搜索

In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.
植物寄生线虫属线虫寄生前及寄生阶段的原位杂交(ISH)   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
New Phytologist
Oct 2017

Abstract

The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.

Keywords: Gene expression pattern (基因表达模式), Plant pathogen (植物病原体), Preparasitic and parasitic stages (寄生前和寄生阶段), mRNAs (mRNAs)

Background

So far, the stable transformation of plant-parasitic nematode(s) has not been successful. ISH enables the analysis of spatio-temporal gene expression in vivo in whole-mount Meloidogyne spp. nematodes. These root-knot nematodes hatch in the soil as microscopic vermiform juveniles (J2) and infect host plant roots. J2s penetrate the root and migrate to the root vascular cylinder cells. The juveniles settle in the root and develop into J3 and J4 parasitic juveniles that induce the differentiation specialized feeding cells. The nematode eventually develops into a pear-shaped female that will release hundreds of eggs on the root surface. Here, we report a detailed protocol to detect single RNA molecules in preparasitic whole mount J2s and parasitic stages. ISH on parasitic stages requires an additional procedure the day before extraction of the nematodes from infected roots. We describe the detection of transcripts using digoxigenin (DIG)-labeled cDNA probes in nematode whole mount tissues.

Materials and Reagents

  1. Nitrile gloves
  2. RNase-free microcentrifuge tubes (1.5 ml) (e.g., Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: AM12450 )
  3. Sieve of 2.5 mm/250 µm/40 µm/10 µm
  4. 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
  5. Paper towel
  6. Microscope slides and cover slip (e.g., Fisher Scientific, catalog number: 12-544-1 )
  7. Autoclaved razor blades
  8. RNase-free filtered tips, e.g.,
    20 µl tips (Mettler-Toledo, Rainin, catalog number: 17007957 )
    200 µl tips (Mettler-Toledo, Rainin, catalog number: 17002927 )
    1,000 µl tips (Mettler-Toledo, Rainin, catalog number: 17014361 )
  9. Root-knot nematodes (e.g., M. incognita Morelos strain, M. enterolobii Godet strain) preparasitic J2s (10,000) or parasitic stages (50)
  10. DIG-labelled ISH probe(s)
    Note: See Procedure A for probe synthesis and storage.
  11. Forward and reverse primers designed to allow an amplicon size around 200 bp (e.g., SePOP Desalted oligos from Eurogentec)
  12. PCR-grade dNTPs (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297117 )
  13. Pfu DNA Polymerase (Promega, catalog number: M7741 )
  14. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
  15. QIAquick Gel extraction kit (QIAGEN, catalog number: 28704 )
  16. Digoxigenin (DIG) DNA Labeling Kit (Roche Diagnostics, catalog number: 11175025910 )
  17. TE buffer pH 8 (10 mM Tris-HCl, 1 mM EDTA) (e.g., Sigma-Aldrich, catalog number: 93283 )
  18. RNaseZap® RNase Decontamination Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  19. 70% ethanol
  20. Tap water
  21. Acetone (e.g., VWR, catalog number: 20065.293 )
  22. Methanol (e.g., VWR, catalog number: 20847.307 )
  23. Anti-Digoxigenin-AP-Fab fragments Labelling Mix (Roche Diagnostics, catalog number: 11093274910 )
  24. BCIP 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (Roche Diagnostics, catalog number: 11383221001 )
  25. NBT 4-Nitro blue tetrazolium chloride (Roche Diagnostics, catalog number: 11383213001 )
  26. Pectinex® (Sigma-Aldrich, catalog number: P2611 )
  27. Celluclast® (Sigma-Aldrich, catalog number: C2730 )
  28. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  29. Potassium phosphate, monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  30. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  31. Magnesium sulfate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138 )
  32. Sucrose (e.g., Sigma-Aldrich, catalog number: S9378 )
  33. 10x PBS (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9624 )
  34. 37% formaldehyde solution (Sigma-Aldrich, catalog number: F15587 )
    Note: This product has been discontinued.
  35. Proteinase K 20 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2546 )
  36. Formamide, deionized (Sigma-Aldrich, catalog number: F9037 )
  37. SSC buffer 20x (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9770 )
  38. Boehringer blocking reagent (Roche Diagnostics, catalog number: 11096176001 )
  39. SDS (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15553027 )
  40. Denhart’s solution (Sigma-Aldrich, catalog number: D2532 )
  41. EDTA pH 8 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9261 )
  42. Salmon sperm DNA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15632011 )
  43. tRNA from baker’s yeast (Sigma-Aldrich, catalog number: R8759 , type X-SA)
  44. Maleic acid (Fisher Scientific, catalog number: 10348843)
    Manufacturer: Acros Organics, catalog number: 125230051 .
  45. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  46. Sodium hydroxide (NaOH) (VWR, catalog number: 28245.298 )
  47. Tris-base (e.g., Sigma-Aldrich, catalog number: T1503 )
  48. Concentrated HCl (VWR, catalog number: 20248.295 , type 35%)
  49. Deionized water
  50. Nuclease-free water (not DEPC-Treated; Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9938 )
  51. For nematode extraction (see Recipes)
    1. Pectinex® and Celluclast® mix
    2. M9-buffer pH 7
    3. 50% sucrose in M9 buffer
    4. RNase-free 1x PBS
  52. For probe, fixation and hybridization, nuclease-free (see Recipes)
    1. Fixative buffer
    2. 1 mg/ml Proteinase K solution
    3. tRNA from Baker’s yeast
    4. Hybridization buffer (HB)
    5. Washing buffer 1 (4x SSC + 0.1% SDS)
    6. Washing buffer 2 (0.1x SSC + 0.1% SDS)
    7. 10% Boehringer blocking reagent
    8. Filtered maleic acid buffer, pH 7.5
    9. Tris-HCl pH 9.5
    10. Alkaline phosphatase detection buffer pH 9.5 (APB)

Equipment

  1. Pipetmens (e.g., Gilson, models: P2 , P20 , P200 , P1000 )
  2. Thermocycler (e.g., Biometra, model: T3000 )
  3. NanoDrop (e.g., Agilent Technologies, model: Agilent 2100 Bioanalyser , catalog number: G2939)
  4. Microcentrifuge at room temperature (RT) (e.g., Hitachi Koki, model: CT15E ) with fixed-angle rotor (e.g., Hitachi Koki, model: T15A61 )
  5. Centrifuge for 50 ml tube (e.g., Eppendorf, model: 5804 R ) with swing-bucket rotor (e.g., Eppendorf, model: A-4-44 )
  6. 50 ml glass beaker (Corning, PYREX®)
  7. Fume hood
  8. Aquarium air pump (Rena Aquatic Supply, model: Model 301 )
  9. Glass plate
  10. Mini hybridization oven (Appligene, catalog number: 001050414 )
  11. Vortexer (e.g., Baxter, catalog number: S8223-1 )
  12. Orbital shaker (VWR)
  13. 37 °C water bath
  14. Fridge at 4 °C
  15. -20 °C freezer
  16. -80 °C freezer
  17. Autoclave
  18. Microscope (ZEISS, model: Axioplan 2 )

Procedure

  1. PCR digoxigenin (DIG)-labeled cDNA antisense and control (sense) probe synthesis and purification
    1. PCR amplify a selected specific fragment from the targeted gene (~200 bp) using cDNA or, in absence of introns, genomic DNA as a matrix, with forward and reverse primers 1 µM each, dNTPs 250 µM and proof-reading Pfu polymerase.
    2. Purify the PCR1 fragment with QIAquick PCR Purification Kit if a single band is obtained or purify the band from the gel with QIAquick gel extraction kit, elute with 10 µl elution buffer and quantify on gel or using NanoDrop.
    3. Perform the DIG labeling step through a second PCR by using DIG labeled dUTPs using 50-100 ng of PCR1, 2 µM forward or reverse primer for sense or anti-sense probe, respectively, DIG-dUTP/dNTP mix (DIG DNA labeling kit).
    4. Purify PCR product with QIAquick PCR Purification Kit and resuspend in 10 µl TE buffer at pH 8.0.
    5. For quality control, run small amount (1 µl) of the labeled probe (PCR2) on an agarose gel. In addition, probes can be tested using dot blot to analyze the sensitivity or Northern blot for specificity (Liao et al., 2014), prior to in situ hybridization (ISH).
    6. DIG-labeled cDNA probes are stored at -20 °C. They are stable for more than a year.

  2. Day 1. Preparasitic or parasitic root-knot nematode (RKN) extraction from roots and fixation for ISH
    1. To extract parasitic stages from infected roots, rinse roots with tap water, cut them into ~1 cm sticks.
    2. Immerse roots in the Pectinex® and Celluclast® mix and incubate the mixture for 4 h (to overnight) at RT under gentle agitation (orbital shaker, 50 rounds per minute).
    3. Prepare 2 Corning tubes containing 40 ml of 50% sucrose/M9 and store them in the fridge at 4 °C.
    4. After digestion, pour the liquid through a 2.5 mm sieve and collect the filtrate.
    5. Pour the liquid through a 250 µm sieve and collect the filtrate.
    6. Pour the liquid through a 40 µm sieve.
    7. Collect the parasitic RKNs on the 40 µm sieve with a minimum volume of tap water (~8 ml).
    8. Purify the nematodes by sucrose gradient centrifugation: add slowly 4 ml of the collected nematode on the top of cold 50% sucrose/M9 mix (2 tubes) (Figure 1).
    9. Centrifuge at 720 x g, at 4 °C for 5 min, without a break.
    10. Collect the layer of nematodes with a P1000 and wash them carefully adding 40 ml M9 buffer in a 50 ml glass beaker (Figure 1).


      Figure 1. Extraction of parasitic stages by sucrose gradient

    11. To remove sucrose, wash them twice with 50 ml M9, on the 10 µm sieve, in a 50 ml glass beaker and finally transfer to a 50-ml Corning tube.
    12. Collected nematodes are at the parasitic juvenile’s stage 2 (J2), 3 (J3) or 4 (J4), or adult females. Freshly hatched RKN preparasitic J2 are obtained from eggs as described in Caillaud and Favery (2016).
    13. Under a fume hood, with gloves, resuspend the nematodes (10,000 J2s or at least 50 parasitic stages) in 10 ml of fixative buffer in a 50-ml Corning tube. The fixation lasts overnight (16 h) for the preparasitic J2 and 48 h for parasitic stages at RT.

  3. Day 2. (or Day 4 for parasitic stages) Nematode cutting and permeabilization for ISH
    1. Wipe down bench top and pipettors with RNaseZap®.
    2. Clean a glass plate with soap, rinse with nuclease-free water, spray RNaseZap® and rinse with 70% ethanol. Dry well with a paper towel.
    3. Centrifuge the 50 ml tube containing nematodes soaked in fixative buffer at 1,620 x g, slow deceleration.
    4. Withdraw fixative buffer but keep 500 µl and transfer to 1.5 ml Eppendorf tubes.
    5. Under the fume hood, wearing gloves, spread the nematodes on the glass plate, and cut the nematodes into two to three pieces using the cutting system (vibrating aquarium pump + autoclaved razor blades) described in Figure 2 and Video 1. Hold it with both hands. Check under the microscope the efficiency of the cutting. Do not cut too much, in particular the parasitic stages.


      Figure 2. Nematode cutting before ISH. Diagram (A) and picture of the experimental device (B).

      Video 1. Nematode cutting for ISH

    6. Add few drops of 1x RNase-free PBS buffer to collect the nematode sections on the glass plate and transfer into an Eppendorf tube.
    7. Spin down the sample (15,000 x g, 1 min, slow deceleration). Remove the buffer.
    8. Suspend in 1 ml of 1x RNase-free PBS, and then centrifuge the tube at 15,000 x g for 1 min with slow deceleration. Repeat Steps C7 and C8 three times.
    9. For nematode permeabilization, add 1 ml of cold 1 mg/ml Proteinase K solution to the nematodes. Keep Proteinase K in a cold block and do not vortex.
    10. Incubate the tube at 37 °C for 1 h under rotation (in a hybridization oven).
    11. Spin down the sample (15,000 x g, 1 min, slow deceleration). Keep the pellet.
    12. Wash the sample in 1 ml of 1x PBS and centrifuge the tube at 15,000 x g for 1 min with slow deceleration. Keep the pellet. Repeat this step three times. In the last wash, remove as much 1x PBS as possible to obtain a dry pellet.
    13. Place the pellet at -80 °C for 15 min.
    14. Add 1 ml cold methanol (precool at -80 °C).
    15. Vortex to suspend the nematodes and leave them 2 min at RT.
    16. Spin down the sample (15,000 x g, 1 min, slow deceleration).
    17. Remove methanol.
    18. Add 1 ml cold acetone (precool at -80 °C).
    19. Place the tube at -80 °C for 15 min.
    20. Spin down the sample (15,000 x g, 1 min, slow deceleration).

  4. Day 2 (or Day 4 for parasitic stages): (in situ) hybridization
    1. Set the temperature of the hybridization oven up to 50 °C
    2. Preheat the hybridization buffer (HB) at 50 °C.
    3. Remove acetone.
    4. Add 500 µl of pre-heat HB on the nematode pellet.
    5. Spin down the sample (15,000 x g, 1 min, slow deceleration) and remove the supernatant.
    6. Add 500 µl of pre-heated HB on the nematode pellet.
    7. Incubate for 30 min at 50 °C under rotation in the oven.
    8. Meanwhile, denature the probes at 100 °C for 5 min (water bath) and transfer onto ice immediately (let the temperature drop swiftly to prevent re-annealing). Keep them on ice for 3 min.
    9. Spin down the sample (15,000 x g, 1 min, slow deceleration).
    10. Set the temperature of the oven at 42 °C.
    11. Suspend the nematodes in 100 µl HB per probe (e.g., 500 µl for 5 probes).
    12. Dispatch 100 µl HB into individual nuclease free tubes.
    13. Add 5 µl of each probe.
    14. Perform the hybridization overnight at 42 °C (40-50 °C, see Notes) under agitation in the oven.

  5. Day 3 (or Day 5 for parasitic stages): Washes and revelation
    1. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    2. Wash the pellet with 1 ml washing buffer 1 (4x SSC, 0.1% SDS), 10 min at RT under rotation.
    3. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    4. Repeat Steps E2 and E3 twice.
    5. Wash the pellet with 1 ml washing buffer 2 (0.1x SSC, 0.1% SDS), 10 min at 50 °C under rotation.
    6. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    7. Repeat Steps E5 and E6 twice.
    8. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    9. Wash 30 sec with 1 ml of filtered maleic acid buffer (1x).
    10. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    11. Incubate the nematodes at 37 °C for 30 min (oven) in 1 ml of 1x Boehringer blocking reagent (see Recipes).
    12. Incubate the nematodes at 37 °C for 3 h in 1 ml of the anti-DIG antibody coupled with Alkaline Phosphatase (1:500) in 1x Boehringer blocking reagent.
    13. Spin down the sample (15,000 x g, 1 min, slow deceleration). Discard supernatant.
    14. Wash three times each with 1 ml 1x filtered maleic acid buffer at 37 °C during 15 min.
    15. Wash briefly with 1 ml Alkaline Phosphatase detection buffer (APB).
    16. Reveal the signal by incubating the nematodes for 6 to 8 h at RT in 1 ml APB + 3.75 µl BCIP + 5 µl NBT without agitation.
    17. Put a drop on a microscope slide, add a cover slip.
    18. Observe under a microscope (Axioplan2, 10x objective).
    19. If no signal is detected, continue the incubation overnight at 4 °C.
    20. Wash the nematode with nuclease-free water once.
    21. Put a drop on a microscope slide, add a cover slip.
    22. Observe under the microscope and image nematodes.

Data analysis

Nematodes are examined with differential interference contrast microscopy. The signal observed should be compared to the images obtained with the control sense probe (negative control) (Figure 3). A positive control should be added. We usually used the MiPG gene encoding a polygalacturonase expressed in the two sub ventral glands (Jaubert et al., 2002a). We recommend taking pictures of at least 40 stained nematodes, analyzing the cellular localization of detected RNA spots, and to count the number of stained nematodes. Each ISH experiment should be reproduced independently. ISH could be performed after siRNA gene silencing of the target gene to ensure specificity and/or to evaluate the silencing effect. Moreover, control experiments using a different set of probes hybridizing the same target gene could be done to ensure the specificity of the probes to the target gene.


Figure 3. Tissue expression of M. incognita EFFECTOR1 (MiEFF1) in preparasitic J2 and parasitic juveniles. A-B. Transcripts from MiEFF1 (Minc17998) were localized in the salivary dorsal gland (arrowhead) of preparasitic J2s. C. The sense MiEFF1 control probe yielded no labeling of nematode tissues. MiEFF1 tissue expression in a parasitic juvenile was presented in Jaouannet et al., 2012. Scale bars = 50 µm.

Notes

  1. For nematode purification and fixation, prepare fresh solutions immediately before use.
  2. Prepare two Corning tubes containing 40 ml of 50% sucrose/M9 and store them in the fridge at 4 °C.
  3. For ISH, wear gloves. All solutions are RNAse Free, do not pipette directly into the Master solutions.
  4. All the experiments performed with the fixative and hybridization buffers should be performed under the fume hood.
  5. Be very careful when using the razor blade.
  6. Day 1 Place aliquots of methanol and acetone at -80 °C for nematode permeabilization step of the next day.
  7. For nematode permeabilization, do not vortex the Proteinase K and keep it in a cold block.
  8. The temperature of hybridization can be adjusted to obtain the best balance between the specificity and quantity of the signal.
  9. Digoxigenin (DIG)-labeled RNA probes can be used instead of cDNA probes as in Rosso et al. (1999).
  10. Use nuclease-free, not DEPC-treated, water.
  11. For revelation, horseradish peroxidase (HRP) can be used in replacement of alkaline phosphatase (AP). We chose AP because the signal from AP substrates gradually increases over time, is very stable and can last several days.

Recipes

For nematode extraction

  1. Pectinex® and Celluclast® mix
    15 ml Celluclast®
    30 ml Pectinex®
    Tap H2O up to 200 ml
  2. M9-buffer
    42.3 mM Na2HPO4
    22 mM KH2PO4
    85.6 mM NaCl
    1 mM MgSO4·7H2O
  3. 50% sucrose in M9
    50 g sucrose
    M9 buffer up to 100 ml
  4. RNase-free 1x PBS
    1 ml 10x PBS
    9 ml nuclease-free distilled water

For probe, fixation and hybridization, nuclease-free
  1. Fixative buffer
    1 ml 37% formaldehyde solution
    1x PBS up to 10 ml
    Notes:
    1. Prepare and manipulate under the fume hood.
    2. If 37% formaldehyde solution was stored at 4 °C; warm it to RT prior to opening.
  2. 1 mg/ml Proteinase K solution
    1 ml Proteinase K solution (20 mg/ml)
    1x PBS up to 20 ml
  3. tRNA from Baker’s yeast
    Dissolve 500 U yeast tRNA in 1 ml of nuclease-free water
    Aliquot and store at -80 °C
  4. Hybridization buffer (HB)
    Note: Prepare and manipulate under the fume hood and store at -20 °C.
    5 ml deionized formamide (99.5%, 4 °C)
    0.5 ml SSC buffer (20x, RT)
    1 ml Boehringer blocking reagent (10%, 4 °C)
    2 ml SDS (10%, RT)
    200 µl Denhart’s solution (50x, -20 °C)
    20 µl EDTA pH 8 (0.5 M, RT)
    200 µl salmon sperm DNA (10 mg/µl, -20 °C)
    62 µl tRNA from Baker’s yeast (500 U/ml, -80 °C)
    Nuclease-free water up to 10 ml
  5. Washing buffer 1 (4x SSC + 0.1% SDS)
    400 µl SSC buffer (20x, RT)
    20 µl SDS (10%, RT)
    Nuclease-free water up to 2 ml
  6. Washing buffer 2 (0.1x SSC + 0.1% SDS)
    10 µl SSC buffer (20x, RT)
    20 µl SDS (10%, RT)
    Nuclease-free water up to 2 ml
  7. 10% Boehringer blocking reagent
    1 g blocking reagent powder in a bottle
    Filtered maleic acid buffer, pH 7.5 up to 10 ml
    Autoclave to dissolve and store at 4 °C
  8. 12. Filtered maleic acid buffer, pH 7.5
    11.61 g maleic acid (100 mM)
    8.76 g NaCl (150 mM)
    Adjust pH to 7.5 by NaOH
    Autoclave, filtrate and store at RT
  9. Tris-HCl 2 M, pH 9.5
    1. Dissolve 121.1 g of Tris-base in 400 ml of deionized water
    2. Adjust the pH to the desired value by adding concentrated HCl
    3. Allow the solution to cool to room temperature before making final adjustments to the pH
    4. Adjust the volume of the solution to 500 ml with deionized water
    5. Dispense into 50 ml aliquots and sterilize by autoclaving
  10. Alkaline phosphatase detection buffer pH 9.5 (APB)
    50 ml Tris-HCl 2 M pH 9.5 (100 mM final)
    5.85 g NaCl (100 mM final)
    10.17 g MgCl2·7H2O (50 mM final)
    Nuclease-free water up to 1 L
    Aliquot, autoclave and store at room temperature

Acknowledgments

This protocol has been adapted from de Boer et al. (1998) and Rosso et al. (1999). It has made possible the study of the expression profile of several dozens of genes (Jaubert et al., 2002a and 2002b; Neveu et al., 2003; Jaubert et al., 2004; Ledger et al., 2006; Dubreuil et al., 2007; Jaouannet et al., 2012; Jaouannet et al., 2013; Danchin et al., 2013; Nguyen et al., 2017). These works were funded by INRA, CNRS, University of Nice Sophia Antipolis, the Region Provence-Alpes-Côte d’Azur, the French Government, the European Union and NATO Collaborative Research Grant. C-N.N. was supported by USTH fellowships, 911-USTH program, from the Ministry of Education and Training of The Socialist Republic of Vietnam. The authors have no conflict of interest or competing interests to declare.

References

  1. Caillaud, M. C. and Favery, B. (2016). In vivo imaging of microtubule organization in dividing giant cell. Methods Mol Biol 1370: 137-144.
  2. Danchin, E. G. J., Arguel, M.-J., Campan-Fournier, A., Perfus-Barbeoch, L., Magliano, M., Rosso, M.-N., Da Rocha, M., Da Silva, C., Nottet, N., Labadie, K., Guy, J., Artiguenave, F. Abad, P. (2013). Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining. PLoS Pathog 9(10): e1003745.
  3. de Boer, J. M., Yan, Y., Smant, G., Davis, E. L. and Baum, T. J. (1998). In-situ hybridization to messenger RNA in Heterodera glycines. J Nematol 30(3): 309-312.
  4. Dubreuil, G., Magliano, M., Deleury, E., Abad, P. and Rosso, M. N. (2007). Transcriptome analysis of root-knot nematode functions induced in the early stages of parasitism. New Phytol 176(2): 426-436.
  5. Jaouannet, M., Magliano, M., Arguel, M. J., Gourgues, M., Evangelisti, E., Abad, P. and Rosso, M. N. (2013). The root-knot nematode calreticulin Mi-CRT is a key effector in plant defense suppression. Mol Plant Microbe Interact 26(1): 97-105.
  6. Jaouannet, M., Perfus-Barbeoch, L., Deleury, E., Magliano, M., Engler, G., Vieira, P., Danchin, E. G., Da Rocha, M., Coquillard, P., Abad, P. and Rosso, M. N. (2012). A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei. New Phytol 194(4): 924-931.
  7. Jaubert, S., Laffaire, J. B., Abad, P. and Rosso, M. N. (2002a). A polygalacturonase of animal origin isolated from the root-knot nematode Meloidogyne incognita. FEBS Lett 522(1-3): 109-112.
  8. Jaubert, S., Laffaire, J. B., Ledger, T. N., Escoubas, P., Amri, E. Z., Abad, P. and Rosso, M. N. (2004). Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita. Int J Parasitol 34(7): 873-880.
  9. Jaubert, S., Ledger, T. N., Laffaire, J. B., Piotte, C., Abad, P. and Rosso, M. N. (2002b). Direct identification of stylet secreted proteins from root-knot nematodes by a proteomic approach. Mol Biochem Parasitol 121(2): 205-211.
  10. Ledger, T. N., Jaubert, S., Bosselut, N., Abad, P. and Rosso, M. N. (2006). Characterization of a new β-1,4-endoglucanase gene from the root-knot nematode Meloidogyne incognita and evolutionary scheme for phytonematode family 5 glycosyl hydrolases. Gene 382: 121-128.
  11. Liao, Y., Fung, T. S., Huang, M., Fang, S., Zhong, Y. and Liu, D. (2014). RNA isolation and Northern blot analysis. Bio-protocol 4(6): e1077.
  12. Neveu, C., Jaubert, S., Abad, P. and Castagnone-Sereno, P. (2003). A set of genes differentially expressed between avirulent and virulent Meloidogyne incognita near-isogenic lines encode secreted proteins. Mol Plant Microbe Interact 16(12): 1077-1084.
  13. Nguyen, C. N., Perfus-Barbeoch, L., Quentin, M., Zhao, J., Magliano, M., Marteu, N., Da Rocha, M., Nottet, N., Abad, P. and Favery, B. (2017). A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism. New Phytol.
  14. Rosso, M. N., Favery, B., Piotte, C., Arthaud, L., De Boer, J. M., Hussey, R. S., Bakker, J., Baum, T. J. and Abad, P. (1999). Isolation of a cDNA encoding a β-1,4-endoglucanase in the root-knot nematode Meloidogyne incognita and expression analysis during plant parasitism. Mol Plant Microbe Interact 12(7): 585-591.

简介

基因的时空表达模式为更好地理解其生物学功能提供了重要的指示。 原位杂交(ISH)使用标记的互补单链RNA或DNA探针来定位整个生物体,整个器官或一部分组织中的基因转录物。 我们将ISH技术应用于植物寄生虫
【背景】到目前为止,植物寄生性线虫的稳定转化尚未成功。 ISH能够在整个装载的Meloidogyne spp中分析体内时空基因表达。线虫。这些根结线虫在土壤中以微小蚓状幼虫(J2)形式孵化并感染宿主植物根部。 J2s穿透根部并迁移到根部维管柱状细胞。幼虫定居在根部,发育成J3和J4寄生幼鱼,诱导分化专化饲养细胞。线虫最终发育成梨形雌性,将在根表面释放数百个卵。在这里,我们报告了一个详细的协议来检测准备性整体安装J2s和寄生阶段中的单个RNA分子。寄生虫阶段的ISH需要在感染根部提取线虫前一天采取额外的程序。我们描述了在线虫整个组织中使用地高辛(DIG)标记的cDNA探针检测转录物。

关键字:基因表达模式, 植物病原体, 寄生前和寄生阶段, mRNAs

材料和试剂

  1. 丁腈手套
  2. 无RNA酶的微量离心管(1.5ml)(例如,Thermo Fisher Scientific,Applied Biosystems TM,目录号:AM12450)。

  3. 2.5 mm / 250μm/ 40μm/ 10μm的筛网
  4. 50毫升试管(Corning,Falcon ,目录号:352070)
  5. 纸巾
  6. 显微镜载玻片和盖玻片(如,Fisher Scientific,目录号:12-544-1)
  7. 高压灭菌刀片
  8. 无RNase滤芯,例如
    20微升的提示(梅特勒 - 托利多,Rainin,目录号:17007957)
    200微升的提示(梅特勒 - 托利多,Rainin,目录号:17002927)
    1,000μl吸头(梅特勒 - 托利多,Rainin,目录号:17014361)
  9. (10,000)或寄生虫阶段(50),根结线虫(例如,,M。incognita 莫雷洛斯菌株,M。enterolobii Godet菌株) )
  10. DIG标记的ISH探针(多种)
    注意:有关探针合成和存储的信息,请参阅程序A.
  11. 正向和反向引物设计为允许大约200bp的扩增子大小(例如来自Eurogentec的SePOP脱盐寡聚体)。
  12. PCR级dNTPs(Thermo Fisher Scientific,Invitrogen TM,目录号:10297117)
  13. Pfu DNA聚合酶(Promega,目录号:M7741)
  14. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  15. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  16. 地高辛(DIG)DNA标记试剂盒(Roche Diagnostics,目录号:11175025910)
  17. TE缓冲液pH 8(10mM Tris-HCl,1mM EDTA)(例如,Sigma-Aldrich,目录号:93283)。
  18. RNaseZap RNase去污溶液(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9780)
  19. 70%乙醇
  20. 自来水
  21. 丙酮(,例如,VWR,目录号:20065.293)
  22. 甲醇(例如,VWR,目录号:20847.307)
  23. 抗地高辛配基-AP-Fab片段标记混合物(Roche Diagnostics,目录号:11093274910)
  24. BCIP 5-溴-4-氯-3-吲哚基 - 磷酸酯,4-甲苯胺盐(Roche Diagnostics,目录号:11383221001)
  25. NBT 4-硝基蓝四唑氯化物(Roche Diagnostics,目录号:11383213001)
  26. Pectinex(Sigma-Aldrich,目录号:P2611)
  27. Celluclast (Sigma-Aldrich,目录号:C2730)
  28. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  29. 磷酸钾一元(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  30. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  31. 硫酸镁(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:63138)
  32. 蔗糖(例如,Sigma-Aldrich,目录号:S9378)
  33. 10x PBS(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9624)
  34. 37%甲醛溶液(Sigma-Aldrich,目录号:F15587)
    注意:此产品已停产。
  35. 蛋白酶K 20mg / ml(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2546)
  36. 去甲基化甲酰胺(Sigma-Aldrich,目录号:F9037)
  37. SSC缓冲液20x(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9770)
  38. 勃林格封闭剂(Roche Diagnostics,目录号:11096176001)
  39. SDS(Thermo Fisher Scientific,Invitrogen TM,目录号:15553027)
  40. Denhart的解决方案(Sigma-Aldrich,目录号:D2532)
  41. EDTA pH 8(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9261)
  42. 鲑鱼精子DNA(Thermo Fisher Scientific,Invitrogen TM,目录号:15632011)
  43. 来自面包酵母(Sigma-Aldrich,目录号:R8759,型号X-SA)的tRNA
  44. 马来酸(Fisher Scientific,目录号:10348843)
    制造商:Acros Organics,产品目录号:125230051。
  45. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  46. 氢氧化钠(NaOH)(VWR,目录号:28245.298)
  47. Tris-碱(例如,Sigma-Aldrich,目录号:T1503)
  48. 浓HCl(VWR,目录号:20248.295,类型35%)
  49. 去离子水
  50. 无核酸酶的水(未经DEPC处理; Thermo Fisher Scientific,Invitrogen TM,目录号:AM9938)
  51. 线虫提取(见食谱)
    1. Pectinex ®和Celluclast® mix
    2. M9缓冲液pH 7
    3. M9缓冲液中有50%蔗糖
    4. 无RNase 1x PBS
  52. 对于探针,固定和杂交,无核酸酶(见食谱)
    1. 固定缓冲液
    2. 1毫克/毫升蛋白酶K溶液
    3. 来自贝克酵母的tRNA
    4. 杂交缓冲液(HB)
    5. 洗涤缓冲液1(4x SSC + 0.1%SDS)
    6. 洗涤缓冲液2(0.1x SSC + 0.1%SDS)
    7. 10%勃林格封闭剂
    8. 过滤马来酸缓冲液,pH 7.5
    9. Tris-HCl pH 9.5
    10. 碱性磷酸酶检测缓冲液pH 9.5(APB)

设备

  1. Pipetmens(例如,Gilson,型号:P2,P20,P200,P1000)
  2. 热循环仪(例如,Biometra,型号:T3000)。
  3. NanoDrop(例如,Agilent Technologies,型号:Agilent 2100生物分析仪,目录号:G2939)
  4. 使用固定角转子(例如,Hitachi Koki,型号:T15A61)在室温下(RT)(例如,Hitachi Koki,型号:CT15E)微量离心。
  5. 用摆动转子(例如Eppendorf,型号:A-4-44)对50ml离心管(例如Eppendorf,型号:5804R) >
  6. 50毫升玻璃烧杯(Corning,PYREX®)。
  7. 通风橱
  8. 水族馆气泵(瑞纳水产供应,型号:301型)
  9. 玻璃板
  10. 小型杂交炉(Appligene,目录号:001050414)
  11. Vortexer( ,Baxter,产品目录号:S8223-1)
  12. 轨道振动筛(VWR)

  13. 37°C水浴
  14. 在4°C的冰箱
  15. -20°C冷冻机
  16. -80°C冷冻机
  17. 高压灭菌器
  18. 显微镜(蔡司,型号:Axioplan 2)

程序

  1. PCR地高辛(DIG)标记的cDNA反义和对照(有义)探针的合成和纯化
    1. 使用cDNA或在不存在内含子的情况下使用cDNA从靶基因(〜200bp)扩增选定的特定片段,基因组DNA作为基质,正向和反向引物各1μM,dNTP250μM和校正读数Pfu聚合酶。 br />
    2. 如果获得单一条带,用QIAquick PCR纯化试剂盒纯化PCR1片段,或用QIAquick凝胶提取试剂盒从凝胶中纯化条带,用10μl洗脱缓冲液洗脱并在凝胶上或使用NanoDrop定量。
    3. 使用DIG标记的dUTP分别使用50-100ngPCR1,2μM正义或反义探针正向或反义探针DIG-dUTP / dNTP混合物(DIG DNA标记试剂盒)通过第二次PCR进行DIG标记步骤, 。
    4. 用QIAquick PCR纯化试剂盒纯化PCR产物,并重悬于pH为8.0的10μlTE缓冲液中。
    5. 为了质量控制,在琼脂糖凝胶上少量(1μl)标记探针(PCR2)。此外,在原位杂交(ISH)之前,可以使用斑点印迹来检测探针以分析灵敏度或RNA特异性的Northern印迹(Liao 。,2014) 。
    6. DIG标记的cDNA探针储存在-20°C。他们稳定了一年多。

  2. 第一天:从根部提取的预处理或寄生根结线虫(RKN)和ISH固定
    1. 为了从受感染的根中提取寄生虫阶段,用自来水冲洗根部,将其切成约1厘米的棒。
    2. 将根部浸入Pectinex®和Celluclast®混合物中,在室温下轻轻搅拌(定轨摇床,每分钟50转),将混合物孵育4小时(至过夜)。
    3. 准备含有40毫升50%蔗糖/ M9的2个康宁管,并将它们储存在4°C的冰箱中。
    4. 消化后,将液体倒入2.5毫米筛并收集滤液。
    5. 将液体倒入250μm筛子并收集滤液。
    6. 将液体倒入40μm筛子。

    7. 采用最小量的自来水(〜8 ml)收集40μm筛上的寄生RKNs。
    8. 通过蔗糖梯度离心纯化线虫:在冷的50%蔗糖/ M9混合物(2管)的顶部缓慢加入4ml所收集的线虫(图1)。
    9. 在720℃下在4℃下离心5分钟,没有中断。
    10. 用P1000收集线虫层,小心地将它们加入50ml玻璃烧杯中,加入40ml M9缓冲液(图1)。


      图1.通过蔗糖梯度提取寄生阶段

    11. 要去除蔗糖,用50毫升M9,在10微米筛上,50毫升玻璃烧杯中洗两次,最后转移到50毫升康宁管中。
    12. 收集的线虫处于寄生青少年的阶段2(J2),3(J3)或4(J4)或成年女性。新鲜孵化的RKN准选择性J2从蛋中获得,如Caillaud和Favery(2016)所述。
    13. 在带有手套的通风橱中,在50ml Corning试管中重悬10 ml固定缓冲液中的线虫(10,000个J2s或至少50个寄生虫阶段)。
      固定持续过夜(16小时),准备阶段J2,寄生阶段48小时
  3. 第2天(或寄生阶段的第4天)ISH的线虫切割和透化
    1. 用RNaseZap ®擦拭台式和移液器。
    2. 用肥皂清洁玻璃板,用不含核酸酶的水冲洗,喷淋RNaseZap ,并用70%乙醇冲洗。
      用纸巾擦干
    3. 缓慢减速离心含有线虫浸泡在固定缓冲液中的50ml试管,放置速度为1,620×g。
    4. 取出固定缓冲液,但保持500μl并转移到1.5ml Eppendorf管中。
    5. 在通风橱中,戴上手套,将线虫铺在玻璃板上,使用图2和视频1中所述的切割系统(振动水族箱泵+蒸压剃须刀片)将线虫切成两到三块。手中。在显微镜下检查切割的效率。切勿过多切割,尤其是寄生阶段。


      图2. ISH之前的线虫切割图(A)和实验装置(B)的图片。

      s视频1

    6. 加入几滴不含1x RNase的PBS缓冲液以收集玻璃板上的线虫部分并转移到Eppendorf管中。
    7. 旋下样品(15,000 em x g,1 min,缓慢减速)。删除缓冲区。
    8. 悬浮于1ml无1x RNase的PBS中,然后以15,000xg g离心1分钟,缓慢减速。
      重复步骤C7和C8三次
    9. 对于线虫透化,向线虫中加入1ml冷1mg / ml蛋白酶K溶液。将蛋白酶K保存在冷冻块中,不要漩涡。

    10. 在旋转下(在杂交炉中)37°C孵育1小时。
    11. 旋下样品(15,000 em x g,1 min,缓慢减速)。保持颗粒。
    12. 将样品在1ml 1x PBS中洗涤并以15,000xg g离心1分钟并缓慢减速。保持颗粒。重复这一步三次。在最后一次洗涤中,尽可能多地去除1x PBS以获得干燥颗粒。

    13. 将沉淀置于-80°C下15分钟
    14. 加入1毫升冷甲醇(-80°C预冷)。
    15. 漩涡以暂停线虫,并在室温放置2分钟。
    16. 旋转样品(15,000 em x g,1 min,慢速减速)。
    17. 去除甲醇。
    18. 加入1 ml冷丙酮(预冷至-80°C)。
    19. 将试管置于-80°C下15分钟。
    20. 旋转样品(15,000 em x g,1 min,慢速减速)。

  4. 第2天(或第4天寄生阶段):(原位杂交)

    1. 将杂交炉的温度设置为50°C

    2. 在50°C预热杂交缓冲液(HB)
    3. 去除丙酮。

    4. 在线虫颗粒上加入500μl预热HB
    5. 旋下样品(15,000×g,1分钟,缓慢减速)并除去上清液。

    6. 在线虫颗粒上加入500μl预热HB

    7. 在50°C下在烤箱中旋转下孵育30分钟
    8. 同时,将探针在100℃下变性5分钟(水浴)并立即转移到冰上(让温度迅速下降以防止再次退火)。将它们放在冰上3分钟。
    9. 旋转样品(15,000 em x g,1 min,慢速减速)。
    10. 将烤箱的温度设置为42°C。
    11. 每个探针以100μlHB悬浮线虫(,例如,500μl用于5个探针)。
    12. 将100μlHB分装到单个无核酸酶的试管中。
    13. 每个探针添加5μl。

    14. 在42°C(40-50°C,请参阅注意事项)下,在烘箱中搅动下进行杂交过夜。

  5. 第3天(或第5天寄生阶段):洗涤和启示
    1. 旋下样品(15,000 em x g,1 min,缓慢减速)。丢弃上清。
    2. 用1ml洗涤缓冲液1(4x SSC,0.1%SDS)洗涤沉淀,在室温下旋转10分钟。
    3. 旋下样品(15,000 em x g,1 min,缓慢减速)。丢弃上清。
    4. 重复步骤E2和E3两次。
    5. 用1ml洗涤缓冲液2(0.1x SSC,0.1%SDS)洗涤沉淀,在50℃旋转10分钟。
    6. 旋下样品(15,000 em x g,1 min,缓慢减速)。丢弃上清。

    7. 重复步骤E5和E6两次
    8. 旋下样品(15,000 em x g,1 min,缓慢减速)。弃上清。
    9. 用1ml过滤的马来酸缓冲液(1x)洗30秒。
    10. 旋下样品(15,000 em x g,1 min,缓慢减速)。丢弃上清。
    11. 在37℃孵育线虫30分钟(烤箱)在1毫升的1倍勃林格封闭试剂(见食谱)。
    12. 在1ml Boehringer封闭剂中加入1ml与碱性磷酸酶(1:500)偶联的抗DIG抗体,37℃孵育线虫3小时。
    13. 旋下样品(15,000 em x g,1 min,缓慢减速)。丢弃上清。

    14. 每次用1ml 1x过滤马来酸缓冲液在37℃洗涤三次,每次15分钟
    15. 用1 ml碱性磷酸酶检测缓冲液(APB)简单冲洗。
    16. 通过将线虫在室温下在1ml APB +3.75μlBCIP +5μlNBT中温育6至8小时而没有搅动显示信号。
    17. 把一滴放在显微镜载玻片上,加一个盖玻片。
    18. 在显微镜下观察(Axioplan2,10倍物镜)。
    19. 如果没有检测到信号,继续在4°C孵育过夜。

    20. 用无核酸酶的水清洗线虫
    21. 把一滴放在显微镜载玻片上,加一个盖玻片。

    22. 在显微镜和图像线虫下观察

数据分析

用微分干涉对比显微镜检查线虫。观察到的信号应该与使用控制检测探针(阴性对照)获得的图像进行比较(图3)。应该增加一个积极的控制。我们通常使用编码在两个亚腹侧腺体中表达的多聚半乳糖醛酸酶的MiPG基因(Jaubert et al。,2002a)。我们建议拍摄至少40条染色的线虫,分析检测到的RNA斑点的细胞定位,并计算染色的线虫数量。每个ISH实验应该独立复制。 ISH可以在siRNA基因沉默靶基因之后进行以确保特异性和/或评估沉默效应。此外,使用不同探针组杂交相同靶基因的对照实验可以完成以确保探针对靶基因的特异性。


图3. M的组织表达。 incognita EFFECTOR1 ( MiEFF1 )在准备性J2和寄生青少年中的应用。 A-B。来自 MiEFF1 的成绩单( Minc17998 )位于准备性J2s的唾液背腺(箭头)。 C.感应MiEFF1对照探针不产生线虫组织的标记。 2012年,Jaouannet等人发表了一项寄生青少年的MiEFF1组织表达。比例尺= 50μm。

笔记

  1. 对于线虫净化和固定,请在使用前立即制备新鲜溶液。
  2. 准备两个含有40毫升50%蔗糖/ M9的康宁管,并将它们存放在4°C的冰箱中。
  3. 对于ISH,戴上手套。所有的解决方案都是无RNAse的,不要直接吸入主解决方案。
  4. 所有使用固定剂和杂交缓冲液进行的实验都应在通风橱中进行。

  5. 使用剃刀刀片时要非常小心
  6. 第1天在-80°C下放置甲醇和丙酮的等分试样,以用于第二天的线虫透化步骤。
  7. 对于线虫透化,不要涡旋蛋白酶K并将其保存在冷冻块中。
  8. 可以调整杂交温度以获得信号特异性和数量之间的最佳平衡。
  9. 地高辛(DIG)标记的RNA探针可以用来代替cDNA探针,如Rosso等人(1999)所述。
  10. 使用无核酸酶,而不是DEPC处理过的水。
  11. 对于启示,辣根过氧化物酶(HRP)可用于替代碱性磷酸酶(AP)。我们选择AP是因为来自AP底物的信号随时间逐渐增加,非常稳定并且可以持续数天。

食谱

提取线虫

  1. Pectinex ®和Celluclast® mix
    15毫升Celluclast
    30毫升Pectinex®

    点击H 2 O至200毫升
  2. M9缓冲区
    42.3mM Na 2 HPO 4 4/2 22 mM KH 2 PO 4 4/2 85.6 mM NaCl
    1mM MgSO 4·7H 2 O
  3. M9中50%蔗糖
    50克蔗糖
    M9缓冲液可达100毫升
  4. 无RNase 1x PBS
    1毫升10x PBS
    9毫升无核酸酶蒸馏水

用于探针,固定和杂交,无核酸酶
  1. 固定缓冲液
    1毫升37%甲醛溶液
    1个PBS最多10毫升
    备注:
    1. 在通风橱下准备和操作。
    2. 如果将37%的甲醛溶液在4℃下储存,在打开之前将其温热至RT。
  2. 1毫克/毫升蛋白酶K溶液
    1毫升蛋白酶K溶液(20毫克/毫升)
    1x PBS最多20 ml
  3. 来自贝克酵母的tRNA

    在1毫升无核酸酶的水中溶解500U酵母tRNA 分装并储存在-80°C。
  4. 杂交缓冲液(HB)
    注意:在通风橱下进行准备和操作,并保存在-20°C。
    5 ml去离子甲酰胺(99.5%,4°C)
    0.5ml SSC缓冲液(20x,RT)
    1 ml Boehringer封闭剂(10%,4°C)
    2ml SDS(10%,RT)
    200μlDenhart's溶液(50x,-20°C)
    20μlEDTA pH 8(0.5M,RT)
    200μl鲑鱼精子DNA(10 mg /μl,-20°C)

    来自Baker's酵母的62μltRNA(500 U / ml,-80°C) 无核酸酶水可达10毫升
  5. 洗涤缓冲液1(4x SSC + 0.1%SDS)
    400μlSSC缓冲液(20x,RT)
    20μlSDS(10%,RT)
    不含核酸酶的水可达2毫升
  6. 洗涤缓冲液2(0.1x SSC + 0.1%SDS)
    10μlSSC缓冲液(20x,RT)
    20μlSDS(10%,RT)
    不含核酸酶的水可达2毫升
  7. 10%勃林格封闭剂
    1克阻塞试剂粉末在一个瓶子里
    过滤马来酸缓冲液,pH值7.5至10毫升

    高压灭菌器在4°C溶解并储存
  8. 12.过滤的马来酸缓冲液,pH 7.5

    11.61 g马来酸(100 mM) 8.76克NaCl(150毫摩尔)
    用NaOH调节pH至7.5
    高压灭菌器,过滤并在RT
    存放
  9. Tris-HCl 2M,pH 9.5

    1. 溶解121.1克Tris-碱于400毫升去离子水中

    2. 通过加入浓HCl来调节pH值至所需值
    3. 在对pH进行最终调整之前,让溶液冷却至室温

    4. 用去离子水调整溶液的体积至500毫升
    5. 分装成50毫升等分试样并通过高压灭菌法灭菌
  10. 碱性磷酸酶检测缓冲液pH 9.5(APB)
    50 ml Tris-HCl 2 M pH 9.5(终浓度100 mM)
    5.85g NaCl(最终100mM)
    10.17克MgCl 2·7H 2 O(50mM最终)
    不含核酸酶的水可达1升

    分装,高压灭菌并在室温下储存

致谢

该协议已从de Boer等人改编而来。 (1998)和Rosso et al。(1999)。它使得研究几十种基因的表达谱成为可能(Jaubert等人,2002a和2002b; Neveu等人,2003; Jaubert等人, 2004; Ledger等人,2006; Dubreuil等人,2007; Jaouannet等人,,,< 2012; Jaouannet et al。,2013; Danchin et al。,2013; Nguyen et al。,2017)。这些作品由INRA,CNRS,尼斯大学Sophia Antipolis,普罗旺斯 - 阿尔卑斯 - 蓝色海岸地区,法国政府,欧盟和北约合作研究资助资助。 C-N,N-。得到了越南社会主义共和国教育和培训部的USTH奖学金,911-USTH计划的支持。作者没有利益冲突或竞争利益声明。

参考

  1. Caillaud,M.C。和Favery,B。(2016)。 体内成像分裂巨细胞中的微管组织 Methods Mol Biol 1370:137-144。
  2. Danchin,EGJ,Arguel,M.-J.,Campan-Fournier,A.,Perfus-Barbeoch,L.,Magliano,M.,Rosso,M.-N.,Da Rocha,M.,Da Silva,C. ,Nottet,N.,Labadie,K.,Guy,J.,Artiguenave,F. Abad,P.(2013)。 鉴定新的靶基因以更安全和更具体地控制泛基因组中的根结线虫采矿 PLoS Pathog 9(10):e1003745。
  3. de Boer,J.M.,Yan,Y。,Smant,G.,Davis,E.L。和Baum,T.J。(1998)。 原生杂交信使RNA在 Heterodera glycines < 。 Nematol 30(3):309-312。
  4. Dubreuil,G.,Magliano,M.,Deleury,E.,Abad,P.和Rosso,M.N。(2007)。 寄生虫早期诱导的根结线虫功能的转录组分析 < New Phytol 176(2):426-436。
  5. Jaouannet,M.,Magliano,M.,Arguel,M. J.,Gourgues,M.,Evangelisti,E.,Abad,P.和Rosso,M.N。(2013)。 根结线虫钙网蛋白Mi-CRT是植物防御抑制的关键效应物。 Mol Plant Microbe Interact 26(1):97-105。
  6. Jaouannet,M.,Perfus-Barbeoch,L.,Deleury,E.,Magliano,M.,Engler,G.,Vieira,P.,Danchin,EG,Da Rocha,M.,Coquillard,P.,Abad,P 。和Rosso,MN(2012)。 将根结线虫分泌蛋白注入巨细胞并靶向细胞核。 a> New Phytol 194(4):924-931。
  7. Jaubert,S.,Laffaire,J.B.,Abad,P.和Rosso,M.N。(2002a)。 动物来源的多聚半乳糖醛酸酶从根结线虫中分离出来
  8. Jaubert,S.,Laffaire,J.B.,Ledger,T.N.,Escoubas,P.,Amri,E.Z.,Abad,P.and Rosso,M.N。(2004)。 两种14-3-3同系物的比较分析及其在根结线虫中的表达模式< Meloidogyne incognita 。 Int J Parasitol 34(7):873-880。
  9. Jaubert,S.,Ledger,T.N.,Laffaire,J.B.,Piotte,C.,Abad,P.和Rosso,M.N。(2002b)。 通过蛋白质组学方法直接鉴定根结线虫的细胞分泌蛋白质 < Mol Biochem Parasitol 121(2):205-211。
  10. Ledger,T.N.,Jaubert,S.,Bosselut,N.,Abad,P.和Rosso,M.N。(2006)。 从根结线虫中鉴定新的β-1,4-内切葡聚糖酶基因 Meloidogyne incognita 和phytonematode家族5糖基水解酶的进化方案。 Gene 382:121-128。
  11. Liao,Y.,Fung,T. S.,Huang,M.,Fang,S.,Zhong,Y.和Liu,D。(2014)。 RNA分离和Northern印迹分析。 Bio-protocol 4(6) :e1077。
  12. Neveu,C.,Jaubert,S.,Abad,P.和Castagnone-Sereno,P.(2003)。 在无毒和有毒的Meloidogyne incognita之间差异表达的一组基因等基因系编码分泌蛋白。 Mol Plant Microbe Interact 16(12):1077-1084。
  13. Nguyen,CN,Perfus-Barbeoch,L.,Quentin,M.,Zhao,J.,Magliano,M.,Marteu,N.,Da Rocha,M.,Nottet,N.,Abad,P.和Favery,B 。(2017)。 根结线虫小甘氨酸和富含半胱氨酸的分泌效应物MiSGCR1参与植物寄生。 新Phytol 。
  14. Rosso,M.N.,Favery,B.,Piotte,C.,Arthaud,L.,De Boer,J.M.,Hussey,R.S.,Bakker,J.,Baum,T.J。和Abad,P。(1999)。 在根结线虫中分离编码β-1,4-内切葡聚糖酶的cDNA > Meloidogyne incognita 和植物寄生过程中的表达分析。 Mol Plant Microbe Interact 12(7):585-591。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jaouannet, M., Nguyen, C., Quentin, M., Jaubert-Possamai, S., Rosso, M. and Favery, B. (2018). In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.. Bio-protocol 8(6): e2766. DOI: 10.21769/BioProtoc.2766.
提问与回复

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

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