Name: quantifying plant soluble protein and digestible carbohydrate content, using corn (zea mays) as an exemplar
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多亏了我们所有的合作伙伴, 他们帮助了甜玉米领域的收集, 包括多米尼克 Reisig 和丹莫特在北卡罗来纳州立大学, 和帕特波特在得克萨斯州拉伯克 & M 大学德克萨斯州。多亏菲奥娜 Clissold 帮助优化了协议, 并为这篇手稿提供了编辑。这项工作的一部分是由德州 A & M c Everette Salyer 奖学金 (昆虫学系) 和生物技术风险评估赠款项目2015-33522-24099 来自美国农业部的竞争性赠款 (授予天然气和机顶盒)。

1. 工厂的收集和加工
<ol><li>收集和处理工厂样品<ol>
<li>采集植物样品后, 用镊子将植物材料浸入液氮中, 并贮存在-80 摄氏度。如果收集的植物样品太大, 不能闪光冷冻, 快速冷却样品使用干冰和转移到一个-80 °c 冷藏库尽快。植物材料的营养素含量在组织分离后会发生变化, 在采集后应尽快冷冻植物标本。 警告:液氮在接触皮肤时会引起严重冻伤。请务必在批准的容器中运输液氮, 并?...

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通过将已建立的色度测定方法与有效的植物特异萃取协议相结合, 本文所展示的化验结果为测定植物可溶性蛋白和可消化碳水化合物含量提供了一种合理、准确的检测手段。我们用玉米作为范例的结果说明了这些协议是如何被用来在不同生物相关的空间尺度上获得精确测量的。例如, 我们能够发现植物可溶性蛋白和可消化的碳水化合物含量在地理区域, 品种 (或基因型), 组织类型, 甚至空间隔离组织...

植物生物量构成了几乎所有陆地食物网的基础。植物通过根系获得土壤中的营养成分, 并利用其叶面组织中的阳光合成生物分子。特别是, 碳和氮被用来制造碳水化合物, 蛋白质 (由氨基酸组成), 以及构建植物生物量所需的脂质 (应该指出, 在植物生理学中, "营养素" 一词通常指土壤元素, 如 N,但是, 在整个本文中, 这一术语将提到生物分子, 如蛋白质、碳水化合物和脂质。当草食动物消耗植物材料时, 植物组织中的营养素被分解成它们的组成部分, 然后用来驱动消费者的生理过程。这样, 植物营养素对消费者的生理有很大的影响, 同时对高阶生态交互作用和食物网络动力学也有重要影响。
在动物王国中, 可溶性蛋白质和可消化碳水化合物是与生存、繁殖和性能1密切相关的营养素。此外, 大多数动物积极调节其摄入这两个营养素, 以满足其生理需求1,2。这对于检测植物组织中糖和氨基酸浓度的昆虫草食动物尤其如此, 这反过来又指导饲养行为。因此, 植物可溶性蛋白和可消化碳水化合物含量在植物-昆虫相互作用的演化过程中起着很强的作用。
虽然有关植物可溶性蛋白和可消化碳水化合物含量的数据相对少见 (但见6、7、8、9、10、11), 但有一个优势可提供植物元素含量 (碳、氮、磷) 的数据。这主要是因为元素在植物矿物营养3,4,5起主要作用。在测量元素时, 相互关系被用来推断可溶性蛋白质和可消化碳水化合物的数量, 但准确的计算往往很难获得。例如, 由于碳在所有有机化合物中无所不在存在, 因此不可能使用碳作为植物可消化碳水化合物含量的指示器。元素氮与植物可溶性蛋白质含量之间存在较强的关系, 常采用广义氮-蛋白质转化因子。然而, 有确凿的证据表明, 氮-蛋白质转化是高度物种特定的12,13,14,15, 使得使用广义转换可能错误。因此, 氮-蛋白质转化因子往往缺乏精确度, 特别是对草食动物营养研究所需的程度。此外, 含有氮的植物化感物质, 如生物碱和芥子油苷, 通常对食草动物有毒, 可以混淆这些转换。
在这里, 我们提供了两种化学化验方法来测定植物组织中可溶性蛋白质和可消化碳水化合物的浓度。这些化验结果分别提出, 但建议它们同时用于分析相同的植物样本, 以实现更全面的分析植物营养素。两者都采用类似的方法, 包括提取步骤, 然后通过吸光度量化。对于这两种协议, 植物样品的准备也是相同的, 这使得两种分析的串联都很容易运行。这些化验的效用并不源于他们的新奇, 因为它们依赖于旧的, (布拉德福德, 琼斯,, 和) 建立的比色测定16,17,18, 但在这里我们组织了一个明确的和易于遵循的将这些方法与更模糊的植物特异萃取技术17、19结合在一起的协议, 以便使这些化验的应用更便于植物相关领域的研究。
对于这两种化验, 植物营养素首先通过冷冻, 冻干和研磨的植物材料的物理提取。对于可溶性蛋白质测定, 在氢氧化钠溶液中通过几轮涡流和加热样品, 进一步进行化学萃取17、19 。众所周知的布拉德福德化验, 利用考马斯灿烂的蓝色 G-250, 然后用于量化可溶性蛋白质和多肽之间的 3,000-5, 000 道尔顿16,17。这一检测范围在1-20 µg 总蛋白每微板块井或 < 25 µg/毫升, 但不测量游离氨基酸。消化性碳水化合物的提取步骤是基于 Smith 的稀酸法. 20 , 并允许分离可溶性糖, 淀粉和果聚糖-但不结构碳水化合物。采用苯酚-硫酸定量方法. 18和测量所有单, 寡糖, 和多糖 (以及甲基衍生物)。这种化验可以量化特定的糖, 但在这里, 我们用它作为一个指标, 总可消化碳水化合物的内容 (见史密斯等。20进行更详细的分析)。这些化验结果表明, 这两种营养素与植物生态生理学和草食动物的表现密切相关, 提供了陆地食物网基地资源质量的重要数据。提出这些协议促进了植物营养素数据集的生成, 以获得对植物生理学、草食动物营养生态学和植物食草动物相互作用的更透彻的理解。

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">microplate reader (spectrophotometer)</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;">Model 680 XR</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Bio-Rad Protein Assay Dye Reagent concentrate</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;">#5000006</td>
			<td>450mL</td>
		</tr>
	</tbody>
</table>

quantifying plant soluble protein and digestible carbohydrate content, using corn (zea mays) as an exemplar

元素数据通常用于推断植物的质量作为草食动物的资源。然而, 生物分子中的碳无处不在, 含氮植物防御化合物的存在, 以及氮与植物蛋白质含量之间的物种特异相关性的变化, 都限制了这些推断的准确性。此外, 针对植物和/或草食动物生理学的研究需要一种不使用广义相关的准确水平。这里提出的方法为研究人员提供了一个明确而快速的协议, 直接测量植物可溶性蛋白和可消化碳水化合物, 这两种植物营养素与动物的生理性能密切相关。这些协议结合了良好的特性比色测定和优化植物特定的消化步骤, 以提供准确和可重现的结果。我们对不同的甜玉米组织的分析表明, 这些化验对检测植物可溶性蛋白和可消化的碳水化合物含量的变化具有敏感性。这包括不同生长区域和植物种类或品种之间的植物差异, 以及在组织类型上的植物差异, 甚至在同一组织内的位置差异。将可溶性蛋白质和可消化碳水化合物含量与元素数据结合起来, 也有可能为植物生物学提供新的机会, 将植物矿质营养与植物生理过程联系起来。这些分析还有助于生成研究营养生态学、植物食草动物相互作用和食物网络动力学所需的可溶性蛋白质和可消化碳水化合物的数据, 这将反过来促进生理学和生态研究。

为了说明这些方法的实用性, 我们分析了四种不同的田间和甜玉米组织的可溶性蛋白和可消化碳水化合物含量, 为草食性昆虫提供了独特的潜在营养资源。我们收集了来自美国 (明尼苏达州, 北卡罗来纳州, 德克萨斯州) 三个农业地区的玉米穗, 其中包括五种不同品种的甜玉米 (即基因型) 和一个外群的田间玉米品种。表 3显示了这些玉米样品的摘要和收集的...

Watch this Scientific Journal Video about Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar at JoVE.com

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

本文所描述的协议提供了一种清晰和平易近人的方法来测量植物组织中可溶性蛋白质和可消化 (非结构) 碳水化合物的含量。量化这两种植物营养素的能力对促进植物生理学、营养生态学、植物食草动物相互作用和食物网络生态学等领域具有重要的意义。

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

This method allows researchers in the field of plant sciences and nutritional ecology to accurately measure concentrations of soluble proteins and digestible carbohydrates in plant tissue. The main advantage of these techniques is that they offer a quick and easy method for quantifying these two incredibly important macronutrients with high accuracy. These techniques have strong implications for the field of ecology because plant proteins and carbohydrates make up the base of terrestrial food webs.Generally, individuals new to this technique may struggle because there are several steps and some techniques which are not commonly performed in a general-use lab. To begin, weigh out replicate samples of each tissue into labeled 1.5 milliliter microcentrifuge tubes. Next, record the precise mass of each sample.Using a micropipetter, add 500 micro-liters of 0.1 molar sodium hydroxide to each tube. Close the lids tightly and sonic heat the tubes for 30 minutes. Next, place the tubes in a preheated hot-water bath for 15 minutes.After this, centrifuge the tubes at 15, 000 G for 10 minutes. Pipette the supernatant into new, labeled microcentrifuge tubes, using a new pipette tip for each sample. Add 300 microliters of 0.1 molar sodium hydroxide to the pellet, and repeat the centrifugation for 10 minutes.After this, remove the supernatant and transfer it to the tubes containing the supernatant from the first centrifugation. To neutralize the pH of the supernatant, add 11 microliters of 5.8 molar hydrochloric acid. Use litmus paper to confirm the pH is approximately seven.Next, add 90 microliters of 100%trichloroacetic acid to each tube. Then incubate the tubes on ice for 30 minutes. Centrifuge the samples at 13, 000 G for 10 minutes at four degrees Celsius.Carefully use a vacuum line and glass micropipette tip to remove the trichloroacetic acid. Be very careful not to disturb the pellet with the suction tip. Next, wash the pellet with 100 microliters of acetone cooled to 20 degrees Celsius, then allow the acetone to evaporate in a fume hood.Dissolve the protein pellet with one milliliter of sodium hydroxide. Additional rounds of heating, vortexing and sonic heating may be required. Add 160 microliters of each IGG standard solution to a 96-well plate in triplicate, starting with the A1 position Next, in a new 1.5 milliliter tube add 50 microliters of each sample solution to 950 microliters of distilled water.Then add 60 microliters of each diluted sample to the well plate in triplicate, starting with the H1 position. Add 100 microliters of distilled water to all of the blank and unknown sample wells. Using a multi-channel pipette, add 40 microliters of Coomassie Brilliant Blue G-250 protein dye to each well of the plate.Using a needle, pop any bubbles present in the wells. After this, allow the plate to incubate at room temperature for five minutes. Then use a microplate spectrophotometer to record the absorbence values for each well at 595 nanometers.First, weigh out replicates from each tissue sample into glass 15 milliliter tubes. Label the tubes and record the exact mass of each sample. Next, add one milliliter of 0.1 molar sulfuric acid to each tube and close the caps tightly.Then place the tubes in a boiling-water bath for one hour. Transfer the tubes to a tepid water bath to cool. Then pour the contents of the tubes into labeled 1.5 milliliter microcentrifuge tubes.After this centrifuge tubes at 15, 000 G for 10 minutes. Use a micro pipette to transfer the supernatant to new labeled 1.5 milliliter tubes. Using a pipette transfer 15 microliters of each unknown sample to its own test tube.Then add 385 microliters of distilled water to each tube. In a fume hood add 400 microliters of 5%phenol to each standard and unknown sample test tube. Immediately thereafter add two milliliters of sulfuric acid to each tube.Make sure to add the sulfuric acid to the surface of the solution. After incubating the tubes for 10 minutes, vortex the tubes. Then incubate the tube for an additional 30 minutes.Transfer 800 microliter from each sample tube to three polystyrene 1.5 milliliter semi-microcuvettes. Then set the spectrophotometer to read at 490 nanometers and calibrated with a blank cuvette. Finally, run each cuvette through the spectrophotometer and record the absorbence.Using this protocol, the soluble protein and digestible carbohydrate content of four different field and sweet corn tissues from three geographical regions were analyzed. Significant differences in soluble protein content were observed between the regions, thus necessitating separate analyses for each region. Across regions there were several differences in soluble protein and digestible carbohydrate content between tissues.In Minnesota, tissues only varied in soluble protein content while North Carolina tissues varied in both soluble protein and digestible carbohydrates. Variability in tissue macronutrient content in Texas depended on the variety. This technique will allow researchers in the field of plant sciences and nutritional ecology to precisely measure soluble plant protein and digestible carbohydrates, rather than extrapolating from elemental measures.Don't forget that working with liquid nitrogen, concentrated acids and bases, can be extremely hazardous. So always use precautions such as gloves, safety goggles, aprons and closed-toed shoes whenever working with these chemicals.

Research

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Environment

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

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