<ol><li>首先，我们需要准备提取2毫升螺丝帽小瓶：加入400ul提取缓冲液（1 - 丙醇：水：浓盐酸2:1:0.002卷/体积/体积）和10μL各自的内部标准（10ng/μl ）一小瓶。在液氮中冷冻，然后添加的陶瓷珠。 </li><li>添加植物材料（50至200毫克），而小瓶仍然定位在液氮。需要注意的是要估计的进一步处理之前，以便精确量化，这是基于对鲜重后，添加植物材料的重量。 </li><li>确保所有的液氮蒸发前紧紧地关闭第小瓶。请注意，上限需要有橡胶密封，以防止泄漏的溶剂，因此，提取的化合物，在同质化。 </li><li>均匀FastPrep或Precellys珠粉碎机匀浆在6000速度（Precellys）在30秒的组织。 </li><li>从匀浆小瓶单独删除，小心地打开瓶盖，添加二氯甲烷1ml到每一个样本。关闭小瓶再次紧密，均质15秒10再次在6000（Precellys）。 </li><li>瓶转移到桌面离心机和10.000xg为60秒的水相有机相分开。 </li><li>相分离后，转移个人小瓶绿色较低层（有机相，含有激素和内部标准）4毫升一个干净的玻璃小瓶。避免调水（上相）。此外，醋酸乙酯，二氯甲烷可以用来代替。在这种情况下ethyacetate阶段将上层（绿色），现在更容易去除，并转移到一个新鲜4毫升的玻璃小瓶。如前所述，避免调水。 </li><li>吹掉溶剂与空气通过多方面的，约10-25分钟（取决于对样本）;检查与一个单一的气流提示，如果样品是干燥的（小心）。不要overdry样本，这可能会导致损失的化合物。 </li><li>样品干燥后，我们就可以开始我们的样品中含有的化合物的羧基甲基酸。首先，100μL一个diethyether /甲醇混合液（9:1体积/体积）被添加到一个2M trimethylsilyldiazomethane的解决方案（正己烷，西格玛Aldrich公司）4μl每一瓶。样品瓶与聚四氟乙烯内衬硅胶隔一个开放的顶部的螺丝盖，立即关闭。轻轻摇动，并在室温下孵育25至30分钟。 </li><li>第9步完成后，打开一个加热块，并设置所需的收集温度。甲基化合物的波动，取决于它们的大小，同时也对其他的，更极像= O - OH和- NH组。对于茉莉酸和水杨酸甲酯的集合温度约80 ° C是足够的，而其他化合物和C18的脂肪酸一样，茉莉酸，氨基酸的结合物，冠毒素，12 - 羰基phytodienoic酸甲酯例如需要温度之间180-200 ° C至有效挥发。 </li><li>经过30分钟孵化甲基化反应需要停止，以避免不必要的二次反应。这是通过加入4μL一个2M的醋酸溶液（正己烷），每一瓶。帽和短期震荡后关闭的小瓶，样品孵育另一个30分钟。 </li><li>这气相提取使用的过滤器都是手工制作，并在另一种形式提出市售。它们由一个外部的聚四氟乙烯管（约7 - 8CM长），合身的长度约4cm的玻璃管插入到这从一个侧面。在另一侧的聚四氟乙烯管，不锈钢丝网光盘压管内，直到它到达内玻璃管上，约20 - 30毫克的吸附剂（无论是80/100的super（已停产）或HayeSep ® Q80/100）添加。另一个不锈钢丝网光盘插入最后一个玻璃尖推入聚四氟乙烯管，从而仔细按吸收剂不锈钢丝网光盘。详细的图片过滤器如图1所示。 <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="图1" /> 图1。 </li><li>在第11步的孵化时间，过滤器收集的甲基化，因此，植物激素的波动，有使用甲醇的第一200ul洗涤，然后与每个200ul二氯甲烷洗涤。溶剂过滤器余下的是由空气吹走。该过滤器现在可以用于通过气相提取的挥发性激素诱捕。 </li><li> 4毫升样品瓶盖​​的橡胶隔孵育30min后用手术刀切开。气相提取的清洁过滤器连接到个人的流速约800ml/min真空线来收集的植物激素。该过滤器，然后插入小瓶通过削减隔。一个小吸管尖也插入作为一个进气口。在这个过程的初始一部分，与插入过滤器的小瓶举行在直立位置，里面的小瓶液体，以避免任何接触过滤嘴，这将污染的过滤器，并随后的GC / MS。小瓶，然后用手举行，直到所有的液体蒸发是通过过滤器。然后附上的过滤器的小瓶放在加热块和热蒸发的化合物为3分钟收集。如图1所示的系统以及设置的原则组件。这最后的气相提取步骤后仍然附着在过滤器的小瓶放置在机架上，并让其冷却下来。 </li><li>当过滤器再次达到室温150毫升二氯甲烷洗脱到1.5毫升的装有插入GC小瓶。改变从小瓶加盖并用GC / MS分析的过滤器吹出来的剩余的溶剂。 </li></ol>气相色谱仪配备分流/不分流进样器（分流模式250℃，进样量为1μ）连接到一台质谱仪，在化学电离模式（CI）的经营应当用于分析）。 HP - 1MS列化合物是分开的（30米x0.25毫米x025μm）在40℃，注射1分钟后举行，然后温度在15编程° C /分钟至250℃（10分钟）用氦气，为载体气体（恒定流速0.7ml/min）。对于基于MS的分析，一个quadropole（如安捷伦HP5973）和离子阱（如瓦里安土星2200）质谱仪可以使用。作为一个CI气体要么异丁烷气罐或甲醇（水库从液体的蒸气用于）可使用，但是，在每种情况下的电离反应的碎片应检查前的选择离子数（SIC）的计划创建。我们用下面的程序：在Varian GC 3900/Saturn的MS 2200系统和甲醇作为CI试剂5.00 - 11.30min离子（M +1）+ 153-158（153水杨酸甲酯，甲基2 H 5 -水杨酸）; 11.30 - 13.30min离子（M +1）+ 207-228（茉莉酸甲酯，甲基二氢茉莉酮209 207和225和227）; 13.30 - 16.30min离子（M +1）+ 237-300（甲基脱落酸，甲基2 261 H 6脱落酸267（脱落酸产生主要是[M + H 2 O +1] +离子），所有的化合物，应确定与地道的市售标准比较。JA，SA和ABA是由相互关联的峰面积进行定量（提取离子）与各自的内部标准的峰面积（提取离子）的使用植物材料的鲜重的基础上。

<ol>
	<li>Schmelz, E. A., Engelberth, J., Tumlinson, J. H., Alborn, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vapor+phase+extraction+in+metabolic+profiling+of+phytohormones+and+other+metabolites.">The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites.</a> Plant Journal. 39, 790-808 (2004).</li><li>Engelberth, J., Alborn, H. T., Schmelz, E. A., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+signals+prime+plants+against+insect+herbivore+attack.">Airborne signals prime plants against insect herbivore attack.</a> PNAS. 101, 1781-1785 (2004).</li><li>Schmelz, E. A., Engelberth, J., Alborn, H. T., O&amp;acute, D. o. n. n. e. l., Sammons, P., Toshima, M., H, J. H. T. u. m. l. i. n. s. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+phytohormones,+phytotoxins,+and+volatile+organic+compounds+in+plants.">Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants.</a> PNAS. 100, 10552-10557 (2003).</li><li>Engelberth, J., Schmelz, E. A., Alborn, H., Cardoza, Y. J., Huang, J., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+Jasmonic+acid+and+Salicylic+acid+by+Vapor+Phase+Extraction+and+Gas+Chromatography+-+Chemical+Ionization-Mass+Spectrometry.">Simultaneous Quantification of Jasmonic acid and Salicylic acid by Vapor Phase Extraction and Gas Chromatography - Chemical Ionization-Mass Spectrometry.</a> Analytical Biochemistry. 312, 242-250 (2003).</li></ol>

这里介绍的方法已被证明了植物信号的大小和被提取，甲基化，而蒸发的化合物的化学性质并不妨碍它的化合物广泛可靠的工作。此外，甲基化过程，因为它是本文所述，是不是衍生化的唯一途径。 Silyllation和乙酰化的替代方法和协议，可以很容易地在网上找到。 如果不能够监测记录在运行过程中应仅限于从过程中排除太多不必要的离子质量范围内的单离子质谱仪。例如，?...

monitoring plant hormones during stress responses

在植物响应各种环境刺激和压力调节植物激素和相关的信令化合物发挥了重要作用。其中最严重的应力是昆虫的取食，病原体的感染，干旱胁迫。对于其中的每一个讲一套特定的激素和/或组合被称为微调的反应，从而保证植物的生存。在这些反应的调控涉及的主要激素是茉莉酸（JA），水杨酸（SA）和脱落酸（ABA）。为了更好地理解个人激素的作用以及他们潜在的相互作用在这些反应，这是必要的，在一个时间以及空间的方式来监测其丰度的变化。对于这些和其他信号的化合物容易，灵敏度高，重现性好量化，我们开发了基于汽相萃取和气相色谱/质谱（GC / MS）分析（1，2，3，4）的方法。这些化合物从植物组织中提取的酸性水溶液后，1 - 丙醇与二氯甲烷混合羧酸化合物的甲基化，高温下挥发，在高分子吸水收集。后成样品瓶洗脱分析物是通过气相色谱分离和化学电离质谱检测。然后使用适当的内部标准，允许有关分析物和内标峰面积的简单量化。

Watch this Scientific Journal Video about Monitoring Plant Hormones During Stress Responses at JoVE.com

Monitoring Plant Hormones During Stress Responses

提供一个简单的方法是，允许小组织样本的多种植物激素的快速提取和分析。该程序使用气相萃取庄严的纯化步骤。 GC / MS化学电离主要生产（M +1）+离子的样品进行了分析。

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

monitoring-plant-hormones-during-stress-responses

Research

JoVE Journal

Biology

16.5K 次观看.  University of Texas. 提供一个简单的方法是，允许小组织样本的多种植物激素的快速提取和分析。该程序使用气相萃取庄严的纯化步骤。 GC / MS化学电离主要生产（M +1）+离子的样品进行了分析。

视频: Monitoring Plant Hormones During Stress Responses

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

科研

生物学

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

翻译起始的分析在应力条件由多聚核糖体剖析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培：一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

结合透明和荧光显微镜，可视化基因表达的变化和对芸苔疟原虫的生理反应

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...