The function of the protein of interest can be modified by fusing it to a transcriptional repressor domain. For example, overexpression of a fusion between AG and an ERF-associated amphiphilic repression (EAR) motif from the protein SUPERMAN (SRDX) was shown to phenocopy a strong ag mutant phenotype (Mitsuda et al., 2006). We independently generated a 35Spro:AG-SRDX fusion that was designed to produce an identical protein to the published version in Mitsuda et al. (2006) (Figure 1C), however, we did not observe any strong ag-like phenotypes. We also overexpressed a fusion of AG with the WUSB repression domain from the transcription factor WUSCHEL in plants (Ikeda et al., 2009) and, in this case, did observe ag-like phenotypes (Figure 1D). However, we also observed plants with leaf-like floral organs (Figure 1D, inset). The reasons behind these discrepancies are unclear to us, however, they highlight the usefulness of generating control lines that constitutively express the chimeric gene prior to generating an inducible version.
    The gene of interest can also be expressed as fusion with a transcriptional activation domain. To our knowledge, such domains have not been fused with AG, however, they have been successfully used to understand the functions of other important floral regulators (e.g. Parcy et al., 1998). As with the gain-of-function experiments, the overexpression of the chimeric protein may alter its behavior.
    To perform this type of perturbation, the protein of interest can be fused to a transcriptional activation [e.g. VP16 (Triezenberg et al., 1988; Cousens et al., 1989)] or repression domain [e.g. SRDX (Hiratsu et al., 2002)]. This should result in the activation or repression of all genes that the protein associates with, respectively (Note 10). The choice of an activation or repression domain would be weighted based on the known activities of the protein of interest and on the presence of endogenous transcriptional effector domains in the protein of interest.
    1. Fuse the protein of interest to the transcriptional effector domain of choice so that the reading frame of the gene and domain remain intact. The chosen domain should be positioned so that it is unlikely to interfere with the activity of the protein of interest (Notes 1-2). To generate a non-inducible version of the fusion protein, place this fusion gene downstream of a chosen promoter (Note 11) in a binary vector.
    2. Transform wild-type plants with the fusion gene by floral dip (Clough and Bent, 1998) and identify transformants using the appropriate selection technique (Miki and McHugh, 2004).
    3. Characterize the phenotypes of the resulting transgenic plants. If they match expectations, proceed to step B4 (Note 12).
    4. To make an inducible version, introduce the fusion gene into the dexamethasone or ethanol-responsive two-component promoter systems (Note 4).
    5. Transform wild-type plants with inducible transgene by floral dip (Clough and Bent, 1998) and identify transformants using the appropriate selection technique (Miki and McHugh, 2004).
    6. Discard T1 plants displaying phenotypes in the absence of the inducing agent.
    7. Treat the inflorescences of the remaining T1 plants with the appropriate inducer (Note 6).
    8. Phenotype the flowers periodically after treatment. A gradient of phenotypes would be expected, depending on the stage of a given flower when it was treated (Note 7).
    9. Alternatively, the plants can also be characterized on a molecular level to select candidates for further analysis (Notes 8-9). However, the phenotype produced after activating the transgene should be carefully examined.
    10. Select plants that produce desirable phenotypes and/or desirable mRNA levels and isolate lines that are homozygous for the transgene.
    11. At this point, an inducible perturbation experiment can be initiated. However, it may be difficult to dissect the stage-specific functions of the factor of interest on a molecular level. Therefore, we recommend crossing the transgenic lines with the appropriate floral induction system.

  3. Loss-of-function-mediated dynamic perturbation
    The use of loss-of-function-mediated perturbation strategies offers several advantages over the perturbation strategies described above. In this case, the activity of the factor of interest would be removed via small RNAs. Therefore, the caveats associated with overexpression of the factor of interest, or a modified version, would not apply. The options available to perform this type of perturbation include the production of short interfering RNAs from a double-stranded RNA (dsRNA) precursor (termed RNA interference, RNAi) or artificial microRNAs (amiRNAs) (Ossowski et al., 2008; Schwab et al., 2006). The RNAi strategy relies on expressing a dsRNA that is homologous to a large portion of the factor of interest. This results in the production of many short RNA molecules, whereas amiRNAs rely on the production of a single type of small RNA to target a specific transcript (Schwab et al., 2006). Notably, RNAi-mediated perturbation is thought to lead to more off-targets than amiRNA-mediated perturbation (Ossowski et al., 2008; Schwab et al., 2006).
    We previously screened seven independent amiRNA constructs before identifying an amiRNA that efficiently targets the AG mRNA, which, when overexpressed, can recapitulate a strong ag null phenotype (Figure 1F) (O’Maoileidigh et al., 2013). We also successfully perturbed AG activity with an RNAi construct, however, the phenotype was somewhat weaker than the phenotype observed for the functional amiRNA (Figure 1E). Furthermore, the proportion of first generation plants that displayed a phenotype in the RNAi lines (~8%) was much lower than the proportion of lines that displayed a phenotype in the amiRNA lines (100%). Given this, and the potential off-target effects associated with RNAi-mediated perturbation, our recommendation would be to utilize amiRNAs to perturb gene activity.
    To perform a loss-of-function-mediated dynamic perturbation experiment, the chemically inducible two-component promoter systems described above can be used. To demonstrate functionality, we recommend making stable transformants that constitutively express the RNAi/amiRNA of interest prior to making an inducible version (Note 13).
    1. Design an RNAi (Note 14) or amiRNA (Note 15) construct and place it under the control of a promoter of choice in a binary vector.
      Notes:
      1. The materials and the cloning procedure to generate RNAi constructs are described in Eamens and Waterhouse (2011).
      2. The cloning procedure to generate amiRNAs is described by Schwab et al. (2006) while the appropriate vectors can be obtained through wmd3.weigelworld.org.
    2. Transform wild-type plants with the construct by floral dip (Clough and Bent, 1998) and identify transformants using the appropriate selection technique (Miki and McHugh, 2004).
    3. If the phenotypes of the transgenic plants match expectations, proceed to step C4 (Note 12). Alternatively, if the phenotype of a corresponding null mutant is unknown, the mRNA levels of the gene of interest can be determined.
    4. To allow an inducible knockdown of gene activities, introduce the RNAi/amiRNA sequence into the dexamethasone or ethanol responsive two-component promoter systems (Note 4).
    5. Transform wild-type plants with inducible transgene by floral dip (Clough and Bent, 1998) and identify transformants using the appropriate selection technique (Miki and McHugh, 2004).
    6. Discard T1 plants displaying phenotypes in the absence of the inducing agent.
    7. Treat the inflorescences of the remaining first generation plants with the appropriate inducer (Note 6).
    8. Phenotype the flowers periodically after treatment. A gradient of phenotypes would be expected, depending on the stage of a given flower when it was treated (Note 16).
    9. Alternatively, the plants can be characterized on a molecular level to select candidates for further analysis (Notes 8-9).
    10. Select plants that produce desirable phenotypes and/or desirable mRNA levels (Note 17) and isolate lines that are homozygous for the transgene.
    11. At this point, an inducible perturbation experiment can be initiated. However, it may be difficult to dissect the stage-specific functions of the factor of interest on a molecular level. Therefore, we recommend crossing the transgenic lines with the appropriate floral induction system.

  4. Inducible perturbation followed by gene expression analysis and phenotypic analysis
    Gene expression profiling using dynamic perturbations with whole inflorescences
    It is possible to perform an inducible gene perturbation experiment using whole inflorescences, however, no temporal information will be available from these data. In fact, the RNA populations isolated are likely to be dominated by RNA from older flowers (Wellmer et al., 2004).
    1. Grow plants so that they bolt in a relatively synchronous manner (Note 18).
    2. Divide plants into two populations: those that will be treated with the inducer and those that will be treated with a mock solution. The same strategy would be applied to any control plants being used.
    3. Treat the inflorescence to activate expression of the dynamic perturbation transgene (Note 6).
    4. Harvest approximately 20 inflorescences per sample from a few hours to days after initiating the dynamic perturbation using liquid nitrogen to keep the samples frozen. Extract RNA and perform qRT-PCRs to measure the responsiveness of i) the effector molecule (e.g. amiRNA precursor, protein fusion) and ii) a gene (or several genes) that will be affected by the induction. These data will allow the assessment of the kinetics of the knockdown, which will inform the subsequent experimental design (Note 19).
    5. Select the time at which the tissue will be harvested after the perturbation construct is activated.


      Figure 2. Response of AP1pro:AP1-GR ap1 cal inflorescence to dexamethasone treatment. A. An untreated inflorescence-like meristem. B-C. An inflorescence meristem 5 days (B) and 8 days (C) after treatment with a solution containing dexamethasone.

    6. Repeat steps 1-3 in Section D, above.
    7. Harvest approximately 20 inflorescences at chosen time-points using liquid nitrogen to keep the samples frozen.
    8. Process tissue for molecular analysis.

    Gene expression profiling in a stage-specific manner using dynamic perturbations
    Combining dynamic perturbation strategies with the FIS, which facilitates the collection of flowers at similar stages of development, affords the user with a low-cost, easy to use approach to identify stage-specific functions of their gene of interest (Figure 2A-C). The inducible transgene of choice can be crossed with the appropriate FIS or can be transformed directly into the FIS (Notes 20-21).
    1. Isolate the inducible transgene in a suitable FIS background (Note 20).
    2. Grow plants so that they bolt in a relatively synchronous manner (Note 18).
    3. Induce synchronous flowering by locally treating the ap1-1 cal-1 inflorescences (Note 22) (O'Maoileidigh et al., 2015).
    4. Divide plants into two populations: those that will be treated with the inducer and those that will be treated with a mock solution. The same strategy would be applied to any control plants being used.
    5. Treat the inflorescence to activate expression of the dynamic perturbation transgene (Note 6).
    6. Harvest whole flowers (Note 23) from approximately 20 plants per sample from hours to days after initiating the dynamic perturbation using liquid nitrogen to keep the samples frozen. Extract RNA and perform qRT-PCRs to measure the responsiveness of i) the effector molecule (e.g. amiRNA precursor, protein fusion) and ii) candidate gene(s) that will be affected by the perturbation to the inducer. These data will allow the assessment of the kinetics of the knockdown, which will inform the subsequent experimental design (Note 19).
    7. To perform the perturbation experiment, select the time-points of flower development you wish to investigate using Table 1.
    8. Select the time at which the tissue will be harvested after the perturbation construct is activated.
    9. Repeat steps 1-4 in Section D, above.
    10. Harvest whole flowers from approximately 20 plants at the chosen time-points from inducer-treated and mock-treated inflorescences using liquid nitrogen to keep the samples frozen.
    11. Process tissue for molecular analysis.

      Table 1. Correlation of days after activation of synchronous flowering in the FIS with stages of flower development. After day 7-8, the flowers developing from the inflorescence are no longer synchronized. At this point, however, it is possible to distinguish them morphologically. The table indicates approximations of the developmental stages that can be harvested on the indicated day after initiation of synchronous flowering using the FIS. These approximations are based on the flower stages described in Smyth et al. (1990) and growth of the plants in continuous light.


    Phenotyping flowers after a dynamic gene perturbation
    1. Grow plants so that they bolt in a relatively synchronous manner (Note 18).
    2. Activate the inducible promoter system with the appropriate inducer (Note 6).
    3. Dissect flowers at anthesis in the following days and phenotype them as desired. Continue this process until the flowers have produced the strongest mutant-like phenotype and the flowers return to a wild-type state.
    4. Correlate the phenotype you observed at anthesis with the stage at which the transgene was activated (Note 24).
    5. Compile a stage-dependent phenotyping series to compare with expression profiling data.

  5. Controls
    We outline the controls below that can be used for each perturbation strategy. In addition, we outline controls that are required for the inducible systems themselves. These latter controls are extremely important, as we have previously characterized the effects of these inducible systems on the transcriptome of A. thaliana (O'Maoileidigh et al., 2015).
    Transcriptional effector-mediated perturbations: Essential sequences in the transcriptional effector domain of choice can be mutated to produce a functionally inactive transcriptional effector domain [e.g. mSRDX (Hiratsu et al., 2004) or mVP16 (Cress and Triezenberg, 1991)]. These inactive domains can be fused to the factor of interest and expressed in an identical way to the primary experimental line. The protein-mEAR fusion should behave as a gain-of-function line. Therefore, it may also be desirable to generate an equivalent gain-of-function line that lacks an EAR motif as a control.
    amiRNA-mediated perturbation: We have previously described a CONTROL-amiRNA sequence that is not predicted to target any annotated A. thaliana transcript (O'Maoileidigh et al., 2015). Therefore, this CONTROL-amiRNA can be expressed in an identical manner to the primary experimental line to control for expression or phenotypic artifacts. An additional control includes generating an amiRNA-resistant version of the factor of interest by introducing synonymous mutations in the amiRNA target site of the corresponding gene. This would require the generation of an equivalent mutant plant whose phenotype is restored by an amiRNA-resistant factor.
    AlcR and GR-LhG4 transcription factors: These chemically responsive transcription factors have been shown to influence the expression of the A. thaliana transcriptome (O'Maoileidigh et al., 2015). Therefore, an essential control is to transform plants with T-DNAs that contain the AlcApro/AlcR or OPpro/GR-LhG4 cassettes and identify plants that express the transcription factors to a similar level to the primary experimental line.
    Treatment controls: All treatment controls should be partnered with a “mock” treatment. This mock treatment solution should consist of an identical mix of the solvents and surfactants used but lacking the active ingredient. An additional control includes the treatment of a wild-type line, or an equivalent line, with the active solution.

Notes

  1. For example, the GR domain was fused to the C-terminus of AG since the DNA binding domain of AG is present at the N-terminus (Ito et al., 2004). It is possible that several versions with the tag of interest in different positions may need to be made.
  2. Often short linkers of approximately 6-10 amino acids are placed between the protein of interest and the domain being fused to it (Sabourin et al., 2007). This may reduce the likelihood of unwanted negative interactions. A repeat of nine alanine amino acids or a mix of glycine and alanine are commonly used in plants (Tian et al., 2004; Heisler et al., 2005).
  3. Often the CMV 35S promoter is used (Benfey and Chua, 1990), however, it may not drive expression sufficiently in the tissues of interest. The promoter of UBIQUITIN10 is frequently used as an alternative for constitutive expression (Grefen et al., 2010). Promoters that drive expression only in certain tissues can also be used. This latter option may be desirable, especially if the inducible transgene is being expressed in its mutant background, as it may reduce the occurrence of false positives.
  4. The promoters upstream of the AlcR and GR-LhG4 chimeric transcription factors can be altered to suit the experimental design.
  5. Mutant plants can be useful in this case in order to show that the fusion protein can restore wild-type activity, as in the case of 35Spro:AG-GR ag-1 plants (Ito et al., 2004). The use of wild-type plants to identify a gain-of-function phenotype is also possible, however, it may be more difficult to interpret.
  6. The concentration of dexamethasone or DHT to use may need to be optimized. As little as 50 nM DHT has been used to activate gene expression (Sun et al., 2009). Ten μM dexamethasone is commonly used, however, the use of a lower concentration may be desirable (Yamaguchi et al., 2015). Silwett L-77 should be used at a concentration of 0.015 % (v/v) to improve the uptake of the chemical. We have used ethanol vapor to activate the AlcApro/AlcR system (O’Maoileidigh et al., 2013; Wuest et al., 2012). Briefly, we enclosed plants in sealed containers (18 cm x 32 cm x 50 cm) with two 50 ml tubes containing 10 ml 100% ethanol for a range of times (O’Maoileidigh et al., 2013; Wuest et al., 2012).
  7. This was elegantly shown by Ito et al. (2007), who used this strategy to dissect the stage-specific activities of AG. Flowers of 35Spro:AG-GR ag-1 plants that were treated with dexamethasone at early stages of development produced mature flowers containing carpels and stamens whereas flowers treated at later contained carpelloid sepals and stamenoid petals (Ito et al., 2004).
  8. If using the GR/AR fusion technique, the mRNA levels of the fusion gene can be determined. If using a two-component inducible system, the mRNA levels of the gene of interest can be determined after treatment with the inducer. Alternatively, the mRNA levels of the AlcR/GR-LhG4 genes can be determined, which should correlate with the expression output of the gene of interest.
  9. If selecting plants based on expression levels, it may be desirable to identify plants with strong, intermediate and weak expression levels of the transgene as the effects of extremely high expression can have negative consequences for downstream applications.
  10. The protein of interest does not necessarily have to be a transcription factor, however, it must be localized in proximity to DNA.
  11. The chimeric protein may not function as expected and the screening of inducible versions can be laborious. Therefore, the production of a constitutively expressed version is advised. Promoters that drive expression only in certain tissues can also be used.
  12. In order to interpret the phenotype produced, it may be necessary to identify null mutants or gain-of-function mutants of the same gene.
  13. Multiple amiRNAs may need to be screened, therefore, the expression of these amiRNAs from a constitutive promoter to select a functional amiRNA is advised.
  14. The region cloned to generate a dsRNA construct should not be homologous to other annotated genes.
  15. We prefer to select amiRNAs from the WMD3 platform (wmd3.weigelworld.org) so that the gene body is tiled with different amiRNAs. This is because the secondary structure of the target mRNA may interfere with amiRNA function.
  16. Strong expression of the AlcR or GR-LhG4 transcription factors exacerbate non-specific effects. Therefore, a balance between the level of the knockdown and the expression of the chemically-responsive transcription factor should be reached.
  17. We rotate our plants in the growth chambers to minimize effects of differing temperature and light gradients that might be present.
  18. The depletion and recovery of the mRNA of the target gene in response to the activation of the RNAi/amiRNA construct will dictate the time at which tissue will be harvested. The optimal time to collect tissue would be when the mRNA levels of the target gene are lowest, however, protein abundance of the target may also need to be measured.
  19. There are dexamethasone and DHT-responsive FISs available. The AlcApro/AlcR two-component system is compatible with either, however, the OPpro/GR-LhG4 system is compatible only with the DHT-responsive FIS.
  20. There are versions of the FIS that are resistant to glufosinate (‘BASTA’) and kanamycin treatments, respectively (O'Maoileidigh et al., 2015). Therefore, these plants can be transformed with a T-DNA containing the inducible transgene of interest with a compatible selection marker.
  21. If the recovery of the mRNA levels of the targeted factor is rapid, then multiple or continuous treatments may be necessary prior to see strong mutant phenotypes.
  22. The AP1pro:AP1-GR ap1 cal FIS can be activated by treating the inflorescences with a solution containing 10 μM dexamethasone, 0.015% (v/v) Silwett L-77. The AP1pro:AP1-AR ap1 cal FIS can be activated by treating the inflorescences with a solution containing from 500 μM DHT (higher concentrations can be used), 0.015% (v/v) Silwett L-77.
  23. The flowers at early stages should be scraped from the surface of the inflorescence with sharp forceps rather than harvesting the entire inflorescence (O'Maoileidigh and Wellmer, 2014).
  24. Smyth et al. (1990) characterized the development of A. thaliana flowers in detail, which includes the approximate timings of each developmental stage. The length of these stages can be correlated with the perturbation series generated using the inducible perturbation line.

Recipes

  1. 10 mM dexamethasone stock solution
    Resuspend dexamethasone powder in 100% EtOH to make a 10 mM stock
    Stored at -20 °C
  2. 100 mM 5α-Androstan-17β-ol-3-one (DHT) stock solution
    Resuspend DHT in 100% EtOH to make a 100 mM stock
    Stored at -20 °C
  3. GR activation solution
    10 μM dexamethasone
    0.015% (v/v) Silwet L-77
    0.1% (v/v) ethanol
  4. AR activation solution
    500 μM DHT
    0.015% (v/v) Silwet L-77
    0.5% (v/v) ethanol

Acknowledgments

This work was supported by grants from Science Foundation Ireland to F. W. and E. G. This protocol was established using the knowledge and reagents from several previous studies (O'Maoileidigh et al., 2015; O'Maoileidigh and Wellmer, 2014; O'Maoileidigh and Wellmer, 2014; O'Maoileidigh et al., 2013). We thank Dr. Jeff Long for the gifts of the OPpro-pBJ36 and 35Spro:GR-LhG4-pML-BART plasmids. We thank the three anonymous reviewers for their helpful comments.

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[简介] 基因活性扰动,随后是下游靶点的表达分析,代表了了解基因功能和解剖调节层次的有力方法。 "静态"突变体系(例如由点突变,转移-DNA插入或组成型表达引起的)的转录组通常与野生型对应序列的转录组相比较,其可以混淆表达的生物学解释数据。例如,转录因子AGAMOUS(AG)的无效突变体(称为"ag-1")缺少雄蕊和心皮,其被萼片和花瓣替代(图1A和1B)(Bowman等人, et al。,1989)。将ag-1花的转录组与野生型花的比较可以导致以下结论:AG调节控制花药和雌蕊中成熟组织的规格的基因的表达。这种解释是不合适的,因为AG是这些器官类型的规范所需要的(Bowman等人,1989; Yanofsky等人,1990)。因此,观察到的表达差异的原因可能是组织偏差的结果(Wellmer等人,2004)。动态扰动规避这些和其他问题:i)收获的组织将在形态上相同,因为来自"模拟处理的"和诱导物处理的植物的组织通常在24小时时间框架内收获,ii)下游靶标的响应可以在分钟和小时的范围内测量,这可以用于推断直接和间接相互作用,iii)可以解析感兴趣的调节物的发育依赖性功能(O'Maoileidigh等人, >,2015)。 ??模型植物拟南芥的花是微小的,特别是在发育的早期阶段。此外,它们以顺序方式启动,使得没有两个花在任何给定花序上处于完全相同的发育阶段(Smyth等人,1990)。直到最近,这些性状抑制了对花发育的最早阶段的分子机制的研究。有许多可用于从年轻花芽分离组织的策略,包括激光捕获显微切割和荧光激活细胞分选(Birnbaum等人,2003; Mantegazza等人,2014; Wuest和Grossniklaus,2014)。我们更喜欢使用花诱导系统(FIS)在特定发育阶段分离花,因为它是一种用户友好的低成本方法(O'Maoileidigh等人,2015; Wellmer < et al。,2006; O'Maoileidigh and Wellmer,2014)。该系统基于将APETALA1(AP1)活性重新引入 apetala1-1 花椰菜-1 ( ap1-1 cal -1)背景通过大鼠糖皮质激素(Wellmer等人,2006; O'Maoileidigh and Wellmer,2014; O'Maoileidigh et al。或分别与AP1融合的小鼠雄激素(O'Maoileidigh等人,2015)受体配体结合结构域(分别表示为GR和AR)。一旦用地塞米松或二氢睾酮(DHT,5α-雄甾烷-17β-醇-3-酮)分别处理FIS em-cal-1 花序,AP1转位到核以调节转录,这导致在处于相似发育阶段的单个花序上形成许多花(Wellmer等人,2006; O'Maoileidigh和Wellmer,2014)。 我们已经表明,阶段特异性花可以从发育的早期到晚期可靠地收获(Wellmer等人,2006; Ryan等人,2015)。我们还证明了这些FIS可以与促进动态扰动的双组分诱导系统组合(O'Maoileidigh等人,2015; O'Maoileidigh等人 ,2013; Wuest ,2012)。我们已经产生了响应于地塞米松和DHT的FIS,其分别与乙醇反应性AlcApro/AlcR和OPpro/GR-LhG4地塞米松反应性双组分系统组合以表达人工微RNA(amiRNA),其干扰基因活性。我们已经成功地使用这些系来评估感兴趣的基因的阶段特异性功能(O'Maoileidigh等人,2015; O'Maoileidigh等人,2013; Wuest ,2012)。值得注意的是,我们还观察到这些双组分诱导系统可以影响α的表达。 (O'Maoileidigh等人,2015年)的非拟南芥基因组中的氨基酸序列。因此,我们开发和描述了必须采取的几种控制措施,以解释这些实验工件(O'Maoileidigh等人,2015年)。 在下面的协议中,我们提供技术建议,实现花卉发展期间的动态扰动策略和实验设计的建立。作为指南,我们简要讨论实施的扰动策略,以了解同源异位基因AG的功能,包括我们发表的和未发表的结果,以及文献中的数据。

材料和试剂

  1. 20cm×32cm×50cm的具有塑料盖的托盘[Romberg& Sohn,目录号:51221K(托盘)和74051K(盖)]
  2. 50ml离心管(Sigma-Aldrich,目录号:CLS430829-500E)
  3. 一次性巴斯德移液管(Thermo Fisher Scientific,目录号:12837625)
  4. 小鼠AR编码序列[从载体pBJ36-mAR(O'Maoileidigh等人,2015)获得]
    注意:只有配体结合域与AP1融合。
  5. 大鼠GR编码序列[从载体pBJ36-GR(O'Maoileidigh等人,2013)获得]
    注意:只有配体结合域与AP1融合。
  6. SRDX结构域融合体[使用载体p35SSRDXG(Mitsuda等人,2011)产生]
    注意:SRDX氨基酸序列是LDLELRLGFA(Ikeda和Ohme-Takagi,2009)。
  7. WUSB编码序列[从载体p35SWUSB(Ikeda et al。,2009)获得]
    注意:WUSB氨基酸序列是HRRTLPLFPMHGED(Ikeda等人,2009)。
  8. VP16结构域融合[使用载体p35SVP16(Mitsuda等人,2011)产生]
    注意:Genbank登录号KM486811用于克隆载体中的VP16激活结构域序列。
  9. AlcA序列[获自AlcApro-pBJ36(Leibfried等人,2005)]
  10.   AlcR编码序列[从35Spro获得:AlcR-pML-BART(Leibfried等人,2005)]
  11. GR-LhG4编码序列[从35Spro获得:GR-LhG4-pML-BART(Wuest等人,2012)]
  12. OPpro序列[从6xOPpro-pBJ36(Wuest等人,2012)获得]
  13. 花诱导系统(FIS)[各种版本的FIS可以从Frank Wellmer博士(O'Maoileidigh等人,2015; Wellmer等人,2006; O'Maoileidigh and Wellmer,2014)]
  14. CONTROL-amiRNA序列[从pBJ36-AlcApro:CTRL-amiRNA获得(O'Maoileidigh等人,2015)]
  15. GR抗体[得自Santa Cruz(sc-1002)(Kaufmann等人,2010)]
  16. 地塞米松(Sigma-Aldrich,目录号:D4902)
  17. α-雄甾烷-17β-醇-3-酮(DHT)(Sigma-Aldrich,目录号:A8380)
  18. 用于分子克隆和表达分析的一般试剂
  19. 液氮
  20. 10 mM地塞米松储备溶液(见配方)
  21. 100 mM DHT储备溶液粉(见配方)
  22. GR激活解决方案(参见配方)
  23. AR激活解决方案(参见配方)

设备

  1. Sharp Forceps(Sigma-Aldrich,目录号:T5790)
  2. 解剖显微镜
  3. 用于分子克隆和表达分析的通用设备

程序

  1. 增益函数介导的动态扰动
    使用诱导型获益功能策略来理解监管层次结构可以是非常有用的。例如,Gomez-Mena等人(2005)在花椰菜花叶病毒(CMV)启动子(Benfey和Chua)的控制下过表达AG-GR融合物,1990),以鉴定AG的靶物(Gomez-Mena等人,2005)。他们鉴定为响应于AG活化而差异表达的许多基因显示为直接靶标(O'Maoileidigh等人,2013; Gomez-Mena等人)。 ,2005)。 Ito等人(2004)还使用35Spro:AG-GR系在蛋白质生物合成抑制剂放线菌酮存在下进行扰动实验,其用于推断蛋白质生物合成抑制剂放线菌酮的直接调节, SPOROCYTELESS 基因。此外,Ito等人(2007)将35Spro:AG-GR转基因引入 ag-1 背景并确定AG活性的时间要求,特别是在雄蕊发育期间,使用AG剂量依赖性拯救试验(Ito等人,2004)。尽管这些功能获得功能介导的扰动策略是非常有用的,但是它们也可以产生人为的结果,因为感兴趣的蛋白质的活性由于其改变的时空表达模式而表现不同(Smith等人。,2011)。
    为了进行功能获得功能介导的动态扰动实验,目的蛋白可以简单地从感兴趣的启动子与糖皮质激素(GR)或雄激素(AR)受体融合技术(O'Maoileidigh)联合表达, et al。,2015; Yamaguchi et al。,2015; Sun et al。,2009)。或者,可以使用化学诱导型双组分启动子系统OPpro/GR-LhG4或AlcApro/AlcR(Roslan等人,2001; Deveaux等人,2003 ; Craft等人,2005)。使用这些启动子系统不需要修饰目标蛋白质,这相对于GR/AR融合技术简化了设计过程并且消除了GR/AR结构域会干扰蛋白质功能的可能性。
    1. 将感兴趣的基因的编码区融合到编码区 GR/AR配体结合域(注释1-2)。一定要保留 所关注基因和GR/AR结构域的开放阅读框。地点 选择启动子下游的基因融合(注3) 向量。或者,使用双组分诱导系统(参见, 以上和注4)。
    2. 转化野生型或突变植物(注5) 与诱导型转基因通过花浸和鉴定转化体 使用适当的选择技术(Clough和Bent,1998)
    3. 丢弃在没有诱导剂的情况下显示表型的第一代(T <1>)植物。
    4. 用适当的诱导剂处理剩余的第一代植物的花序(注6)
    5. 处理后至少每隔一天对花进行表型。一个 将期望表型的梯度,这取决于a的阶段 (注7)。
    6. 或者, 植物也可以在分子水平上进行表征以进行选择 进一步分析的候选人(注8-9)。然而,表型 激活转基因后生产的产品应仔细检查
    7. 选择几个独立的线,如果使用GR/AR融合 技术,进行Western印迹分析,使用抗GR或 抗AR抗体,在第二代测定水平 融合蛋白的表达
    8. 选择生成的行 全长蛋白,期望的表型和/或期望的mRNA水平。 ?分离对于转基因是纯合的系
    9. 在这 点,可以启动可诱导的扰动实验。然而, 可能很难剖析阶段的具体功能 分子水平上的目的因子。因此,我们建议 使转基因品系与适当的花诱导杂交 系统

      图1. AG活性的扰动 野生型Landsberg erecta 花; B.一种ag-1花; C.一朵花从a ?含有35Spro:AG-SRDX转基因的植物(未公开); D.一朵花 来自含有35Spro:AG-WUSB转基因的植物(未公开)。插图: ?来自含有35Spro:AG-WUSB的独立品系的花序 ?转基因(未发表); E.从包含a的植物的花 35Spro:RNAi-AG转基因(未发表); F.从植物的花 含有35Spro:AG-4-amiRNA转基因(O'Maoileidigh等人,2013)。

  2. 转录效应域介导的动态扰动
    感兴趣的蛋白质的功能可以通过将其融合到转录阻遏物结构域来修饰。例如,在AG和来自蛋白SUPERMAN(SRDX)的ERF相关两亲阻遏(EAR)基序之间的融合的过表达显示表现出强的突变体表型(Mitsuda等人。,2006)。我们独立地产生了35Spro:AG-SRDX融合物,其设计为产生与Mitsuda等人(2006)(2006)(图1C)中公开的版本相同的蛋白质,然而,我们没有观察到任何强类似表型。我们还过表达AG与来自植物中转录因子WUSCHEL的WUSB抑制结构域的融合物(Ikeda等人,2009),并且在这种情况下,观察到 样表型(图1D)。然而,我们还观察到具有叶状花器官的植物(图1D,插图)。这些差异背后的原因不清楚我们,然而,他们强调生成控制线组成型表达嵌合基因之前生成诱导版本的有用性。
    感兴趣的基因也可以表达为与转录激活结构域的融合。据我们所知,这样的结构域没有与AG融合,然而,它们已经成功地用于理解其它重要的花调节剂的功能(例如,Parcy等人 1998)。与功能获得的实验一样,嵌合蛋白的过表达可能改变其行为 为了进行这种类型的扰动,目标蛋白质可以融合到转录激活上[例如, VP16(Triezenberg等人,1988; Cousens等人,1989)]或抑制结构域[例如 SRDX(Hiratsu et al。,2002)]。这应该导致分别与蛋白质缔合的所有基因的激活或抑制(注10)。激活或抑制结构域的选择将基于目的蛋白质的已知活性和目标蛋白质中内源性转录效应结构域的存在来加权。
    1. 融合感兴趣的蛋白质的转录效应结构域 选择,使得基因和结构域的阅读框保持完整。 选择的域应该被定位,以便不可能 干扰目标蛋白质的活性(注释1-2)。至 产生融合蛋白的非诱导型,置于此 融合基因下游选择的启动子(注11)在二进制 向量。
    2. 用融合基因转化野生型植物 花落(Clough和Bent,1998),并且鉴定使用的转化体 适当的选择技术(Miki和McHugh,2004)
    3. 表征所得转基因植物的表型。如果它们符合期望,则进行步骤B4(注12)。
    4. 为了制作诱导型,将融合基因导入 地塞米松或乙醇响应双组分启动子系统(注 ?4)。
    5. 转化具有诱导型转基因的野生型植物 花落(Clough和Bent,1998),并且鉴定使用的转化体 适当的选择技术(Miki和McHugh,2004)
    6. 在不存在诱导剂的情况下,丢弃显示表型的T 1植物
    7. 用适当的诱导剂处理剩余的T sub 1植物的花序(注6)。
    8. 处理后定期对花进行表型。梯度 根据给定花的阶段,预期表型 (注7)。
    9. 或者,植物也可以 在分子水平上表征以进一步选择候选物 分析(注8-9)。然而,活化后产生的表型 应仔细检查转基因
    10. 选择植物 产生期望的表型和/或期望的mRNA水平并分离 对于转基因是纯合的
    11. 在这一点上, 可以启动诱导型扰动实验。 然而,它可能很难剖析阶段的具体功能 在分子水平上的兴趣因子。因此,我们建议 使转基因品系与适当的花诱导杂交 系统。

  3. 失去功能介导的动态扰动
    使用损失函数介导的扰动策略提供了优于上述扰动策略的几个优点。在这种情况下,感兴趣的因子的活性将通过小RNA去除。因此,与感兴趣的因子或修饰版本的过表达相关的注意事项将不适用。可用于执行这种类型的扰动的选项包括从双链RNA(dsRNA)前体(称为RNA干扰,RNAi)或人工微小RNA(amiRNA)产生短干扰RNA(Ossowski等人。 >,2008; Schwab等人,2006)。 RNAi策略依赖于表达与感兴趣的因子的大部分同源的dsRNA。这导致许多短RNA分子的产生,而amiRNA依赖于单一类型的小RNA的产生以靶向特异性转录物(Schwab等人,2006)。值得注意的是,RNAi介导的扰动被认为导致比amiRNA介导的扰动更多的脱靶(Ossowski等人,2008; Schwab等人,2006)。
    我们先前筛选7个独立的amiRNA构建体,然后鉴定有效靶向mRNA的amiRNA,当过表达时,可以重现强的无效表型(图1F)(图1F)(O 'Maoileidigh ,,2013)。我们还成功地用RNAi构建体扰动AG活性,然而,表型略微弱于对于功能性amiRNA观察到的表型(图1E)。此外,在RNAi系(?8%)中显示表型的第一代植物的比例远低于在amiRNA系(100%)中显示表型的系的比例。鉴于此,以及与RNAi介导的扰动相关的潜在脱靶效应,我们的建议是利用amiRNA扰乱基因活性。
    为了进行功能丧失介导的动态扰动实验,可以使用上述化学诱导的双组分启动子系统。为了演示功能,我们建议在制作可诱导版本之前制备组成型表达目的RNAi/amiRNA的稳定转化体(注13)。
    1. 设计RNAi(注14)或amiRNA(注15)构建并放置 在二元载体中选择的启动子的控制下 注意:
      1. 产生RNAi构建体的材料和克隆程序在Eamens和Waterhouse(2011)中描述。
      2. 产生amiRNA的克隆程序由Schwab等人描述 et al。 (2006),而适当的向量可以通过 wmd3.weigelworld.org 获取。
    2. 转化野生型植物与 (Clough和Bent,1998)和识别 使用适当的选择技术(Miki和 McHugh,2004)。
    3. 如果转基因植物的表型匹配 期望,进行步骤C4(注12)。或者,如果 表型的相应无效突变体是未知的,mRNA水平 可以确定感兴趣的基因
    4. 允许诱导 基因活性的敲低,将RNAi/amiRNA序列引入 地塞米松或乙醇响应双组分启动子系统 (注4)。
    5. 转化具有诱导型转基因的野生型植物 花落(Clough和Bent,1998),并且鉴定使用的转化体 适当的选择技术(Miki和McHugh,2004)
    6. 在不存在诱导剂的情况下,丢弃显示表型的T 1植物
    7. 用适当的诱导剂处理剩余的第一代植物的花序(注6)
    8. 处理后定期对花进行表型。梯度 根据给定花的阶段,预期表型 (注16)。
    9. 或者,可以在分子水平上表征植物以选择候选物用于进一步分析(注8-9)
    10. 选择产生期望的表型和/或期望的植物 mRNA水平(注释17),并分离纯合的品系 转基因
    11. 在这一点上,可诱导的扰动实验 可以启动。但是,可能很难 解剖感兴趣的因子的阶段特异性功能 分子水平。因此,我们建议跨越转基因株系 与适当的花诱导系统。

  4. 诱导干扰后基因表达分析和表型分析
    使用整个花序的动态扰动进行基因表达谱分析
    有可能使用整个花序进行诱导型基因扰动实验,然而,没有时间信息可从这些数据。事实上,分离的RNA群体可能由来自老花的RNA主导(Wellmer等人,2004)。
    1. 种植植物,使它们以相对同步的方式螺栓连接(注18)
    2. 将植物分成两个种群:将被处理的种群 诱导剂和将用模拟溶液处理的那些。的 相同的策略将应用于所使用的任何对照植物
    3. 处理花序以激活动态扰动转基因的表达(注6)
    4. 每个样品从几个小时收获约20个花序 到使用液氮启动动态扰动后的几天 以保持样品冷冻。提取RNA并进行qRT-PCR以测量 i)效应分子(例如amiRNA前体)的响应性, 蛋白质融合)和ii)将受影响的基因(或几个基因) 通过感应。这些数据将允许动力学的评估 的敲除,这将通知后续的实验设计 (注19)。
    5. 选择在扰动构建体激活后收获组织的时间。


      图2. AP1pro:AP1-GR ap1 cal 花序对 地塞米松处理。 A.未处理的花序样分生组织。 公元前。处理后5天(B)和8天(C)的花序分生组织 ?用含地塞米松的溶液
    6. 重复上述D节中的步骤1-3。
    7. 在选定的时间点使用液氮收获约20个花序以保持样品冷冻。
    8. 用于分子分析的工艺组织。

    使用动态扰动以阶段特定方式进行基因表达谱分析
    将动态扰动策略与FIS组合,其有利于在相似发育阶段收集花,为用户提供了低成本,易于使用的方法来鉴定其感兴趣的基因的阶段特异性功能(图2A-C) 。选择的诱导型转基因可以与适当的FIS杂交,或者可以直接转化为FIS(注20-21)。
    1. 在适当的FIS背景中隔离诱导型转基因(注20)
    2. 种植植物,使它们以相对同步的方式螺栓连接(注18)
    3. 通过局部处理ap1-1 cal-1花序诱导同步开花(注22)(< em> ,2015)。
    4. 将植物分成两个种群:将被处理的种群 诱导剂和将用模拟溶液处理的那些。的 相同的策略将应用于所使用的任何对照植物
    5. 处理花序以激活动态扰动转基因的表达(注6)
    6. 收获全花(注23)从约20植物每 样品在开始动态扰动后从几小时到几天 使用液氮保持样品冷冻。提取RNA和 执行qRT-PCR以测量i)效应物的反应性 分子(例如amiRNA前体,蛋白质融合)和ii)候选物 将受到对诱导物的扰动影响的基因。这些 数据将允许评估击倒的动力学,其中 将通知后续实验设计(注19)
    7. 要执行扰动实验,请使用表1选择要调查的花发育的时间点。
    8. 选择在扰动构建体激活后收获组织的时间。
    9. 重复上述D节中的步骤1-4。
    10. 从选择的大约20棵植物收获整个花 时间点从诱导物处理和模拟处理的花序使用 液氮保持样品冷冻
    11. 处理用于分子分析的组织。

      表1.同步开花激活后的天数相关性 在具有花发育阶段的FIS中。 7-8天后,花 ?从花序发育不再同步。在这 点,但是,可以在形态上区分它们。的 表格表示可以是发展阶段的近似值 在开始同步开花后的指定日收获 ?使用FIS。这些近似是基于花阶段 描述在Smyth等人(1990)和连续的植物生长中 光。


    动态基因干扰后对花进行表型分析
    1. 种植植物,使它们以相对同步的方式螺栓连接(注18)
    2. 用适当的诱导剂激活诱导型启动子系统(注6)。
    3. 在接下来的几天在花期解剖花,并对它们进行表型 ?如预期的。继续这个过程,直到花产生了 最强的突变体样表型和花返回到野生型 状态
    4. 将您在开花期观察到的表型与转基因激活的阶段相关联(注24)。
    5. 编译阶段依赖表型系列与表达谱分析数据进行比较。

  5. 控制
    我们概述下面的控制可以用于每个扰动策略。此外,我们概述了诱导系统本身所需的控制。这些后面的控制是非常重要的,因为我们以前的特征的这些诱导系统的效应的转录组的。 thaliana (O'Maoileidigh ,,2015)。
    转录效应物介导的扰动:选择的转录效应子结构域中的基本序列可以突变以产生功能失活的转录效应结构域[例如mSRDX(Hiratsu等人。,2004)或mVP16(Cress和Triezenberg,1991)]。这些无活性结构域可以融合到感兴趣的因子并以与主实验线相同的方式表达。蛋白-mEAR融合物应当表现为功能获得线。因此,也可能希望产生缺少作为控制的EAR图案的等效功能获得线。
    amiRNA介导的扰动:我们先前已经描述了不预测靶向任何经注释的 A的CONTROL-amiRNA序列。 thaliana 成绩单(O'Maoileidigh ,,2015)。因此,该CONTROL-amiRNA可以以与初级实验线相同的方式表达以控制表达或表型人工产物。另外的对照包括通过在相应基因的amiRNA靶位点中引入同义突变来产生目的因子的amiRNA-抗性版本。这将需要产生一个相当的突变植物,其表型由amiRNA抗性因子恢复 AlcR和GR-LhG4转录因子:这些化学反应性转录因子已经显示影响α的表达。 thaliana transcriptome(O'Maoileidigh ,,2015)。因此,基本控制是用含有AlcApro/AlcR或OPpro/GR-LhG4盒的T-DNA转化植物,并将表达转录因子的植物鉴定为与主实验品系相似的水平。
    治疗控制:所有治疗对照应与"模拟"治疗合作。该模拟处理溶液应由所用的溶剂和表面活性剂的相同混合物组成,但缺少活性成分。另外的控制包括用活性溶液处理野生型品系或等效品系

笔记

  1. 例如,GR结构域与AG的C末端融合,因为AG的DNA结合结构域存在于N末端(Ito等人,2004)。可能需要对具有不同位置的感兴趣标签的多个版本进行设置
  2. 通常,在感兴趣的蛋白质和与其融合的结构域之间放置大约6-10个氨基酸的短接头(Sabourin等人,2007)。这可以减少不期望的负相互作用的可能性。在植物中通常使用九种丙氨酸氨基酸的重复或甘氨酸和丙氨酸的混合物(Tian等人,2004; Heisler等人,2005)。 br />
  3. 通常使用CMV启动子(Benfey和Chua,1990),然而,其可能不能在感兴趣的组织中充分驱动表达。 UBIQUITIN10的启动子经常用作组成型表达的替代物(Grefen等人,2010)。也可以使用仅在某些组织中驱动表达的启动子。后一种选择可能是可取的,特别是如果诱导型转基因在其突变背景中表达,因为它可以减少假阳性的发生。
  4. 可以改变AlcR和GR-LhG4嵌合转录因子上游的启动子以适合实验设计。
  5. 在这种情况下,突变植物可以是有用的,以便显示融合蛋白可以恢复野生型活性,如在Spro:AG-GR em-ag-1 >植物(Ito等人,2004)。使用野生型植物鉴定功能获得表型也是可能的,然而,它可能更难以解释。
  6. 可能需要优化使用的地塞米松或DHT的浓度。使用少至50nM的DHT来激活基因表达(Sun等人,2009)。通常使用10μM地塞米松,然而,可能需要使用较低浓度(Yamaguchi等人,2015)。 Silwett L-77应以0.015%(v/v)的浓度使用以改善化学品的吸收。我们已经使用乙醇蒸气来活化AlcApro/AlcR系统(O'Maoileidigh等人,2013; Wuest等人,2012)。简而言之,我们将植物在密封容器(18cm×32cm×50cm)中用两个含有10ml 100%乙醇的50ml管密封一段时间(O'Maoileidigh等人,2013 ; Wuest ,2012)。
  7. 这由Ito等人(2007)优雅地显示,他们使用该策略来解剖AG的阶段特异性活性。在发育的早期阶段用地塞米松处理的35Spro:AG-GR em-1 植物的花产生包含心皮和雄蕊的成熟花,而在稍后处理的花包含carpelloid萼片和花萼瓣> et al。,2004)。
  8. 如果使用GR/AR融合技术,可以确定融合基因的mRNA水平。如果使用双组分诱导型系统,可以在用诱导物处理后测定目标基因的mRNA水平。或者,可以确定AlcR/GR-LhG4基因的mRNA水平,其应该与目标基因的表达输出相关。
  9. 如果基于表达水平选择植物,可能需要鉴定具有转基因的强,中和弱表达水平的植物,因为极高表达的作用可对下游应用具有负面影响。
  10. 感兴趣的蛋白质不一定是转录因子,然而,它必须位于DNA附近
  11. 嵌合蛋白可能不如预期地发挥功能,并且可诱导型的筛选可能是费力的。因此,建议生成一个表达式的版本。也可以使用仅在某些组织中驱动表达的启动子
  12. 为了解释产生的表型,可能有必要鉴定同一基因的空突变体或功能获得突变体。
  13. 多个amiRNA可能需要筛选,因此,建议从组成型启动子表达这些amiRNA以选择功能性amiRNA。
  14. 克隆以产生dsRNA构建体的区域不应与其他注释的基因同源。
  15. 我们更喜欢从WMD3平台选择amiRNA( wmd3.weigelworld.org )使得基因体与不同的amiRNA平铺。这是因为靶mRNA的二级结构可能干扰amiRNA功能
  16. 强表达的AlcR或GR-LhG4转录因子加剧非特异性效应。因此,应达到敲低水平与化学反应性转录因子表达之间的平衡
  17. 我们在生长室中旋转我们的植物,以尽量减少可能存在的不同温度和光梯度的影响
  18. 响应于RNAi/amiRNA构建体的激活,靶基因的mRNA的消耗和回收将决定将收获组织的时间。收集组织的最佳时间是目标基因的mRNA水平最低时,但是目标的蛋白质丰度也可能需要测量。
  19. 有地塞米松和DHT反应FISs可用。 AlcApro/AlcR双组分系统与任一种兼容,然而,OPpro/GR-LhG4系统仅与DHT响应FIS相容。
  20. 有一些版本的FIS分别对草铵膦('BASTA')和卡那霉素处理有抗性(O'Maoileidigh等人,2015)。因此,这些植物可以用含有相关选择标记的感兴趣诱导型转基因的T-DNA转化。
  21. 如果靶因子的mRNA水平的恢复快速,则在看到强突变表型之前可能需要多次或连续的治疗。
  22. 可以通过用含有10μM地塞米松,0.015%(v/v)Silwett L-77的溶液处理花序来激活AP1pro:AP1-GR ap1 cal FIS。可以通过用含有500μMDHT(可以使用更高浓度),0.015%(v/v)Silwett L-77的溶液处理花序来活化AP1pro:AP1-AR em1c1c1 FIS 。
  23. 早期阶段的花应该用锋利的镊子从花序表面刮掉,而不是收获整个花序(O'Maoileidigh和Wellmer,2014)。
  24. Smyth等人(1990)描述了A的发展。 thaliana 花,其中包括每个发展阶段的大致时间。这些阶段的长度可以与使用可诱导扰动线产生的扰动序列相关联

食谱

  1. 10 mM地塞米松储备液
    将地塞米松粉末重悬于100%EtOH中,得到10mM储备液
    储存于-20°C
  2. 100mM5α-雄甾烷-17β-醇-3-酮(DHT)储液
    将DHT重悬于100%EtOH中以制备100mM储液
    储存于-20°C
  3. GR激活解决方案
    10μM地塞米松
    0.015%(v/v)Silwet L-77
    0.1%(v/v)乙醇
  4. AR激活解决方案
    500μMDHT
    0.015%(v/v)Silwet L-77
    0.5%(v/v)乙醇

致谢

这项工作得到了来自科学基金会爱尔兰给FW和EG的赠款的支持。该方案是使用来自几个先前研究的知识和试剂建立的(O'Maoileidigh等人,2015; O'Maoileidigh和Wellmer, 2014; O'Maoileidigh and Wellmer,2014; O'Maoileidigh ,2013)。我们感谢Jeff Long博士的OPpro-pBJ36和35Spro:GR-LhG4-pML-BART质粒的礼物。我们感谢三位匿名审稿人的有益意见。

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How to cite this protocol: Graciet, E., Ó’Maoiléidigh, D. S. and Wellmer, F. (2016). Strategies for Performing Dynamic Gene Perturbation Experiments in Flowers. Bio-protocol 6(7): e1774. DOI: 10.21769/BioProtoc.1774; Full Text



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