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Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
植物总提取物中泛素蛋白酶体活性测定   

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Abstract

The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to meet the current demands of protein degradation via the UPS within the cell. We describe the adaptation of an intramolecular quenched fluorescence assay utilizing substrate-mimic peptides for the measurement of proteasome activity in total plant extracts. The peptide substrates contain donor-quencher pairs that flank the scissile bond. Following cleavage, the increase in dequenched donor emission of the product is subsequently measured over time and used to calculate the relative proteasome activity.

Keywords: Proteasome(蛋白酶体), Immunity(免疫力), Plant defense(植物防御), Targeted proteolysis(靶向蛋白水解), Fluorogenic substrate(荧光底物)

Background

The ubiquitin proteasome system (UPS) is the major protein degradative machinery in eukaryotic cells and as such the UPS is essential for the regulation of many cellular processes, including signaling, cell cycle, vesicle trafficking, and immunity. Proteins destined for turnover are marked by the covalent attachment of ubiquitin and then degraded by the 26S proteasome. The 26S proteasome is composed of two subparticles, the 20S core protease (CP) that compartmentalizes the protease active sites and the 19S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Proteasome activity is modulated in order to maintain proteostasis in response to fluctuating internal and external conditions. We have recently shown that the UPS is involved in several aspects of plant immunity and a range of plant and animal pathogens subvert the UPS to enhance their virulence (Üstün et al., 2013; Üstün et al., 2014; Üstün and Börnke, 2015; Üstün et al., 2016). In plants, proteasome activity is strongly induced during basal defense and adapted bacterial pathogens can interfere with this induction using specific virulence factors. This protocol describes an assay to assess the relative chymotrypsin-like proteolytic activity of the proteasome in total plant extracts using a fluorogenic substrate. This assay is carried out in a 96-well format using a plate reader and thus is amendable to medium to high throughput and can easily be modified to measure additional proteolytic activities of the proteasome by exchanging the substrate accordingly.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml microcentrifuge tubes
  3. Black walled 96-well plates for proteasome activity measurements (Corning, catalog number: 3603 )
  4. Clear 96-well plates for protein measurements (Greiner Bio One International, catalog number: 655101 )
  5. Plant material (the protocol is optimized for leaf samples from Nicotiana benthamiana, Capsicum annuum and Arabidopsis thaliana but can also be applied to other plant species or tissues)
  6. Murashige and Skoog (MS) agar (Sigma-Aldrich)
  7. Sucrose (MP Biomedicals, catalog number: 04821713 )
  8. Liquid nitrogen (N2)
  9. Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, catalog number: 5000002 )
  10. Proteasome substrate
    Suc-LLVY-AMC (chymotrypsin-like activity substrate) (Sigma-Aldrich, catalog number: S6510 )
    Note: Other proteolytic activities of the proteasome can be measured using the same protocol by using appropriate substrates (Z-ARR-AMC for measuring the trypsin-like activity [Bachem, catalog number: I-1125.0050 ] and Z-LLE-AMC for the determination of the peptidylglutamyl-peptide-hydrolyzing [PGPH] activity of the 20S proteasome [Bachem, catalog number: I-1945.0005 ]).
  11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  12. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A3377 )
  13. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  14. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
  15. 1,4-Dithiothreitol (DTT) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15508013 )
  16. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  19. Proteasome substrate solution (see Recipes)
  20. Extraction buffer (see Recipes)
  21. Proteasome lysis/assay buffer (see Recipes)

Equipment

  1. Pipettes
  2. pH-meter
  3. Cork borer 0.7 cm diameter (Sigma-Aldrich, catalog number: Z165220 )
  4. Overhead stirrer (Heidolph Instruments, model: RZR 1 )
  5. Cooled microcentrifuge (4 °C)
  6. Adjustable thermoblock for 96-well plates (Eppendorf, model: ThermoMixer® C )
  7. Plate reader spectrophotometer (Absorbance, Fluorescence) (e.g., BioTek Instruments, model: Synergy HT )

Software

  1. Gen5 software (Biotek Instruments)

Procedure

  1. Plant cultivation
    1. Arabidopsis thaliana: Arabidopsis seeds (ecotype Columbia-0) were sown on Murashige and Skoog (MS) agar (Sigma-Aldrich) plates supplemented with 2% (w/v) sucrose and cultivated in tissue culture under a 16-h-light/8-h-dark regime (irradiance of 150 μE m-2 sec-1) at 70% humidity. Arabidopsis seeds germinated on soil were grown under short-day conditions (8 h of light/16 h of dark [23 °C/21 °C], irradiance of 150 μE m-2 sec-1, 70% humidity).
    2. Nicotiana benthamiana and Capsicum annuum plants were grown in soil in a greenhouse with daily watering and subjected to a 16-h-light/8-h-dark cycle (25 °C/21 °C) at 300 μE m-2 sec-1 light and 40% relative humidity.

  2. Sample preparation
    1. Punch out two to four leaf discs with a cork borer (diameter 0.7 cm), combine discs from the same sample in a single 1.5 ml microcentrifuge tube and immediately freeze leaf discs in liquid nitrogen. Samples can be stored at -80 °C until further processing.
    2. Grind frozen leaf material in 200 µl extraction buffer (see Recipes) in an overhead stirrer until the material is completely thawed (be sure to prevent heating of the sample). Alternatively, leaf discs can be ground to a fine powder in a pre-cooled mortar. Add extraction buffer and continue to grind until the material is completely thawed. Pour the suspension into a microcentrifuge tube.
    3. Centrifuge samples at 17,000 x g for 10 min at 4 °C and subsequently transfer the supernatant to a fresh tube.
    4. Determine the protein content of the extracts with the Bio-Rad Assay Dye according to the manufacturer’s instructions using BSA as a standard.
    5. Adjust the protein concentration of the supernatant to 1 mg/ml with extraction buffer. Keep samples on ice until use. Proceed to measurement as soon as possible. It is generally not recommended to store the samples for a longer period of time as the proteasome activity is relative unstable even if stored at -80 °C.

  3. Assay
    1. Prepare assay buffer (see Recipes).
    2. Add 25-50 μg of plant extract into the wells of a black walled 96-well plate and subsequently mix with 220 μl assay buffer. The amount of extract used is plant species and tissue dependent but should be kept the same within a single experiment. Empirically, 50 µg from Nicotiana tabacum or Capsicum annuum and 25 µg from Arabidopsis thaliana leaf samples work best. Process at least three biological replicates per sample/experimental condition (that is material harvested from different plants receiving the same treatment).
    3. Incubate for 15 min at 30 °C.
    4. Add proteasome substrate into the wells (15-30 μl/per sample; final concentration should be 100 μM to 200 μM). Add at least three blank wells including all solutions except protein extract for later background subtraction.
    5. Measure the release of amino-methyl-coumarin at 30 °C (A360ex/A460em) on a fluorescent plate reader every 1.5 min over a period of 120 min.
      The protocol details in Gen5 (Biotek Instruments) are as follows:
      Plate Type: 96-WELL PLATE
      Set Temperature: Setpoint 30 °C, Preheat before moving to next step
      Start Kinetic: Runtime 2:00:00 (HH:MM:SS), Interval 0:01:30, 81 Reads
      Read: Fluorescence Endpoint, Full Plate, Filter Set1: Excitation: 360/40, Emission: 460/40, Optics: Top, Gain: 60, Readspeed: Normal, Readheight: 1 mm
      End Kinetic

Data analysis

The representation of the results can be done in relative fluorescent units (RFUs) by subtracting the RFU levels of the blank wells (containing all solutions except protein extract) from all values. Calculate proteasome activity from the mean linear slope of the emission curve (Figure 1) and express it as fluorescence units per minute (RFU min-1) or as percentage relative to controls, respectively. The mean slope (Mean V in Gen5) is calculated by a linear regression on points in the calculation zone (marked by a green bar in Figure 1) by the software. In Gen5 (Biotek Instruments) go to Protocols > Data Reduction > Well Analysis and tic the Mean V box. For Mean V, the calculation zone is typically adjusted automatically to ignore misleading data points generated at the beginning of a kinetic assay due to noise. The Mean V values can then be exported to Excel by pressing the ‘Edit Table’ button within the ‘Statistics’ view for further analysis (calculation of mean values and statistics). Refer to the user manual of your plate reader for specific details of your instrument.
A typical result of data analysis is shown in Figure 2. Here, pepper plants were infected with different bacterial strains alongside with MgCl2, as a control. For the experiments it is important to use such controls and leaf material from different plants (at least n = 3 to be able to perform statistics) that are of the same age.


Figure 1. Screenshot of a typical output of a proteasome activity measurement in a leaf sample from Arabidopsis thaliana using the above described procedure. The sample was measured on a Biotek Synergy HT plate reader controlled by the Gen5 software (Biotek Instruments). The green bar indicates the part of the linear slope that has been used to calculate the relative fluorescence units per minute (RFU min-1).


Figure 2. XopJ reduces proteasome activity during Xanthomonas-pepper interaction. Pepper leaves were infiltrated with Xanthomonas campestris pv vesicatoria (Xcv) strains indicated in the figure (WT, wild type; ∆xopJ, strain lacking the type-III effector protein XopJ; ∆xopD strain lacking the type-III effector protein XopD). At 3 dpi proteasome activity in total leaf extracts was determined by monitoring the breakdown of the fluorogenic peptide suc-LLVY-NH-AMC in a fluorescence spectrophotometer. Data represent the mean SD (n = 5). Significant differences were calculated using Student’s t-test and are indicated by: *P < 0.05; **P < 0.01.

Recipes

  1. Proteasome substrate solution
    Dissolve Suc-LLVY-AMC in DMSO to a final concentration of 2 mM and store frozen at -20 °C (stable for several months)
  2. Extraction buffer (always prepare fresh and keep on ice until use)
    2 mM ATP
    50 mM HEPES-KOH (adjust to pH 7.2 with 1 N KOH)
    2 mM DTT
    0.25 M sucrose
  3. Proteasome lysis/assay buffer (always prepare fresh and keep on ice until use)
    100 mM HEPES-KOH (adjust to pH 7.8 with 1 N KOH)
    5 mM MgCl2
    10 mM KCl
    2 mM ATP

Acknowledgments

This work was supported by the Federation of the European Biochemical Societies (long-term fellowship to S.Ü.), and by the Deutsche Forschungsgemeinschaft (grant Nos. CRC973 and BO1916/5-2 to F.B.). This protocol is modified from previous work described in Reinheckel et al. (2000).

References

  1. Reinheckel, T., Ullrich, O., Sitte, N. and Grune, T. (2000). Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress. Arch Biochem Biophys 377(1): 65-68.
  2. Üstün, S., Bartetzko, V. and Börnke, F. (2013). The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence. PLoS Pathog 9(6): e1003427.
  3. Üstün, S. and Börnke, F. (2015). The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6. Plant Physiol 168(1): 107-119.
  4. Üstün, S., König, P., Guttman, D. S. and Börnke, F. (2014). HopZ4 from Pseudomonas syringae, a member of the HopZ type III effector family from the YopJ superfamily, inhibits the proteasome in plants. Mol Plant Microbe Interact 27(7): 611-623.
  5. Üstün, S., Sheikh, A., Gimenez-Ibanez, S., Jones, A., Ntoukakis, V. and Börnke, F. (2016). The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type III effectors. Plant Physiol 172(3): 1941-1958.

简介

细胞内的蛋白质水平,构象和位置的微调平衡对于细胞对给定刺激作出反应所需的动态变化是至关重要的。 这需要通过泛素蛋白酶体系统(UPS)调节受损或短寿命蛋白质的周转。 因此,调节蛋白酶体的蛋白酶活性以满足目前通过细胞内的UPS的蛋白质降解的需求。 我们描述了使用底物 - 模拟肽进行分子内淬灭荧光测定来测定总植物提取物中蛋白酶体活性的适应性。 肽底物含有侧向于剪切键的供体 - 猝灭剂对。 切割后,随后随时测量产物的去质子供体发射的增加,并用于计算相对蛋白酶体活性。
【背景】泛素蛋白酶体系统(UPS)是真核细胞中主要的蛋白质降解机制,因此,UPS对许多细胞过程的调节至关重要,包括信号传导,细胞周期,囊泡运输和免疫。用于营业额的蛋白质通过泛素的共价连接标记,然后被26S蛋白酶体降解。 26S蛋白酶体由两个亚颗粒组成,即20S核心蛋白酶(CP),其分隔蛋白酶活性位点和19S调节颗粒,其将适当的底物识别并转移到CP腔中进行分解。调节蛋白酶体活性以维持蛋白酶抑制以响应内部和外部条件的波动。我们最近显示,UPS涉及植物免疫的几个方面,一系列植物和动物病原体颠覆了UPS来增强其毒力(Üstünet al。,2013;üstünet al。,2014;ÜstünandBörnke,2015 ;Üstünet al。,2016)。在植物中,在基础防御期间强烈诱导蛋白酶体活性,并且适应性细菌病原体可以使用特异性毒力因子干扰该诱导。该方案描述了使用荧光底物评估总植物提取物中蛋白酶体的相对糜蛋白酶样蛋白水解活性的测定法。该测定使用平板阅读器以96孔格式进行,因此可修改为中至高通量,并且可以容易地修饰以通过相应地更换基底来测量蛋白酶体的额外的蛋白水解活性。

关键字:蛋白酶体, 免疫力, 植物防御, 靶向蛋白水解, 荧光底物

材料和试剂

  1. 移液器提示
  2. 1.5 ml微量离心管
  3. 用于蛋白酶体活性测量的黑色的96孔板(Corning,目录号:3603)
  4. 清除96孔板进行蛋白质测量(Greiner Bio One International,目录号:655101)
  5. 植物材料(该方案针对本发明的烟草样本,辣椒和/或拟南芥进行优化,但也可应用于其他植物物种或组织)
  6. Murashige和Skoog(MS)琼脂(Sigma-Aldrich)
  7. 蔗糖(MP Biomedicals,目录号:04821713)
  8. 液氮(N 2 2)
  9. Bio-Rad蛋白测定试剂盒II(Bio-Rad Laboratories,目录号:5000002)
  10. 蛋白酶体底物
    Suc-LLVY-AMC(胰凝乳蛋白酶样活性底物)(Sigma-Aldrich,目录号:S6510)
    注意:蛋白酶体的其他蛋白水解活性可以使用相同的方案通过使用适当的底物(Z-ARR-AMC测量胰蛋白酶样活性[Bachem,目录号:I-1125.0050]和Z-LLE-用于测定20S蛋白酶体的肽基谷氨酰肽水解[PGPH]活性的AMC [Bachem,目录号:I-1945.0005])。/ / em>
  11. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  12. 腺苷5'-三磷酸二钠盐水合物(ATP)(Sigma-Aldrich,目录号:A3377)
  13. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H3375)
  14. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60377)
  15. 1,4-二硫苏糖醇(DTT)(Thermo Fisher Scientific,Invitrogen TM,目录号:15508013)
  16. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  19. 蛋白酶体底物溶液(参见食谱)
  20. 提取缓冲液(见配方)
  21. 蛋白酶体裂解/测定缓冲液(参见食谱)

设备

  1. 移液器
  2. pH计
  3. 软木钻孔直径0.7厘米(Sigma-Aldrich,目录号:Z165220)
  4. 顶置式搅拌器(Heidolph Instruments,型号:RZR 1)
  5. 冷冻微量离心机(4℃)
  6. 96孔板的可调节热块(Eppendorf,型号:ThermoMixer C)
  7. 读板器分光光度计(吸光度,荧光)(例如,BioTek Instruments,型号:Synergy HT)

软件

  1. Gen5软件(Biotek Instruments)

程序

  1. 植物栽培
    1. 拟南芥拟南芥种子(Ecotype Columbia-0)播种在补充有2%(w / v)的藻酸盐的Murashige和Skoog(MS)琼脂(Sigma-Aldrich)蔗糖,并在组织培养物中在70%湿度下在16小时光/ 8小时黑暗的条件(150μEm -2至 sec 的辐照度)下培养。 。在土壤上发芽的种子在短时间条件下生长(8小时的光/ 16小时的黑暗[23℃/ 21℃],150微米/平方米的辐照度< / sup> sec -1 ,70%湿度)
    2. 烟草烟草和辣椒植物在日常浇水的温室中在土壤中生长并进行16小时光照/ 8小时黑夜循环(25℃) / 21℃),在300μEm sec -1 光和40%相对湿度。

  2. 样品制备
    1. 用一个软木钻孔机(直径为0.7厘米)打出两到四片叶盘,将同一样品的圆盘与单个1.5毫升的微量离心管混合,立即在液氮中冷冻叶盘。样品可以储存在-80°C直到进一步处理。
    2. 在顶置式搅拌器中研磨200μl提取缓冲液(参见食谱)中的冷冻叶子材料,直到材料完全解冻(确保防止样品加热)。或者,叶片可以在预冷的研钵中研磨成细粉末。加入提取缓冲液,继续研磨,直到材料完全解冻。将悬浮液倒入微量离心管中
    3. 在4℃下以17,000×g离心样品10分钟,然后将上清液转移到新管中。
    4. 使用Bio-Rad Assay Dye根据制造商的说明书使用BSA作为标准确定提取物的蛋白质含量。
    5. 用提取缓冲液调节上清液的蛋白质浓度为1 mg / ml。将样品保存在冰上直至使用。尽快进行测量。通常不推荐将样品储存较长时间,因为蛋白酶体活性相对不稳定,即使储存在-80°C。

  3. 化验
    1. 准备测定缓冲液(参见食谱)。
    2. 将25-50μg植物提取物加入到黑色的96孔板的孔中,随后与220μl测定缓冲液混合。使用的提取物的数量是植物物种和组织依赖性,但应在单个实验中保持相同。经验地,来自烟草烟草或辣椒辣椒的50μg和来自拟南芥叶片样品的25μg的效果最好。每个样品/实验条件(即从接受相同处理的不同植物收获的材料)至少进行三次生物重复。
    3. 在30°C孵育15分钟。
    4. 将蛋白酶体底物加入孔(15-30μl/每个样品;最终浓度应为100μM至200μM)。添加至少三个空白孔,包括蛋白质提取物以外的所有溶液,以进行背景减除
    5. 在120分钟的时间内,每隔1.5分钟在荧光板读数器上测量30℃下氨基甲基香豆素的释放(A 360°/ A / 460mm)。 br /> Gen5(Biotek Instruments)的协议细节如下:
      板型:96孔板
      设定温度:设定值30°C,预热,然后再转到下一步骤
      启动Kinetic:运行时间2:00:00(HH:MM:SS),间隔0:01:30,81读取
      阅读:荧光端点,全板,过滤器组1:激发:360/40,发射:460/40,光学:顶部,增益:60,读取速度:正常,高度:1 mm
      End Kinetic

数据分析

通过从所有值中减去空白孔(含有蛋白质提取物以外的所有溶液)的RFU水平,可以在相对荧光单位(RFU)中进行结果的表示。从发射曲线的平均线性斜率计算蛋白酶体活性(图1),并将其表示为每分钟荧光单位(RFU min -1 )或相对于对照的百分数。平均斜率(Gen5中的平均V)通过软件通过计算区域(图1中的绿色条标示)上的点的线性回归计算。在Gen5(Biotek Instruments)中,转到Protocols&gt;数据缩减&gt;井分析和平均V箱。对于平均值V,计算区域通常会自动调整,以忽略由于噪声在动力学测定开始时产生的误导性数据点。然后可以在"统计"视图中按"编辑表"按钮将平均值V导出到Excel,以进一步分析(平均值和统计的计算)。有关仪器的具体细节,请参阅读板器的用户手册。
数据分析的典型结果如图2所示。在这里,作为对照,将胡椒植物与不同的细菌菌株和MgCl 2一起感染。对于实验,使用来自不同植物(至少n = 3能够执行统计学)的这种对照和叶材料是相同年龄的重要的。


图1.使用上述方法从拟南芥的叶样品中的蛋白酶体活性测量的典型输出的屏幕截图。在Biotek Synergy HT板上测量样品读者由Gen5软件(Biotek Instruments)控制。绿色条表示用于计算每分钟相对荧光单位(RFU min -1 )的线性斜率部分。


图2. XopJ在黄单胞菌属 -pepper相互作用期间减少蛋白酶体活性。 使用图中所示的野生型Xanthomonas campestris (Xcv)菌株渗透胡椒叶(WT,野生型;ΔxopJ ,缺乏缺乏III型效应子蛋白XopD的III型效应子蛋白XopJ;ΔxopD菌株的菌株)。通过在荧光分光光度计中监测荧光肽suc-LLVY-NH-AMC的分解来测定总叶提取物中的3dpi蛋白酶体活性。数据表示平均SD(n = 5)。使用Student's 测试计算出显着差异,并用以下表示:* 0.05; ** 0.01。

食谱

  1. 蛋白酶体底物溶液
    将Suc-LLVY-AMC在DMSO中溶解至终浓度为2mM,并在-20℃下冷冻(稳定数月)
  2. 提取缓冲液(始终准备新鲜并保持在冰上直到使用)
    2 mM ATP
    50 mM HEPES-KOH(用1N KOH调节至pH 7.2)
    2 mM DTT
    0.25 M蔗糖
  3. 蛋白酶体裂解/测定缓冲液(始终准备新鲜并保持在冰上直到使用)
    100mM HEPES-KOH(用1N KOH调节至pH 7.8)
    5mM MgCl 2
    10 mM KCl
    2 mM ATP

致谢

这项工作得到了欧洲生物化学学会联合会(与S.Ü.的长期研究金)以及德意志丰业集团(授权号CRC973和BO1916 / 5-2至F.B.)的支持。该协议从Reinheckel等人描述的以前的工作中进行了修改。 (2000)。

参考

  1. Reinheckel,T.,Ullrich,O.,Sitte,N。和Grune,T。(2000)。氧化应激期间人造血K562细胞中20S和26S蛋白酶体活性的差异性损伤。 Arch Biochem Biophys 377(1):65-68 。
  2. Üstün,S.,Bartetzko,V.和Börnke,F。(2013)。&nbsp; III型Xanthomonas campestris III型效应物XopJ靶向宿主细胞蛋白酶体以抑制水杨酸介导的植物防御。 PLoS Pathog 9(6 ):e1003427。
  3. Üstün,S.和Börnke,F.(2015)。&lt; a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/25739698"target ="_ blank" > Xanthomonas campestris III型效应物XopJ蛋白水解降解蛋白酶体亚基RPT6。植物生理学168(1):107-119。
  4. Üstün,S.,König,P.,Guttman,DS andBörnke,F。(2014)。&nbsp; 来自丁香假单胞菌的HopZ4,抑制植物中的蛋白酶体。植物微生物相互作用 27(7):611-623。
  5. Üstün,S.,Sheikh,A.,Gimenez-Ibanez,S.,Jones,A.,Ntoukakis,V.andBörnke,F。(2016)。&nbsp; 蛋白酶体作为植物免疫的中枢,是由三态效应子假单胞菌靶向的。植物生理学 172(3):1941-1958。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Üstün, S. and Börnke, F. (2017). Ubiquitin Proteasome Activity Measurement in Total Plant Extracts. Bio-protocol 7(17): e2532. DOI: 10.21769/BioProtoc.2532.
  2. Üstün, S., Sheikh, A., Gimenez-Ibanez, S., Jones, A., Ntoukakis, V. and Börnke, F. (2016). The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type III effectors. Plant Physiol 172(3): 1941-1958.
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