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Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells
剑菌介导的拟南芥根系转化(E-ART): 分析利于剑菌介导植物细胞转化(EMT)因素的实验方案   

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Abstract

Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of crop species. In this protocol, we adapted a previously published account that describes the use of Arabidopsis thaliana roots to investigate the interaction of A. thaliana and Agrobacterium tumefaciens. In our laboratory, we routinely use A. thaliana root explants to examine the factors that enhance the utility of EMT. In addition, the E-ART protocol can be used to study the transcriptional response of E. adhaerens and host plant following exposure to explant tissue, the transformability of different Ensifer adhaerens strains/mutants as well as testing the susceptibility of A. thaliana mutant lines as a means to decipher the mechanisms underpinning EMT.

Keywords: Ensifer adhaerens strain OV14(附着剑菌菌株OV14), Plant transformation(植物转化), A. thaliana(拟南芥), Root assay(根分析), Transient GUS expression(GUS瞬时表达)

Background

The advancement of Ensifer-mediated transformation (EMT) technology to successfully transform dicots viz., Arabidopsis thaliana, Solanum tuberosum, Nicotiana tabacum, Manihot esculenta, Brassica napus, and the monocot; Oryza sativa, has been previously reported (Wendt et al., 2012; Zuniga-Soto et al., 2015; Chavarriaga-Aguirre et al., 2016; Rathore et al., 2016). Additionally, genomic analysis of E. adhaerens OV14 by Rudder et al. (2014) revealed the bacterium possessed a 7.7 Mb genome comprised of two circular chromosomes (3.96 Mb and 2.01 Mb) and two plasmids (1.61 Mb and 125 Kb). A comparative analysis of the genome of E. adhaerens OV14 with Agrobacterium tumefaciens C58 (classic genetic engineer) and Sinorhizobium meliloti 1021 (a rhizobia with a propensity for low rates of genetic transformation; Broothaerts et al., 2005), highlighted that both E. adhaerens OV14 and S. meliloti 1021 contain homologs to several chromosomal-based genes essential for Agrobacterium-mediated transformation (AMT). However, a suite of genes which positively influence the successful transformation of a plant genome were found to be only present in the genome of E. adhaerens OV14 but absent from S. meliloti 1021 (Rudder et al., 2014). Overall, the sequence analysis of the E. adhaerens OV14 genome has significantly expanded the knowledge base describing the genetic systems that regulate the transformation of plant genomes via EMT.

To date several transient transformation systems have been proposed to study gene function in plants in regard to the use of A. tumefaciens (Wroblewski et al., 2005; Gelvin, 2006; Bhaskar et al., 2009; Li et al., 2009; Van Loock et al., 2010; Hwang et al., 2013; Krenek et al., 2015). Whereas, the stable transformations of any plant species are lengthy processes to test the utility of a bacterial transfection system to transform the plants. The advantages of transient transformation methods include rapid production of results, functional genomic studies and recombinant protein production (Van Loock et al., 2010; Krenek et al., 2015). The model plant A. thaliana is a powerful research tool with which to study the molecular, genetic and biochemical processes that support genetic transformation of somatic tissues as well as in planta (Provart et al., 2016). In contrast to the several months typically required for the transformation of primary crop species, these investigations require a rapid, reproducible and easy quantification method to determine the rate of transient transformation. Previously, Gelvin (2006) reported an efficient and reproducible quantitative assay for Agrobacterium-mediated Transformation using A. thaliana roots. This assay has well served the purpose of testing either Agrobacterium strains or A. thaliana ecotypes/mutants in the authors’ laboratory for more than two decades, reflecting the significance and competency of the assay in publications such as Shi et al. (2014). Conversely, a transient transformation method to facilitate comparable studies via EMT is not available. In response, the Ensifer-mediated A. thaliana Root Transformation (E-ART) protocol presented here is designed to address this deficit so that specific genetic and microbiological factors that support/enhance EMT can be identified to support the application of the technology to agronomically important crop species. The protocol is a modified version of an existing AMT based quantitative A. thaliana roots assay (Gelvin, 2006). While developing the E-ART protocol, we learnt that it is possible to improve transient GUS expression in A. thaliana root segments by adjusting several experimental factors (e.g., time of co-cultivation, acetosyringone concentrations, etc.) involved in the early stages of E. adhaerens transfection. E-ART, being the first quantitative method of transient gene expression for EMT will facilitate the rapid evaluation of novel E. adhaerens strains in plant transformation while also providing a platform to assess the genetic response of plants to EMT.

Materials and Reagents

  1. 2 ml centrifuge tubes
  2. Square Petri-dishes (Greiner Bio One International, catalog number: 688161 )
  3. Parafilm (Bemis, catalog number: PM992 )
  4. 50 ml Falcon tube
  5. Scalpel blades (NO. 10A, Swan Morton, catalog number: 0302 )
  6. Sterile filter papers (GE Healthcare, catalog number: 1004-090 )
  7. Petri dish 92 x 16 mm w/o cams (SARSTEDT, catalog number: 82.1472 )
  8. A. thaliana seed (in this case ecotype Columbia, Col-0)
    Note: A. thaliana seed is no more than 6 months old being stored at 4 °C.
  9. E. adhaerens strain OV14 harbouring plasmid of choice (in this case pCambia5105/pCambia5106 plasmids [Jefferson et al., 2006])
  10. 70% ethanol
  11. Distilled sterile water (DSW)
  12. Bleach (5% sodium hypochlorite; final concentration used is 50% Bleach, i.e., 1:1 Bleach:water)
  13. Tween-20
  14. Agarose (0.1%, Sigma-Aldrich, catalog number: A9539-500G )
  15. Antibiotics for bacterial selection: kanamycin, streptomycin, spectinomycin (Duchefa Biochemie)
  16. Sodium chloride (0.9% NaCl solution)
  17. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  18. Cefotaxime sodium (Duchefa Biochemie, catalog number: C0111.0005 )
  19. Tryptone (Oxoid, catalog number: LP0042 )
  20. Yeast extract (Oxoid, catalog number: LP0021 )
  21. Calcium chloride dehydrate (Duchefa Biochemie, catalog number: C0504 )
  22. Agar No. 1 (Oxoid, catalog number: LP0011 )
  23. MS basal salts (Duchefa Biochemie, catalog number: M0221 )
  24. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  25. 2,4-Morpholino-ethane sulfonic acid (MES monohydrate) (Duchefa Biochemie, catalog number: M1503 )
  26. Myo-inositol (Duchefa Biochemie, catalog number: I0609 )
  27. Nicotinic acid (Duchefa Biochemie, catalog number: N0611 )
  28. Pyridoxine (Duchefa Biochemie, catalog number: P0612 )
  29. Thiamine-HCl (Duchefa Biochemie, catalog number: T0614 )
  30. D-Glucose monohydrate (Duchefa Biochemie, catalog number: G0802 )
  31. Indole-3-acetic acid (IAA) (Duchefa Biochemie, catalog number: I0901 )
  32. 2,4-Dicholorophenocxyacetic acid (2,4-D) (Duchefa Biochemie, catalog number: D0911 )
  33. Kinetin (Duchefa Biochemie, catalog number: K0905 )
  34. X-GlcA cyclohexylammonium salt (Duchefa Biochemie, catalog number: X1405 )
  35. Dimethyl sulfoxide (DMSO) (Duchefa Biochemie, catalog number: D1370 )
  36. Teagasc-Tryptone Yeast-extract (TTY) medium (Rathore et al., 2015) (see Recipes)
  37. MS based media (see Recipes)
    1. Seed germination media (SGM)
    2. Co-cultivation media (CCM)
    3. Callus Induction media (CIM)
    4. Vitamin stock
  38. X-GlcA solution (see Recipes)
  39. Histochemical GUS stain solution (Jefferson et al., 1987) (see Recipes)

Equipment

  1. Pipettes (P1000, P100, P10)
  2. Controlled environment room/chamber to grow healthy A. thaliana plants (24 °C, 16 h light, 8 h dark), abbreviated as CT room
  3. Conical flasks (250 ml, sterile)
  4. Centrifuge
  5. Incubator (28 °C and 37 °C)
  6. Shaker incubator (28 °C, 220 rpm)
  7. Fridge (4 °C) and freezers (-20 °C and -80 °C)
  8. Laminar flow to perform aseptic work
  9. Bead sterilizer/flame to sterilize forceps
  10. Scalpel blades handles (No.7 S/S, Swan Morton, catalog number: 0907 )
  11. Autoclave (15 min at 121 °C and 15 psi)
  12. pH meter
  13. Weighing balance
  14. NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  15. Elga water purifier (Veolia Water Solution & Technologies, model: PURELAB® OPTION-R 7, catalog number: OR007BPM1 )
  16. Stereo-microscope

Software

  1. SAS system (Version 9.3, copyright 2002-2010 by SAS Institute Inc., Cary, NC, USA)

Procedure

  1. Seed sterilization and germination
    1. Surface sterilize A. thaliana seeds (~5 mg) in a 2 ml tube with 70% ethanol (EtOH) by vortexing the tube for 2 min. Discard the EtOH with an adjustable micropipette (1,000 µl) and wash seeds twice with DSW. All the steps were performed under a laminar flow hood to ensure aseptic conditions throughout.
    2. Add 50% Bleach containing 0.1% Tween-20 to the seeds and sterilize for 10 min with vigorous shaking. Once complete, decant Bleach with a 1,000 µl micropipette (Figure 1A).
    3. Rinse the seeds for five times with DSW to remove any residue of Bleach.
    4. Post-sterilization process, add 0.1% agarose (previously autoclaved for 15 min at 121 °C) to seeds for ease of handling.
    5. Using a micropipette (1,000 µl), place ~4 seeds per drop aligned in a row leaving ~1 inch distance from the top end of square Petri-dishes (Figures 1B and 1C) containing MS based seed germination medium (SGM, see Recipes). Double wrap the Petri-dishes using Parafilm.
    6. Incubate the Petri-dishes vertically in a stand (to ensure elongated root formation) in a CT (controlled temperature) room set at 24 °C, 16 h light ~4,000 lux photoperiod and 8 h dark for 15-20 days (Figure 1D).
    7. Root harvesting should be achieved before roots reach the end of dish, at which stage ~20 segments of < 0.5 cm length each should be obtained per plant.


      Figure 1. Arabidopsis thaliana seed sterilisation and germination. A. The A. thaliana ecotype Col-0 in 50% Bleach in a 2 ml centrifuge tube; B. Placing sterilised seeds on square Petri-dishes leaving ~1 inch top space for plants to grow; C. Plated seeds on SGM; D. Vertically aligned Petri-dishes in CT room (at 24 °C, 16 h photoperiod) showing plantlets with roots.

  2. Preparation of Ensifer inoculums
    1. Grow E. adhaerens OV14_pCambia5105 (here onwards E5105) on a TTY agar (see Recipes) containing 100 mg/L kanamycin, 200 mg/L streptomycin and 200 mg/L spectinomycin to select for the unitary plasmid containing T-DNA as well as virulence (vir) genes, overnight at 28 °C (Figure 2A).
    2. Pick a single colony to inoculate 10 ml TTY broth containing appropriate antibiotics (100 mg/L kanamycin, 200 mg/L streptomycin and spectinomycin) in a sterile conical flask and grow overnight at 28 °C and 220 rpm to reach 0.8 OD600 nm (Figures 2B and 2C).
    3. Pellet the cells in 50 ml Falcon tubes by centrifugation and wash them using 0.9% NaCl to remove any antibiotic residues. Centrifuge bacterial cultures at 3,750 x g (rcf) for 25-30 min for the inoculum preparation. Re-suspend the cells in 0.9% NaCl to maintain 0.8 OD600 nm (Figure 2D).
    4. Add 200 µM acetosyringone and incubate at 28 °C, 220 rpm for additional 1.5 h to induce virulence.


      Figure 2. Steps involved in preparing E5105 cultures for transfection of root segments. A. Overnight grown E5105 on TTY agar containing 100 mg/L kanamycin + 200 mg/L streptomycin + 200 mg/L spectinomycin to obtain single colonies. B. Single colony derived E5105 culture in TTY broth with appropriate antibiotics. C. E5105 culture in a 50 ml Falcon tube for centrifugation to remove TTY broth + antibiotics. D. E5105 resuspended in 0.9% NaCl and supplemented with 200 µM acetosyringone for vir induction at 28 °C, 1.5 h.

  3. Preparation and transfection of A. thaliana root segments
    1. Separate the A. thaliana roots from plants using a sterile scalpel blade before laying roots down on a Petri-dish containing a small amount (~500 µl) of DSW to ensure adequate root hydration (Figures 3A and 3B).
    2. Align roots together and cut into 0.3 to 0.5 cm long segments (Figure 3C). Place bundles of ~30 root segments on MS based co-cultivation medium (CCM, see Recipes) containing 200 µM acetosyringone.
    3. Using a P1000 pipette place 2-3 drops of E5105 on each the A. thaliana root bundle and allow a transfection time of 10 min (Figure 3D). Untreated root segments (no bacteria) should be used as a negative control.
    4. Remove any excess bacterial solution after the 10 min transfection time using the P1000 pipette. Double wrap the Petri-dishes with Parafilm.
    5. Co-culture the bacteria and A. thaliana roots at 20 °C, in the dark for 5 days.


      Figure 3. Processing of Arabidopsis thaliana plants and inoculations. A. Moving A. thaliana plants from SGM to square Petri-dish lid containing DSW; B. Separating the roots from the shoots; C. Cutting A. thaliana roots into ~3 mm segments; D. Inoculating the root segments with E5105 post 1.5 h vir induction.

  4. Transient GUS expression assay
    1. Post 5 days co-cultivation; wash treated/untreated root segments in DSW containing 500 mg/L cefotaxime to mitigate bacterial overgrowth.
    2. GUS staining: The activity of β-glucuronidase in putative transgenic root segments is performed as described by Jefferson et al. (1987) using one of two ways:
      1. For faster results, the washed roots are treated in GUS stain solution (see Recipes) with an overnight incubation at 37 °C. To remove the excess stain, wash root segments in 90% (v/v) ethanol (Figures 4A and 4B).
      2. To allow further proliferation of root segments and to facilitate ease of handling, transfer the roots onto callus induction medium (CIM, see Recipes) containing 250 mg/L cefotaxime. Seal the plates with Parafilm and incubate for additional 5-6 days at 24 °C in the dark, before staining in GUS stain solution, overnight at 37 °C (Figures 4C and 4D).


        Figure 4. Histochemical GUS activity via E-ART. Root segments were immediately transferred to GUS stain post 5 days co-cultivation (A) untreated control and (B) treated with E5105. GUS staining of root segments showing callus formation (indicated with red arrows) when segments were transferred to CIM containing 250 mg/L cefotaxime for 6 days post co-cultivation (C) untreated control and (D) treated with E5105.

Data analysis

Perform all experiments in triplicate with three technical replicates (one Petri-dish containing 9 bundles of root explants was treated as one technical replicate). The transient expression of GUS in A. thaliana roots treated with E5105 (E. adhaerens OV14 carrying pCAMBIA5105) is measured based on GUS activity i.e., blue colouration. Count blue roots/spots containing root segments for each treatment in each replicate under a stereo-microscope and record in MS Excel (Microsoft, USA). For statistical significance, analyse data using the General Linear Model (GLM) in the SAS system (Version 9.3, copyright 2002-2010 by SAS Institute Inc., Cary, NC, USA). The GLM procedure enables t-test (LSD) and Tukey’s studentized Range (HSD) test using the SAS system.

Notes

  1. Store seeds in Falcon tubes, properly labelled for the ecotype at 4 °C.
  2. All steps are strictly performed under aseptic conditions in a laminar flow hood. All media are autoclaved (15 min at 121 °C) and media supplements such as growth hormones, antibiotics, are filter sterilised and added after autoclaving.
  3. Seal the Petri-dishes with a double layer of Parafilm to maintain humidity to grow healthy plants. It is primarily important for high transient GUS expression that the plants are young to provide healthy roots quickly.
  4. Always process the A. thaliana plants for obtaining root explants before a flower bolt emerges as older plants will lead to lower transformation rates as detailed by Gelvin (2006).
  5. More importantly, always use fresh bacterial cultures to obtain higher rates of transformations. Do not allow bacterial cultures (E5105) to grow over 0.8 OD600 nm. Higher OD cultures will overgrow bacteria during co-cultivation that will negatively influence the GUS expression.
  6. The transfection of roots should be performed within 30 min of cutting the root segments. Leaving cut root segments for a longer time will affect the transfection and transformation negatively.
  7. Always include untreated root explants (no bacterial inoculum) as a negative control in the experiment.
  8. Current work shows that a combination of 5 days co-cultivation time and 200 µM acetosyringone improved rate of transient GUS expression in the A. thaliana root segments.

Recipes

Note: The following media are prepared, autoclaved and stored at room temperature.

  1. Teagasc-tryptone yeast extract (TTY) medium
    1.0% tryptone
    0.5% yeast extract
    After autoclaving add 20 ml of 1 M sterile CaCl2 per litre of medium
    To make TTY agar, add 1.5% agar to TTY broth prior to autoclaving
  2. MS based media (Table 1)
    Note: The MS based media are poured in Petri-dishes and can be stored at 4 °C for up to 15 days. Prepare vitamin stock, filter sterilise and store aliquots of 1 ml at -20 °C for up to 1 month.

    Table 1. Media recipe used for Ensifer-mediated Arabidopsis thaliana root transformation (E-ART)


  3. X-GlcA solution
    To prepare 1 M of X-GlcA solution:
    Add 521.8 mg of X-GlcA to 10 ml DMSO and vortex to dissolve
    Aliquot 1 ml in centrifuge tubes to store at -20 °C for long-term use
    Note: This substance is hygroscopic and light sensitive.
  4. Histochemical GUS stain solution
    Prepare GUS solution by adding below listed chemicals to reach the final concentrations as:
    50 mM sodium phosphate buffer pH 7.0
    1 mM EDTA
    0.1 mM of ferri- and ferro-cyanide, individually
    0.1% SDS
    40 mM X-GlcA
    Store aliquots at -20 °C

Acknowledgments

This research work was supported by the Teagasc Walsh Fellowship Scheme which funded DSR. Authors gratefully acknowledge Prof. Stanton B. Gelvin for the Agrobacterium transformation of Arabidopsis thaliana Roots: A Quantitative Assay (2006) published as a chapter in Methods in Molecular Biology, Agrobacterium protocols, which was adapted to develop E-ART protocol.

References

  1. Bhaskar, P. B., Venkateshwaran, M., Wu, L., Ane, J. M. and Jiang, J. (2009). Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS One 4(6): e5812.
  2. Broothaerts, W., Mitchell, H. J., Weir, B., Kaines, S., Smith, L. M., Yang, W., Mayer, J. E., Roa-Rodriguez, C. and Jefferson, R. A. (2005). Gene transfer to plants by diverse species of bacteria. Nature 433(7026): 629-633.
  3. Chavarriaga-Aguirre, P., Brand, A., Medina, A., Prias, M., Escobar, R., Martinez, J., Diaz, P., Lopez, C., Roca, W. M. and Tohme, J. (2016). The potential of using biotechnology to improve cassava: a review. In Vitro Cell Dev Biol Plant 52(5): 461-478.
  4. Gelvin, S. B. (2006). Agrobacterium transformation of Arabidopsis thaliana roots: a quantitative assay. Methods Mol Biol 343: 105-113.
  5. Hwang, H. H., Wu, E. T., Liu, S. Y., Chang, S. C., Tzeng, K. C. and Kado, C. I. (2013). Characterization and host range of five tumorigenic Agrobacterium tumefaciens strains and possible application in plant transient transformation assays. Plant Pathology 62: 1384-1397.
  6. Jefferson, R. A., Jefferson, O. A., Smith, L., Baillie, B. K., Raines, S., Ulkir, B., Tassie, A. and Tian, L. (2006). Freedom to co-operate: Transbacter as a Biological Open Source (BIOS) Tool for Gene Transfer. 8th international congress of plant molecular biology abstracts. Plant Mol Biol Rep 24: 141-160.
  7. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13): 3901-3907.
  8. Krenek, P., Samajova, O., Luptovciak, I., Doskocilova, A., Komis, G. and Samaj, J. (2015). Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 33(6 Pt 2): 1024-1042.
  9. Li, J. F., Park, E., von Arnim, A. G. and Nebenfuhr, A. (2009). The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5: 6.
  10. Provart, N. J., Alonso, J., Assmann, S. M., Bergmann, D., Brady, S. M., Brkljacic, J., Browse, J., Chapple, C., Colot, V., Cutler, S., Dangl, J., Ehrhardt, D., Friesner, J. D., Frommer, W. B., Grotewold, E., Meyerowitz, E., Nemhauser, J., Nordborg, M., Pikaard, C., Shanklin, J., Somerville, C., Stitt, M., Torii, K. U., Waese, J., Wagner, D. and McCourt, P. (2016). 50 years of Arabidopsis research: highlights and future directions. New Phytol 209(3): 921-944.
  11. Rathore, D. S, Doohan, F. and Mullins, E. (2016). Capability of the plant-associated bacterium, Ensifer adhaerens strain OV14, to genetically transform its original host Brassica napus. Plant Cell Tiss Organ Cult 127: 85-94.
  12. Rathore, D. S., Lopez-Vernaza, M. A., Doohan, F., Connell, D. O., Lloyd, A. and Mullins, E. (2015). Profiling antibiotic resistance and electrotransformation potential of Ensifer adhaerens OV14; a non-Agrobacterium species capable of efficient rates of plant transformation. FEMS Microbiol Lett 362(17): fnv126.
  13. Rudder, S., Doohan, F., Creevey, C. J., Wendt, T. and Mullins, E. (2014). Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes. BMC Genomics 15: 268.
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  15. Van Loock, B., Markakis, M. N., Verbelen, J. P. and Vissenberg, K. (2010). High-throughput transient transformation of Arabidopsis roots enables systematic colocalization analysis of GFP-tagged proteins. Plant Signal Behav 5(3): 261-263.
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简介

OV14;土壤传播的根瘤菌科的α-变形细菌强化了新型植物转化技术平台,称为“插入式”介导的转化(EMT)。 EMT可以稳定地转化单子叶植物和双子叶植物,并且EMT的宿主范围在不同范围的作物种类上不断扩大。在这个协议中,我们调整了一个以前发布的帐户,描述了使用拟南芥根系来研究 A的相互作用。 thaliana 和根癌土壤杆菌。在我们的实验室,我们通常使用 A。 thaliana 根外植体,以检查增强EMT效用的因素。此外,E-ART协议可用于研究E的转录反应。接种外植体组织后的寄主植物,宿主植物,不同的引物菌株/突变体的可变性以及测试A的易感性。作为破译支持EMT的机制的手段。
【背景】推进“Ensifer”介导的转化(EMT)技术以成功地转化双子叶菊,即拟南芥,马铃薯Solanum tuberosum ,Nicotiana tabacum ,Manihot esculenta ,欧洲油菜和单子叶植物;之前曾报道过(Wendt等人,2012; Zuniga-Soto等人),2015; Chavarriaga-Aguirre et al。,2016; Rathore等人,2016)。另外,E的基因组分析。 (2014)发现,该细菌具有7.7Mb的基因组,其包含两条环状染色体(3.96Mb和2.01Mb)和两条质粒(1.61Mb和125Kb) )。 E的基因组的比较分析。具有根癌土壤杆菌的过氧化氢酶(OV14),C58(经典基因工程师)和中华根瘤菌(Sinorhizobium meliloti)1021(具有低遗传转化率倾向的根瘤菌; Broothaerts et al。,2005),强调了E。贵重物品 OV14和 S。 meliloti 1021含有对于农杆菌介导的转化(AMT)必需的若干基于染色体的基因的同系物。然而,发现一组正面影响植物基因组成功转化的基因只存在于E基因组中。 。ns ns ns OV OV OV but but but but but。。。 meliloti 1021(Rudder等人,,2014)。总的来说,E的序列分析。 adhaerens OV14基因组显着扩展了描述通过EMT调节植物基因组转化的遗传系统的知识库。
&NBSP;迄今为止,已经提出了几种瞬态转化系统来研究植物中使用A的基因功能。 (Wroblewski et al。,2005; Gelvin,2006; Bhaskar等人,2009; Li等人, 2009; Van Loock等人,2010; Hwang等人,2013; Krenek等人,2015)。而任何植物物种的稳定转化是漫长的过程,以测试细菌转染系统转化植物的效用。瞬时转化方法的优点包括快速生成结果,功能基因组研究和重组蛋白质生产(Van Loock等人,2010; Krenek等人,2015)。模型工厂thaliana 是一个强大的研究工具,用于研究支持体细胞组织以及植物中遗传转化的分子,遗传和生化过程(Provart等人,,2016)。与初级作物物种转化通常需要的几个月相比,这些研究需要快速,可重复和容易的量化方法来确定瞬时转化的速率。此前,Gelvin(2006)报道了使用A的农杆菌介导的转化的有效且可重复的定量分析。 thaliana 根。该测定很好地用于测试农杆菌菌株或A型菌株的目的。作者实验室的生态型/突变体二十多年,反映了诸如Shi等人(2014)等出版物中测定的重要性和能力。相反,通过EMT促进可比较研究的瞬时转化方法是不可用的。作为回应, Ensifer 介绍的 A。本文提出的根系转化(E-ART)方案旨在解决这一缺陷,从而可以确定支持/增强EMT的特定遗传和微生物因子,以支持将该技术应用于农业重要作物。该协议是现有的基于AMT的量化的修改版本。 thaliana 根测定(Gelvin,2006)。在开发E-ART协议的同时,我们了解到可以改善A中的瞬时GUS表达。通过调整参与早期阶段的几个实验因素(例如,/或共同培养的时间,乙酰丁香酮浓度,等) E. adhaerens 染。作为EMT瞬时基因表达的第一种定量方法,E-ART将有助于快速评价新型E。 adherens菌株在植物转化过程中,同时也提供了一个评估植物对EMT遗传反应的平台。

关键字:附着剑菌菌株OV14, 植物转化, 拟南芥, 根分析, GUS瞬时表达

材料和试剂

  1. 2 ml离心管
  2. 方格培养皿(Greiner Bio One International,目录号:688161)
  3. 石蜡膜(Bemis,目录号:PM992)
  4. 50ml Falcon tube
  5. Scalpel刀片(NO。10A,Swan Morton,目录号:0302)
  6. 无菌滤纸(GE Healthcare,目录号:1004-090)
  7. 培养皿92 x 16毫米w / o凸轮(SARSTEDT,目录号:82.1472)
  8. 甲。 thaliana 种子(在这种情况下是生态型Columbia,Col-0)
    注意:拟南芥种子在4℃下不超过6个月。
  9. 电子。具有选择性质粒(在这种情况下为pCambia5105 / pCambia5106质粒[Jefferson等人,2006])的菌株OV14菌株
  10. 70%乙醇
  11. 蒸馏无菌水(DSW)
  12. 漂白剂(5%次氯酸钠;最终浓度为50%漂白剂,即1:1漂白剂:水)
  13. Tween-20
  14. 琼脂糖(0.1%,Sigma-Aldrich,目录号:A9539-500G)
  15. 细菌选择抗生素:卡那霉素,链霉素,壮观霉素(Duchefa Biochemie)
  16. 氯化钠(0.9%NaCl溶液)
  17. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  18. 头孢噻肟钠(Duchefa Biochemie,目录号:C0111.0005)
  19. 胰蛋白胨(Oxoid,目录号:LP0042)
  20. 酵母提取物(Oxoid,目录号:LP0021)
  21. 氯化钙脱水(Duchefa Biochemie,目录号:C0504)
  22. 琼脂1号(Oxoid,目录号:LP0011)
  23. MS基础盐(Duchefa Biochemie,目录号:M0221)
  24. 蔗糖(Duchefa Biochemie,目录号:S0809)
  25. 2,4-吗啉代 - 乙磺酸(MES一水合物)(Duchefa Biochemie,目录号:M1503)
  26. 肌醇(Duchefa Biochemie,目录号:I0609)
  27. 烟酸(Duchefa Biochemie,目录号:N0611)
  28. 吡哆素(Duchefa Biochemie,目录号:P0612)
  29. 硫胺素-HCl(Duchefa Biochemie,目录号:T0614)
  30. D-葡萄糖一水合物(Duchefa Biochemie,目录号:G0802)
  31. 吲哚-3-乙酸(IAA)(Duchefa Biochemie,目录号:I0901)
  32. 2,4-二氯代苯氧基乙酸(2,4-D)(Duchefa Biochemie,目录号:D0911)
  33. Kinetin(Duchefa Biochemie,目录号:K0905)
  34. X-GlcA环己基铵盐(Duchefa Biochemie,目录号:X1405)
  35. 二甲基亚砜(DMSO)(Duchefa Biochemie,目录号:D1370)
  36. Teagasc-Tryptone酵母提取物(TTY)培养基(Rathore et al。,2015)(见配方)
  37. 基于MS的媒体(见配方)
    1. 种子发芽媒体(SGM)
    2. 共培养媒体(CCM)
    3. 愈伤组织诱导培养基(CIM)
    4. 维生素库存
  38. X-GlcA溶液(参见食谱)
  39. 组织化学GUS染色溶液(Jefferson等人,1987)(参见食谱)

设备

  1. 移液器(P1000,P100,P10)
  2. 控制环境室/室可以健康发展。 (24℃,16小时光照,8小时黑暗),缩写为CT室
  3. 锥形瓶(250毫升,无菌)
  4. 离心机
  5. 培养箱(28°C和37°C)
  6. 摇床培养箱(28°C,220 rpm)
  7. 冰箱(4°C)和冷柜(-20°C和-80°C)
  8. 层层流动执行无菌工作
  9. 珠子消毒器/火焰消毒镊子
  10. Scalpel刀片柄(No.7 S / S,Swan Morton,目录号:0907)
  11. 高压灭菌器(121°C和15 psi)15分钟)
  12. pH计
  13. 称重平衡
  14. NanoDrop2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  15. Elga净水器(Veolia Water Solution&amp; Technologies,型号:PURELAB OPTION-R 7,目录号:OR007BPM1)
  16. 立体显微镜

软件

  1. SAS系统(版本9.3,SAS Institute Inc.版权所有,2002-2010,Cary,NC,USA)

程序

  1. 种子灭菌和发芽
    1. 在具有70%乙醇(EtOH)的2ml试管中,通过涡旋管将表面消毒拟南芥种子(〜5mg)2分钟。用可调微量移液管(1000μl)弃去EtOH,用DSW洗涤两次。所有步骤均在层流罩下进行,以确保无菌条件。
    2. 向种子中加入含有0.1%Tween-20的50%漂白液,并剧烈振荡灭菌10分钟。一旦完成,用1000μl微量移液管滗出漂白剂(图1A)
    3. 用DSW冲洗种子五次,以清除任何残留的漂白剂。
    4. 灭菌后,加入0.1%琼脂糖(先前在121℃高压灭菌15分钟),使种子易于处理。
    5. 使用微量移液管(1,000μl),每滴放置约4粒种子,每行一滴,距离包含基于MS的种子发芽培养基(SGM,参见食谱)的方形培养皿(图1B和1C)顶端离离约1英寸的距离, 。使用Parafil双重包装Petri菜。
    6. 在设置在24℃,16小时光照〜4,000勒克斯光周期和8小时黑暗中的15-20天的CT(控制温度)室中垂直孵育培养皿(以确保细根形成)(图1D) 。
    7. 在根到达菜的末端之前应该实现根收获,在该阶段〜每株植株需要0.5厘米长度。


      图1.拟南芥种子灭菌和萌发。 :一种。在2ml离心管中的50%漂白液中的拟南芥生态型Col-0; B.将灭菌的种子放置在方形培养皿上,留下约1英寸的顶部空间用于植物生长; C. SGM上的种子; D. CT房间垂直排列的培养皿(24°C,16 h光周期),显示有根的小植物。

  2. 准备接种物接种物
    1. 成长E在含有100mg / L卡那霉素,200mg / L链霉素和200mg / L壮观霉素的TTY琼脂(参见食谱)上的OV14_pCambia5105(这里是E5105),以选择含有T-DNA的单质粒,以及毒素( vir )基因在28°C过夜(图2A)
    2. 挑取单个菌落,在无菌锥形瓶中接种含有适当抗生素(100mg / L卡那霉素,200mg / L链霉素和壮观霉素)的10ml TTY肉汤,并在28℃和220rpm下生长过夜,达到0.8 OD 600nm(图2B和2C)。
    3. 通过离心将细胞置于50ml Falcon管中,用0.9%NaCl洗涤以除去任何抗生素残留物。将细菌培养物以3,750×g(rcf)离心25-30分钟用于接种物制剂。将细胞重新悬浮在0.9%NaCl中以维持0.8 OD 600 nm(图2D)。
    4. 加入200μM乙酰丁香酮,并在28℃,220rpm下孵育另外1.5小时以诱导毒力。


      图2.用于转染根部的E5105培养物的制备步骤 A.在含有100mg / L卡那霉素+ 200mg / L链霉素+ 200mg / L壮观霉素的TTY琼脂上过夜生长E5105,得到单个殖民地。 B.使用适当的抗生素在TTY肉汤中产生单个菌落来源的E5105培养物。 C.E5105培养在50ml Falcon管中,用于离心以除去TTY肉汤+抗生素。 D.E5105重悬于0.9%NaCl中,补充200μM乙酰丁香酮以在28℃,1.5小时诱导感染。

  3. 拟南芥根系的准备和转染
    1. 分开 A。在使用无菌手术刀叶片的植物根部从根部放下含有少量(〜500μl)DSW的培养皿,以确保足够的根部水合(图3A和3B)。
    2. 将根对齐并切成0.3至0.5厘米长的段(图3C)。在含有200μM乙酰丁香酮的MS基础培养基(CCM,参见食谱)上放置约30个根部的束。
    3. 使用P1000移液器在每个 A上放置2-3滴E5105。 thaliana 根束,并允许10分钟的转染时间(图3D)。未处理的根部(无细菌)应用作阴性对照。
    4. 使用P1000移液管在10分钟的转染时间后,除去任何多余的细菌溶液。双面包裹石蜡玻璃。
    5. 共培养细菌,而A。 thaliana 根在20°C,黑暗中5天。


      图3.拟南芥植物和接种的处理。 :一种。将拟南芥植物从SGM移植到包含DSW的方形培养皿; B.将根与芽分开; C.切割A. thaliana 根到〜3毫米段; D.使用E5105后1.5小时接种根部段感染。

  4. 瞬时GUS表达测定
    1. 邮政5天合作;在含有500mg / L头孢噻肟的DSW中洗涤处理/未处理的根部段以减轻细菌过度生长。
    2. GUS染色:使用以下两种方法之一,如Jefferson等人(1987)所述进行推定的转基因根节段中β-葡糖醛酸糖苷酶的活性:
      1. 为了更快的结果,将洗涤的根在GUS染色溶液(参见食谱)中在37℃下过夜孵育处理。为了去除多余的污渍,洗涤90%(v / v)乙醇中的根部分(图4A和4B)
      2. 为了进一步增殖根部并促进易于处理,将根转移到含有250mg / L头孢噻肟的愈伤组织诱导培养基(CIM,参见食谱)上。用Parafilm密封板,并在24℃下在黑暗中孵育5-6天,然后在GUS染色溶液中染色,37℃过夜(图4C和4D)。


        图4.通过E-ART的组织化学GUS活性根部分段在5天共培养(A)未处理对照和(B)用E5105处理后立即转移到GUS染色体上。 (C)未经处理的对照组和(D)用E5105处理,将片段转移至含有250mg / L头孢噻肟的CIM(C)未处理对照组(D)处理6天时,显示愈伤组织形成(表示为红色箭头)的根部段的GUS染色。

数据分析

通过三次技术重复进行一式三份的所有实验(一个含有9根根外植体的培养皿作为一个技术重复进行处理)。基于GUS活性测量E5105(携带pCAMBIA5105的OV14)载体的GUS在基因组中的瞬时表达,蓝色着色。在立体显微镜下,在每个重复的每个处理中计数含有根部分的蓝色根/斑点,并在MS Excel(Microsoft,USA)中记录。对于统计学意义,使用SAS系统中的通用线性模型(GLM)分析数据(版本9.3,SAS Institute Inc.,Cary,NC,USA的版权所有2002-2010)。 GLM程序使用SAS系统实现了 -test(LSD)和Tukey的学生范围(HSD)测试。

笔记

  1. 将种子储存在Falcon管中,在4°C下适当标记生态型。
  2. 所有步骤都严格在层流罩内的无菌条件下进行。将所有培养基进行高压灭菌(121℃15分钟),培养基补充剂如生长激素,抗生素进行过滤灭菌,并在高压灭菌后加入。
  3. 用双层Parafilm密封培养皿,以保持湿度,养成健康植物。对于高瞬态GUS表达来说,植物很年轻,能够迅速提供健康的根源,这一点至关重要
  4. 始终处理 A。由于Gelvin(2006)详细描述,较年长的植物将导致较低的转化率,因此在花螺栓出现之前获得根外植体的拟南芥植物。
  5. 更重要的是,总是使用新鲜的细菌培养物来获得更高的转化率。不允许细菌培养物(E5105)生长超过0.8 OD 600 nm。更高的OD培养将在共培养期间过度生长细菌,这将对GUS表达产生负面影响
  6. 根部的切割应在30分钟内进行。使切割根部分更长时间会对转染和转化产生负面影响。
  7. 在实验中始终包括未处理的根外植体(无细菌接种物)作为阴性对照
  8. 目前的工作表明,5天共培养时间和200μM乙酰丁香酮的组合提高了A期瞬时GUS表达的速率。 thaliana 根段。

食谱

注意:以下介质已准备好,高压灭菌,并在室温下储存。

  1. Teagasc-tryptone酵母提取物(TTY)培养基
    1.0%胰蛋白胨
    0.5%酵母提取物
    经高压灭菌后,每升培养基中加入20ml无菌CaCl 2/2 在制作TTY琼脂之前,先加入1.5%琼脂给TTY肉汤,然后再进行高压灭菌
  2. 基于MS的媒体(表1)
    注意:将基于MS的培养基倒入培养皿中,可以在4℃下储存长达15天。准备维生素库存,过滤消毒,并在-20°C储存1 ml的等分试样长达1个月。

    表1.用于 的拟媒体拟南芥根系转换(E-ART)


  3. X-GlcA解决方案
    准备1 M的X-GlcA溶液:
    加入521.8mg X-GlcA至10ml DMSO并涡旋以溶解
    在离心管中分装1毫升,储存于-20°C,长期使用
    注意:该物质具有吸湿性和光敏性。
  4. 组织化学GUS染色液
    通过添加以下列出的化学品来制备GUS溶液,以达到最终浓度:
    50mM磷酸钠缓冲液pH 7.0
    1 mM EDTA
    0.1mM铁氰化铁和氰化铁,分别为
    0.1%SDS
    40 mM X-GlcA
    在-20°C储存等分试样

致谢

这项研究工作得到资助DSR的Teagasc Walsh奖学金计划的支持。作者衷心感谢Stanton B.Gelvin教授为拟南芥拟南芥进行农杆菌转化根:定量测定(2006),作为分子生物学方法一章出版, >农杆菌协议,适用于开发E-ART协议。

参考

  1. Bhaskar,PB,Venkateshwaran,M.,Wu,L.,Ane,JM和Jiang,J.(2009)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/19503835"target ="_ blank"> 农杆菌介导的瞬时基因表达和沉默:用于马铃薯功能基因测定的快速工具 PLoS One < / 4(6):e5812。
  2. Broothaerts,W.,Mitchell,HJ,Weir,B.,Kaines,S.,Smith,LM,Yang,W.,Mayer,JE,Roa-Rodriguez,C.and Jefferson,RA(2005) class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15703747"target ="_ blank">通过不同种类的细菌将基因转移到植物上。 >自然 433(7026):629-633。
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引用:Rathore, D. S., Doohan, F. M. and Mullins, E. (2017). Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells. Bio-protocol 7(19): e2564. DOI: 10.21769/BioProtoc.2564.
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