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Jun 2020

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Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers
应用碳纳米载体对烟草基因进行高效瞬时敲减   

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Abstract

Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader range of plant species than biotic methods, and iii) there are fewer regulatory complications when using abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant genome.

Graphic abstract:

Keywords: Plant gene silencing (植物基因沉默), RNA interference (RNA干扰), siRNA delivery (siRNA转运), Gene knock-down (基因敲减), Nicotiana benthamiana (本生烟草), Carbon nanotubes (碳纳米管), Single-walled carbon nanotubes (SWNT) (单壁碳纳米管( SWNT )), Nanomaterials (纳米材料)

Background

Gene silencing through RNA interference (RNAi) was discovered in the early 1990s by plant researchers studying petunia flower coloring (Van der Krol et al., 1990). In RNAi, specifically in post-transcriptional gene silencing (PTGS), gene expression level is reduced through mRNA degradation caused by small RNA molecules – micro (miRNA) or small interfering (siRNA) RNA. RNAi has been a breakthrough technology, not only in plant research and biotechnology applications, but also for many other organisms, including human therapy applications (Sierakowska et al., 1996).

The first step of siRNA-mediated RNAi in plants is the delivery of siRNA molecules into plant cells. Delivery is a big bottleneck in plant biotechnology, given the presence of plant cell wall that acts as a physical barrier for the delivery of biotechnology-relevant cargoes such as DNA, RNA, and protein. In plants, siRNA delivery is most commonly accomplished through viral vector delivery via Agrobacterium tumefaciens. However, most plant viruses are limited in their host range (Silva et al., 2010) and the size of cargo they can efficiently deliver (Burch-Smith et al., 2004). Agrobacterium-mediated delivery is also limited in terms of plant host species, causes uncontrolled DNA integration into the plant nuclear genome, and results in constitutive expression of siRNA, which limits temporal control over gene silencing (Baltes et al., 2017).

Carbon nanotubes are one dimensional high-aspect-ratio nanomaterials that have many advantageous features for siRNA delivery in plants. First, given their needle-like structure with a small diameter (~1 nm), long length (~500 nm) and high stiffness, single-walled carbon nanotubes (SWNTs) have shown to transport across the plant cell wall and localize inside plant cells (Demirer et al., 2019b). Second, high surface area and diverse surface chemistry options of SWNTs enable delivery of diverse biological cargoes (Beyene et al., 2016; Del Bonis-O’Donnell et al., 2017; Demirer et al., 2020). Lastly, SWNTs have the ability to delay the intracellular degradation of biomolecular cargoes (Demirer et al., 2019a and 2020), which is especially valuable when working with fragile molecules like RNA.

Recently, we have developed a method to deliver siRNA molecules targeting the silencing of a transgenic GFP gene in Nicotiana benthamiana leaves, and an endogenous stress gene, ROQ1, using SWNTs (Demirer et al., 2020). In this approach, we first load sense and antisense strands of siRNA onto two separate SWNT nanoparticle solutions via pi-pi interactions that form between the sp2 carbon nanotube surface lattice and the aromatic bases of single stranded RNA (ssRNA). Next, we introduce an equimolar mixture of these RNA-SWNT solutions into intact plant leaves for GFP silencing. Our results demonstrate efficient silencing of GFP as assessed by confocal microscopy imaging, quantitative PCR (qPCR), and Western blotting, both for transgenic GFP and also for the endogenous ROQ1 gene, with disease-resistance applications (Demirer et al., 2020). This transient gene knock-down approach could be applied to other plant species, tissues, and target genes with minimal modifications. Additionally, the RNA loading method used in this study is not specific to siRNA, and thus, it can be adapted for the delivery of other types of nucleic acids with some optimization (e.g., guide RNA or messenger RNA for CRISPR genome editing applications).

Below, we provide a step-by-step protocol for the synthesis and characterization of siRNA loaded SWNTs, and the measurement of gene silencing efficiency in tobacco leaves through confocal imaging, qPCR and Western blotting (Figure 1).


Figure 1. Overview of the siRNA-SWNT gene silencing procedure

Materials and Reagents

  1. SunGro Sunshine LC1 Grower soil mix (SUN52128CFLP)

  2. Delicate task wipes (Kimberly-Clark, catalog number: 06-666)

  3. 100K MWCO Amicon spin filters (MilliporeSigma, catalog number: UFC510024)

  4. PVDF Membrane, Precut, 7 x 8.4 cm (Bio-Rad, catalog number: 1620174)

  5. Sterile syringe filter (0.45 μm; VWR, catalog number: 28145-481)

  6. Microcentrifuge tubes (1.5 ml; VWR, catalog number: 89000-028)

  7. Conical tubes (50 ml; Olympus, catalog number: 28-106)

  8. Pipette tips (Low retention 10 μl, 200 μl, 1,000 μl filter tips; USA Scientific, catalog numbers: 1181-3710, 1180-8710, 1182-1730)

  9. Extended-length pipette tips (1,000 μl; Eppendorf, catalog number: 0030073614)

  10. #1 Microscopy cover glass (Fisher Scientific, catalog number: 12-542B)

  11. Microscope slides (VWR, catalog number: 16004-422)

  12. Syringe (1 ml; BD, catalog number: 14-823-434)

  13. Mini Trans-Blot Filter paper (Bio-Rad, catalog number: 1703932)

  14. EasyStrip‚ Plus PCR Tube (Thermo Scientific, catalog number: AB2005)

  15. Plant seeds (mGFP5 Nicotiana benthamiana is obtained from the Staskawicz lab at UC Berkeley, mGFP5 plants constitutively express GFP targeted to the ER under the control of the Cauliflower mosaic virus 35S promoter)

  16. Goat anti-rabbit horseradish peroxidase-conjugated antibody (Abcam, catalog number: ab205718)

  17. Anti-GFP antibody, ChIP Grade (Abcam, catalog number: ab290)

  18. HiPCO SWNTs (NanoIntegris, Super purified, catalog number: HS28-037)

  19. MilliQ water

  20. Nuclease-free water (Qiagen, catalog number: 129114)

  21. Sodium chloride, NaCl (Sigma-Aldrich, catalog number: S9888-500G)

  22. Hydrochloric acid, HCl (37% [vol/vol]; Sigma, catalog number: 320331)

  23. Single-stranded RNA oligonucleotides, including sense and antisense siRNA strands – 21 nucleotides (Integrated DNA Technologies, IDT)

  24. Sodium dodecyl sulfate, molecular biology grade (Sigma-Aldrich, catalog number: 436143-100G)

  25. Tris/HCl (Sigma-Aldrich, catalog number: 10812846001)

  26. EDTA (Sigma-Aldrich, catalog number: E9884-100G)

  27. NP-40 (Sigma-Aldrich, catalog number: 492016-100ML)

  28. Glycerol (Sigma-Aldrich, catalog number: G5516-500ML)

  29. Pierce 660 nm Protein Assay (Thermo, catalog number: 22660)

  30. iScript cDNA synthesis kit (Bio-Rad, catalog number: 1708891)

  31. PowerUp SYBR green master mix (Applied Biosystems, catalog number: A25742)

  32. Qubit Protein Assay (ThermoFisher Scientific, catalog number: Q33211)

  33. RNeasy plant mini kit (QIAGEN, catalog number: 74904)

  34. BSA (Sigma-Aldrich, catalog number: A4737-25G)

  35. TWEEN20 (Sigma-Aldrich, catalog number: P9416-100ML)

  36. Ammonium persulphate, APS (Sigma, catalog number: 248614-100G)

  37. Low range ultra agarose (Bio-Rad, catalog number: 1613107)

  38. ECL Prime Western Blotting System (MilliporeSigma, catalog number: GERPN2232)

  39. TEMED (N,N,N,N'-tetramethylethylenediamine; Sigma, catalog number: T9281)

  40. Glycine (Sigma, catalog number: G8898)

  41. Methanol (Sigma, catalog number: 179957)

  42. 4x Laemmli sample Buffer (Bio-Rad, 10 ml, catalog number: 1610747)

  43. Liquid nitrogen

  44. SYBR Gold Nucleic Acid Gel Stain (Invitrogen, catalog number: S11494)

  45. 30% Acrylamide/Bis solution 19:1 (Bio-Rad, catalog number: 1610154)

  46. Protease inhibitor cocktail (Sigma, catalog number: P9599-1ML)

  47. 0.1 M NaCl (see Recipes)

  48. 10% (wt/vol) Ammonium persulphate solution (APS) (see Recipes)

  49. 10x Transfer buffer (see Recipes)

  50. 1x Transfer buffer (see Recipes)

  51. 10x Tris-Buffered Saline (TBS) buffer (1 M Tris, 1.5 M NaCl, pH 7.4) (see Recipes)

  52. 1x TBST buffer (see Recipes)

  53. Lysis buffer (see Recipes)

Equipment

  1. Analytical balance (Radwag, model: AS 60/220.R2)

  2. Ultrasonic bath (Branson, model: 15-336-100)

  3. Ultrasonic homogenizer with 6-mm tip (Cole-Parmer, models: UX-04711-70, UX-04712-14)

  4. Vortex mixer (Fisher Scientific, model: 02-215-365)

  5. pH meter (Spectrum, model: 242-97839)

  6. Orbital shaker (Waverly, model: S1CE)

  7. NanoVue Plus spectrophotometer (GE Life Sciences, model: 28-9569-61)

  8. Visible spectrophotometer (Thermo Scientific, model: 14-385-445)

  9. Near-infrared spectrometer (Princeton Instruments IsoPlane 320 coupled to a liquid nitrogen-cooled Princeton Instruments PyLoN-IR 1D array of InGaAs pixels)

  10. UV-Vis-NIR Spectrophotometer (Shimadzu, model: UV-3600 Plus)

  11. Tabletop centrifuge (Eppendorf, catalog number: 5418000017)

  12. Centrifuge (Eppendorf, model: 5424R)

  13. Tweezers (VWR, catalog number: 63042-518)

  14. Scissors (VWR, catalog number: 82027-582)

  15. Mortar and pestle (Cole-Parmer, catalog number: EW-63100-54)

  16. Pant growth chamber (HiPoint, model: 740 FHLED)

  17. Gel image-analysis system (Typhoon FLA 9500, GE Healthcare Services)

  18. Electrophoresis power supply (PowerPac basic power supply; Bio-Rad, catalog number: 1645050)

  19. Mini Trans-Blot Cell (Bio-Rad, catalog number: 1703811)

  20. Mini-Protein TGX gels (Bio-Rad, catalog number: 456-1094)

  21. ChemiDoc XRS+ System (Bio-Rad, catalog number: 1708265)

  22. Confocal Microscope (Zeiss, model: LSM 710)

  23. Thermal Cycler CFX96 Touch Real-Time PCR Detection System (Bio-Rad, catalog number: 1855195)

  24. Thermal Cycler PCR (Applied Biosystems Veriti 96-Well, catalog number: 4375786)

Software

  1. GraphPad Prism 7.0a (https://www.graphpad.com/scientific-software/prism/)

  2. Fiji ImageJ 2.0.0 (https://imagej.net/Fiji/Downloads)

  3. Zen Blue 2.6 (https://www.zeiss.com/microscopy/us/downloads.html)

Procedure

  1. Plant growth

    Germinate transgenic mGFP5 Nicotiana benthamiana seeds (see Note 1) and grow seeds in SunGro Sunshine LC1 Grower soil mix in a growth chamber for four to six weeks before experiments. Use 12-h light at 24 °C and 12-h dark at 18 °C cycle for growing plants.

    Note: Different plant species may require different germination and growth conditions.


  2. siRNA design and generation

    1. Currently, there are many software to design gene specific siRNA sequences with minimal off-target effects. A recently developed software called “siRNA-Finder (si-Fi) Software” can be used in plants (Lück et al., 2019).

    2. After the design of siRNA sequences, sense and antisense RNA strands can be purchased from Integrated DNA Technologies (IDT) as single-stranded oligonucleotides.


  3. RNA-SWNT preparation

    1. Dissolve sense and antisense siRNA strands in 0.1 M NaCl at a concentration of 100 mg/ml.

    2. Add 1 mg dry HiPCO SWNTs to 20 μl of dissolved sense RNA, and complete the solution volume to 1 ml with 0.1 M NaCl (see Note 2).

    3. Bath sonicate the mixture for 10 min at room temperature in Ultrasonic bath (Branson).

    4. Probe-tip sonicate the mixture with a 3-mm tip at 50% amplitude (~7W) for 30 min in an ice bath. Renew ice bath if it starts melting during the sonication to prevent heating.

    5. Rest the solution at room temperature for 30 min.

    6. Centrifuge the solution at 16,100 x g for 1 h in room temperature to remove bundled SWNTs. The supernatant contains the individually suspended sense-RNA-SWNTs. Keep the supernatant and discard the SWNT pellet to the hazardous nanomaterials waste.

    7. Repeat the same protocol for the antisense RNA strand (see Note 3). Store RNA-SWNT solutions at 4 °C.


  4. Removal of unbound siRNA

    1. Add 500 μl sense-RNA-SWNT and 500 μl antisense-RNA-SWNT into two separate 100K Amicon spin filters that are placed in 2 ml collection tubes. Centrifuge 4 min at 8,000 x g in room temperature.

    2. Collect the flow-throughs from sense-RNA-SWNT and antisense-RNA-SWNT in separate tubes and place the spin filters into the same collection tubes.

    3. Add 0.1 M NaCl into the spin filters until the volume reaches 500 μl. Repeat the wash step.

    4. Perform a total of 8 washes to remove all unbound RNA molecules. Merge and accumulate all flow-through solutions (separately for sense-RNA-SWNT and antisense-RNA-SWNT) for later measurement of removed RNA amount.

    5. Calculate the SWNT concentration by measuring the carbon nanotube absorbance at 632 nm using a spectrophotometer (use 2 μl for NanoVue Plus spectrophotometer or dilute to 1 ml for Thermo Scientific). Divide the absorbance value by SWNT extinction coefficient of 0.036 to obtain SWNT concentration in the unit of μg/ml (If the sample is diluted, multiply the absorbance value also by the dilution factor).

    6. Calculate the concentration of RNA loaded on SWNTs by measuring the absorbance of collected flow-through solutions at 260 nm (use 2 μl for NanoVue Plus spectrophotometer), and subtracting the total amount of RNA removed from the total amount of RNA added (2 mg in this case).

    7. (Optional) For additional characterization, record sense-RNA-SWNT and antisense-RNA-SWNT absorbance spectra with Shimadzu UV-3600 Plus, and fluorescence spectra with a near-infrared spectrometer (Princeton Instruments IsoPlane 320 coupled to a liquid nitrogen-cooled Princeton Instruments PyLoN-IR 1D array of InGaAs pixels). See Demirer et al., 2020 for representative examples of SWNT absorbance and fluorescence spectra.


  5. Infiltration of leaves with RNA-SWNTs

    1. Select healthy and fully-developed leaves from mGFP5 Nicotiana benthamiana (4-6 weeks old) plants for experiments.

    2. Merge 100 μl of 200 nM sense-RNA-SWNTs with 100 μl of 200 nM antisense-RNA-SWNTs in a 1.5 ml Eppendorf tube (see Note 4). Mix well.

    3. Immediately after mixing, make a small puncture on the abaxial (bottom) surface of the leaf with a pipette tip, and infiltrate ~100-200 μl of the siRNA-SWNT mixture from the hole with a 1 ml needleless syringe with caution not to damage the leaf (see Note 5).

    4. Use a Kimwipe tissue to remove the excess siRNA-SWNT solution on the leaf surface. Mark the infiltrated area with a Sharpie pen without damaging the leaf.

    5. Infiltrate a negative control solution, such as the free siRNA without SWNTs or scrambled RNA suspended SWNTs that does not target the gene of interest. If possible, also infiltrate a positive control solution, such as viral siRNA delivery sample (see Note 6).

    6. Return the infiltrated plant(s) into the growth chamber until the measurement of gene silencing.


  6. Gene silencing determination through quantitative PCR (qPCR)

    1. 24-h after infiltration, cut the infiltrated leaf areas (maximum of 100 mg leaf tissue per sample) and extract total RNA with a RNeasy plant mini kit. After cutting the leaf, immediately proceed with the first step of the RNA extraction protocol (i.e., grinding the tissue in liquid nitrogen using mortar and pestle) to make sure gene expression levels do not change after cutting. Follow the protocol of the RNeasy plant mini kit (see Note 7).

    2. Following RNA extraction, measure the RNA concentration and purity with a spectrophotometer. Reverse transcribe 1 μg total RNA into complementary DNA (cDNA) using an iScript cDNA synthesis kit. Follow the protocol of the iScript cDNA synthesis kit.

    3. Use PowerUp SYBR green master mix for the qPCR step with 2 μl cDNA from Step 2 and specific primers for the target and housekeeping genes. Follow the protocol of the PowerUp SYBR green kit for relative quantification of target mRNA in the siRNA-SWNT infiltrated leaf compared to negative controls of free siRNA without SWNTs or scrambled RNA-SWNTs.

      Example: The target gene in our qPCR was mGFP5 (GFP transgene inserted into Nb), and EF1 (elongation factor 1) as our housekeeping (reference) gene.

    4. Analyze the qPCR data using the ddCt method (Rao et al., 2013). See Figure 2B for representative qPCR results.


  7. Gene silencing determination through confocal fluorescence microscopy imaging

    1. If silencing a fluorescent protein, such as GFP, confocal fluorescence microscopy can be used to detect approximate silencing efficiency.

    2. After infiltration, leave the plants with intact infiltrated leaves in the growth chamber for 72 h.

    3. 72-h after infiltration, cut a small flat piece (0.5-1 cm x 0.5-1 cm) of the infiltrated leaf around the infiltration point and prepare a glass slide with cover slip (thickness #1). Add 50-100 μl water in between the glass slide and cover slip for imaging. Image samples before the leaf piece dries out (optimally within 15 to 30 min).

    4. Image the plant tissue with 488 nm laser excitation with a GFP filter cube (in the case of GFP silencing), and also capture brightfield with a transmitted light if available (see Note 8).

    5. Capture at least 10 to 15 fields of view with same optical settings per sample, including non-treated leaf, and any negative and positive control samples.

    6. For each sample, compare the mean fluorescence intensity value with the mean GFP fluorescence intensity of a non-treated leaf, which can be used to determine silencing efficiency of siRNA-SWNTs. Pay attention to use the same imaging parameters and quantification analyses for samples imaged on different days. See Figure 2A for representative confocal imaging results.



      Figure 2. Representative gene silencing results. A. Confocal microscopy images of non-treated and free-siRNA treated control leaves and siRNA-SWNT infiltrated sample leaves of Nicotiana benthamiana. Imaged after 3 days post-infiltration, scale bars are 50 μm. B. Quantitative PCR analysis results for GFP gene silencing in Nicotiana benthamiana leaves with siRNA-SWNTs.


  8. Gene silencing determination through Western blotting

    1. 72-h after infiltration, harvest infiltrated leaves and grind them in liquid nitrogen using mortar and pestle to recover dry frozen leaf powders.

    2. Transfer the frozen leaf powder into a tube with 400 μl pre-chilled lysis buffer (see Recipes).

    3. Lyse tissue on ice for 1 h. Centrifuge the tubes at 10,000 x g at 4 °C for 20 min. Following centrifugation, gently transfer the supernatants containing whole proteins to a new clean tube. Quantify total extracted proteins with a Pierce 660 nm Protein Assay.

    4. Mix the samples with the appropriate loading buffer for gel electrophoresis, and boil the mixture at 95-100 °C for 5 min either using a heat block or thermal cycler. Load 0.5 µg of normalized total proteins from each sample and analyze with SDS-PAGE gel (Bio-Rad precast tris/glycine gel, 4-20% gradient). Run the gel at 120 V for 60 min.

    5. Transfer the gel to a PVDF membrane in cold transfer buffer (see Recipes) and run at 400 mA in 1x transfer buffer with methanol for no more than 60 min in a cold room with an ice block.

    6. Block the membrane for 1 h using 7.5% BSA in 1x TBST buffer (see Recipes) followed by overnight incubation at 4 °C with the primary GFP antibody. After extensive washing, probe the corresponding protein bands with a goat anti-rabbit horseradish peroxidase-conjugated antibody for 30 min. After washing, develop the bands by incubation with chemiluminescence (Amersham ECL prime kit) in less than 2 min and image with a ChemiDoc XRS+ System.

    7. Quantify the intensity of GFP bands with ImageJ software.

Data analysis

  1. For all experiments, perform at least 3 biological replicates. Biological replicates are defined as experiments consisting of independent infiltrations with RNA-SWNTs into different plants.

  2. For quantitative analysis of confocal imaging, capture at least 10-15 non-overlapping fields of view per sample and per biological replicate, which is defined as technical replicates. Average the fluorescence intensity of these technical replicates to obtain the mean fluorescent intensity for a given biological replicate. Confocal images can be analyzed with either the ImageJ or Zen Blue software.

  3. For the qPCR assay, perform at least 3 technical replicates, which are defined as reactions from the same isolated RNA batch obtained from the same infiltration. To analyze qPCR results for gene silencing efficiency, used the previously developed ddCt method. The details of this method can be found in Rao et al. (2013).

  4. When comparing more than 2 samples with each other statistically for a single independent variable (e.g., silencing efficiency), use one-way ANOVA with Tukey’s multiple comparisons test. Report both the P value and F. When comparing more than 2 samples with each other statistically for multiple independent variables (e.g., silencing efficiency over multiple time points), use two-way ANOVA with Sidak’s multiple comparisons test. Report both the P value and F. When comparing two samples only, Student’s t-test can be used. Report the P value. GraphPad Prism can be used to plot the data and perform statistical significance analysis.

Notes

  1. This protocol is provided for the silencing of GFP gene in transgenic tobacco (Nicotiana benthamiana) leaves, but can be adapted for the silencing of other genes and in other plant species, including other transgenes or endogenous genes.

  2. RNA and SWNT amounts can be scaled up or down depending on the experimental need. In scaling, keep the mass ratio of RNA:SWNT at 2. Different final siRNA concentration may be needed for different gene targets, or in different plant species or tissues.

  3. Typically, if one RNA strand suspends SWNTs well, its complementary strand has a lower suspension efficiency. This is expected given the complementary base pairs of the sense and antisense strands which can lead to varying adsorption ability of RNA bases to the SWNT surface. We typically obtain 20-50 μg/ml RNA-SWNT suspensions after centrifugation and purification steps.

  4. Pay attention that 200 nM is the RNA concentration on SWNTs, and not the SWNT concentration. Depending on the suspension efficiency, this corresponds to ~2 μg/ml SWNTs.

  5. 100-200 μl infiltration is suggested for confocal imaging studies, whereas larger volume or multiple infiltrations should be performed for qPCR and Western blot to cover the full leaf area.

  6. It is preferred to infiltrate the SWNT and control samples on the same leaf to avoid leaf to leaf variation in gene expression and imaging.

  7. RNA work must be performed carefully on a clean bench (optimally dedicated to RNA-based experiments) to prevent RNA degradation. Wipe the surfaces and equipment with RNase solution and change gloves frequently. Keep the RNA on ice during the protocol, and transfer to -20 °C or -80 °C when the protocol is completed.

  8. If the leaf is too thick or wavy to get the entire field of view into focus, image with z-stack and perform the silencing analysis on the stacked image. Keep the imaging parameters same between samples that will be compared with each other.

Recipes

  1. 0.1 M NaCl

    Weigh 0.5844 g of NaCl and dissolve in 100 ml nuclease free water

    Sterile filter with 0.22-micron syringe filter

  2. 10% (wt/vol) Ammonium persulphate solution (APS)

    Add 5 g of APS to 50 ml of MilliQ water, and mix to dissolve

    The solution can be stored at 4 °C for up to 3 months

  3. 10x Transfer buffer

    To make 1 L of 10x Transfer buffer, add 30.3 g Tris base, 144 g Glycine to 800 ml MilliQ water, and mix to dissolve

    Then add MilliQ water to a final volume of 1 L

  4. 1x Transfer buffer

    To make 1 L of 1x Transfer buffer, add 100 ml 10x Transfer buffer, 200 ml methanol to 700 ml MilliQ water and mix the solution

    The buffer needs to be stored at 4 °C, and it is better to prepare the buffer before running each experiment

  5. 10x Tris-Buffered Saline (TBS) buffer (1 M Tris, 1.5 M NaCl, pH 7.4)

    To make 250 ml 10x TBS buffer, add 30.3 g Tris base, 21.9 g NaCl to 200 ml MilliQ water and mix to dissolve

    Use HCl to adjust the pH of the solution to 7.4, then add MilliQ water to a final volume of 250 ml

    The solution can be stored at 4 °C for up to 6 months

  6. 1x TBST buffer

    Add 50 ml 10x TBS buffer, and 500 μl of Tween 20 (0.1%) to 500 ml MilliQ water and mix the solution

    The buffer can be stored at 4 °C for at least 1 month

  7. Lysis buffer

    10 mM Tris/HCl

    150 mM NaCl

    1 mM EDTA

    0.1% NP-40

    5% glycerol

    1% protease inhibitor cocktail

    pH 7.5

    To make 100 ml lysis buffer, add 1.21 g of Tris base, 0.877 g NaCl, 29.24 mg EDTA, 0.1 g NP-40, and 5 ml glycerol to 80 ml nuclease free water, mix to dissolve, adjust the pH to 7.5 by HCl, add 1 ml protease inhibitor cocktail, and fill the final volume to 100 ml using the nuclease free water.

    Note: The lysis buffer can be stored at -20 °C for at least 6 months.

Acknowledgments

This protocol is derived from Demirer et al. (2020).

G.S.D. is supported by the Schlumberger Foundation Faculty for the Future Program and Caltech Resnick Sustainability Institute. We acknowledge support of a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Stanley Fahn PDF Junior Faculty Grant with Award # PF-JFA-1760, a Beckman Foundation Young Investigator Award, a USDA AFRI award, a USDA NIFA award, the Moore Foundation, and an FFAR New Innovator Award (M.P.L). M.P.L. is a Chan-Zuckerberg Biohub investigator.

Competing interests

Authors declare no competing interest.

References

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  12. van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. and Stuitje, A. R. (1990). Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2(4): 291-299.

简介

[摘要]植物基因敲低是研究基因型与表型关系,提高作物对病害的抵抗力以及实现植物分子高效生物合成的有用方法。小干扰RNA(siRNA)介导的基因沉默是在植物中实现基因敲低的最常见方法之一。传统上,通过将siRNA序列编码到DNA载体中,将siRNA传递到完整的植物细胞中,然后通过病毒和/或细菌方法传递。在这个协议中,我们提供的siRNA分子的替代直接递送方法为完整的植物细胞的高效瞬时根Ë击倒在模型的烟草植物,烟草本塞姆氏烟草,叶子。我们的方法使用一维碳基纳米材料,单壁碳纳米管(SWNTs)来传递siRNA,而不依赖于病毒/细菌的传递。我们方法的独特优势在于:i )不需要对siRNA序列进行DNA编码; ii)与非生物方法相比,这种非生物方法可在更广泛的植物物种中起作用,并且iii)使用非生物递送时,调节并发症更少方法,其中基因沉默是瞬时的,而无需对植物基因组进行永久性修饰。

图形摘要:

图形抽象标题

[背景技术[ 0002 ]在1990年代初,植物研究人员研究矮牵牛花的着色发现了通过RNA干扰(RNAi)引起的基因沉默(Van der Krol等,1990)。在RNAi中,特别是在转录后基因沉默(PTGS)中,由于小RNA分子–微小(miRNA)或小干扰(siRNA)RNA引起的mRNA降解,基因表达水平降低。RNAi已经成为一项突破性技术,不仅在植物研究和生物技术应用中,而且在许多其他生物中,包括在人类治疗中的应用(Sierakowska et al。,1996)。

siRNA介导的RNAi在植物中的第一步是将siRNA分子传递到植物细胞中。考虑到植物细胞壁的存在对生物技术相关货物(如DNA,RNA和蛋白质)的传递具有物理障碍,因此传递是植物生物技术的一大瓶颈。在植物中,最通常通过根癌土壤杆菌的病毒载体递送来完成siRNA的递送。但是,大多数植物病毒的寄主范围有限(Silva等,2010)以及它们可以有效传递的货物大小(Burch - Smith等,2004)。农杆菌介导的传递在植物宿主物种方面也受到限制,导致不受控制的DNA整合到植物核基因组中,并导致siRNA组成型表达,从而限制了基因沉默的暂时控制(Baltes等,2017)。

碳纳米管是一维高纵横比纳米材料,具有许多在植物中传递siRNA的有利特征。首先,由于它们的针状结构具有小直径(〜1 nm),长长度(〜500 nm)和高刚度,因此单壁碳纳米管(SWNTs)已显示出能够跨植物细胞壁运输并定位于植物内部细胞(Demirer等人,2019b)。其次,单壁碳纳米管的高表面积和多样的表面化学选择使人们能够运送各种生物货物(Beyene等,2016; Del Bonis-O'Donnell等,2017; Demirer等,2020)。最后,单壁碳纳米管具有延迟生物分子货物的细胞内降解的能力(Demirer等人,2019a和2020),这在处理易碎分子(如RNA)时特别有价值。

最近,我们开发了递送siRNA分子靶向在转基因GFP基因的沉默的方法烟草本塞姆氏叶,并内源基因应力,ROQ1,使用单壁碳纳米管(Demirer等人,2020) 。在这种方法中,我们首先通过在sp 2碳纳米管表面晶格和单链RNA(ssRNA )的芳香族碱基之间形成的pi-pi相互作用将siRNA的有义链和反义链加载到两个单独的SWNT纳米颗粒溶液上。接下来,我们将这些RNA-SWNT解决方案的等摩尔混合物引入完整的植物叶片中,以进行GFP沉默。我们的结果表明,通过共聚焦显微镜成像,定量PCR(qPCR)和W酯印迹法评估的GFP对转基因GFP和内源ROQ1基因均具有有效的抗病性(Demirer et al。,2020)。。这种瞬时基因敲低方法可以以最小的修改应用于其他植物物种,组织和靶基因。此外,本研究中使用的RNA加载方法并非针对siRNA,因此,它可以进行一些优化(例如,用于CRISPR基因组编辑应用的指南RNA或信使RNA),适用于递送其他类型的核酸。。

在下文中,我们提供了用于合成和表征siRNA的单壁碳纳米管的分步方案,以及通过共聚焦成像,qPCR和W酯化印迹法(图1)测量了烟叶中基因沉默的效率。

图1. siRNA-SWNT基因沉默程序概述

关键字:植物基因沉默, RNA干扰, siRNA转运, 基因敲减, 本生烟草, 碳纳米管, 单壁碳纳米管( SWNT ), 纳米材料

材料和试剂
1. SunGro Sunshine LC1种植者土壤混合料(SUN52128CFLP)       
2.精美的任务湿巾(Kimberly-Clark,目录号:06-666)       
3. 100K MWCO Amicon旋转过滤器(MilliporeSigma ,目录号:UFC510024)       
4. PVDF膜,预切割,7 × 8.4 cm(Bio-Rad,目录号:1620174)       
5.无菌注射器式滤器(0.45 μ米; VWR,目录号:28145-481)     
6.微量离心管(1.5 ml; VWR,目录号:89000-028)     
7.锥形管(50毫升; Olympus,目录号:28-106)     
8.移液管头(低保留10 μ升,200 μ升,1000个μ升过滤嘴;美国科学,产品目录号:1181-3710,1180-8710,1182年至1730年)     
9.扩展长度吸管头(1000 μ升;的Eppendorf,目录号:0030073614)     
10. #1显微镜盖玻片(Fisher Scientific,目录号:12-542B) 
11.显微镜载玻片(VWR,目录号:16004-422) 
12.注射器(1 ml; BD,目录号:14-823-434) 
13. Mini Trans-Blot滤纸(Bio-Rad,目录号:1703932) 
14. EasyStrip ,Plus PCR试管(Thermo Scientific,目录号:AB2005) 
15.植物种子(mGFP5本氏烟草是从加州大学伯克利分校的Staskawicz实验室获得的,mGFP5植物在花椰菜花叶病毒35S启动子的控制下组成型表达靶向ER的GFP)   
16.山羊抗兔辣根过氧化物酶结合抗体(Abcam,目录号:ab205718)   
17.抗GFP抗体,ChIP级(Abcam,目录号:ab290)   
18. HiPCO SWNT(NanoIntegris ,超纯,目录号:HS28-037)   
19. MilliQ水   
20.无核酸酶的水(Qiagen,目录号:129114)   
21.氯化钠NaCl(Sigma-Aldrich,目录号:S9888-500G)   
22.盐酸HCl(37%[ vol / vol ] ; Sigma,目录号:320331)   
23.单链RNA寡核苷酸,包括有义和反义siRNA链– 21个核苷酸(Integrated DNA Technologies,IDT)   
24.十二烷基硫酸钠,分子生物学等级(Sigma-Aldrich,目录号:436143-100G)   
25. Tris / HCl(Sigma-Aldrich,目录号:10812846001)   
26. EDTA(Sigma-Aldrich,目录号:E9884-100G)   
27. NP-40(Sigma-Aldrich,目录号:492016-100ML)   
28.甘油(西格玛奥德里奇,目录号:G5516-500ML)   
29. Pierce 660 nm蛋白测定(Thermo,目录号:22660)   
30. iScript cDNA合成试剂盒(Bio-Rad,目录号:1708891)   
31. PowerUp SYBR绿色预混料(Applied Biosystems,目录号:A25742)   
32. Qubit蛋白测定(ThermoFisher Scientific,目录号:Q33211)   
33. RNeasy植物迷你套件(QIAGEN,目录号:74904)   
34. BSA(Sigma-Aldrich,目录号:A4737-25G)   
35. TWEEN20(Sigma-Aldrich,目录号:P9416-100ML)   
36.过硫酸铵,APS(Sigma,目录号:248614-100G)   
37.低范围超琼脂糖(Bio- R ad,目录号:1613107)   
38. ECL Prime Western印迹系统(MilliporeSigma ,目录号:GERPN2232)   
39. TEMED(N,N,N,N'-四甲基乙二胺; Sigma,目录号:T9281)   
40.甘氨酸(西格玛,目录号:G8898)   
41.甲醇(西格玛,目录号:179957)   
42. 4 × Laemmli样品缓冲液(Bio- R ad,10 ml,目录号:1610747)   
43.液氮   
44. SYBR金核酸凝胶染色剂(Invitrogen,目录号:S11494)   
45. 30%丙烯酰胺/双酚19:1(Bio- R ad,目录号:1610154)   
46.蛋白酶抑制剂混合物(Sigma,目录号:P9599-1ML )   
47. 0.1 M NaCl(请参阅食谱)   
48. 10%(wt / vol)过硫酸铵溶液(APS)(请参阅食谱)   
49. 10 ×传输缓冲区(请参阅食谱)   
50. 1 ×传输缓冲区(请参阅食谱)   
51. 10 × Tris缓冲盐(TBS)缓冲液(1 M Tris,1.5 M NaCl,pH 7.4)(请参见食谱)   
52. 1 × TBS T缓冲区(请参阅食谱)   
53.裂解缓冲液(请参见食谱)   

设备



分析天平(Radwag ,型号:AS 60 / 220.R2)
超声波浴(Branson,型号:15-336-100)
超声波均化器以6毫米尖端(Cole-Parmer公司,米Odel等小号:UX-04711-70,UX-04712-14)
涡旋混合器(Fisher Scientific,型号:02-215-365)
pH计(Spectrum,型号:242-97839)
轨道振动筛(Waverly,型号:S1CE)
NanoVue Plus分光光度计(GE生命科学,型号:28-9569-61)
可见分光光度计(Thermo Scientific,型号:14-385-445)
近红外光谱仪(Princeton Instruments IsoPlane 320与液氮冷却的InGaAs像素的PyLoN -IR 1D阵列进行液氮冷却)
紫外可见近红外分光光度计(Shimadzu ,型号:UV-3600 Plus)
台式离心机(Eppendorf,目录号:5418000017)
离心机(Eppendorf,型号:5424R)
镊子(VWR,目录号:63042-518)
剪刀(VWR,货号:82027-582)
研钵和杵(Cole-Parmer,目录号:EW-63100-54)
裤子生长室(HiPoint ,型号:740 FHLED)
凝胶图像分析系统(台风FLA 9500,GE Healthcare Services)
电泳电源(PowerPac基本电源; Bio-Rad,目录号:1645050)
Mini Trans-Blot Cell(Bio- R ad,货号:1703811)
迷你蛋白TGX凝胶(Bio- R ad ,目录号:456-1094)
ChemiDoc XRS +系统(Bio- R ad,目录号:1708265)
共焦显微镜(Zeiss ,型号:LSM 710)
Thermal Cycler CFX96触摸实时PCR检测系统(Bio- R ad,目录号:1855195)
热循环PCR(Applied Biosystems Veriti 96-Well,目录号:4375786)

软件



GraphPad Prism 7.0a(https://www.graphpad.com/scientific-software/prism/)
斐济ImageJ 2.0.0(https://imagej.net/Fiji/Downloads)
Zen Blue 2.6(https://www.zeiss.com/microscopy/us/downloads.html)

程序



植物生长
发芽转基因mGFP5烟草本塞姆氏种子(见注1),并生长在种子SunGro阳光LC1种植者土壤结构实验之前,在生长室4至6周。对于生长中的植物,在24 °C下使用12 h光照,在18 °C下使用12 h黑暗。


注意:不同的植物物种可能需要不同的发芽和生长条件。



siRNA设计和生成
当前,有许多软件可以设计出具有最小脱靶效应的基因特异性siRNA序列。可以在植物中使用最近开发的称为“ siRNA-Finder(si -Fi)软件”的软件(Lück等,2019)。
在设计siRNA序列后,可以从Integrated DNA Technologies(IDT)购买有义和反义RNA链,作为单链寡核苷酸。

RNA-SWNT制备
将有义和反义siRNA链溶于浓度为100 mg / ml的0.1 M NaCl中。
添加1毫克干燥的HiPCO单壁碳纳米管以20微升溶解义RNA,并完成溶液体积为1 ml的用0.1M的NaCl(见注2)。
在室温下在超声浴(布兰森)中将混合物浴超声处理10分钟。
探针尖端在冰浴中以3 %尖端以50%振幅(〜7W)超声处理混合物30分钟。如果在超声处理期间冰浴开始融化,请对其进行更新以防止加热。
将溶液在室温下静置30分钟。
离心该溶液在16,100 ×克为1个小时在室温下除去捆绑的SWNT。上清液包含单独悬浮的正义RNA-SWNT。保留上清液并将SWNT沉淀物丢弃到有害的纳米材料废物中。
对反义RNA链重复相同的方案(请参见注释3)。将RNA-SWNT解决方案存储在4 ° C。

去除未结合的siRNA
加入500微升有义RNA-SWNT和500微升反义RNA-SWNT成两个单独的100K的Amicon旋转过滤器被放置在2个毫升收集管中。离心机在8 4分钟,000 ×克在室温。
将有义RNA-SWNT和反义RNA-SWNT的流通物收集在单独的试管中,然后将自旋过滤器放入同一收集管中。
添加0.1M NaCl的入旋转过滤器,直到体积达到500微升。重复洗涤步骤。
总共执行8次洗涤以去除所有未结合的RNA分子。合并并累积所有流通液(对于有义RNA-SWNT和反义RNA-SWNT分别使用),以便以后测量去除的RNA量。
计算SWNT concent配给通过测量碳纳米管的吸光度在632nm处用分光光度计(使用2微升用于NanoVue加分光光度计或稀到1ml为Thermo Scientific)进行。用吸光度值除以0.036的SWNT消光系数,得出以μg / ml为单位的SWNT浓度(如果将样品稀释,则将吸光度值e也乘以稀释倍数)。
计算浓度的RNA上的SWNT装载通过测量收集的吸光度流通在260nm(使用2种溶液微升为NanoVue加分光光度计),并减去RNA的总量从RNA的总量除去加入(2毫克在这个案例)。
(可选)为进一步表征,请使用Shimadzu UV-3600 Plus记录有义RNA-SWNT和反义RNA-SWNT吸收光谱,并使用近红外光谱仪(Princeton Instruments IsoPlane 320与液氮冷却的Princeton耦合,记录荧光光谱)仪器InGaAs像素的PyLoN -IR 1D数组)。参见Demirer等。,2020年为SWNT吸光度和荧光光谱的代表性示例。

RNA-SWNTs浸润叶片
选择健康和充分发展的叶mGFP5烟草本塞姆氏(4-6周龄)植物的实验。
合并100微升的200 nM的有义RNA的单壁碳纳米管具有100微升的200 nM的反义RNA的单壁碳纳米管在1.5 ml的Eppendorf管中(见注4)。拌匀
在混合后立即使用移液管尖端的叶,并浸润〜100-200的远轴(下)表面上的小穿刺微升从与1空穴所述siRNA-SWNT的混合物毫升无针注射器小心不损坏叶子(请参阅注释5)。
用Kimwipe纸巾去除叶面上多余的siRNA-SWNT溶液。用沙皮笔标记渗透区域,不要损坏叶子。
渗入阴性对照溶液,例如不含SWNT的游离siRNA或未靶向目标基因的加扰的RNA悬浮SWNT。如果可能,还应浸入阳性对照溶液,例如病毒siRNA输送样品(请参见注释6)。
将已渗入的植物放回生长室,直到测量基因沉默为止。

通过定量PCR(qPCR)确定基因沉默
1.渗透后24小时,切开渗透的叶区域(每个样品最大叶组织为100 mg),并使用n RNeasy plant mini试剂盒提取总RNA 。切割叶后,立即与RNA提取协议的第一步骤中继续进行(即,使用研钵和研杵研磨该组织在液氮中),以确保基因表达水平切割后不改变。遵循RNeasy工厂迷你套件的规程(请参见注释7)。     
2. RNA提取后,用分光光度计测量RNA浓度和纯度。反转录1 μ克使用的总RNA为互补DNA(cDNA)的iScript cDNA合成试剂盒。遵循iScript cDNA合成试剂盒的协议。     
3.使用的上电SYBR绿色主混合物用于qPCR的步骤用2 μ升的cDNA从第2步和特异性引物对靶和管家基因。遵循PowerUp SYBR绿色试剂盒的规程,与没有SWNT或加扰的RNA-SWNT的游离siRNA阴性对照相比,对siRNA-SWNT浸润的叶子中靶mRNA的相对定量。     
示例:我们qPCR的目标基因是mGFP5 (将GFP转基因插入Nb中),以及EF1 (延伸因子1)作为我们的管家(参考)基因。


4.使用ddCt方法分析qPCR数据(Rao等人,2013)。代表性的qPCR结果参见图2B。     

通过共聚焦荧光显微镜成像确定基因沉默
如果使荧光蛋白(如GFP)沉默,则可使用共聚焦荧光显微镜检测近似的沉默效率。
渗透后,将完整叶浸润的植物留在生长室中72小时。
渗透后72 h,在渗透点附近切一小片(0.5-1 cm × 0.5-1 cm)的渗透叶,并准备一个带盖玻片的玻片(厚度#1)。添加50-100 μ升水在玻璃载玻片和盖玻片用于成像之间。叶片变干之前的图像样本(最佳在15到30分钟内)。
用GFP滤光片在488 nm激光激发下对植物组织成像(在GFP沉默的情况下),如果可用,还可以用透射光捕获明场(请参见注释8)。
每个样品(包括未处理的叶片以及任何阴性和阳性对照样品)使用相同的光学设置捕获至少10至15个视野。
对于每个样品,将平均荧光强度值与未处理叶片的平均GFP荧光强度进行比较,这可用于确定siRNA-SWNT的沉默效率。注意对不同日期成像的样品使用相同的成像参数和定量分析。有关共焦成像的代表性结果,请参见图2A。





图2.代表性的基因沉默结果。A.未处理和游离-siRNA处理的对照叶片和烟草本氏烟草的siRNA-SWNT浸润样品叶片的共聚焦显微镜图像。渗透后3天成像,比例尺为50μm 。B.定量PCR分析结果在GFP基因沉默烟草本塞姆氏叶用siRNA-单壁碳纳米管。



基因沉默DET通过ermination W¯¯西部时代印迹
渗透72小时后,收获渗透的叶片,并用研钵和研杵在液氮中研磨,以回收干燥的冷冻叶片粉末。
转移冷冻叶粉末成管400微升预冷的溶解缓冲液(见ř ecipes)。
在冰上溶解组织1小时。将试管在10,000 × g下于4 °C离心20分钟。离心后,将含有完整蛋白质的上清液轻轻转移至新的干净试管中。使用Pierce 660 nm蛋白分析定量提取的总蛋白。
将样品与适当的上样缓冲液混合以进行凝胶电泳,然后使用加热块或热循环仪在95-100°C下将混合物煮沸5分钟。从每个样品中加载0.5 µg标准化总蛋白,并用SDS-PAGE凝胶(Bio- R和预制的tris /甘氨酸凝胶,4-20%梯度)进行分析。在120 V下运行凝胶60分钟。
转移凝胶至PVDF膜在冷转移缓冲液(见ř ecipes)和以400mA在1个运行×用甲醇转移缓冲液用于在冷室中在用冰块不超过60分钟。
使用在1 × TBST缓冲液中的7.5%BSA封闭膜1小时(参见R ecipes),然后在4 °C下与一抗GFP一起孵育过夜。彻底洗涤后,用山羊抗兔辣根过氧化物酶结合的抗体探测相应的蛋白条带30分钟。洗涤后,通过在不到2分钟的时间内用化学发光(Amersham ECL试剂盒)孵育并用ChemiDoc XRS +系统成像来扩增条带。
使用ImageJ软件量化GFP谱带的强度。

数据分析



对于所有实验,至少要进行3次生物学重复。生物学重复定义为由RNA-SWNTs独立浸润到不同植物中的实验。
对于共聚焦成像的定量分析,每个样品和每个生物重复至少捕获10-15个不重叠的视野,这被定义为技术重复。对这些技术复制品的荧光强度取平均值,以获得给定生物学复制品的平均荧光强度。共焦图像可以使用ImageJ或Zen Blue软件进行分析。
对于qPCR分析,至少要进行3次技术重复,这被定义为来自相同渗透的相同分离RNA批次的反应。为了分析qPCR结果的基因沉默效率,使用了以前开发的ddCt方法。该方法的细节可以在Rao等人的文章中找到。(2013)。
当针对单个自变量(例如,沉默效率)在统计学上比较两个以上样本时,请使用单向方差分析与Tukey的多重比较测试。报告P值和F。在统计上比较两个以上样本的多个独立变量(例如,多个时间点的沉默效率)时,请使用双向ANOVA和Sidak的多重比较测试。报告P值和F。仅比较两个样本时,可以使用Student t检验。报告P值。GraphPad Prism可用于绘制数据并执行统计显着性分析。

笔记



提供该协议用于沉默转基因烟草(Nicotiana benthamiana )叶片中的GFP基因,但可以适用于沉默其他基因和其他植物物种,包括其他转基因或内源基因。
RNA和SWNT的数量可以根据实验需要扩大或缩小。在缩放过程中,将RNA:SWNT的质量比保持在2。对于不同的基因靶标,或在不同的植物物种或组织中,可能需要不同的最终siRNA浓度。
通常,如果一条RNA链很好地悬浮SWNT,则其互补链的悬浮效率会降低。考虑到有义和反义链的互补碱基对,这可能导致RNA碱基对SWNT表面的吸附能力变化。我们通常获得20-50微克/米升后离心和纯化步骤RNA-SWNT悬浮液。
注意200 nM是SWNT上的RNA浓度,而不是SWNT浓度。根据悬浮液效率,这相当于〜2μg / ml SWNT。
100-200微升浸润建议共聚焦成像研究,而较大的体积或多个渗透应用于qPCR来执行,并且w ^西部时代印迹,以覆盖全叶面积。
优选在同一叶片上渗入SWNT和对照样品,以避免基因表达和成像中叶片间的差异。
必须在干净的工作台(最好专门用于基于RNA的实验)上小心进行RNA工作,以防止RNA降解。擦拭表面和设备RN一个本质的解决方案和变化频繁的手套。在方案操作期间将RNA置于冰上,并在方案完成后转移至-20 ° C或-80 ° C。
如果叶子太厚或波浪状以至于无法获得整个视野,请使用z-stack成像,然后对堆叠的图像进行沉默分析。将要相互比较的样本之间的成像参数保持相同。

菜谱



0.1 M氯化钠
称取0.5844 g NaCl,溶于100 ml无核酸酶的水中


带0.22微米针头过滤器的无菌过滤器


10%(wt / vol)过硫酸铵溶液(APS)
将5克APS加到50毫升密尔中稀释水,混合溶解


解决方案可以存储在 4°C长达3个月


10 ×传输缓冲区
制作1公升的 10倍转移缓冲液,将30.3 g Tris碱,144 g甘氨酸添加到800 ml MilliQ水中,混合溶解


然后加 MilliQ水的最终体积为1 L


1 ×传输缓冲区
要制成1升1 ×转移缓冲液,请添加100 ml 10 ×转移缓冲液,200 ml甲醇至700 ml MilliQ水并混合溶液


缓冲液需要保存在4°C,最好在每次实验前准备好缓冲液


10 × Tris缓冲盐(TBS)缓冲液(1 M Tris,1.5 M NaCl,pH 7.4)
要制成250毫升10 × TBS缓冲液,请在200毫升MilliQ水中加入30.3克Tris碱,21.9克NaCl并混合以溶解


使用HCl将溶液的pH值调节至7.4,然后添加MilliQ水至250 ml的最终体积


该溶液可在4°C下保存长达6个月


1 × TBS T缓冲器
加入50毫升10 × TBS缓冲液,和500 μ升吐温20(0.1%)至500ml的米尔LIQ水并混合溶液


缓冲液可在4°C下储存至少1个月


裂解缓冲液
10毫米Tris / HCl


150毫米氯化钠


1毫米EDTA


0.1%NP-40


5%甘油


1%蛋白酶抑制剂混合物


pH值7.5


要制备100 ml裂解缓冲液,请在80 ml无核酸酶水中加入1.21 g Tris碱,0.877 g NaCl,29.24 mg EDTA,0.1 g NP-40和5 ml甘油,混合溶解,用HCl调节pH值至7.5 ,加入1 ml蛋白酶抑制剂混合物,并使用无核酸酶的水将最终体积填充至100 ml 。


注意:Ť他裂解缓冲液可以储存在-20℃下至少6个月。



致谢



该协议源自Demirer等。(2020)。


GSD得到了斯伦贝谢基金会未来计划学院和Caltech Resnick Sustainability Institute的支持。我们感谢科学接口(CASI)获得的Burroughs Wellcome基金职业奖,斯坦福·法恩PDF初级教师奖,PF-JFA-1760奖,贝克曼基金会青年研究者奖,USDA AFRI奖,USDA NIFA奖,摩尔基金会和FFAR新创新奖(MP L)。MPL是Chan-Zuckerberg Biohub的调查员。



利益争夺



作者声明没有竞争利益。



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引用:Demirer, G. S. and Landry, M. P. (2021). Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers. Bio-protocol 11(1): e3897. DOI: 10.21769/BioProtoc.3897.
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