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Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice
从带病毒的灰飞虱中分离水稻条纹病毒制备物并机械接种于水稻   

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本实验方案简略版
New Phytologist
Apr 2016

Abstract

Tenuiviruses can infect the plants of the family Poaceae, and cause serious loss of crops, particularly rice and maize, in South-Eastern Asian countries. Tenuiviruses usually depend on insect vectors for their transmission and cannot be transmitted between plants through wounds or abrasions. Rice stripe virus (RSV), a typical member of tenuiviruses, is efficiently transmitted by the small brown planthopper Laodelphax striatellus in a persistent-propagative manner to cause rice stripe disease. Here we presented a convenient method, the midrib micro-injection, to mechanically inoculate insect-derived RSV into rice leaves for conducting pathogenicity assay on rice plants.

Keywords: Rice stripe virus (水稻条纹病毒), Mechanical inoculation (机械接种), Micro-injection (微量注射), Small brown planthopper (灰飞虱)

Background

Tenuiviruses cannot be mechanically inoculated into plants, unless through vascular puncture inoculation with quite different transmission rates ranging from 1% to 90% according to different experimental details (Louie, 1995; Hogenhout et al., 2008). As to RSV, mechanical transmission usually fails or yields a low infectious rate (Ling, 1972). In particular, the transmission rate was only 6% after the injection of the RSV crude extraction from diseased plants (Okuyama and Asuyama, 1959). The midrib micro-injection method mentioned in this work promotes the RSV transmission rate to 17%. Though the incidence of RSV by mechanical transmission is still much lower than that by insect vector transmission (53%), our method provides a convenient way for mechanical inoculation of persistent-propagative plant viruses. Moreover, based on this method, replication and gene expression of a persistent-propagative plant virus can be determined more accurately in infected plant hosts without the interference of insects, i.e., the inoculation doses and the insect proteins.

Materials and Reagents

  1. 15 ml centrifuge tube, RNase-/DNase-free (Corning, catalog number: 430791 )
  2. 1.5 ml clear Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
  3. Plastic tissue grinder pestles (Tiangen Biotech, catalog number: OSE-Y001 )
  4. Pipettes with tips of 10 μl, 100 μl, and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
  5. PVDF Western blotting membranes (Sigma-Aldrich, catalog number: 03010040001 )
    Note: Currently, it is ‘Merck, catalog number: 03010040001 ’.
  6. Extra thick blot paper filter paper (Bio-Rad Laboratories, catalog number: 1703965 )
  7. 96-well ELISA Microplates (Greiner Bio One International, catalog number: 650001 )
  8. Adhesive plastic
  9. 25 G ⅝ to 30 G ½ gauge needle
  10. Drummond replacement glass capillaries, 100/vial (Drummond Scientific, catalog number: 3-000-203-G/X )
  11. Parafilm® M (Sigma-Aldrich, catalog number: P7793 )
    Note: Currently, it is ‘Merck, catalog number: P7793 ’.
  12. Insect vectors (Laodelphax striatellusi, Fallen) (collected from a field population in Hai’an, Jiangsu Province, China)
  13. RSV cp gene was amplified with the primers: 5’-ATGGGTACCAACAAGCCAGC-3’, and 5’-CTAGTCATCTGCACCTTCTG-3’. PCR was run on the ProFlexTM PCR System under cycling conditions of 95 °C for 5 min, followed by 30 cycles of 95 °C for 20 sec, 50 °C for 30 sec and 72 °C for 30 sec. The purified 969 bp of PCR products were cloned into pET-28a (Merck, Novagen, catalog number: 69865-3 ) for Cp protein expression. The recombinant cp plasmid was sent to Beijing Genomics Institute for monoclonal anti-Cp antibody production
  14. Host plants (Oryza sativa L. spp. japonica var. Nippobare)
    Note: Healthy 2-week-old rice leaves with the length of approximately 15 cm were used for microinjection.
  15. Protein loading buffer (5x) (Adipogen International, catalog number: AG-10T-0020-L001 )
  16. ExpressPlusTM PAGE gel, 4-20% (GenScript, catalog number: M42010 )
  17. PageRulerTM prestained protein ladder, 10 to 180 kDa (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 ), used for SDS-PAGE
  18. HEPES (Fisher Scientific, catalog number: BP310-100 ), used for SDS-PAGE
  19. EMPLURA® methanol (Merck, catalog number: 822283 )
  20. ELISA coating buffer, 1x (Solarbio, catalog number: C1050 )
  21. SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34095 )
  22. Goat anti-mouse IgG (H+L) secondary antibody, HRP (Thermo Fisher Scientific, InvitrogenTM, catalog number: 32430 )
  23. Anti-Cp antibody (Beijing Genomics Institute)
  24. 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, catalog number: T0440 )
    Note: Currently, it is ‘Merck, catalog number: T0440 ’.
  25. Sulfuric acid (H2SO4) (5 N) (Merck, catalog number: 480364 ), used for ELISA
  26. Sodium dodecyl sulfate (SDS) (AMRESCO, catalog number: 0227 ), used for ELISA
  27. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
    Note: Currently, it is ‘Merck, catalog number: M8410 ’ .
  28. Tween® 20 (Sigma-Aldrich, catalog number: P1379 )
  29. Phosphate buffer saline (PBS), pH 7.4, basic (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
  30. OxoidTM Skim milk power (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0031B )
  31. Phosphate buffer saline with Tween 20 (1x) (PBST) (see Recipes)
  32. Transfer buffer (see Recipes)
  33. Blocking buffer (see Recipes)
  34. Diluted monoclonal anti-Cp antibody (see Recipes)
  35. Diluted Goat anti-mouse IgG, HRP-conjugated antibody (see Recipes)

Equipment

  1. TGrinder High Speed Tissue grinder (Tiangen Biotech, catalog number: OSE-Y30 )
  2. Centrifuge 5424R (Eppendorf, model: 5424 R )
  3. Decolorizing Orbital Shaker (T-Bota Scietech Instruments & Equipment, model: TS-200 ), used for antibody incubation in ELISA and Western blots
  4. Chemiluminescent Imaging System (Tanon, model: Tanon 5200 )
  5. SpectraMax® Paradigm® Multi-Mode Detection Platform (Molecular Devices, model: SpectraMax Paradigm Multi-Mode )
  6. Micropipette Puller (Sutter Instrument, model: P-97 )
  7. ProFlexTM PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: ProFlexTM 3 x 32-Well PCR System, catalog number: 4484073 )
  8. Glass incubators (Φ55 x 150 mm, Φ110 x 150 mm), used for planthopper incubation
    Note: Each glass incubator must be sealed with a nylon mesh in order to protect the cultured plants or insects from other interference sources. The culture condition is 25 °C, with 16 h of light daily (Figure 1).


    Figure 1. Plant culture and insect rearing. The small brown planthoppers and rice seedlings are cultured in glass incubators sealed with nylon meshes. The viruliferous or nonviruliferous planthoppers are geographically isolated, which are cultured in different insectaries.

  9. MixMate® , the 3-in-1 mixer (Eppendorf, catalog number: 5353000014 )
  10. H2O3 dry bath (Coyote Bioscience, model: H2O3-100C )
  11. PowerPacTM universal power supply (Bio-Rad Laboratories, catalog number: 1645070 )
  12. Trans-Blot® TurboTM transfer system (Bio-Rad Laboratories, catalog number: 1704150 )
  13. Nanoject II Programmable Nanoliter injector (World Precision Instruments, model: Nanoliter 2000 )
  14. Backfilling Needle for Nanoject II Auto-Nanoliter Injector, 2” Length (Drummond Scientific, model: 3-000-027 )
  15. SZ61 Stereo microscope (Olympus, model: SZ61 ), used for microinjection

Procedure

  1. Preparation of RSV crude extractions
    1. For RSV extraction, use 100-200 whole bodies of viruliferous small brown planthoppers as samples.
      Note: Insects should be directly grinded upon collection.
    2. Brush down the viruliferous planthoppers into a 15 ml tube, and put it on ice for 2-3 min in order to freeze them.
    3. Fully grind total planthoppers in 800 μl of PBS-buffer (pH 7.2) (v:v, 1:1) with TGrinder, and maintain the tube in ice.
      Note: No research has been reported how to extract plant viruses from insect vectors, whereas several researches suggested that RSV-infected plants should be extracted in PBS buffer at 4 °C (Ishikawa et al., 1982; Ishikawa et al., 1989). So in this work, we grind the planthoppers at the same condition.
    4. Shake the mixture thoroughly until well blended, and then place the tube in ice for 30 min.
    5. Centrifuge the mixture at 1,344 x g for 5 min at 4 °C, and transfer the supernatant into a 1.5 ml pre-cooling RNA-free tube.
    6. Repeat step A5.
    7. Centrifuge at 8,400 x g for 15 min at 4 °C, and store the supernatant at 4 °C for further usage.
      Note: The supernatant is RSV crude extraction.
    8. Use the same protocol to isolate the crude extractions from nonviruliferous small brown planthoppers as negative controls.
      Note: Both RSV crude extractions and the extractions from nonviruliferous planthoppers that used for mechanical inoculation should be microinjected into rice leaves as soon as possible.

  2. Detection of RSV through Western blots
    1. For RSV detection, mix 20 µl 5x protein loading buffer with 80 µl extractions from both viruliferous and nonviruliferous planthoppers, and boil the mixture for 5 min.
    2. Load 20 µl of the treated samples into each well of the ExpressPlus TMPAGE gel, along with prestained marker.
      Note: Make sure that the treated samples from viruliferous and nonviruliferous planthoppers will not become cross-contamination.
    3. Run the gel for 40 min at 140 V.
    4. Prepare a PVDF membrane which was slightly bigger than the size of the gel.
    5. Place the PVDF membrane in methanol for 5 min.
    6. Place the PVDF membrane and gel in 1x transfer buffer (see Recipes) for 10 min.
    7. Prepare the transfer sandwich and place it in a transfer tank with 25 mA for 30 min.
      Note: The transfer sandwich includes a layer of filter paper at each end, the PVDF membrane, and gel. The PVDF membrane is placed between the gel surface and the positive electrode in the sandwich.
    8. Block the membrane in 5% skimmed milk in TBST at room temperature (RT) for 0.5-1 h.
    9. Wash the membrane with PBST (see Recipes) three times for 5 min each time.
    10. Incubate the membrane in the monoclonal anti-Cp antibody solution (see Recipes) by using Decolorizing Orbital Shaker at RT for 1.5 h or at 4 °C overnight.
    11. Repeat step B9.
    12. Incubate in the HRP-conjugated antibody solution by using Decolorizing Orbital Shaker at RT for 1.5 h.
    13. Wash the membrane with PBST five times for 5 min each time.
    14. Use the West Femto Substrate Trial Kit to the blot according to the manufacturer’s instruction.
      Note: Prepare working solution by mixing 100 µl of the stable peroxide solution and the luminol/enhancer solution. Use 100 µl working solution per cm2 of membrane.
    15. Capture the chemiluminescent signals through Chemiluminescent Imaging System.
      Note: Cp could be only detected from the extractions of the viruliferous small brown planthoppers (Figure 2).


      Figure 2. RSV detection of the crude extractions from nonviruliferous (lane 1) and viruliferous (lane 2) small brown planthoppers. The monoclonal anti-Cp antibody was used as the primary antibody.

  3. Quantification of RSV crude extractions
    1. Prepare the Cp standards in PBS-buffer (pH 7.2) according to previously optimized standard ELISA protocol (Nemzek et al., 2001). Nine dilutions are made (0, 0.0045, 0.045, 0.45, 4.5, 12.5, 25, 45, 90 ng/ml).
    2. Use RSV crude extractions as samples.
      Note: Samples for ELISA should be tested in duplicate.
    3. Add 100 µl of the standards and samples into each well of a 96-well ELISA plate, and mix them with 100 µl 1x coating buffer. Cover the plate with an adhesive plastic and incubate at 4 °C overnight.
    4. Wash the plate three times by filling each well with 200 µl PBST, at least for 3 min each time.
    5. Block the remaining protein-binding sites in the wells by adding 200 µl blocking buffer (see Recipes) per well. Cover the plate with an adhesive plastic and incubate at RT for no less than 30 min.
    6. Repeat step C4.
    7. Add 100 µl of diluted monoclonal anti-Cp antibody to each well. Cover the plate with an adhesive plastic and incubate at RT for 1.5 h.
    8. Repeat step C4.
    9. Add 100 µl of diluted Goat anti-mouse IgG, HRP-conjugated antibody to each well (see Recipes). Cover the plate with an adhesive plastic and incubate at RT for 1.5 h.
    10. Remove the supernatant and wash the plate five times with PBST, for 1 min each time.
    11. Dispense 100 µl of TMB solution into each well.
    12. Gently mix on Decolorizing Orbital Shaker at RT for 30 min.
      Note: This reaction should be protected from light.
    13. Add 100 µl of 2 M H2SO4 or 1% SDS to stop the reaction.
      Note: Make sure that the color has changed from blue to yellow in all the wells.
    14. Read the optical density (OD) at 450 nm on the SpectraMax® Paradigm® Multi-Mode Detection Platform within 5 min.
      Note: You can also read the absorbance value of OD630 after chromogenic reaction without adding stop solution.

  4. Microinjection of RSV into rice leaves
    1. Prepare the needles for microinjection on a Micropipette Puller (Dean, 2006) (Video 1).
      Note: Needles for microinjection are pulled from glass capillaries, and 25 G ⅝ to 30 G ½ gauge needle is ideal for injection.

      Video 1. Preparing the needles for microinjection on the Micropipette Puller

    2. Fill the glass capillary with mineral oil before installation.
    3. Dilute the RSV crude extraction with PBS buffer to a concentration of Cp around 10 ng/ml.
    4. Empty the needle and fill with the diluted RSV crude extraction.
      Note: The diluted RSV crude extraction should be placed on a Parafilm and then be inhaled into the needle.
    5. Microinject 23 nl of the diluted RSV crude extraction into the midribs of the healthy 2-week-old rice leaves for five times through a glass needle at slow speed using Nanoliter 2000. The interval between each injection site is 1 cm (Video 2). Both sides of the leaves were OK for microinjection.
      Note: The midrib should be observed to be filled with RSV crude extraction upon microinjection (Figure 3).


      Figure 3. RSV microinjection and symptom observation. A. RSV microinjection. RSV crude extractions were microinjected into the midribs of rice leaves through Nanoliter 2000. The red arrow indicates the injection site. Symptoms of rice leaves inoculated with extractions from nonviruliferous planthoppers (B) and from viruliferous planthoppers (C) were observed two weeks later. The RSV extractions from viruliferous planthoppers induced typical disease symptoms.

      Video 2. Microinjection of RSV through Nanoject II Programmable Nanoliter injector

    6. Microinject the extractions from nonviruliferous small brown planthoppers into the midribs of healthy rice leaves as negative control.
      Note: The negative control is very important as it can tell you whether or not the experimental system works.
    7. Evaluate the development of disease symptoms of both the injected leaves and systemic leaves after about 2-3 weeks post inoculation. The disease symptoms are evaluated as described by Toriyama and Sakurai (1969).
      Note: Make at least three replicates, and each group contains at least 30 seedlings. The incidence of RSV by mechanical transmission is about 17% after the midrib microinjection of insect-derived RSV crude extractions.

Data analysis

Calculation of ELISA results

  1. Calculate the mean of OD630 or OD450 values for each group of standards and samples.
    Note: In this experiment, OD630 was measured after chromogenic reaction without adding stop solution.
  2. Build a standard curve by plotting the mean OD value obtained from each standard against its concentration in ng/ml on linear graph paper. X-axis shows the concentrations of standard proteins, and Y-axis shows the corresponding absorbance values (Figure 4).
  3. Determine the corresponding concentration of each sample in ng/ml by using the mean OD values based on the standard curve.
  4. Multiply the derived concentrations by the dilution factor to determine the actual concentration of Cp protein in the RSV crude extraction samples.
    Note: Make sure that samples are diluted appropriately, the OD values of which are within the scope of the standard curve.


    Figure 4. Data analysis of ELISA result by using CurveExpert 1.4. A. Data input. The concentrations of Cp and the OD630 values were listed as the X and Y axis. B. Standard curve construction. The standard curve was built based on data plot. S and r value were listed in the top right corner. C. The equation of the standard curve. The equation was automatically calculated. The parameters were listed in the right pane.

Notes

  1. It is recommended that during the ELISA and microinjection step, extractions from healthy insect samples should be used as negative controls, in order to evaluate the experimental systems.
  2. RSV crude extractions from nonviruliferous or viruliferous planthoppers are stored at 4 °C for no longer than two days. Generally, the RSV crude extractions with the OD value above 1.00 (Cp concentration around 10 ng/ml) is suitable for microinjection.

Recipes

  1. Phosphate buffer saline with Tween 20 (1x) (PBST)
    0.2% Tween 20
    Add to PBS buffer (pH 7.2)
    Filter sterilize using filtering device
  2. Transfer buffer (semi-dry)
    48 mM Tris
    39 mM glycine
    20% methanol
    0.04% SDS
  3. Blocking buffer
    1 or 5% skim milk
    Add to TBST buffer
    Mix well and filter
    Store at 4 °C for no longer than 3 days
  4. Diluted monoclonal anti-Cp antibody
    Add 5 μl monoclonal anti-Cp antibody into 10 ml blocking buffer (5% skim milk)
    Store at 4 °C for no longer than 1 day
  5. Diluted Goat anti-mouse IgG, HRP-conjugated antibody
    Add 2 μl monoclonal anti-Cp antibody into 10 ml blocking buffer (5% skim milk)
    Store at 4 °C for no longer than 1 day

Acknowledgments

This work is adapted from previously published papers (Zhao et al., 2016). We acknowledge the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB11040200) and the Major State Basic Research Development Program of China (973 Program) (No. 2014CB13840402) for funds. The authors declare no conflict of interests.

References

  1. Dean, D. A. (2006). Preparation (pulling) of needles for gene delivery by microinjection. CSH Protoc 7.
  2. Hogenhout, S. A., Ammar el, D., Whitfield, A. E. and Redinbaugh, M. G. (2008). Insect vector interactions with persistently transmitted viruses. Annu Rev Phytopathol 46: 327-359.
  3. Ishikawa, K., Omura, T. and Hibino, H. (1982). Characterization of rice stripe virus a heavy component carrying infectivity. J Gen Virol 61(2): 187-195.
  4. Ishikawa, K., Omura, T. and Tsuchizaki, T. (1989). Association of double-and single-stranded RNAs with each four components of rice stripe virus. Jan J Phytopathol 55: 315-323.
  5. Ling, K. C. (1972). Rice virus diseases. 148.
  6. Louie, R. U. (1995). Vascular puncture of maize kernels for the mechanical transmission of maize white line mosaic virus and other viruses of maize. v.85. (ARS, Corn and Soybean Research Unit, Wooster).
  7. Nemzek, J. A., Siddiqui, J. and Remick, D. G. (2001). Development and optimization of cytokine ELISAs using commercial antibody pairs. J Immunol Methods 255(1-2): 149-157.
  8. Okuyama, S. and Asuyama, H. (1959). Mechanical transmission of rice stripe virus to rice plant. Ann PSJ 24: 35.
  9. Toriyama, K. and Sakurai, Y. (1969). Breeding rice varieties for resistance to stripe virus disease. Jpn Agr Res Q 4: 5.
  10. Zhao, W., Yang, P., Kang, L. and Cui, F. (2016). Different pathogenicities of Rice stripe virus from the insect vector and from viruliferous plants. New Phytol 210(1): 196-207.

简介

细菌病毒可以感染禾本科植物,并在东南亚国家造成严重的作物损失,特别是稻米和玉米。 病毒通常依靠昆虫载体传播,不能通过伤口或擦伤传播。 水稻条纹病毒(RSV)是典型的tenuiviruses的成员,以持续繁殖的方式被小型褐飞虱灰飞虱高效率地传播,导致水稻条纹病。 在这里,我们提出了一种方便的方法,即中微量注射,以机械方式将昆虫来源的RSV接种到水稻叶中,以对水稻植物进行致病性测定。
【背景】除非通过根据不同的实验细节从1%至90%的完全不同的传输速率进行血管穿刺接种(Louie,1995; Hogenhout等人,2008),否则不能将机器接种到植物中。至于RSV,机械传播通常失败或产生低感染率(Ling,1972)。特别地,从病变植物注射RSV粗提物后,传播率仅为6%(Okuyama and Asuyama,1959)。在这项工作中提到的中微注射方法将RSV传播率提高到17%。虽然机械传播RSV的发生率仍远低于昆虫载体传播(53%),但是我们的方法为持续繁殖的植物病毒的机械接种提供了便利的方法。此外,基于这种方法,可以在受感染的植物宿主中更精确地确定持续增殖植物病毒的复制和基因表达,而不受昆虫即接种剂量和昆虫蛋白质的干扰。

关键字:水稻条纹病毒, 机械接种, 微量注射, 灰飞虱

材料和试剂

  1. 15 ml离心管,RNase- / DNase-free(Corning,目录号:430791)
  2. 1.5毫升透明微管(Corning,Axygen ,产品目录号:MCT-150-C)
  3. 塑料组织研磨机杵(天根生物,目录号:OSE-Y001)
  4. 移液器的尖端10微升,100微升和1000微升(Eppendorf,目录号码:3120000020,3120000046和31200000623)
  5. PVDF Western印迹膜(Sigma-Aldrich,目录号:03010040001)
    注意:目前它是'默克,目录号:03010040001'。
  6. 超厚印迹纸滤纸(Bio-Rad Laboratories,目录号:1703965)

  7. 96孔ELISA微孔板(Greiner Bio One International,目录号:650001)
  8. 胶粘剂
  9. 25 G⅝至30 G½规格的针头
  10. Drummond替代玻璃毛细管,100 /小瓶(Drummond Scientific,目录号:3-000-203-G / X)
  11. Parafilm M(Sigma-Aldrich,目录号:P7793)
    注意:目前它是'默克,目录号:P7793'。
  12. 昆虫载体( Laodelphax striatellusi ,Fallen)(从中国江苏省海安市的田间种群收集)
  13. 用引物5'-ATGGGTACCAACAAGCCAGC-3'和5'-CTAGTCATCTGCACCTTCTG-3'扩增RSV cp基因。在ProFlex TM PCR系统上在95℃的循环条件下进行PCR 5分钟,然后进行30个循环的95℃20秒,50℃30秒和72℃ 30秒将纯化的969bp的PCR产物克隆到pET-28a(Merck,Novagen,目录号:69865-3)中以进行Cp蛋白表达。将重组质粒送到北京基因组研究所进行单克隆抗Cp抗体生产。
  14. 寄主植物(Oryza sativa L。spp。 japonica var。Nippobare )
    注:健康的2周龄水稻叶片长约15厘米,用于显微注射。
  15. 蛋白上样缓冲液(5x)(Adipogen International,目录号:AG-10T-0020-L001)
  16. ExpressPlus TM PAGE凝胶,4-20%(GenScript,目录号:M42010)
  17. PageRuler TM预染蛋白梯度,10至180kDa(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26616),用于SDS-PAGE
  18. HEPES(Fisher Scientific,目录号:BP310-100),用于SDS-PAGE
  19. EMPLURA甲醇(Merck,目录号:822283)
  20. ELISA包被缓冲液,1x(Solarbio,目录号:C1050)
  21. SuperSignal TM West Femto最大灵敏度底物(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:34095)
  22. 山羊抗小鼠IgG(H + L)二抗HRP(Thermo Fisher Scientific,Invitrogen TM,目录号:32430)
  23. 抗Cp抗体(北京基因组研究所)
  24. 用于ELISA的3,3',5,5'-四甲基联苯胺(TMB)液体底物系统(Sigma-Aldrich,目录号:T0440)
    注意:目前是“默克,目录号:T0440”。
  25. 用于ELISA的硫酸(H 2 SO 4)(5N)(Merck,目录号:480364)
  26. 十二烷基硫酸钠(SDS)(AMRESCO,目录号:0227),用于ELISA
  27. 矿物油(Sigma-Aldrich,目录号:M8410)
    注意:目前是'默克,目录号:M8410'。
  28. Tween 20(Sigma-Aldrich,目录号:P1379)
  29. 磷酸盐缓冲盐水(PBS),pH7.4,碱性(1x)(Thermo Fisher Scientific,Gibco TM,目录号:10010031)
  30. Oxoid TM脱脂牛奶粉(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:LP0031B)
  31. 吐温20磷酸盐缓冲液(1x)(PBST)(见食谱)
  32. 传输缓冲区(见食谱)
  33. 阻塞缓冲区(见食谱)
  34. 稀释的单克隆抗Cp抗体(见食谱)
  35. 稀释山羊抗鼠IgG,HRP结合抗体(见食谱)

设备

  1. TGrinder高速组织研磨机(天根生物科技,目录号:OSE-Y30)
  2. 离心机5424R(Eppendorf,型号:5424 R)
  3. 用于ELISA和Western印迹的抗体孵育的脱色轨道振荡器(T-Bota Scietech Instruments& Equipment,型号:TS-200)
  4. 化学发光成像系统(Tanon,型号:Tanon 5200)
  5. SpectraMax™Paradigm®多模式检测平台(Molecular Devices,型号:SpectraMax Paradigm Multi-Mode)
  6. 微管拉拔器(萨特仪器,型号:P-97)
  7. ProFlex TM PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:ProFlex TM 3×32-Well PCR System,目录号:4484073 )
  8. 用于飞虱孵化的玻璃培养箱(Φ55×150mm,Φ110×150mm)
    注:每个玻璃培养箱都必须用尼龙网密封,以保护培养的植物或昆虫免受其他干扰源的侵害。培养条件是25°C,每天光照16小时(图1)。


    图1.植物培养和昆虫饲养小褐飞虱和水稻幼苗在用尼龙网密封的玻璃培养箱中培养。有毒或无毒的飞虱在地理上是分离的,在不同的昆虫中培养。

  9. MixMate ®,三合一搅拌器(Eppendorf,产品目录号:5353000014)
  10. H 2 O 3干浴(Coyote Bioscience,型号:H 2 O 3 -10℃) >
  11. PowerPac TM通用电源(Bio-Rad Laboratories,目录号:1645070)
  12. Trans-Blot TM Turbo TM转移系统(Bio-Rad Laboratories,目录号:1704150)
  13. Nanoject II可编程纳升注射器(世界精密仪器公司,型号:Nanoliter 2000)
  14. Nanoject II自动纳升注射器回填针,2“长(Drummond Scientific,型号:3-000-027)
  15. SZ61立体显微镜(Olympus,型号:SZ61),用于显微注射

程序

  1. RSV粗提取物的制备
    1. 对于RSV提取,使用100〜200个全毒的小褐飞虱作为样本。
      注意:昆虫应该直接收集。
    2. 将病毒飞虱刷入15毫升的试管中,放在冰上2-3分钟以冻结。
    3. 用TGrinder在800μlPBS缓冲液(pH7.2)(v:v,1:1)中充分研磨总飞虱,并将管保持在冰中。
      注:关于如何从昆虫载体中提取植物病毒的研究尚未见报道,而一些研究表明RSV感染的植物应在4℃的PBS缓冲液中提取(Ishikawa et al。,1982; Ishikawa et al。 ,1989)。所以在这项工作中,我们在相同的条件下研磨飞虱。
    4. 彻底摇匀混合物至混匀,然后将试管置冰中30分钟。
    5. 在4℃下将混合物在1,344×g下离心5分钟,并将上清液转移到1.5ml预冷的无RNA管中。
    6. 重复步骤A5。
    7. 在4℃下离心8,400×g 15分钟,并将上清液保存在4℃以备进一步使用。
      注意:上清液是RSV粗提物。
    8. 使用相同的协议来分离非疣状小褐飞虱的粗提取物作为阴性对照。
      注:RSV粗提物和用于机械接种的非飞虱飞虱提取物应尽快显微注射到水稻叶中。

  2. 通过Western印迹检测RSV
    1. 对于RSV检测,将20μl5x蛋白上样缓冲液与80μl来自毒性和非毒性飞虱的提取物混合,并将混合物煮沸5分钟。
    2. 将20μl处理过的样品加入ExpressPlus TMPAGE凝胶的每个孔中,同时加入预染记号。
      注意:确保来自病毒和非病毒飞虱的处理样品不会交叉污染。

    3. 在140 V运行凝胶40分钟。
    4. 准备一个略大于凝胶尺寸的PVDF膜。
    5. 将PVDF膜置于甲醇中5分钟。
    6. 将PVDF膜和凝胶放入1x转移缓冲液中(参见配方)10分钟。
    7. 准备转移三明治,并将其放置在25 mA的转印槽中30分钟。
      注意:转印三明治包括两端的一层滤纸,PVDF膜和凝胶。将PVDF膜置于凝胶表面和夹层中的正极之间。
    8. 在室温(RT)下在TBST中的5%脱脂牛奶中封闭膜0.5-1小时。
    9. 用PBST清洗膜(参见食谱)3次,每次5分钟。
    10. 通过使用脱色轨道振荡器在室温下1.5小时或在4℃过夜孵育单克隆抗Cp抗体溶液中的膜(参见食谱)。
    11. 重复步骤B9。
    12. 通过使用脱色轨道振荡器在RT孵育HRP缀合的抗体溶液1.5小时。
    13. 每次用PBST清洗5次,每次5分钟。
    14. 根据制造商的说明使用西方Femto底物试剂盒进行印迹。
      注意:通过混合100μl稳定的过氧化物溶液和鲁米诺/增强剂溶液来制备工作溶液。使用100μl工作溶液/ cm 2膜。
    15. 通过化学发光成像系统捕获化学发光信号。
      注:Cp只能从病毒性小褐飞虱的提取物中检测到(图2)。


      图2. RSV检测非疣状(1道)和含毒的(2道)小型褐飞虱的粗提取物。使用单克隆抗Cp抗体作为一抗。

  3. RSV粗提取物的量化
    1. 根据先前优化的标准ELISA方案(Nemzek等人,2001),在PBS缓冲液(pH7.2)中制备Cp标准品。进行9次稀释(0,0.0045,0.045,0.45,4.5,12.5,25,45,90ng / ml)。
    2. 使用RSV粗提取物作为样品。
      注意:用于ELISA的样品应重复测试。
    3. 加入100μl的标准品和样品到96孔ELISA板的每个孔中,并与100μl1x包被缓冲液混合。用粘性塑料盖住平板并在4℃下孵育过夜。
    4. 每次用200μlPBST填充每个孔,每次至少洗3分钟,洗板3次。
    5. 通过每孔添加200μl封闭缓冲液(参见食谱)来阻断孔中剩余的蛋白质结合位点。用粘性塑料盖住平板并在室温下孵育不少于30分钟。
    6. 重复步骤C4。
    7. 每孔加100μl稀释的单克隆抗Cp抗体。用粘性塑料盖住平板并在室温下孵育1.5小时。
    8. 重复步骤C4。
    9. 添加100μL稀释的山羊抗小鼠IgG,辣根过氧化物酶标记的抗体每孔(见食谱)。用粘性塑料盖住平板并在室温下孵育1.5小时。
    10. 去除上清液,用PBST洗板5次,每次1分钟。
    11. 向每个孔中分配100μl的TMB溶液。

    12. 在室温下轻轻混合脱色轨道振荡器30分钟 注意:这个反应应该避光。
    13. 加入100μl2M H 2 SO 4或1%SDS以终止反应。
      注意:确保所有孔中的颜色从蓝色变为黄色。
    14. 在5分钟内,在SpectraMax?Paradigm?多模式检测平台上读取450 nm的光密度(OD)。
      注意:您也可以读取显色反应后OD的吸光度值,而不添加终止溶液。 />
  4. RSV在水稻叶片中的微量注射
    1. 在Micropipette Puller上准备显微注射针(Dean,2006)(视频1)。
      注意:显微注射针是从玻璃毛细管中抽出的,25 G⅝到30 G½针针是注射的理想之选。

      视频1

    2. 安装前用玻璃毛细管填充矿物油。
    3. 用PBS缓冲液稀释RSV粗提物至约10ng / ml的Cp浓度。
    4. 清空针头并用稀释的RSV粗提物填充。
      注:稀释后的RSV粗提物应置于石蜡膜上,然后吸入针中。
    5. 使用Nanoliter 2000,将稀释的RSV粗提物微量注射到健康的2周龄水稻叶的中脉中,缓慢通过玻璃针5次,每个注射位点之间的间隔为1cm(视频2)。
      叶子的两面都可以进行微量注射 注意:显微注射时应观察到中脉充满了RSV粗提物(图3)。


      图3. RSV显微注射和症状观察A. RSV显微注射。通过Nanoliter 2000将RSV粗提物显微注射到水稻叶中部。红色箭头指示注射部位。两周后观察接种了来自非疣绿飞虱(B)和病毒飞虱(C)的稻叶的症状。
      来自病毒性飞虱的RSV提取物引起典型的疾病症状
      视频2

    6. 微量注射从非疣状小褐飞虱到健康稻叶中部作为阴性对照。
      注:阴性对照非常重要,因为它可以告诉你实验系统是否工作。
    7. 在接种后约2-3周后评估注射叶和全身叶的疾病症状的发展。按照鸟山(Toriyama)和樱井(Sakurai)(1969)的描述评估疾病症状。
      注意:至少重复三次,每组至少包含30个幼苗。在中脉显微注射昆虫来源的RSV粗提物后,机械传播的RSV发生率约为17%。

数据分析

计算ELISA结果

  1. 计算每组标准品和样品的OD值或OD值的平均值。
    注意:在这个实验中,在发色反应之后,不添加终止溶液,测量OD 630。
  2. 建立一个标准曲线,绘制从每个标准获得的平均OD值与线性方格纸上的ng / ml的浓度。 X轴显示标准蛋白质的浓度,Y轴显示相应的吸光度值(图4)。
  3. 根据标准曲线,用平均OD值确定每个样品的相应浓度(ng / ml)。
  4. 将衍生浓度乘以稀释倍数,以确定RSV粗提物样品中Cp蛋白的实际浓度。
    注意:确保样品被适当稀释,其OD值在标准曲线范围内。


    图4.使用CurveExpert 1.4进行ELISA结果的数据分析。 :一种。数据输入。 Cp和OD 630值的浓度被列为X和Y轴。 B.标准曲线结构。标准曲线是基于数据图建立的。 S和r值被列在右上角。 C.标准曲线的方程。等式自动计算。这些参数在右侧窗格中列出。

笔记

  1. 建议在ELISA和显微注射步骤中,健康昆虫样品的提取应作为阴性对照,以评估实验系统。
  2. 来自非疣状或病毒性飞虱的RSV粗提物在4℃下储存不超过两天。通常,OD值高于1.00(Cp浓度约10ng / ml)的RSV粗提物适用于显微注射。

食谱

  1. 吐温20磷酸盐缓冲液(1x)(PBST)
    0.2%吐温20
    加入PBS缓冲液(pH7.2)
    使用过滤设备过滤消毒
  2. 转移缓冲液(半干)
    48 mM Tris
    39 mM甘氨酸
    20%甲醇
    0.04%SDS
  3. 阻塞缓冲区
    1或5%的脱脂牛奶
    添加到TBST缓冲区
    充分混合并过滤

    在4°C下保存不超过3天
  4. 稀释的单克隆抗Cp抗体
    将5μl单克隆抗Cp抗体加入10 ml封闭缓冲液(5%脱脂牛奶)中

    在4°C储存不超过1天
  5. 稀释山羊抗小鼠IgG,HRP标记的抗体

    加入2μl单克隆抗Cp抗体到10 ml封闭缓冲液(5%脱脂牛奶)中
    在4°C储存不超过1天

致谢

这项工作是从以前发表的文章(赵等人,2016年)改编。我们承认中国科学院战略重点研究计划(XDB11040200)和国家重大基础研究发展计划(973计划)(No. 2014CB13840402)资助。作者声明没有利益冲突。

参考

  1. Dean,D.A。(2006)。 通过显微注射法制备(拉动)基因递送针 CSH Protoc 7。
  2. Hogenhout,S.A.,Ammar el,D.,Whitfield,A.E。和Redinbaugh,M.G。(2008)。 昆虫媒介与持续传播的病毒的相互作用 Annu Rev Phytopathol 46:327-359。
  3. Ishikawa,K.,Omura,T。和Hibino,H。(1982)。 水稻条纹病毒的特征是一种携带传染性的重组分。
  4. Ishikawa,K.,Omura,T。和Tsuchizaki,T。(1989)。 双链和单链RNA与水稻条纹病毒。 Jan J Phytopathol 55:315-323。
  5. Ling,K.C。(1972)。 水稻病毒疾病 148.
  6. Louie,R.U.(1995)。 用于玉米白线花叶病毒等机械传播的玉米籽粒的血管穿刺玉米病毒。 (ARS,玉米和大豆研究部,Wooster)。
  7. Nemzek,J.A。,Siddiqui,J。和Remick,D.G。(2001)。 使用商业抗体对开发和优化细胞因子ELISA。免疫学方法 255(1-2):149-157。
  8. Okuyama,S.和Asuyama,H.(1959)。水稻条纹病毒在水稻上的机械传播。 Ann PSJ 24:35.
  9. Toriyama,K.和Sakurai,Y。(1969)。 培育抗条纹病毒病的水稻品种 Jpn Agr Res Q 4:5。
  10. Zhao,W.,Yang,P.,Kang,L.和Cui,F。(2016)。 来自昆虫载体和病毒植物的水稻条纹病毒的不同致病性 新Phytol 210(1):196-207。
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引用:Zhao, W., Kang, L. and Cui, F. (2017). Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice. Bio-protocol 7(21): e2597. DOI: 10.21769/BioProtoc.2597.
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