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Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology
无创显微技术测定水稻中的H+流   

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Abstract

Rice plants release proton (H+) from root cells into rhizosphere area leading to the acidification of the rhizosphere and increased solubility of ferric iron complexes on the cell membrane, which is important for iron uptakes. Here, we present a detailed protocol to measure H+ flux in root hairs of transgenic rice seedlings and transgenic rice protoplasts by the Non-invasive Micro-test Technique (NMT). The NMT system is based on a non-invasive microelectrode technology that is automatically controlled by a computer, to achieve a three-dimensional, real-time, dynamic characterization of the concentration, velocity, and direction of a variety of molecules or ions. Because there is no need to directly contact the measured cells that could cause cell damage, we are able to obtained accurate and real time information on ion concentration. This is the first protocol that describes the non-invasive micro measurement technique of both root hairs and protoplasts in rice. In NMT, voltage differences are measured at two excursion points that are manipulated using a computer. Voltage differences can be converted into H+ fluxes using the ASET 2.0 (The imFlux® software) and JCal v3.2.1 Software. Analysis of the H+ fluxes provides a simultaneous measure of the crossing of a localized region of the root surface in response to stress, which provides real-time in-situ detection of net ion transport across membranes. This method will promote use of NMT in plant biology.

Keywords: Proton flux(质子通量), Non-invasive Micro-test Technique (NMT)(非损伤微测技术(NMT)), Rice(水稻), Microelectrode(微电极)

Materials and Reagents

  1. Cell strainer (400 mesh)
  2. Centrifuge tube (1.5 ml and 10 ml) (Corning Inc., catalog number: MC-150-C )
  3. Round Petri dish (35 mm in diameter) (Corning Inc.)
  4. Square Petri dish (100 mm, pym-100-10) (Xinshengke)
  5. Plastic strips
  6. Constructed vector (Li et al., 2015)
  7. Transgenic Secretory 24 (OsSEC24) (Li et al., 2015) rice seedlings and plasma membrane (PM) ATPase 2 (OsPMA2)-mCherry (Li et al., 2015) plus OsSEC24-GFP double-transgenic rice protoplasts
  8. Cellulase (Onozuka, catalog number: 130419-01 )
  9. Macerozyme (Japan) (catalog number: 121207-01)
  10. M519 (catalog number: 14C0519105B)
  11. Mannitol
  12. MES (pH 5.7)
  13. KCl
  14. NaCl
  15. CaCl2
  16. MgCl2
  17. Sucrose
  18. 1%-1.5% agar
  19. Calibration medium buffer (pH 7.0) for NMT (see Recipes)
  20. Calibration medium buffer (pH 5.0) for NMT (see Recipes)
  21. Incubation solution (see Recipes)
  22. Mmg solution (see Recipes)
  23. MS medium (see Recipes)
  24. NT medium (see Recipes)
  25. Protoplasting enzyme solution (see Recipes)
  26. Polyethylene glycol (PEG)-calcium solution (see Recipes)
  27. Standard medium buffer (pH 6.0) for NMT (see Recipes)
  28. Washing buffer (see Recipes)

Equipment

  1. Centrifuge (SIGMA Laborzentrifugen GmbH, model: 3K15 )
  2. Glass beaker (100 ml) (Bomex)
  3. Growth chamber
  4. Liquid ion exchanger (LIX) Holder (YoungerChina)
  5. Microelectrodes (1-2 μm, 4-5 μm) (YoungerUSA)
  6. Micropipettor (100-1,000 μl and 10-100 μl) (Eppendorf)
  7. Non-invasive Micro-test System (YoungerUSA, model: NMT100 series )
  8. Shaker (Focus Technology Co., Shanghai Meditry Instrument, model: THZ-C-1 )

Software

  1. NIS-Elements AR software (available at http://www.youngerusa.com)
  2. ASET 2.0 software (The imFlux® software) (available at http://www.youngerusa.com)
  3. JCal v3.2.1 (available at http://www.youngerusa.com)

Procedure

  1. Measurement of transgenic rice root hair system
    1. Culture of transgenic rice seedlings
      Fourteen-day-old transgenic rice (Oryza sativa L. ‘Japonica’) seedlings that were transformed with pCAMBIA1302-OsSEC24-GFP were germinated on MS medium under controlled conditions in a 16 h light/8 h dark cycle at 28 degrees centigrade (°C). Secretory 24 (OsSEC24) is a component of the coat protein II (OsCOPII) vesicles that are required for secretory protein sorting, including of plasma membrane (PM) ATPase 2 (OsPMA2). Further information regarding OsSec24 may be found in Li et al. (2015).
    2. Non-invasive measurement
      1. Preparation and calibration of the microelectrode
        1. Chloridize the silver for 20 sec with 100 mM KCl by using a silver chloride device, as shown in Figure 1.


          Figure 1. Schematic diagram of the silver chloride device. A silver is connected to the positive electrode of the battery and a platinum wire is connected to the negative electrode of the battery.

        2. Fill the holder and microelectrode with 0.5 cm H+ liquid ion exchanger (LIX) and 1-1.5 cm NaCl/K2HPO4 buffer.
        3.  Place the holder and microelectrode under the microscope lens and fill the tip of the microelectrode with 15-25 μm H+ LIX, as shown below and in Figures 2 and 7.


          Figure 2. Schematic diagram of a position of the microelectrode and LIX holder. Microelectrode (right) and LIX holder (left) are in the same field of a microscope. The picture is come from YoungerUSA (Xuyue, Beijing) NMT Service Center.

        4. Install the microelectrode into the Non-invasive Micro-test device and manually place the microelectrode in the ready position, as shown in Figure 3.


          Figure 3. Connection diagram of microelectrodes and Non-invasive Micro-test device. The prepared microelectrode is inserted into the device. The picture is come from YoungerUSA (Xuyue, Beijing) NMT Service Center.

        5. Calibrate each ion-specific microelectrode against a set of known pH buffers before and after each experiment with the ASET 2.0 software to prevent electrode drift (Figure 4). Click the Technique button and ensure ISP/PVP is set at X-30 before calibration, thus, setting the dx at 30 µm. Click the Technique button and choose Calibration, and input 0.0001 in the space for solution 1 and 0.01 in solution 2. Place the reference electrode and microelectrode into the standard medium buffer and ensure that the whole tips of reference electrode and microelectrode are immersed in the standard medium buffer. Calibrate proton (H+) microelectrodes in calibration medium (pH 7.0 and pH 5.0) by clicking Solution 1 and Solution 2 button, respectively, following the same procedure and standards (Figure 7). Only use H+ electrodes with Nernst slope between 53 and 63 mV/decade for measurement. Data must be discarded if the calibration is incorrect.


          Figure 4. Interface of the electrode calibration in ASET 2.0 software. The picture is come from YoungerUSA (Xuyue, Beijing) NMT Service Center.

  2. Measurement of H+ flux in the root hairs of transgenic rice seedlings
    1. Wash and incubate transgenic rice roots in standard medium buffer for 5 min at room temperature.
    2. Place whole transgenic rice roots in the Petri dish with standard medium buffer using plastic strips (Figure 5).


      Figure 5. Schematic diagram of how to place whole transgenic rice roots

    3. View root hairs and microelectrodes under high magnification (40x) with the Zeiss compound microscope by using the NIS-Elements AR software and position as shown in Figure 8C.
    4. Position microelectrodes close to the root hairs belonging to the mature region of the lateral roots, as shown in Figure 8C. Click the Mode button and choose Watch, then click the Record and Resume button to start computer measurement. Microvolt differences are measured at two excursion points at a frequency of 0.05 Hertz (Hz) manipulated with the help of NIS-Elements AR and ASET 2.0 software, as shown in Figure 6. The distance between the two excursion points (dx) is 30 micron (μm). Monitor kinetics of net H+ fluxes near each root hair for 15 min. Two roots per rice seedling and two root hairs per root were assayed.


      Figure 6. Interface of the watch mode in ASET 2.0 software. The picture is come from Younger USA (Xuyue, Beijing) NMT Service Center.

    5. Microvolt differences are exported as raw data before conversion into net H+ fluxes by using JCal v3.2.1. Choose the H+ measured and the dr equal to the dx used, and input the slope and intercept measured during calibration. Finally, enter the data Origin(1) millivolts (mV) and AvgOrigin-X microvolts (µV) as measured by ASET 2.0 software(The imFlux® Software). H+ flux is then calculated directly by using the JCal v3.2.1 software. A positive number represents the outflow of H+ whereas a negative number represents the inflow of H+. The H+ flux assay of each group of transgenic rice root hairs consists of four independent replications (Figure 8).


      Figure 7. Schematic diagram of H+ flux detection. The microelectrode tip is filled with H+ LIX and NaCl/K2HPO4 buffer. The chloridized silver (Ag/AgCl) is immersed in the NaCl/K2HPO4 buffer. A voltage gradient (dV = V2 - V1) is measured by the electrometer between two positions over the travel range (dx). A concentration gradient (dc) is calculated using dV. D, H+ diffusion constant; J, net ion flux of H+.


      Figure 8. Measurement of H+ efflux in rice root hair by using NMT (Li et al., 2015). The results indicate the effects of OsSEC24 on H+ extrusion in transgenic rice roots under -Fe conditions. A. Kinetics of H+ flux in OsSEC24 transgenic (left) and wild type (WT) (right) rice root hair grown on -Fe or +Fe medium for two weeks. B. The mean rates of H+ flux in (A), using the mean value of four independent measurements (mean ± SE). Two roots in each plant and two root hairs in each root were assayed. **indicates P < 0.01 using Student’s t-test. (C) Measuring position using the H+-selective microelectrode during NMT.

  3. Measurement in transformed rice protoplasts
    1. Rice cell suspension culture
      Incubate rice (Oryza sativa L. cv. Japonica) cells (in this case, courtesy of the Institute of Genetics and Development of the Chinese Academy of Sciences) suspended in NT medium at 28 °C at 150 revolutions per min (rpm) for 5 days under dark conditions.
    2. Preparation and transient transformation of rice protoplasts
      Perform protoplast isolation from rice suspension cells and transient expression similar to Li et al. (2015).
      1. Preparation of rice protoplasts
        1. Harvest rice suspension cells into a centrifuge tube and allow to stand for 15 min before discarding the NT medium.
        2. Incubate rice cells in the protoplasting enzyme solution for 1-2 h at 26 °C in an orbital shaker with gentle agitation at 50-75 rpm.
        3. Isolate protoplasts from the undigested material into a glass beaker with a cell strainer and centrifuge at 2,000 rpm for 15 min at 4 °C.
        4. Resuspend cells in 5 ml of washing buffer, followed by pelleting again by centrifugation for 15 min at 2,000 rpm at 4 °C.
      2. Transient transformation of rice protoplasts
        1. Resuspend in 0.3 ml of Mmg solution at room temperature before adding 0.03 ml of plasmids including pBIN20-OsPMA2-mcherry and pBI221-OsSEC24-GFP and 0.33 ml of PEG-calcium solution.
        2. Incubate protoplasts at room temperature for 15 min in the dark for transformation.
        3. Wash transgenic protoplasts with 0.3 ml of washing buffer and centrifuge for 15 min at 2,000 rpm at 26 °C before gentle resuspension in 1 ml incubation solution in a Petri dish and incubation at room temperature for 20-25 h.
        4. Proton secretion is mediated from the cell to the environment by the plasma membrane (PM) and is typically conducted by H+-ATPases in plants. OsPMA2 is a rice PM-H+-ATPase and is sorted to the PM by vesicular trafficking. Further information concerning Ospma2 is available in Li et al. (2015).
    3. Non-invasive measurement
      1. Preparation and calibration of the microelectrode
        To prepare and calibrate the microelectrode, follow the instructions given in section A2a, above, altering step A2a-v such that ISP/PVP is set at X-10.
      2. Measurement of H+ flux in root hairs of transgenic rice seedlings
        1. Place transformed protoplasts in a Petri dish with standard medium buffer and incubate for 5 min at room temperature.
        2. View transformed protoplasts and microelectrodes under high magnification (40x) with the Zeiss compound microscope using the NIS-Elements AR software, positioned as shown in Figure 9C.
        3. Position microelectrodes close to three equally sized transformed protoplasts as shown in Figure 9C. Press the Mode button and choose Watch in ASET 2.0 software (The imFlux® Software). Press the Record and Resume button to start computer measurement. Microvolt differences are measured at two excursion points at a frequency of 0.05 Hz and manipulated with the aid of NIS-Elements AR and ASET 2.0 software (The imFlux® Software). The distance between the two excursion points (dx) is 10 μm. Monitor the kinetics of net H+ fluxes near each protoplast for 15 min.
        4. Export raw microvolt differences and convert to net H+ fluxes using JCal v3.2.1. Select the measured H+ and the dr equal to the dx used and enter the slope and intercept measured during calibration. Finally, paste the values for Origin(1) millivolts(mV) and AvgOrigin-X µV as measured by the ASET 2.0 software (The imFlux® Software). H+ flux is calculated directly by the JCal v3.2.1 software. A positive number represents H+ outflow whereas a negative number represents H+ inflow. The H+ flux assay of each type of transformed protoplast consists of three independent replications (Figure 3).


          Figure 9. Measurement of H+ efflux in transformed rice protoplasts using NMT (Li et al., 2015). The results indicate the effects of OsSEC24 on the transport of OsPMA2 in a rice protoplast. A. Kinetics of H+ flux in mCherry plus GFP double-transgenic rice protoplasts (empty vector on the left), OsPMA2-mCherry single-transgenic protoplasts (in the middle) and OsPMA2-mCherry plus OsSEC24-GFP double-transgenic protoplasts (on the right). The experiment was performed three times. B. The mean rates of H+ flux in A, using the mean value of three independent measurements (mean ± SE). *indicates P < 0.05 using Student’s t-test whereas **indicates P < 0.01 using Student’s t-test. C. Location of OsSEC24 (in vesicle, colored green) and PMA2 (in PM, colored red) in transformed rice protoplasts using confocal microscopy and measuring position using the H+-selective microelectrode of NMT.

Recipes

  1. Calibration medium buffer (pH 7.0) for NMT
    0.6 M mannitol
    4 mM MES (pH 5.7)
    4 mM KCl
    154 mM NaCl
    125 mM CaCl2
    pH adjusted to 7.0 with HCl or NaOH
    Stored at 4 °C
  2. Calibration medium buffer (pH 5.0) for NMT
    0.6 M mannitol
    4 mM MES (pH 5.7)
    4 mM KCl
    154 mM NaCl
    125 mM CaCl2
    pH adjusted to 5.0 with HCl or NaOH
    Stored at 4 °C
  3. Incubation solution
    0.6 M mannitol
    4 mM KCl
    154 mM NaCl
    125 mM CaCl2
    4 mM MES (pH 5.7)
    Stored at 4 °C after autoclave sterilization (121 °C, 15 min)
  4. Mmg solution
    15 mM MgCl2
    0.6 M mannitol
    4 mM MES (pH 5.7)
    Stored at 4 °C after autoclave sterilization (121 °C, 15 min)
  5. MS medium
    4.43 g/L M519
    88 mM sucrose
    pH adjusted to 5.7 with HCl or NaOH
    1%-1.5% agar
    Distribute medium to square petri dishes and stored at 4 °C after autoclave sterilization at 121 °C for 15 min
  6. NT medium
    4.43 g/L M519
    88 mM sucrose
    4.5 mM 2,4-dichlorophenyloxyacetic acid (2, 4-D)
    pH adjusted to 5.8 by using HCl or NaOH
    Stored at 4 °C after autoclave sterilization (121 °C, 15 min)
  7. Protoplasting enzyme solution
    0.6 M mannitol
    10 mM MES (pH 5.7)
    Storage at 4 °C after autoclave sterilization at 121 °C for 15 min and add 1 mM CaCl2, 0.1% bovine serum albumin, 1.5% cellulase RS and 0.75% macerozyme before plating enzymolysis
  8. Polyethylene glycol (PEG)-calcium solution
    40% PEG3350
    0.6 M mannitol
    0.1 M CaCl2
    Stored at 4 °C
  9. Standard medium buffer (pH 6.0) for NMT
    0.6 M mannitol
    4 mM MES (pH 5.7)
    4 mM KCl
    154 mM NaCl
    125 mM CaCl2
    pH adjusted to 6.0 with HCl or NaOH
    Stored at 4 °C
  10. Washing buffer
    154 mM NaCl
    5 mM KCl
    125 mM CaCl2
    2 mM MES (pH 5.7)
    Stored at 4 °C after autoclave sterilization (121 °C, 15 min)

Acknowledgments

The protocol was adapted from our previously published paper Li et al. (2015). We wish to thank Younger USA (Xuyue, Beijing) NMT Service Center for technical support. This study was supported by the National Natural Science foundation of China (Grant Nos. 30770178 and 30971856), Beijing Municipal National Science Foundation Key Fund Projects (B) (Grant No. KZ200710028013), Beijing Municipal National Science Foundation (No. 5042004), and the Seventh Beijing Municipal Outstanding Teacher Award (2011).

References

  1. Li, S., Pan, X. X., Berry, J. O., Wang, Y., Naren, Ma, S., Tan, S., Xiao, W., Zhao, W. Z., Sheng, X. Y. and Yin, L. P. (2015). OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H+ secretion mediated by PM-H+-ATPase. Plant Sci 233: 61-71.
  2. Newman, I. A., Kochian, L. V., Grusak, M. A. and Lucas, W. J. (1987). Fluxes of H+ and K+ in corn roots : characterization and stoichiometries using ion-selective microelectrodes. Plant Physiol 84(4): 1177-1184.
  3. Sun, T., Li, P., Yin, L., Xu, Y. and Yu, S. (2007). Non-invasive scanning ion-selective electrode technique and its applications to the research of higher plants. Progress in Natural Science 17(6): 625-629.
  4. Tan, S., Han, R., Li, P., Yang, G., Li, S., Zhang, P., Wang, W. B., Zhao, W. Z. and Yin, L. P. (2015). Over-expression of the MxIRT1 gene increases iron and zinc content in rice seeds. Transgenic Res 24(1): 109-122.
  5. Xu. Y, Sun. T and Yin. L. P. (2006). Application of non-invasive microsensing system to simultaneously measure both H+ and O2 fluxes around the pollen tube. Journal of Integrative Plant Biology 48 (7): 1-5.
  6. Yang, G., Ma, F., Wang, Y., Feng, C. G., Li, P., Xu, Y., Zhao, W. Z. and Yin, L. P. (2010). Vesicle-related OsSEC27P enhances H+ secretion in the iron deficient transgenic tobacco root. Chinese Science Bulletin 55(13): 3298-3304.

简介

稻植物从根细胞释放质子(H sup +)到根际区域,导致根际的酸化和增加三价铁络合物在细胞膜上的溶解度,这对于铁摄取是重要的。在这里,我们提出了一个详细的协议,测量转基因水稻幼苗和转基因水稻原生质体的根毛通过非侵入性微测试技术(NMT)的H +通量。 NMT系统基于由计算机自动控制的非侵入性微电极技术,以实现多种分子或离子的浓度,速度和方向的三维,实时,动态表征。因为不需要直接接触可能导致细胞损伤的测量细胞,我们能够获得关于离子浓度的准确和实时的信息。这是描述水稻根毛和原生质体的非侵入性微测量技术的第一个方案。在NMT中,在使用计算机操纵的两个偏移点处测量电压差。使用ASET 2.0(imFlux 软件)和JCal v3.2.1软件,可将电压差转换为H +通量。 H sup +通量的分析提供了响应于应力的根表面的局部区域的交叉的同时测量,其提供跨越膜的净离子迁移的实时原位检测。这种方法将促进植物生物学中NMT的使用。

关键字:质子通量, 非损伤微测技术(NMT), 水稻, 微电极

材料和试剂

  1. 细胞过滤器(400目)
  2. 离心管(1.5ml和10ml)(Corning Inc.,目录号:MC-150-C)
  3. 圆形培养皿(直径35mm)(Corning Inc.)
  4. 方形培养皿(100mm,pym-100-10)(Xinshengke)
  5. 塑料条
  6. 构造的向量(Li ,,2015)
  7. 转基因分泌物24(OsSEC24)(Li等人,2015)水稻幼苗和质膜(PM)ATP酶2(OsPMA2)-mCherry(Li等人,2015 )加OsSEC24-GFP双转基因水稻原生质体
  8. 纤维素酶(Onozuka,目录号:130419-01)
  9. Macerozyme(日本)(目录号:121207-01)
  10. M519(目录号:14C0519105B)
  11. 甘露醇
  12. MES(pH 5.7)
  13. KCl
  14. NaCl
  15. CaCl <2>
  16. MgCl 2
  17. 蔗糖
  18. 1%-1.5%琼脂
  19. NMT的校准介质缓冲液(pH 7.0)(参见配方)
  20. NMT的校准介质缓冲液(pH 5.0)(参见配方)
  21. 孵化解决方案(参见配方)
  22. Mmg解决方案(参见配方)
  23. MS介质(参见配方)
  24. NT介质(见配方)
  25. 原生质酶溶液(参见配方)
  26. 聚乙二醇(PEG) - 钙溶液(参见配方)
  27. 用于NMT的标准培养基缓冲液(pH 6.0)(参见配方)
  28. 洗涤缓冲液(见配方)

设备

  1. 离心机(SIGMA Laborzentrifugen GmbH,型号:3K15)
  2. 玻璃烧杯(100ml)(Bomex)
  3. 生长室
  4. 液体离子交换器(LIX)支架(YoungerChina)
  5. 微电极(1-2μm,4-5μm)(YoungerUSA)
  6. 微量移液器(100-1,000μl和10-100μl)(Eppendorf)
  7. 非侵入式微测试系统(YoungerUSA,型号:NMT100系列)
  8. Shaker(Focus Technology Co.,Shanghai Meditry Instrument,型号:THZ-C-1)

软件

  1. NIS-Elements AR软件(可从 http://www.youngerusa.com 获得)
  2. ASET 2.0软件(imFlux ?软件)(可从 http://www.youngerusa.com获取
  3. JCal v3.2.1(可在 http://www.youngerusa.com 查看)

程序

  1. 转基因水稻根系系统的测量
    1. 转基因水稻幼苗的培养 十四天的转基因 转化的水稻('Oryza sativa L.'Japonica' pCAMBIA1302-OsSEC24-GFP在受控的MS培养基上发芽 在28摄氏度(℃)下16小时光照/8小时黑暗循环中的条件。 ?分泌24(OsSEC24)是外壳蛋白II(OsCOPII)的组分, 囊泡是分泌蛋白分选所必需的,包括 质膜(PM)ATP酶2(OsPMA2)。关于 OsSec24 的更多信息可以在Li 等(2015)中找到。
    2. 无创测量
      1. 微电极的准备和校准
        1. 使用氯化银装置用100mM KCl将银氯化20秒,如图1所示。


          图1.氯化银装置的示意图。 银色 连接到电池的正电极和铂丝 连接到电池的负极。

        2. 用0.5cm H +液体离子交换剂(LIX)和1-1.5cm NaCl/K 2 HPO 4缓冲液填充保持器和微电极。
        3.  将支架和微电极放在显微镜镜头下 ?用15-25μmH + LIX填充微电极的尖端,如下所示 ?和图2和图7中

          图2.位置的示意图 微电极和LIX夹。 微电极(右)和LIX支架 (左)在显微镜的同一个领域。图片来自 YoungerUSA(徐岳,北京)NMT服务中心。

        4. 安装 将微电极进入非侵入性微测试装置并手动 将微电极放置在就绪位置,如图3所示。


          图3.微电极和非侵入性的连接图 微测试装置。 准备的微电极插入 设备。图片来自YoungerUSA(Xuyue,北京)NMT 服务中心。

        5. 校准每个离子特异性微电极 在每次实验之前和之后用一组已知的pH缓冲液 ASET 2.0软件防止电极漂移(图4)。单击技术 按钮,确保在校准前将ISP/PVP设置为X-30, 将dx设置为30μm。单击技术按钮并选择 校准,在溶液1和0.01 in的空间中输入0.0001 溶液2.将参比电极和微电极放入 标准介质缓冲并确保整个参考提示 电极和微电极浸没在标准介质缓冲液中。 ?在校准培养基(pH 7.0和pH 7.4)中校准质子(H + +) ?pH 5.0),分别通过点击溶液1和溶液2按钮, 遵循相同的程序和标准(图7)。仅使用具有介于53和63 mV/decade之间的能斯特斜率的H + 电极 测量。如果校准不正确,则必须丢弃数据。


          图4. ASET 2.0软件中电极校准的界面。 图片来自YoungerUSA(北京徐越)NMT服务 中央。

  2. 测量转基因水稻苗的根毛中的H + 通量
    1. 在标准培养基缓冲液中在室温下洗涤和温育转基因水稻根5分钟
    2. 使用塑料条将标准培养基缓冲液置于培养皿中的整个转基因水稻根(图5)

      图5.如何放置整个转基因水稻根的示意图

    3. 查看根毛和微电极在高放大倍率(40x) 用Zeiss复合显微镜通过使用NIS-Elements AR软件 ?和位置如图8C所示。
    4. 位置微电极 接近属于侧生的成熟区域的根毛 根,如图8C所示。单击模式按钮,然后选择观看, 然后单击"记录并恢复"按钮启动计算机测量。 在两个偏移点处测量微伏差 借助于NIS元件操作的0.05赫兹(Hz)的频率 AR和ASET 2.0软件,如图6所示。两者之间的距离 偏移点(dx)为30微米(μm)。监测每根根毛附近的净H + 通量的动力学15分钟。两个根每个水稻幼苗和 测定每个根的两根根毛

      图6. 观看模式在ASET 2.0软件中。图片来自Younger USA (徐岳,北京)NMT服务中心。

    5. 微伏差异 在通过使用转换为净H + 通量之前被导出为原始数据 JCal v3.2.1。选择H + 测量,dr等于使用的dx,和 ?输入校准期间测量的斜率和截距。最后, 输入数据Origin(1)毫伏(mV)和AvgOrigin-X微伏(μV) ?如通过ASET 2.0软件(The imFlux Software)所测量。 H + 通量 ?使用JCal v3.2.1软件直接计算。积极 数字表示H + 的流出,而负数表示 ?H + 的流入。每组转基因水稻的H +通量测定 根毛由四个独立的重复组成(图8)

      图7. H + 通量检测示意图。微电极尖端 ?充满H sup + LIX和NaCl/K 2 HPO 4缓冲液。氯化银 (Ag/AgCl)浸没在NaCl/K 2 HPO 4缓冲液中。电压梯度(dV = ?通过静电计在两个位置之间测量静电电势(V 1 -v 2 -v 1) ?行程范围(dx)。使用以下公式计算浓度梯度(dc) dV。 D,H + 扩散常数; J,H + 的净离子通量

      图8。 通过使用NMT测量水稻根毛中的H + +/+外流(Li等人, 2015)。 结果表明OsSEC24对H + 挤出的影响 转基因水稻根系在-Fe条件下。 A. H + flux in的动力学 OsSEC24转基因(左)和野生型(WT)(右)水稻根毛 在-Fe或+ Fe培养基上生长两周。 B. H + 通量的平均速率 在(A)中,使用四次独立测量的平均值(平均值± SE)。每个植物中有两个根,每个根中有两个根毛 测定。 **表示p < 0.01使用Student's t 测试。 (C)测量 位置在NMT期间使用H +选择性微电极。

  3. 在转化的水稻原生质体中的测量
    1. 水稻细胞悬浮培养
      孵育水稻(水稻 L.cv。 Japonica)细胞(在这种情况下,由遗传学研究所和 ?中国科学院开发)暂停在NT培养基中 在28℃下以150转/分钟(rpm)在黑暗下5天 条件。
    2. 水稻原生质体的制备和瞬时转化 从水稻悬浮细胞进行原生质体分离和类似于Li等的瞬时表达(2015)。
      1. 水稻原生质体的制备
        1. 收获稻悬浮细胞进入离心管,并允许静置15分钟,然后丢弃NT介质。
        2. 孵育水稻细胞在原生质体酶溶液中1-2小时 ?在26℃下在轨道振荡器中以50-75rpm温和搅拌
        3. 将未消化的材料中的原生质体分离成玻璃 烧杯用细胞过滤器并在4℃下以2,000rpm离心15分钟 C。
        4. 将细胞重悬于5ml洗涤缓冲液中,然后通过在4℃下以2,000rpm离心15分钟进行沉淀。
      2. 水稻原生质体的瞬时转化
        1. 在室温下重悬于0.3ml的Mmg溶液中 加入0.03ml质粒,包括pBIN20- OsPMA2-mcherry 和 pBI221-OsSEC24-GFP和0.33ml PEG-钙溶液。
        2. 孵育原生质体在室温下15分钟在黑暗中进行转化。
        3. 洗涤转基因原生质体用0.3毫升的洗涤缓冲液和 在26℃下在2,000rpm下离心15分钟,然后温和再悬浮 在培养皿中的1ml孵育溶液中并在室温下孵育 温度20-25小时。
        4. 质子分泌是由介导的 ?细胞通过质膜(PM)到环??境并且通常 由植物中的H sup +ΔP酶进行。 OsPMA2是水稻PM-H sup + +/-ATPase 通过水泡贩运排序到PM。更多信息 关于 Ospma2 的文章在Li 等人(2015)中提供。
    3. 无创测量
      1. 微电极的准备和校准
        要准备和校准微电极,请按照说明进行操作 在上面的部分A2a中给出,改变步骤A2a-v,使得ISP/PVP被设置为 ?X-10。

      2. 测量转基因水稻苗的根毛中的H + 通量
        1. 将转化的原生质体放在带有标准培养基缓冲液的培养皿中,并在室温下孵育5分钟
        2.  在高处观察转化的原生质体和微电极 放大(40x)用Zeiss复合显微镜使用 NIS-Elements AR软件,如图9C所示定位。
        3. 位置微电极接近三个相等大小的转换 原生质体,如图9C所示。按模式按钮并选择 观看ASET 2.0软件(imFlux ?软件)。按记录和 恢复按钮启动计算机测量。微伏差异 在两个偏移点以0.05Hz的频率测量 借助于NIS-Elements AR和ASET 2.0软件(The imFlux ?软件)。两个偏移点(dx)之间的距离为 ?10μm。监测每个原生质体附近的净H + 通量的动力学 15分钟。
        4. 使用JCal v3.2.1输出原始微伏差异并转换成净H + 通量。选择测量的H + ,dr等于 ?dx,并输入校准期间测量的斜率和截距。 最后,粘贴Origin(1)毫伏(mV)和AvgOrigin-X的值 μV,通过ASET 2.0软件(the imFlux Software)测量。 H + 通量 由JCal v3.2.1软件直接计算。一个正数 表示H + 流出,而负数表示H + 流入。 每种类型的转化原生质体的H sup +通量测定由 三次独立重复(图3)

          图9.测量 使用NMT(Li等人,2015)在转化的水稻原生质体中表达OsPMA2/H + 外排。结果表明OsSEC24对OsPMA2 在水稻原生质体中。 A.mCherry加GFP中H 通量的动力学 双转基因水稻原生质体(左侧的空载体), OsPMA2-mCherry单转基因原生质体(中间)和 OsPMA2-mCherry加OsSEC24-GFP双转基因原生质体 对)。实验进行三次。 B.平均率 H + 通量,使用三次独立测量的平均值 (平均值±SE)。 *表示p < 0.05使用Student's t检验,而 **表示p < 0.01使用Student's t 测试。 C. OsSEC24的位置 (在囊泡中,有色绿色)和PMA2(在PM中,有色红色) ?水稻原生质体使用共聚焦显微镜和测量位置 ?NMT的H +选择性微电极。

食谱

  1. NMT的校准介质缓冲液(pH 7.0)
    0.6 M甘露糖 4mM MES(pH 5.7)
    4 mM KCl
    154 mM NaCl 125mM CaCl 2。 用HCl或NaOH将pH调节至7.0 储存在4°C
  2. NMT的校准介质缓冲液(pH 5.0)
    0.6 M甘露糖 4mM MES(pH 5.7)
    4 mM KCl
    154 mM NaCl 125mM CaCl 2。 用HCl或NaOH将pH调节至5.0 储存在4°C
  3. 孵育溶液
    0.6 M甘露糖 4 mM KCl
    154 mM NaCl 125mM CaCl 2。 4mM MES(pH 5.7)
    高压灭菌消毒(12±1℃,15分钟)后,在4℃保存
  4. Mmg解决方案
    15mM MgCl 2·h/v 0.6 M甘露糖 4mM MES(pH 5.7)
    高压灭菌(121℃,15分钟)后,在4℃下保存
  5. MS介质
    4.43 g/L M519
    88mM蔗糖 用HCl或NaOH将pH调节至5.7 1%-1.5%琼脂 分配培养基到正方形培养皿,并在121℃高压灭菌15分钟后在4℃下贮存
  6. NT介质
    4.43 g/L M519
    88mM蔗糖 4.5mM 2,4-二氯苯氧基乙酸(2,4-D) 使用HCl或NaOH将pH调节至5.8 高压灭菌(121℃,15分钟)后,在4℃下保存
  7. 原生质酶溶液
    0.6 M甘露糖 10mM MES(pH 5.7)
    在121℃下高压灭菌消毒15分钟后在4℃下储存,并在电镀酶解之前加入1mM CaCl 2,0.1%牛血清白蛋白,1.5%纤维素酶RS和0.75%大肠杆菌酶。
  8. 聚乙二醇(PEG) - 钙溶液
    40%PEG3350
    0.6 M甘露糖 0.1M CaCl 2
    储存在4°C
  9. 用于NMT的标准培养基缓冲液(pH 6.0)
    0.6 M甘露糖 4mM MES(pH 5.7)
    4 mM KCl
    154 mM NaCl 125mM CaCl 2。 用HCl或NaOH将pH调节至6.0 储存在4°C
  10. 洗涤缓冲液
    154 mM NaCl 5 mM KCl
    125mM CaCl 2。 2mM MES(pH 5.7)
    高压灭菌(121℃,15分钟)后,在4℃下保存

致谢

该协议改编自我们以前发表的论文Li (2015)。我们要感谢美国(北京徐越)NMT服务中心的技术支持。本研究由中国国家自然科学基金(基金编号:30770178和30971856),北京市国家科学基金重点基金项目(B)(编号KZ200710028013),北京市国家科学基金(5042004),和第七届北京市优秀教师奖(2011)。

参考文献

  1. Li,S.,Pan,X.X,Berry,J.O.,Wang,Y.,Naren,Ma,S.,Tan,S.,Xiao,W.,Zhao,W.Z.,Sheng,X.Y.and Yin, OsSEC24是水稻中功能性的SEC24样蛋白,通过增强抗氧化能力来提高对铁缺乏和高pH的耐受性H + -ATPase介导的分泌和/或分泌。植物科学233:61-71。
  2. Newman,I.A.,Kochian,L.V.,Grusak,M.A。和Lucas,W.J。(1987)。 Fluxes of H + 和K + 在玉米根中:表征和使用离子选择性微电极的化学计量学。植物生理学84(4):1177-1184。
  3. Sun,T.,Li,P.,Yin,L.,Xu,Y.and Yu,S。(2007)。 非侵入性扫描离子选择性电极技术及其在高等植物研究中的应用。 自然科学进展 17(6):625-629
  4. Tan,S.,Han,R.,Li,P.,Yang,G.,Li,S.,Zhang,P.,Wang,W.B.,Zhao,W.Z.and Yin,L.P。 MxIRT1基因的过表达增加了水稻种子中的铁和锌含量。 em> Transgenic Res 24(1):109-122。
  5. 徐先生。 Y,Sun。 T和Yin。 (2006)。 应用非侵入式微型传感系统同时测量H < 。 Journal of Integrative Plant Biology 48(7):1-5。
  6. Yang,G.,Ma,F.,Wang,Y.,Feng,C.G.,Li,P.,Xu,Y.,Zhao,W.Z.and Yin,L.P。(2010)。 囊泡相关的OsSEC27P增强了H + 分泌缺铁转基因烟草根。 中国科学公报 55(13):3298-3304。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, X., Zhang, X. and Yin, L. (2015). Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology. Bio-protocol 5(23): e1649. DOI: 10.21769/BioProtoc.1649.
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