欢迎您, 登录 | 注册

首页 | English

X
加载中

Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70 this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were infiltrated, using a syringe, with either quercetin (dissolved in DMSO) or DMSO (control plants). The plants were mechanically inoculated with PepMV virus inocula. The accumulation of Hsp70 and PepMV were analyzed on local leaves by immunoblot analysis 4 days post inoculation.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants
采用槲皮素研究本氏烟草植株中Hsp70沉默对植株病毒感染的影响

微生物学 > 微生物-宿主相互作用 > 病毒
作者: Matthaios M. Mathioudakis
Matthaios M. MathioudakisAffiliation: Μediterranean Agronomic Institute of Chania, Department of Sustainable Agriculture, Chania, Greece
For correspondence: manth82@yahoo.gr
Bio-protocol author page: a2767
 and Ioannis Livieratos
Ioannis LivieratosAffiliation: Μediterranean Agronomic Institute of Chania, Department of Sustainable Agriculture, Chania, Greece
Bio-protocol author page: a2768
Vol 5, Iss 23, 12/5/2015, 1730 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1675

[Abstract] Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70 this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were infiltrated, using a syringe, with either quercetin (dissolved in DMSO) or DMSO (control plants). The plants were mechanically inoculated with PepMV virus inocula. The accumulation of Hsp70 and PepMV were analyzed on local leaves by immunoblot analysis 4 days post inoculation.

[Abstract]

Materials and Reagents

  1. 1 ml syringes without a needle (BD, Nipro, catalog number: SY3 1 C100U ET )
  2. Mortar and pestles (Carl Roth GmbH + Co., catalog number: XL96.1 and XP01.1 )
  3. Pelet pestles (Nippon Genetics, catalog number: NG006 )
  4. 13 ml round base tubes (SARSTEDT AG & Co, catalog number: 62.515.006 )
  5. 1.5 ml micro tubes (SARSTEDT AG & Co, catalog number: 72.690.001 )
  6. Nicotiana benthamiana leaves from 2 to 3 weeks old seedlings
  7. PepMV-Sp13 isolate infected material
  8. Quercetin hydrate (Sigma-Aldrich, catalog number: 337951 )
  9. Dimethyl sulfoxide (DMSO) (AppliChem GmbH, catalog number: A3009 )
  10. 10 mM Sodium carbonate (Na2CO3) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31432 )
  11. Sodium dihydrogen phosphate monohydrate (0.5 M, pH 7.0) (AppliChem GmbH, catalog number: A3559 )
  12. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
  13. Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore Corporation, catalog number: 104873 )
  14. Potassium chloride (KCl) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31248 )
  15. Sodium Chloride (NaCl) (AppliChem GmbH, catalog number: A1149 )
  16. Magnesium Chloride 6-hydrate (MgCl2) (AppliChem GmbH, catalog number: A4425 )
  17. Silicon carbide (carborundum) 400 mesh (Sigma-Aldrich, catalog number: 357391 )
  18. Bromophenol blue (Sigma-Aldrich, catalog number: B-8026 )
  19. Coomassie Brilliant Blue (AppliChem GmbH, catalog number: A1092 )
  20. Glycerol (Sigma-Aldrich, catalog number: G6279 )
  21. Tris ultrapure (1.5 M, pH 8.8; 1 M, pH 6.8) (AppliChem GmbH, catalog number: A1086 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  23. 30% 37.5:1 Acrylamide/Bis solution (Bio-Rad Laboratories, AbD Serotec®, catalog number: 161-0158 )
  24. 10% Ammonium persulfate (APS) (AppliChem GmbH, catalog number: A2941 )
  25. N, N, N´, N´-tetramethylethylenediamine (TEMED) (AppliChem GmbH, catalog number: A1148 )
  26. 10% Sodium dodecyl sulfate (SDS) (AppliChem GmbH, catalog number: A2263 )
  27. Glycine (AppliChem GmbH, catalog number: A1067 )
  28. Phenyl methanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  29. Methanol (Thermo Fisher Scientific, catalog number: M/4000/17 )
  30. Acetic acid (Sigma-Aldrich, catalog number: 33209 )
  31. PepMV CP polyclonal antibody (Neogen/Adgen Phytodiagnostics, catalog number: 1127-01 )
  32. HSP70 monoclonal antibody (Enzo Life Sciences, Stressgen, catalog number: N27F3-4 )
  33. Anti-rabbit IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3731 )
  34. Anti-mouse IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3721 )
  35. BCIP/NBP Color Development Substrate (Promega Corporation, catalog number: S3771 )
  36. PVDF membrane, Westran Clear Signal (Thermo Fisher Scientific, Whatmann, catalog number: 10485289 )
  37. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  38. Phosphate buffer saline (PBS) (see Recipes)
  39. Protein extraction buffer (see Recipes)
  40. 4x Laemmli buffer (see Recipes)
  41. SDS-PAGE buffers: Separation and Stacking gel buffers (see Recipes)
  42. Western Blot buffers: Running, Transfer, Wash and Detection buffers (see Recipes)

Equipment

  1. Plant growth chamber (25 °C, light:dark =16:8 h)
  2. Biofuge stratos highspeed table centrifuge (Thermo Fisher Scientific, Heraeus, catalog number: 75005282 )
  3. Fixed angle rotor #3334 (Thermo Fisher Scientific, Heraeus, catalog number: 75003334 )
  4. Fixed angle microliter rotor #3332 (Thermo Fisher Scientific, Heraeus, catalog number: 75003332 )
  5. Mini-PROTEAN 3 Cell Electrophoresis System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-3301 )
  6. Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-3930 )
  7. Power Pack Supply model 200/2.0 (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-4761 )
  8. Western blot membranes were visualized using the Gel DocTM XR Molecular Imager & System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-8195 EDU )
  9. Julabo Water-bath TW12 (Sigma-Aldrich, catalog number: Z615498 )
    Note: Pricing & availability is not currently available.

Software

  1. Quantification of the bands intensity as absorbance units was conducted by Quantity One analysis software (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-9600)

Procedure

  1. Preparation of PepMV inocula
    Infected freeze dried material with the PepMV-Sp13 isolate (Aguilar et al., 2002, kindly provided from Dr. M. Aranda, CSIC-CEBAS Murcia, Spain) was homogenized with a ratio 1:10 (w/v) in NaH2PO4 0.5 M phosphate buffer using a mortar and pestle, and used as a fresh virus inocula.

  2. Silencing of Hsp70 by quercetin application
    1. Quercetin was easily dissolved in DMSO in order to prepare an initial stock of 200 mM. The quercetin stock was subsequently diluted in a final concentration of 1 mM using a 10 mM Na2CO3 solution (e.g. 5 μl of 200 mM quercetin per 1 ml). DMSO alone was diluted accordingly to quercetin using the 10 mM Na2CO3 solution. Stock and working solutions of quercetin were always freshly prepared.
    2. One fully expanded leaf of 2 to 3 weeks old N. benthamiana plants (Figure 1) was totally (entirely) infiltrated, from the bottom side, using a syringe (Figure 2) either with 1 mM quercetin solution or diluted DMSO (control plants). Approximately to infiltrate an entire fully expanded leaf 1 ml of the corresponding solution was used.

  3. Viral infection by rub inoculaton
    1. One hour after the plants infiltration (kept at room temperature) the treated leaves with quercetin or DMSO were slightly dusted with carborundum.
    2. Five minutes later the leaves were mechanically inoculated with equal amount (~30 μl) of freshly prepared virus inocula. After the inoculation plants were kept at growth chambers for four days at 25 °C and 16-h light and 8-h dark cycle.


      Figure 1. N. benthamiana leaf before infiltration with DMSO or quercetin (left panel), and DMSO- or quercetin-treated N. benthamiana plants 4 days after PepMV rub inoculation (arrows indicate the DMSO- or quercetin-infiltrated leaves)


      Figure 2. The left panel shows the infiltration method by syringe in N. benthamiana leaves (bottom side) and the right panel the leaves after infiltration

  4. Analysis of the Hsp70 expression levels in Hsp70-silenced plants and the effect in viral Infection
    1. Four days post PepMV inoculation the leaves of N. benthamiana plants treated with quercetin or DMSO were collected (Figure 1). Control plants did not show any phenotype.
    2. Total proteins were extracted from 0.1 g of plant tissue using 300 μl of protein extraction buffer. Briefly: after the grinding of the infiltrated leaves to a fine powder using liquid nitrogen with a mortar and pestle, 0.1 g of tissue powder was homogenized using 300 μl of extraction buffer in 1.5 ml micro tube (using pellet pestle sticks) and good vortex (2 min). 150 μl were mixed with 50 μl of 4x Laemmli buffer and boiled at 95 °C for 5 min. After a centrifugation at 12,000 rpm for 1 min the supernatant of the samples was used in the following steps.
    3. Small Rubisco unit protein was used for quantification of equal loading of total protein extracts in Coomassie Brilliant Blue staining (Figure 3C). After the SDS-polyacrylamide gel electrophoresis (PAGE) on 12% gels, the proteins were electrophoretically transferred onto PVDF membrane. Western Blot analysis was performed under standard conditions. Briefly, the blotted membranes were firstly incubated with the blocking buffer (1x TBS-T, 4% milk) for 1 hour at room temperature and subsequently with the primary antibodies (all conjugated to alkaline phosphatase [AP]), overnight at 4 °C. The following morning the membranes were washed 3 times and then incubated with the secondary AP-conjugated antibodies for 1 h at room temperature. Finally, after washing the membranes 3 times, the blotted proteins were detected using the NBT/BCIP substrate.
    4. In this given example the results showed that the application of quercetin for silencing Hsp70 reduced its protein levels up to 73% (Figure 3A). This reduction of Hsp70 in the case of our example on PepMV infection had a corresponding negative effect on viral accumulation up to 92% (Figure 3B).


      Figure 3. Immunoblot analysis of Hsp70 expression levels and PepMV accumulation in PepMV-inoculated leaves treated either with DMSO (lane 1) or quercetin (lanes 2 to 4, corresponding to different replicate plants), using α-Hsp70 (A panel) and α-PepMV CP (B1 panel) antibodies. B2 panel is an overexposure of B1 and the asterisk indicates the faint CP band. In panel C the small unit of Rubisco protein stained with Coomassie brilliant blue served as protein loading control (LC).

Recipes

  1. 10x PBS
    40 g NaCl
    1 g KCl
    7.2 g Na2HPO4
    1.2 g KH2PO4
    dd H2O to 1 L
    Fix pH to 6.8
  2. Protein extraction buffer
    For 10 ml: Mix 1 ml 10x PBS
    (40 g NaCl, 1 g KCl, 7.2 g Na2HPO4, 1.2 g KH2PO4, dd H2O to 1 L, fix pH to 6.8)
    2% β-mercaptoethanol
    1 mM PMSF
  3. 4x Laemmli buffer
    250 mM Tris-HCl (pH 6.8)
    8% SDS
    40% glycerol
    20% β-mercaptoethanol
    0.02% Bromophenol Blue
  4. SDS-PAGE buffers
    1. Separation gel:
      0.375 M Tris-HCl (pH 6.8)
      12% Acrylamide/Bis solution
      0.1% SDS
      0.1% APS
      0.01% TEMED
    2. Stacking gel:
      0.125 M Tris-HCl (pH 6.8)
      4% acrylamide/Bis solution
      0.1% SDS
      0.1 M APS
      0.01% TEMED
  5. Western Blot buffers
    1. Running buffer
      3 g Tris base
      14.4 g glycine
      1 g SDS
      dd H2O to 1 L, fix pH to 8.3
    2. Transfer buffer (1x TBS)
      3.03 Tris base
      14.4 g glycine
      200 ml methanol
      dd H2O to 1 L
    3. 1x TBS buffer
      6.57 g Tris base
      8.76 g NaCl
      dd H2O to 1 L, fix pH to 7.4
    4. Wash buffer (1x TBS-T)
      1x TBS
      10% Tween-20
      Antibodies were diluted in 1x TBS-T and 4% milk powder
    5. Detection buffer
      0.1 M Tris base
      0.15 M NaCl
      1 mM MgCl2
      33 μl NBT and 16.5 μl BCIP substrates were mixed in 5 ml of detection buffer

Acknowledgments

This protocol was adopted from previous work (Hafrén et al., 2010; Wang et al., 2009). Matthaios Mathioudakis was a recipient of an Onassis Foundation doctoral fellowship.

References

  1. Aguilar, J. M., Hernandez-Gallardo, M. D., Cenis, J. L., Lacasa, A. and Aranda, M. A. (2002). Complete sequence of the Pepino mosaic virus RNA genome. Arch Virol 147(10): 2009-2015.
  2. Hafren, A., Hofius, D., Ronnholm, G., Sonnewald, U. and Makinen, K. (2010). HSP70 and its cochaperone CPIP promote potyvirus infection in Nicotiana benthamiana by regulating viral coat protein functions. Plant Cell 22(2): 523-535.
  3. Hosokawa, N., Hirayoshi, K., Nakai, A., Hosokawa, Y., Marui, N., Yoshida, M., Sakai, T., Nishino, H., Aoike, A., Kawai, K. and et al. (1990). Flavonoids inhibit the expression of heat shock proteins. Cell Struct Funct 15(6): 393-401.
  4. Mathioudakis, M. M., Rodriguez-Moreno, L., Sempere, R. N., Aranda, M. A. and Livieratos, I. (2014). Multifaceted capsid proteins: multiple interactions suggest multiple roles for Pepino mosaic virus capsid protein. Mol Plant Microbe Interact 27(12): 1356-1369.
  5. Mathioudakis, M. M., Veiga, R., Ghita, M., Tsikou, D., Medina, V., Canto, T., Makris, A. M. and Livieratos, I. C. (2012). Pepino mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70. Virus Res 163(1): 28-39.
  6. Verchot, J. (2012). Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection. Front Plant Sci 3: 275.
  7. Wang, R. Y., Stork, J. and Nagy, P. D. (2009). A key role for heat shock protein 70 in the localization and insertion of tombusvirus replication proteins to intracellular membranes. J Virol 83(7): 3276-3287.

材料和试剂

  1. 1ml无针注射器(BD,Nipro,目录号:SY3 1 C100U ET)
  2. 砂浆和杵(Carl Roth GmbH + Co.,目录号:XL96.1和XP01.1)
  3. 小杵(Nippon Genetics,目录号:NG006)
  4. 13ml圆底管(SARSTEDT AG& Co,目录号:62.515.006)
  5. 1.5ml微量管(SARSTEDT AG& Co,目录号:72.690.001)
  6. 本塞姆氏烟草离开2至3周龄的幼苗
  7. PepMV -Sp13隔离受感染的材料
  8. 槲皮素水合物(Sigma-Aldrich,目录号:337951)
  9. 二甲基亚砜(DMSO)(AppliChem GmbH,目录号:A3009)
  10. 10mM碳酸钠(Na 2 CO 3)(Sigma-Aldrich,Riedel-de Haen,目录号:31432)
  11. 磷酸二氢钠一水合物(0.5M,pH7.0)(AppliChem GmbH,目录号:A3559)
  12. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S0876)
  13. 磷酸二氢钾(KH 2 PO 4)(Merck Millipore Corporation,目录号:104873)
  14. 氯化钾(KCl)(Sigma-Aldrich,Riedel-de Haen,目录号:31248)
  15. 氯化钠(NaCl)(AppliChem GmbH,目录号:A1149)
  16. 氯化镁6水合物(MgCl 2)(AppliChem GmbH,目录号:A4425)
  17. 碳化硅(金刚砂)400目(Sigma-Aldrich,目录号:357391)
  18. 溴酚蓝(Sigma-Aldrich,目录号:B-8026)
  19. 考马斯亮蓝(AppliChem GmbH,目录号:A1092)
  20. 甘油(Sigma-Aldrich,目录号:G6279)
  21. Tris超纯(1.5M,pH8.8; 1M,pH6.8)(AppliChem GmbH,目录号:A1086)
  22. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  23. 30%37.5:1丙烯酰胺/双溶液(Bio-Rad Laboratories,AbD Serotec ,目录号:161-0158)
  24. 10%过硫酸铵(APS)(AppliChem GmbH,目录号:A2941)
  25. N,N,N',N'-四甲基乙二胺(TEMED)(AppliChem GmbH,目录号:A1148)
  26. 10%十二烷基硫酸钠(SDS)(AppliChem GmbH,目录号:A2263)
  27. 甘氨酸(AppliChem GmbH,目录号:A1067)
  28. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  29. 甲醇(Thermo Fisher Scientific,目录号:M/4000/17)
  30. 乙酸(Sigma-Aldrich,目录号:33209)
  31. PepMV CP多克隆抗体(Neogen/Adgen Phytodiagnostics,目录号:1127-01)
  32. HSP70单克隆抗体(Enzo Life Sciences,Stressgen,目录号:N27F3-4)
  33. 抗兔IgG,碱性磷酸酶缀合的抗体(Promega Corporation,目录号:S3731)
  34. 抗小鼠IgG,碱性磷酸酶缀合的抗体(Promega Corporation,目录号:S3721)
  35. BCIP/NBP显色底物(Promega公司,目录号:S3771)
  36. PVDF膜,Westran Clear Signal(Thermo Fisher Scientific,Whatmann,目录号:10485289)
  37. Tween-20(Sigma-Aldrich,目录号:P2287)
  38. 磷酸盐缓冲盐水(PBS)(见配方)
  39. 蛋白质提取缓冲液(参见配方)
  40. 4x Laemmli缓冲区(参见配方)
  41. SDS-PAGE缓冲液:分离和堆叠凝胶缓冲液(参见配方)
  42. Western印迹缓冲液:运行,转移,清洗和检测缓冲液(参见配方)

设备

  1. 植物生长室(25℃,光照:黑暗= 16:8h)
  2. Biofuge stratos高速离心机(Thermo Fisher Scientific,Heraeus,目录号:75005282)
  3. 定角转子#3334(Thermo Fisher Scientific,Heraeus,目录号:75003334)
  4. 定向微升转子#3332(Thermo Fisher Scientific,Heraeus,目录号:75003332)
  5. Mini-PROTEAN 3细胞电泳系统(Bio-Rad Laboratories,AbD Serotec ,目录号:165-3301)
  6. 微型透印电泳转移细胞装置(Bio-Rad Laboratories,AbD Serotec ,目录号:170-3930)
  7. Power Pack Supply型号200/2.0(Bio-Rad Laboratories,AbD Serotec ,目录号:165-4761)
  8. 使用Gel Doc TM XR Molecular Imager&系统(Bio-Rad Laboratories,AbD Serotec ,目录号:170-8195 EDU)
  9. Julabo水浴TW12(Sigma-Aldrich,目录号:Z615498) 注意:可用性目前不可用。

软件

  1. 通过Quantity One分析软件(Bio-Rad Laboratories,AbD Serotec ,目录号:170-9600)进行带强度作为吸光度单位的定量,

程序

  1. 制备 PepMV接种体
    使用PepMV-Sp13分离物(Aguilar等人,2002,由Dr.A.Aranda博士,CSIC-CEBAS Murcia,Spain提供的)感染的冷冻干燥材料均质化在NaH 2 PO 4 0.5M磷酸盐缓冲液中的比例为1:10(w/v),使用研钵和杵,并用作新鲜病毒接种物。

  2. 通过槲皮素应用沉默Hsp70
    1. 槲皮素容易溶解在DMSO中以制备初始 库存200 mM。随后将槲皮素原液在最终稀释 ?使用10mM Na 2 CO 3溶液(例如,5μl的200μl),浓度为1mM, mM槲皮素/1ml)。将DMSO单独相应??地稀释成槲皮素 使用10mM Na 2 CO 3溶液。库存和工作解决方案 槲皮素总是新鲜制备的
    2. 一个完全展开的叶 的2至3周龄的N??。本生烟草植物(图1) (完全)从底侧渗入,使用注射器(图2) ?用1mM槲皮素溶液或稀释的DMSO(对照植物)。 大约浸润整个完全膨胀的叶1毫升 使用相应的溶液。

  3. 通过擦伤接种的病毒感染
    1. 植物浸润(保持在室温)后1小时 用槲皮素或DMSO处理的叶子稍微撒粉 金刚砂。
    2. 五分钟后,叶子是机械的 接种等量(约30μl)新鲜制备的病毒接种物。 ?接种后,将植物在生长室中保持四天 ?在25℃和16小时光照和8小时黑暗周期

      图1. N。 本生烟叶在用DMSO或槲皮素浸润之前(左图) panel),和DMSO-或槲皮素处理的N.本生植物4天 之后


      PepMV 摩擦接种(箭头指示DMSO-或 槲皮素渗透叶)


      图2.左侧面板显示 通过注射器在渗透法中。本生烟叶叶(底部) 和右面板浸润后的叶子

  4. Hsp70沉默植物中Hsp70表达水平的分析和病毒感染中的作用
    1. PepMV 后四天接种N的叶。本生植物 用槲皮素或DMSO处理(图1)。控制植物 ?没有显示任何表型
    2. 从中提取总蛋白 0.1g植物组织使用300μl蛋白提取缓冲液。 简言之:在将浸润的叶研磨成细粉末之后 使用液氮用研钵和研杵,0.1g组织粉 使用在1.5ml微量管中的300μl提取缓冲液匀浆 (使用颗粒杵棒)和良好的涡旋(2分钟)。 150μl混合 与50μl的4x Laemmli缓冲液,并在95℃煮沸5分钟。之后 在12,000rpm离心1分钟样品的上清液 用于以下步骤。
    3. 小Rubisco单位蛋白 用于定量等量加载的总蛋白提取物 考马斯亮蓝染色(图3C)。之后 在12%凝胶上的SDS-聚丙烯酰胺凝胶电泳(PAGE),蛋白质 电泳转移到PVDF膜上。蛋白质印迹 在标准条件下进行分析。简言之,印迹 膜首先用封闭缓冲液(1×TBS-T,4% 牛奶)在室温下培养1小时,随后用原代培养基培养 抗体(全部与碱性磷酸酶[AP]缀合)在4℃过夜 ?C。第二天早晨将膜洗涤3次,然后 与辅助AP缀合的抗体在室温下孵育1小时 温度。最后,洗涤膜3次后,吸干 使用NBT/BCIP底物检测蛋白质
    4. 在这里 给出实施例的结果表明,槲皮素的应用 沉默Hsp70降低其蛋白水平高达73%(图3A)。这个 在我们的例子中,在 PepMV 感染的情况下,Hsp70的减少具有 对病毒累积的相应负效应高达92%(图 3B)。


      图3.用DMSO处理的接种PepMV的接种叶中Hsp70表达水平和PepMV积累的免疫印迹分析 (泳道1)或槲皮素(泳道2至4,对应于不同的 使用α-Hsp70(A组)和α-PepMV CP(B1组) 抗体。 B2面板是B1的过度暴露,星号表示 ?微弱的CP波段。在图C中,Rubisco蛋白的小单位染色 ?用考马斯亮蓝作为蛋白质上样对照(LC)。

食谱

  1. 10x PBS
    40克NaCl
    1克KCl
    7.2g Na 2 HPO 4
    1.2克KH 2 PO 4 4/
    dd H 2 O至1L
    将pH值固定为6.8
  2. 蛋白提取缓冲液
    对于10ml:混合1ml 10x PBS
    (40克NaCl,1克KCl,7.2克Na 2 HPO 4,1.2克KH 2 PO 4, ,dd H 2 O至1L,将pH固定为6.8) 2%β-巯基乙醇 1mM PMSF
  3. 4x Laemmli缓冲区
    250mM Tris-HCl(pH 6.8)
    8%SDS
    40%甘油 20%β-巯基乙醇 0.02%溴酚蓝
  4. SDS-PAGE缓冲液
    1. 分离胶:
      0.375M Tris-HCl(pH 6.8)
      12%丙烯酰胺/双溶液
      0.1%SDS
      0.1%APS
      0.01%TEMED
    2. 堆叠胶:
      0.125M Tris-HCl(pH 6.8)
      4%丙烯酰胺/双溶液
      0.1%SDS
      0.1 M APS
      0.01%TEMED
  5. 西方墨水缓冲区
    1. 运行缓冲区
      3克Tris碱
      14.4g甘氨酸
      1克SDS
      dd H 2 O至1L,将pH固定为8.7
    2. 传输缓冲区(1x TBS)
      3.03 Tris碱
      14.4g甘氨酸
      200 ml甲醇
      dd H 2 O至1L
    3. 1x TBS缓冲区
      6.57克三碱值
      8.76g NaCl
      dd H 2 O至1L,将pH固定至7.4
    4. 洗涤缓冲液(1×TBS-T)
      1x TBS
      10%Tween-20
      将抗体在1×TBS-T和4%奶粉中稀释
    5. 检测缓冲区
      0.1 M Tris碱
      0.15 M NaCl
      1mM MgCl 2
      将33μlNBT和16.5μlBCIP底物在5ml检测缓冲液中混合

致谢

该协议是从以前的工作中采用的(Hafrén等人,2010; Wang等人,2009)。 Matthaios Mathioudakis是Onassis基金会博士研究生的获得者。

参考文献

  1. Aguilar,J.M.,Hernandez-Gallardo,M.D.,Cenis,J.L.,Lacasa,A.and Aranda,M.A。(2002)。 Pepino花叶病毒RNA基因组的完整序列 Arch Virol


    em> 147(10):2009-2015。
  2. Hafren,A.,Hofius,D.,Ronnholm,G.,Sonnewald,U.and Makinen,K。 HSP70及其伴侣CPIP通过调节病毒来调节烟草中的马铃薯Y病毒感染外植蛋白功能。 植物细胞 22(2):523-535
  3. Hosokawa,N.,Hirayoshi,K.,Nakai,A.,Hosokawa,Y.,Marui,N.,Yoshida,M.,Sakai,T.,Nishino,H.,Aoike,A.,Kawai, et al。 (1990)。 类黄酮抑制热休克蛋白的表达。细胞结构功能 em> 15(6):393-401。
  4. Mathioudakis,M.M.,Rodriguez-Moreno,L.,Sempere,R.N.,Aranda,M.A。和Livieratos,I.(2014)。 多面蛋白衣壳蛋白:多重相互作用表明Pepino花叶病毒衣壳蛋白有多种作用。 em> Mol Plant Microbe Interact 27(12):1356-1369。
  5. Mathioudakis,M. M.,Veiga,R.,Ghita,M.,Tsikou,D.,Medina,V.,Canto,T.,Makris,A. M. and Livieratos,I.C。 Pepino花叶病毒衣壳蛋白与番茄热休克蛋白同源物70相互作用。 Virus Res 163(1):28-39。
  6. Verchot,J。(2012)。 细胞分子伴侣和折叠酶是植物RNA病毒感染期间膜结合复制和运动复合体的重要贡献者。 Front Plant Sci 3:275.
  7. Wang,R.Y.,Stork,J。和Nagy,P.D。(2009)。 热休克蛋白70在定位和插入墓志病毒中的关键作用 replication proteins to intracellular membranes。 J Virol 83(7):3276-3287。
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。

X


How to cite this protocol: Mathioudakis, M. M. and Livieratos, I. (2015). The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants. Bio-protocol 5(23): e1675. DOI: 10.21769/BioProtoc.1675; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)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google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook