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, 并在搬运过程中佩戴适当的 PPE, 包括冷冻手套、眼谷歌、面罩、围裙和实验室大衣。</li>
		<li>Lyophilize 植物材料使用冷冻干燥器, 以确保组织在新陈代谢不活跃, 而水被删除。干燥, 直到样品的质量稳定, 以确保所有的水已被删除。</li>
		<li>样品干燥后, 用研磨机或磨粒研磨成细粉。将样品储存在干燥柜中, 直到分析。</li>
	</ol>
</li>
</ol>2. 可溶性蛋白测定法
<ol><li>样品准备<ol>
<li>每个组织的复制样品, 大约20毫克每个, 成1.5 毫升聚苯乙烯离心管和标签管。这些样品随后将被称为未知样品。记录每个样本的确切质量, 因为这些信息将需要计算每个未知样本中的%soluble 蛋白。使用离心管与锁定盖子, 因为非锁定盖可能会打开在随后的加热步骤, 导致样品解决方案的损失。</li>
	</ol></li>
	<li>溶解和分离未知样本蛋白<ol>
<li>使用微 pipettor, 在每管中添加500µL 0.1 米氢氧化钠。关闭盖子严密和油脂实验30分钟。预热热水浴90°c。 警告:氢氧化钠是一种很强的碱基, 在接触皮肤时会引起烧伤。虽然0.1 米氢氧化钠代表低浓度, 佩戴防护手套, 以避免皮肤发炎。</li>
		<li>把管子放在热水浴的架子上15分钟。</li>
		<li>离心管在 1.5万 x g 10 分钟。将上清液注入新标记的离心管, 使用新的吸管尖端为每个样品。将300µL 0.1 米的氢氧化钠添加到含有颗粒和离心机的管中, 再以 1.5万 x g 为10分钟。再次, 收集上清液, 并将其与其他上清液相结合, 从相同的样品。这是一个潜在的停止点, 样品可以存储在4°c 隔夜, 如果需要的话。</li>
	</ol></li>
	<li>沉淀植物蛋白<ol>
<li>加入11µL 5.8 米盐酸盐, 中和上清液的 ph 值. 通过将试金石浸在每个样品溶液中, 确认 ph 为7。 警告:盐酸是一种强腐蚀性酸, 在接触皮肤 (皮肤应用或吸入) 时会引起烧伤。在处理 HCl 时, 请戴上手套防止皮肤发炎。</li>
		<li>添加90µL 100% 乙酸酸 (TCA) 到每管和孵化管在冰上30分钟。蛋白质的沉淀会使溶液从清澈变为不透明。如果30分钟后不发生这种情况, 请将管子冷藏1小时。这是一个潜在的停止点, 样品可以存储在4°c 隔夜, 如果需要的话。 警告:乙酸酸具有腐蚀性。在处理时请戴上手套, 防止皮肤接触, 避免吸入。</li>
		<li>离心机样品在 1.3万 x g 10 分钟在4摄氏度。小心地删除 TCA 上清, 用真空线连接到一个精细的玻璃微提示 (相同的提示可以在样品中使用)。不要干扰蛋白颗粒, 避免重吸力和保持适当的距离之间的吸管尖端和颗粒.
</li>
		<li>用100µL-20 摄氏度丙酮快速清洗颗粒。此步骤从颗粒中除去任何残留的 TCA, 这可能会干扰随后的布拉德福德化验;然而, 丙酮会开始溶解蛋白颗粒如果在接触太长, 所以丙酮应该添加然后迅速提取从颗粒在5秒内。</li>
		<li>通过将管子放在通风罩或冰箱中, 使丙酮蒸发。然后, 在每管中加入1毫升0.1 米氢氧化钠, 溶解蛋白颗粒。充分溶解颗粒可能需要在热水浴, 涡流和 sonicating 几轮加热。 注意: 管子放在油烟机或冰箱里的时间越长, 颗粒的干燥越多, 重新溶解的难度就越大。建议将颗粒干燥30分钟, 然后每10-15 分钟检查一次丙酮的存在, 直到发现丙酮已经蒸发 (没有丙酮烟雾是可检测的)。</li>
	</ol></li>
	<li>混合标准溶液, 稀释未知样本解决方案, 并使用布拉德福德化验定量分析未知样品的总蛋白质含量。<ol>
<li>根据表 1中列出的浓度, 制备牛免疫球蛋白 G (igg) 标准溶液 (冻干或 IgG 溶液可与去离子水混合以达到每浓度)。商店标准在4°c。在96井板中, 在 A1 位置 (0 µg/µL A1、A2、A3 位置、0.0125 µg/µL、B1、B2 位置等) 中, 添加每个 IgG 标准溶液的160µL。</li>
		<li>将50µL 0.1 米氢氧化钠加入950µL 蒸馏水中, 制备稀释的氢氧化钠溶液。作为一个空白的负控制, 添加60µL 的这个解决方案的油井板块 (G1, G2, G3 位置)。</li>
		<li>在新的1.5 毫升离心管中加入50µL, 将每个样品溶液中的950µL 蒸馏水稀释, 制备未知样品。然后, 从 H1 位置开始, 将每个稀释样品的60µL 加成三份。96井板应允许分析6标准, 1 空白, 25 未知样本时, 三个三读。每个井板分析应包含自己的 IgG 标准和空白样品。</li>
		<li>将100µL 的蒸馏水添加到所有空白和未知的样品井中。现在每个井都应该包含160µL. 采用多通道吸管 (8 通道), 将考马斯亮蓝 G-250 蛋白染料的40µL 添加到井板第一柱的每一个井中。将染料与样品混合, 多次暴跌。重复所有其他列, 更换吸管提示, 以避免污染。 警告:考马斯灿烂的蓝色 G-250 是一个刺激性和接触皮肤, 眼睛, 或肺部应避免。请戴上手套防止皮肤接触。如果接触皮肤发生, 此产品将染色。</li>
		<li>使用针来弹出在水井中的任何气泡, 因为它们可能会干扰微板块分光光度计阅读。清洁针头以避免水井之间的污染。让微板块在室温下孵化5分钟。</li>
		<li>使用微板块分光光度计, 记录每个井的吸光度值为 595 nm。</li>
		<li>记录三个空白井的平均吸光度, 并从每个标准和未知样本读数中减去这个值。然后, 记录每个标准和未知样本的平均吸光度。 注意: 要用标准曲线计算每个未知样本中的蛋白质含量, 最容易的方法是将每个标准的平均吸光度与每种标准中的蛋白质微克进行回归, 但你可以绘制蛋白质浓度 (µµL) 每个标准如果可取的。但是, 下面的计算是指基于微克的标准曲线, 而不是浓度 (µg/µL)。</li>
		<li>根据每个标准井中的蛋白质总量, 绘制每个标准的平均吸收量 (表 1)。适合于这些数据的线性回归线, 并根据平均吸收量 (图 1a) 计算每个未知样本中蛋白质 (µg) 的数量。 注: 此线的相关系数 (r值) 应大于 0.98, 通常接近0.99。标准回归将特定于每个板块, 因此每个板块中的未知样本井必须分开分析。</li>
		<li>一旦每个未知样本的平均蛋白质量被计算好, 使用下面的等式来计算每个原始样品中蛋白质 (µg) 的总数量: Mp = ((Wp/60) x 1000)/50) * 1000 Mp = 原样品中蛋白质的µg µg在未知样品井中的蛋白质含量<ol>
<li>然后, 使用以下等式确定每个样本中蛋白质的百分比: pp = ((mp/1000)/Mi) * 100 pp =%protein 在样品中 Mi = 样品的初始质量 (毫克) 注: 在某些情况下, 样品可能超过上吸光度阈值。如果是这样, 样品解决方案可能需要在步骤2.4.3 进一步稀释。 在随后的计算中, 必须对附加的稀释进行核算。一些微板块的读者品牌还提供计算机软件, 将根据标准曲线数据自动计算每个未知样本的平均蛋白质浓度。</li>
		</ol>
</li>
	</ol></li>
</ol>3. 消化碳水化合物的测定
<ol><li>样品准备<ol>
<li>称每个组织的复制样品, 大约20毫克每, 成玻璃15毫升管与橡胶衬里的螺丝盖帽 (100 毫米长度)。这些样本随后将被称为未知样本。标签管与防水标志或标签胶带的盖子和记录的确切质量每个样本, 因为这一信息将需要计算%carbohydrates 在每个未知的样品。</li>
	</ol>
</li>
	<li>从每个未知样品中提取易消化的碳水化合物。<ol>
<li>添加1毫升0.1 米2, 所以4到每个管和螺丝帽上管紧密。在沸腾的水浴中放置1小时。 警告:硫酸是非常腐蚀性的。在处理 H2至4时, 请戴上手套、眼谷歌和围裙, 并在通风罩上执行此步骤。</li>
		<li>在温水浴中冷却管。将管的内容倒入标记为1.5 毫升的离心管 (如果某些植物材料留在玻璃管中, 不要担心)。</li>
		<li>离心管在 1.5万 x g 10 分钟。用微 pipettor 去除上清液, 放入新标记的1.5 毫升离心管。在这一点上, 管子可以冷藏一夜。如果继续化验, 请参阅步骤3.3.2。</li>
	</ol>
</li>
	<li>混合标准溶液并量化未知样品的总易消化碳水化合物含量。<ol>
<li>用表 2中列出的浓度制备 D (+) 葡萄糖标准溶液。准备六玻璃试管与每标准溶液的400µL。</li>
		<li>将每个未知样品的吸管15µL 到其自己的试管中, 并在每个试管中添加385µL 的蒸馏水, 每一个总容积为400µL。在油烟机中, 在每个标准和未知样品试管中加入400µL 5% 苯酚, 然后迅速将2毫升浓缩的 H2如此4注入每管。不要接触试管的内容与吸管尖端, 只是添加到解决方案表面 (一个中继吸管的工作最好的)。</li>
		<li>让管子孵化10分钟, 然后仔细地涡旋管子以混合内容。再孵化30分钟。 警告:苯酚和硫酸是腐蚀性刺激性物质。为了防止暴露于皮肤, 眼睛和吸入, 这一步骤应在化学油烟机使用适当的 PPE, 其中包括: 面罩或护目镜, 橡胶手套, 实验室围裙, 和靴子。苯酚与硫酸混合也会产生放热反应, 这将导致管子变热。</li>
		<li>吸管800µL 样品从每管入每3聚苯乙烯1.5 毫升半微小试管 (每样品3技术复制)。将分光光度计设置为 490 nm。在阅读标准或未知样品之前, 校准到含有蒸馏水的空白试管, 并间歇性地贯穿样品读数。通过分光光度计运行每个试管并记录吸光度。每个未知示例的跨技术复制的平均值。 注: 在某些情况下, 样品可能超过上吸光度阈值。如有, 应在步骤3.2.3 中稀释样品。稀释还必须在随后的计算中加以核算。</li>
		<li>计算每个标准的平均吸光度。 注: 要计算每一个未知样品中的总可消化碳水化合物的含量, 使用标准曲线, 最容易的是将每种标准的平均吸光度从每个标准中的 D (+) 葡萄糖的微克中退去, 但也可以如果需要, 绘制每个标准的 D (+) 葡萄糖浓度 (µg/µL)。但是, 下面的计算是指基于微克的标准曲线, 而不是浓度 (µg/µL)。</li>
		<li>通过绘制每个标准溶液中 D (+) 葡萄糖 (µg) 总量的平均吸光度计算标准曲线。将线性回归线与这些数据匹配, 然后使用以下公式计算每个未知样本中的碳水化合物总量 (µg): Mc = ((Ax -b)/米)/(15)) * 1000 样品中碳水化合物的µg x = 未知样本的平均吸光度 b = y 截距 (标准回归线) m = 斜率 (标准回归线) 该线的相关系数应大于 0.98, 通常接近0.99。然后, 使用以下等式确定每个样本中碳水化合物的百分比: Pc = ((mc/1000)/(mi)) * 100 Pc =%carbohydrates Mi = 样品的初始质量 (毫克)</li>
	</ol>
</li>
</ol>

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A reappraisal of its definition and determination. Variation according to species and seed protein content.</a> Journal of Agricultural and Food Chemistry. 38, 18-24 (1990).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Jones, C. G., Hare, J. D., Compton, S. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food carbohydrates: Chemistry, physical properties, and applications. , (2005).</li><li>Chow, P. S., Landh&#228;usser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+routine+measurements+of+total+sugar+and+starch+content+in+woody+plant+tissues.">A method for routine measurements of total sugar and starch content in woody plant tissues.</a> Tree Physiology. 24 (10), 1129-1136 (2004).</li><li>Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., Lee, Y. 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P., Berding, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+applications+of+near+infrared+reflectance+spectroscopy-+a+tool+for+rapid,+cost-effective+prediction+of+the+composition+of+plant+and+animal+tissues+and+aspects+of+animal+performance.">Ecological applications of near infrared reflectance spectroscopy- a tool for rapid, cost-effective prediction of the composition of plant and animal tissues and aspects of animal performance.</a> Oecologia. 116 (3), 292-305 (1998).</li><li>Kokaly, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+a+physical+basis+for+spectroscopic+estimates+of+leaf+nitrogen+concentration.">Investigating a physical basis for spectroscopic estimates of leaf nitrogen concentration.</a> Remote Sensing of Environment. 75 (2), 153-161 (2001).</li><li>Schulz, H., Baranska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+quantification+of+valuable+plant+substances+by+IR+and+Raman+spectroscopy.">Identification and quantification of valuable plant substances by IR and Raman spectroscopy.</a> Vibrational Spectroscopy. 43 (1), 13-25 (2007).</li><li>Cozzolino, D., Mor&#243;n, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+near-infrared+reflectance+spectroscopy+to+analyse+soil+chemical+and+physical+characteristics.">The potential of near-infrared reflectance spectroscopy to analyse soil chemical and physical characteristics.</a> The Journal of Agricultural Science. 140, 65-71 (2003).</li><li>Simpson, S. J., Sword, G. A., Lorch, P. D., Couzin, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibal+crickets+on+a+forced+march+for+protein+and+salt.">Cannibal crickets on a forced march for protein and salt.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (11), 4152-4156 (2006).</li><li>Lihoreau, M., Buhl, J., Sword, G. A., Raubenheimer, D., Simpson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+ecology+beyond+the+individual:+a+conceptual+framework+for+integrating+nutrition+and+social+interactions.">Nutritional ecology beyond the individual: a conceptual framework for integrating nutrition and social interactions.</a> Ecology Letters. 18 (3), 273-286 (2015).</li><li>Deans, C. A., Behmer, S. T., Tessnow, A., Tamez-Guerra, P., Pusztai-Carey, M., Sword, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrition+affects+insect+susceptibility+to+Bt.">Nutrition affects insect susceptibility to Bt.</a> Scientific Reports. 7, 39705 (2017).</li></ol>

作者没有什么可透露的。

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 ...

quantifying-plant-soluble-protein-digestible-carbohydrate-content

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

19.7K 次观看.  Texas A&M University. 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。

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

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

科研

环境科学

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

成绩单及代谢物谱烟草树和杨树的评价作为原料的生物基工业

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Method to Produce Durable Pellets at Lower Energy Consumption Using High Moisture Corn Stover and a Corn Starch Binder in a Flat Die Pellet Mill

A major challenge in the production of pellets is the high cost associated with drying biomass from 30 to 10% (w.b.) moisture content. At Idaho National Laboratory, a high-moisture pelleting process was developed to reduce the drying cost. In this process the biomass pellets are produced at higher feedstock moisture contents than conventional methods, and the high moisture pellets produced are further dried in energy efficient dryers. This process helps to reduce the feedstock moisture content by about 5-10% during pelleting, which is mainly due to frictional heat developed in the die. The objective of this research was to explore how binder addition influences the pellet quality and energy consumption of the high-moisture pelleting process in a flat die pellet mill. In the present study, raw corn stover was pelleted at moistures of 33, 36, and 39% (w.b.) by addition of 0, 2, and 4% pure corn starch. The partially dried pellets produced were further dried in a laboratory oven at 70 &#176;C for 3-4 hr to lower the pellet moisture to less than 9% (w.b.). The high moisture and dried pellets were evaluated for their physical properties, such as bulk density and durability. The results indicated that increasing the binder percentage to 4% improved pellet durability and reduced the specific energy consumption by 20-40% compared to pellets with no binder. At higher binder addition (4%), the reduction in feedstock moisture during pelleting was &#60;4%, whereas the reduction was about 7-8% without the binder. With 4% binder and 33% (w.b.) feedstock moisture content, the bulk density and durability values observed of the dried pellets were &#62;510 kg/m3 and &#62;98%, respectively, and the percent fine particles generated was&#160;reduced to &#60;3%.

In this study, a protocol was developed to produce good quality pellets using a flat die pellet mill at reduced specific energy consumption testing high-moisture corn stover and a starch based binder. The results indicated that adding a corn starch binder improved the pellet durability, reduced percent fines and decreased specific energy consumption.

法生产在降低能耗耐用颗粒使用高水分玉米秸秆和玉米淀粉粘合剂在平模制粒机

A major challenge in the production of pellets is the high cost associated with drying biomass from 30 to 10% (w.b.) moisture content. At Idaho National ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

玉米加工参数和营养品质分析的高通量、微尺度协议 (玉蜀黍)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

海上双壳贝类悬浮饲料定量装置的设计与应用

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. Eye-tracking informatics are known to connect to a range of feelings, health conditions, mental states and actions. But because vision is the result of constant eye-movements, teasing out what is important from what is noise is complex and data intensive. Furthermore, a significant challenge is controlling for what people look at compared to what is presented to them.
The following presents a methodology for combining and analyzing eye-tracking on a video of a natural and complex scene with a machine learning technique for analyzing the content of the video. In the protocol we focus on analyzing data from filmed videos, how a video can be best used to record participants&#39; eye-tracking data, and importantly how the content of the video can be analyzed and combined with the eye-tracking data. We present a brief summary of the results and a discussion of the potential of the method for further studies in complex environments.

The objective of the protocol is to detail how to collect video data for use in the laboratory; how to record eye-tracking data of participants looking at the data and how to efficiently analyze the content of the videos that they were looking at using a machine learning technique.

将眼动追踪数据与从自由观看城市公园环境中的步行视频中获得的视频内容进行分析

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

利用本地绿色凝胶电泳分离菠菜类黄体蛋白配合物, 用时间相关单光子计数表征

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Manufacturing Simple and Inexpensive Soil Surface Temperature and Gravimetric Water Content Sensors

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. However, at the soil surface these variables are highly dynamic and standard sensors do not explicitly measure temperature or moisture in the upper few millimeters of the soil profile. This paper describes methods for manufacturing simple, inexpensive sensors that simultaneously measure the temperature and moisture of the upper 5 mm of the soil surface. In addition to sensor construction, steps for quality control, as well as for calibration for various substrates, are explained. The sensors incorporate a Type E thermocouple to measure temperature and assess soil moisture by measuring the resistance between two gold-plated metal probes at the end of the sensor at a depth of 5 mm. The methods presented here can be altered to customize probes for different depths or substrates. These sensors have been effective in a variety of environments and have endured months of heavy rains in tropical forests as well as intense solar radiation in deserts of the southwestern U.S. Results demonstrate the effectiveness of these sensors for evaluating warming, drying, and freezing of the soil surface in a global change experiment.

Accurately measuring temperature and water content of the upper 5 mm of the soil surface can improve our understanding of environmental controls on biological, chemical, and physical processes. Here we describe a protocol for manufacturing, calibrating, and conducting measurements with soil surface temperature and moisture sensors.

制造简单、廉价的土壤表面温度和重力含水量传感器

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. ...