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Transient Gene Expression for the Characteristic Signal Sequences and the Estimation of the Localization of Target Protein in Plant Cell

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Academic Radiology
Oct 2013



We have proposed and tested a method for characterization of the signal sequences and determinations of target protein localization in a plant cell. This method, called the AgI-PrI, implies extraction of protoplasts from plant tissues after agroinfiltration. The suggested approach combines the advantages of two widely used methods for transient gene expression in plants–agroinfiltration and transfection of isolated protoplasts. The AgI-PrI technic can be applied to other plant species.

Keywords: Agroinfiltration (农杆菌渗透), Protoplast isolation (原生质体分离), Tobacco (烟草), Transient expression (瞬态表达), Signal sequences (信号序列), Subcellular localization (亚细胞定位)


To date, the following techniques for transient expression of genes in plants have been developed and widely used: agroinfiltration, biolistics of plant explants and transfection of protoplasts using polyethylene glycol or electroporation. The effectiveness of these approaches has been clearly demonstrated. Each strategy for transient expression of genes in plants, along with benefits, has its limitations and disadvantages, such as the difficulties in the fine imaging of recombinant reporter proteins in plant cell compartments owing to intricate shapes of plant epidermal cells (agroinfiltration), a low efficiency of transformation and the necessity of specialized equipment and auxiliary material (for biolistics), as well as complex preparatory procedures required for a high yield of viable protoplasts and their effective transfection. This is the reason for developing and testing new methods for transient expression of genes in a plant cell, preferably by improving the experimental protocols and preserving the physiological significance of the results of the studies. Since the cellular localization of proteins in living organisms, including plants, is closely interrelated with their functions, a fine visualization of proteins in living cells becomes an important tool for assessing the functions of the proteins.

Materials and Reagents

  1. Pipettes (Corning, Costar®, catalog number: 4101 )
  2. Inoculation loop
  3. Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101IRR )
  4. Syringe without a needle (B. Braun Melsungen, catalog number: 4645103C )
  5. Scalpel
  6. Nylon mesh, pore size, 40 µm (Sterile Cell Strainers, Corning, catalog number: 431750 )
  7. 10-ml tubes (Corning, Axygen®, catalog number: SCT-10ML )
  8. Agrobacterium tumefaciens strain GV3101 (Mohamed et al., 2004; strain is available in the collection of the Institute of Plant Physiology and can be provided to researchers for experiments)
  9. Nicotiana benthamiana (Sheludko et al., 2007; seeds are available in the collection of the Institute of Plant Physiology and can be provided to researchers for experiments)
  10. LB medium (MP Biomedicals, catalog number: 113002042 )
  11. Rifampicin (Fisher Scientific, catalog number: BP26791 )
  12. Gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
  13. Kanamycin (Thermo Fisher Scientific, GibcoTM, catalog number: 11815024 )
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  15. Tris-HCl, pH 7.0 (Roche Diagnostics, catalog number: 10812846001 )
  16. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  17. Acetosyringone (Abcam, catalog number: ab146533 )
  18. MES (Sigma-Aldrich, catalog number: 69892 )
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: 1058860500 )
  20. Calcium chloride dihydrate (CaCl2·2H2O) (AMRESCO, catalog number: 0556-500G )
  21. Ammonium phosphate monobasic (NH4H2PO4) (Sigma-Aldrich, catalog number: A3048 )
  22. Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  23. Potassium hydroxide (KOH) (AppliChem, catalog number: 211514 )
  24. Cellulase Onozuka R10 (Kinki Yakult)
  25. Pectinase Macerozyme R10 (Kinki Yakult)
  26. Driselase (Sigma-Aldrich, catalog number: D9515 )
  27. Calcium nitrate tetrahydrate (Са(NО3)2·4H2O) (Sigma-Aldrich, catalog number: C2786 )
  28. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
  29. Magnesium sulfate (MgSO4) (Acros Organics, catalog number: AC413485000 )
  30. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  31. Ferric chloride (FеСl3) (Sigma-Aldrich, catalog number: 157740 )
  32. MS medium (Sigma-Aldrich, catalog number: M5519 )
  33. Sucrose (MP Biomedicals, catalog number: 04802536 )
  34. Solution 1 (see Recipes)
  35. Solution 2 (see Recipes)
  36. Solution 3 (see Recipes)
  37. Solution 4 (see Recipes)
  38. Solution 5 (see Recipes)
  39. Knop’s solution (see Recipes)
  40. Agroinfiltration buffer (see Recipes)


  1. Incubator Shaker (Biosan, model: ES-20 , catalog number: BS-010111-AAA)
  2. Centrifuge (Eppendorf)
  3. Transilluminator (Vilber, model: ETX-F26.M , catalog number: Vilber Lourmat 2131 2600 1)
  4. Microscope Axio Imager Z2 (ZEISS, model: Axio Imager Z2 ) equipped with digital camera (ZEISS, model: AxioCam MRc5 ), filter set No. 38 (38 Endow GFP shift free (EX BP 470 nm/40 nm, BS FT 495 nm, EM BP 525 nm/50 nm), ZEISS, catalog number: 000000-1031-346 ) and module ApoTome (ZEISS, model: ApoTome )


  1. ZEN, AxioVision 4.8 (ZEISS)


For this protocol, we used plant expression vectors, with leader signal sequences providing their localization in various compartments of plant cells. The vectors were obtained according to the procedure described earlier (Tyurin et al., 2017).

  1. Cultivation, transformation and selection of A. tumefaciens
    1. Seed cells of the A. tumefaciens strain GV3101 strain using an inoculation loop in 5 ml оf LB medium containing rifampicin (50 μg/ml), gentamicin (25 μg/ml), and carbenicillin (50 μg/ml), (antibiotics do not affect the efficiency of plant transformation) and grow overnight at 28 °C in a shaker incubator (~140 pm).
    2. Transfer the bacterial culture to 95 ml of LB medium without antibiotics and grow to an optical density of OD600 = 0.5-0.6, at 28 °C in a shaker incubator (~140 pm).
    3. Centrifuge cells at 3,500 x g for 5 min, at room temperature. Resuspend the pellet in 20 ml of cold (0 °C) solution 1 (see Recipes).
    4. Centrifuge cells at 3,500 x g for 5 min, at room temperature. Resuspend the pellet in 2 ml of solution 2 (see Recipes).
    5. Competent cells should be used for transformation immediately.
    6. Add 1 µg of an expression vector (with the kanamycin resistance gene) to the competent cells. Incubate cells on ice for 20 min. Heat the solution for 5 min at 37 °C in the shaker incubator. Place cells for 5 min on ice, add 2 ml of LB medium without antibiotics and incubate at 28 °C in the shaker incubator (~140 rpm) for 2 h.
    7. Plate transformed cells on Petri dishes in the medium containing 50 µg/ml of the selective antibiotic kanamycin (use the appropriate selective agent at concentrations recommended by the manufacturer). Isolate colonies and resuspend the cells in 100-150 μl of LB medium without antibiotics, and then freeze them.

  2. Plant material
    Grow Nicotiana benthamiana at 20 ± 2 °C, a photoperiod of 8 h, and an illumination of 100 μmol quanta/(m2 sec) in soil or hydroponics using Knop’s solution (see Recipes) as a nutrient medium for 6 weeks. Frequency of use: 2 times per week.

  3. Agroinfiltration of the N. benthamiana plants
    1. Thaw frozen A. tumefaciens cells and grow them for 48 h at 27 °C in LB medium containing rifampicin (50 μg/ml), gentamicin (25 μg/ml), and carbenicillin (50 μg/ml) in the shaker incubator (~140 pm). Replace the medium with LB medium containing the same antibiotics and solution 3 (see Recipes). Grow the cells to an optical density of OD600 = 0.8.
    2. Centrifuge the cells at 3,000 x g for 5 min, at room temperature. Resuspend the pellet in agroinfiltration buffer (see Recipes) to an optical density of OD600 = 2.4.
    3. Infiltrate the cells into abaxial epiderm of 6-week-old N. benthamiana leaves using a syringe without a needle (Figure 1).
    4. Estimate the quality of transformation on the 4th day after agroinfiltration by imaging of the zones with an expression of the desC-egfp hybrid gene in tobacco leaves at 312 nm, using an ETX transilluminator (Vilber Lourmat). Cut the high fluorescence regions of tobacco leaves out for protoplast extraction (Figure 2).

      Figure 1. Procedure of agroinfiltration into abaxial epiderm of N. benthamiana leaves using a syringe without a needle

      Figure 2. GFP fluorescence in agroinfiltrated Nicotiana benthamiana leaves. The agrobacteria carrying the expression cassettes of GFP fusion protein were syringe-infiltrated into an N. benthamiana leaf, and GFP fluorescence was observed 4 days after infiltration (dpi) in UV light. Fluorescence fields are marked with dashed lines. Scale bar = 20 mm.

  4. Protoplast isolation
    1. Prepare solution 4 (see Recipes) for the cell wall maceration immediately before use and clarify it by centrifugation.
    2. Use the leaf fragments with fluorescence for protoplast isolation according to Nosov et al. (2014). Mince the leaves (about 500 mg) with a scalpel. Add 5 ml of solution 4.
    3. Isolate the protoplasts at 15 °C in a shaker (50 rpm) for 12 h.
    4. Filtrate the protoplast suspension through nylon mesh (pore size, 40 µm). Transfer the protoplast suspension into 10-ml centrifuge tubes and centrifuge at 100 x g for 5 min at room temperature.
    5. Resuspend the pellet of protoplasts in 10 ml of solution 5 and incubate for 5 min at room temperature, and then centrifuge at 100 x g for 5 min, repeat the procedure two times. Resuspend the pellet of protoplasts in 1.5 ml of solution 5 (see Recipes).

  5. Protoplast imaging
    Perform the imaging of zones with target protein in freshly isolated protoplasts with an Axio Imager Z2 microscope (ZEISS) equipped with an AxioCam MR digital camera and filter units (Figure 3).

    Figure 3. Results of analysis of subcellular localization of GFP fusion proteins in tobacco protoplasts isolated from the cells of an agroinfiltrated leaf area. Transient gene expression of plant expression vectors, with leader signal sequences providing their localization in various compartments of plant cells, demonstrates that GFP fusion protein is localized to the chloroplasts (I), endoplasmic reticulum (II), and cytoplasm (III). The merged images (first column) include the green channel (last column) and the chloroplast autofluorescence channel (middle column). Scale bars = 10 μm.

Data analysis

All experiments were performed in at least three independent replicates, each replicate being analyzed at least three times. To determine how well transformation was done we checked the level of GFP fluorescence in agroinfiltrated leaves of N. benthamiana in UV light. For estimation of subcellular localization of GFP fusion proteins in the tobacco protoplasts, we analyzed about 100 protoplasts and visually determined positive transient gene expression in at least 80% of protoplasts.


This protocol can be applied to other plant species (Figure 4).

Figure 4. Potential application of the AgI–PrI technique to different plant species (Tyurin et al., 2017, supplementary data)


  1. Solution 1
    150 mМ NaCl
    10 mМ Tris-HCl (pH 7.0)
  2. Solution 2
    20 mМ CaCl2
    10 mМ Tris-HCl (pH 7.0)
  3. Solution 3
    20 μM acetosyringone
    10 mМ MES
  4. Solution 4
    200 mg/L MgSO4·7H2O
    100 mg/L CaCl2·2H2O
    150 mg/L NH4H2PO4
    0.4 M sorbitol
    4 mМ CaCl2
    12.5 mМ MES-KOH (pH 5.7)
    1% Cellulase Onozuka R10
    0.15% Pectinase Macerozyme R10
    0.4% Driselase
  5. Solution 5
    0.5 М sorbitol
    2.5 mМ CaCl2
  6. Knop’s solution 1 L
    1 g Са(NО3)2
    0.25 g KH2РO4
    0.25 g MgSO4
    0.125 g KСl
    0.0125 g FеСl3
  7. Agroinfiltration buffer
    1x MS medium
    10 mM MES-KOH (pH 5.6)
    2% sucrose
    200 μM acetosyringone


This protocol has been adapted from an earlier study (Tyurin et al., 2017). The authors confirm that there are no known conflicts of interest associated with this publication.


  1. Mohamed, Sh., Boehm, R., Binsfeld, P. C. and Schnabl, H. (2004). Agrobacterium-mediated transformation of two high oleic sunflower (Helianthus annuus L.) genotypes assessment and optimization of important parameters. Helia 27(40): 25-40.
  2. Nosov, А. V., Fomenkov, A. A., Mamaeva, A. S., Solovchenko, A. E., Novikova, G. V. (2014). Extra perspectives of 5-ethynyl-2’-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism. Russ J Plant Physiol 61(6): 899-909.
  3. Sheludko, Y. V, Sindarovska, Y. R., Gerasymenko, I. M., Bannikova, M. A. and Kuchuk, N. V. (2007). Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression. Biotechnol Bioeng 6(3): 608-14.
  4. Tyurin, A. A., Kabardaeva, K. V., Berestovoy, M. A., Sidorchuk, Yu. V., A. Fomenkov, A. A., Nosov, A. V., Goldenkova-Pavlova, I. V. (2017). Simple and reliable system for transient gene expression for the characteristic signal sequences and the estimation of the localization of target protein in plant cell. Russ J Plant Physiol 64(5): 672-679.


我们已经提出并测试了用于表征信号序列和确定植物细胞中目标蛋白质定位的方法。 这种称为AgI-PrI的方法意味着在农杆菌浸润后从植物组织中提取原生质体。 所提出的方法结合了两种广泛使用的用于植物中瞬时基因表达的方法的优点 - 农杆菌浸润和转分离的原生质体。 AgI-PrI技术可应用于其他植物物种。


关键字:农杆菌渗透, 原生质体分离, 烟草, 瞬态表达, 信号序列, 亚细胞定位


  1. 移液器(Corning,Costar ®,目录号:4101)
  2. 接种循环
  3. 培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:101IRR)
  4. 没有针头的注射器(B. Braun Melsungen,目录号:4645103C)
  5. 手术刀
  6. 尼龙网孔,孔径40微米(无菌细胞过滤器,康宁,目录号:431750)
  7. 10毫升试管(Corning,Axygen ,目录号:SCT-10ML)
  8. 根癌土壤杆菌菌株GV3101(Mohamed等人,2004;菌株可在植物生理学研究所的集合中获得,并可提供给研究人员进行实验) >
  9. 烟草(Nicotiana benthamiana)(Sheludko等人,2007年;种子可在植物生理学研究所收集并可提供给研究人员进行实验)
  10. LB培养基(MP Biomedicals,目录号:113002042)
  11. 利福平(Fisher Scientific,目录号:BP26791)
  12. 庆大霉素(Thermo Fisher Scientific,Gibco TM,目录号:15750060)
  13. 卡那霉素(Thermo Fisher Scientific,Gibco TM,目录号:11815024)
  14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  15. Tris-HCl,pH 7.0(Roche Diagnostics,目录号:10812846001)
  16. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  17. Acetosyringone(Abcam,目录号:ab146533)
  18. MES(Sigma-Aldrich,目录号:69892)
  19. 硫酸镁七水合物(MgSO 4•7H 2 O)(Merck,目录号:1058860500)
  20. 氯化钙二水合物(CaCl 2•2H 2 O)(AMRESCO,目录号:0556-500G)
  21. 磷酸二氢铵(NH4H2PO4)(Sigma-Aldrich,目录号:A3048)
  22. 山梨醇(Sigma-Aldrich,目录号:S1876)
  23. 氢氧化钾(KOH)(AppliChem,目录号:211514)
  24. 纤维素酶Onozuka R10(近畿养乐多)

  25. 果胶酶Macerozyme R10(近畿养乐多)
  26. Driselase(Sigma-Aldrich,目录号:D9515)
  27. 硝酸钙四水合物(Са(N°3)2•4H 2 O)(Sigma-Aldrich,目录号:C2786)
  28. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285)
  29. 硫酸镁(MgSO 4)(Acros Organics,目录号:AC413485000)
  30. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  31. 三氯化铁(Fluor III)(Sigma-Aldrich,目录号:157740)
  32. MS培养基(Sigma-Aldrich,目录号:M5519)
  33. 蔗糖(MP Biomedicals,目录号:04802536)
  34. 解决方案1(见食谱)
  35. 解决方案2(请参阅食谱)
  36. 解决方案3(请参阅食谱)
  37. 解决方案4(请参阅食谱)
  38. 解决方案5(请参阅食谱)
  39. Knop的解决方案(见食谱)
  40. 农业浸润缓冲液(见食谱)


  1. 培养箱振荡器(Biosan,型号:ES-20,目录号:BS-010111-AAA)
  2. 离心机(Eppendorf)
  3. Transilluminator(Vilber,型号:ETX-F26.M,目录号:Vilber Lourmat 2131 2600 1)
  4. 装备有数字照相机(ZEISS,型号:AxioCam MRc5)的显微镜Axio Imager Z2(ZEISS,型号:Axio Imager Z2),38号滤光片套装(38 Endow GFP无换色(EX BP 470nm / 40nm,BS FT 495nm ,EM BP 525nm / 50nm),ZEISS,目录号:000000-1031-346)和模块ApoTome(蔡司,型号:ApoTome)


  1. ZEN,AxioVision 4.8(蔡司)



  1. A的培养,改造和选择。癌农杆菌
    1. 使用接种环在含有利福平(50μg/ ml),庆大霉素(25μg/ ml)和羧苄青霉素(50μg/ ml)的5ml LB培养基中的根瘤农杆菌菌株GV3101菌株的种子细胞。 ml)(抗生素不影响植物转化的效率),并在摇床(〜140pm)中在28℃下过夜生长。
    2. 将细菌培养物转移至95ml不含抗生素的LB培养基中,并在振荡培养箱(〜140μm)中在28℃下生长至OD 600 = 0.5-0.6的光密度。 >
    3. 在室温下,将细胞在3,500×g下离心5分钟。
    4. 在室温下,将细胞在3,500×g下离心5分钟。

    5. 应该使用感应细胞进行转化。
    6. 向感受态细胞中加入1μg表达载体(具有卡那霉素抗性基因)。在冰上孵育细胞20分钟。在振荡培养箱中于37℃加热溶液5分钟。将细胞放置在冰上5分钟,加入2毫升不含抗生素的LB培养基,并在振荡培养箱(〜140rpm)中于28℃孵育2小时。
    7. 在含有50μg/ ml选择性抗生素卡那霉素(使用制造商推荐浓度的合适选择剂)的培养基中,将培养皿中的细胞转化到培养皿中。分离菌落并将细胞重悬于100-150μl不含抗生素的LB培养基中,然后冷冻它们。

  2. 植物材料
    使用Knop's溶液在20±2℃下培养本氏烟草,光周期为8小时,光照为100μmol量/(m 2 s) (见食谱)作为6周的营养培养基。使用频率:每周2次。

  3. N的农业渗滤。本生烟草植物
    1. 解冻冻结 A。在含有利福平(50μg/ ml),庆大霉素(25μg/ ml)和羧苄青霉素(50μg/ ml)的LB培养基中于27℃下在摇床培养箱中培养48小时(〜 140 pm)。用含有相同抗生素和溶液3的LB培养基替换培养基(见食谱)。将细胞培养至OD 600 = 0.8的光密度。
    2. 在室温下将细胞在3000×g下离心5分钟。在农杆菌渗滤缓冲液中重悬沉淀(参见食谱)至光密度OD 600 = 2.4。
    3. 将细胞渗入6周龄的N背根表皮。使用不带针头的注射器将本生叶叶片(图1)离开。
    4. 使用ETX透照仪(Vilber Lourmat)通过在烟草叶中在312nm下表达具有desC-egfp杂合基因表达的区域,估计农杆菌渗入后第4天的转化质量。


      图2.农杆菌浸润的本氏烟叶中的GFP荧光。将携带GFP融合蛋白表达盒的农杆菌注射器渗入到N液中。本氏烟叶,并且在紫外光下渗入(dpi)后4天观察到GFP荧光。荧光场用虚线标出。比例尺= 20毫米。

  4. 原生质体分离
    1. 使用前立即制备溶液4(参见食谱)进行细胞壁浸渍,并通过离心澄清。
    2. 按照Nosov等人的方法,将具有荧光的叶片用于原生质体分离(2014)。用手术刀剁碎叶子(约500毫克)。加5毫升溶液4.

    3. 在15°C振荡器(50 rpm)中分离原生质体12小时。
    4. 通过尼龙网孔(孔径40μm)过滤原生质体悬浮液。将原生质体悬浮液转移到10ml离心管中,并在室温下以100×g离心5分钟。
    5. 在10ml溶液5中重悬原生质体的沉淀并在室温下孵育5分钟,然后在100gxg离心5分钟,重复该程序两次。在1.5ml溶液5中重悬原生质球(参见食谱)。

  5. 原生质体成像
    使用配有AxioCam MR数码相机和过滤器的Axio Imager Z2显微镜(ZEISS)(图3)对新鲜分离的原生质体进行靶蛋白区域成像。

    图3.从农杆菌浸润的叶面积的细胞中分离的烟草原生质体中GFP融合蛋白的亚细胞定位分析结果。植物表达载体的瞬时基因表达,其中前导信号序列提供其在植物细胞的各种区室中的定位,表明GFP融合蛋白定位于叶绿体(i strong),内质网( II )和细胞质( III )。合并的图像(第一列)包括绿色通道(最后一列)和叶绿体自发荧光通道(中间列)。比例尺= 10微米。





图4. AgI-PrI技术在不同植物物种中的潜在应用(秋林等人,2017,补充数据)


  1. 解决方案1
    10毫摩尔Tris-HCl(pH 7.0)
  2. 解决方案2
    10毫摩尔Tris-HCl(pH 7.0)
  3. 解决方案3
    10 m MES
  4. 解决方案4
    200mg / L MgSO 4•7H 2 O 100mg / L CaCl 2•2H 2 O 0 150mg / L NH 4 H 2 PO 4←>
    0.4 M山梨醇
    12.5毫克MES-KOH(pH 5.7)
    1%纤维素酶Onozuka R10
    0.15%果胶酶Macerozyme R10
  5. 解决方案5
  6. Knop的解决方案1 L
    1 gСа(NО 3 ) 2
    0.25克KH 2 / CrO 4/2 0.25克MgSO 4
  7. 农业过滤缓冲液
    1x MS媒体
    10 mM MES-KOH(pH 5.6)




  1. Mohamed,Sh。,Boehm,R.,Binsfeld,P.C。和Schnabl,H。(2004)。通过农杆菌介导的两种高油酸向日葵(Helianthus annuus )基因型评估和重要参数的优化。 Helia 27(40):25-40。
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引用:Berestovoy, M. A., Tyurin, A. A., Kabardaeva, K. V., Sidorchuk, Y. V., Fomenkov, A. A., Nosov, A. V. and Goldenkova-Pavlova, I. V. (2018). Transient Gene Expression for the Characteristic Signal Sequences and the Estimation of the Localization of Target Protein in Plant Cell. Bio-protocol 8(4): e2738. DOI: 10.21769/BioProtoc.2738.