Name: estimation of plant biomass lignin content using thioglycolic acid (tga)
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Description: Watch this Scientific Journal Video about Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA at JoVE.com

我们感谢植物与土壤科学与棉花公司对这项研究的部分支持。

1. 植物样本的制备
<ol><li>从温室收集两个月大的棉花植物（图1A）。</li>
	<li>通过松开植物周围的土壤（图1B），轻轻翻转植物盆，将土壤和根部与完整的横向根分开。</li>
	<li>将收集的植物彻底清洗在装满水的托盘中，以清除所有污垢（用于根部样本）（图1C）。</li>
	<li>使用纸巾干燥分离的根、茎和叶组织，并贴上标签（<strong ...

<ol>
	<li>Freudenberg, K., Neish, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Constitutionand Biosynthesis of Lignin. , 129 (1968).</li><li>Chio, C., Sain, M., Qin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+utilization:+A+review+of+lignin+depolymerization+from+various+aspects.">Lignin utilization: A review of lignin depolymerization from various aspects.</a> Renewable and Sustainable Energy Reviews. 107, 232-249 (2019).</li><li>Sun, Z., Fridrich, B., de Santi, A., Elangovan, S., Barta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+Side+of+Lignin+Depolymerization:+Toward+New+Platform+Chemicals.">Bright Side of Lignin Depolymerization: Toward New Platform Chemicals.</a> Chemical Reviews. 118, 614-678 (2018).</li><li>Xu, F., Sun, R. 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木质素在植物生长发育中起着重要作用，最近已广泛研究生物燃料、生物能源和生物制品的应用。木质素富含芳香化合物，储存在所有血管植物的二级细胞壁中。它具有多种工业应用，如木板产品，生物分散剂，絮凝剂，聚氨酯泡沫和在电路板29，30，31的脂。大部分来自纸张和纸浆行业的木质素被作为废物释放或燃烧用于热生...

木质素是植物细胞壁的重要承重成分之一，也是地球上第二丰富的聚合物1。从化学上讲，木质素是一种由高分子量复合酚类化合物组成的交联异质体，形成芳香聚合物的天然可再生来源，并合成生物材料2、3。这种天然聚合物在植物生长、发育、生存、机械支持、细胞壁刚性、水运、矿物运输、耐食性、组织和器官发育、能量沉积以及生物和生物应力4、5、6、7等方面起着重要作用。利格宁主要由三种不同的单酚组成：针叶醇、西纳皮尔和p-coumaryl酒精，它们来自苯丙酮通路8，9。木质素的数量和单体的组成因植物种类、组织/器官类型和植物发育的不同阶段而异。根据来源和单酚成分，木质素大致分为软木、硬木和草木质素。软木主要由95%的针叶醇和4%的p-coumaryl和1%的甲基醇组成。硬木有针叶醇和甲基醇的比例相等，而草木质素是由针叶醇，锡那皮尔和p-coumaryl酒精11，12的不同比例组成。单体的组成至关重要，因为它决定了细胞壁的木质素强度、分解和降解，以及确定分子结构、分支和与其他多糖13、14的交叉连接。
由于其成本低、丰度高，在觅食、纺织业、造纸业以及生物乙醇、生物燃料和生物制品方面，木质素研究越来越受到重视。各种化学方法（如乙酰溴化物、酸洗涤剂、克拉森和高锰酸盐氧化）以及仪器方法（如近红外（NIR）光谱、核磁共振（NMR）光谱和紫外线（UV）光谱仪）用于木质素定量9、17。木质素的分析方法一般根据电磁辐射、重力和溶解度进行分类。电磁辐射对木质素的估计原理是基于木质素的化学特性，木质素吸收特定波长的光。这些结果的估计依据是，木质素比碳水化合物具有更强的紫外线吸收性。1962年，博尔克和萨默维尔使用氯化钾颗粒来估计木材中的木质素含量。然而，由于存在非木质素酚类化合物，且缺乏适当的灭绝系数，这种方法在草本样品中木质素含量的估计存在缺陷。1970年，弗格斯和戈林发现瓜亚西尔和注射器的复合吸收最大度为280纳米和270纳米，这纠正了博尔克和萨默维尔方法19的灭绝系数问题。后来，红外光谱学，一种高度敏感的表诺菌特征技术，也用于木质素估计与少量植物生物质样品。这种技术的一个例子是扩散反射傅立雅转换光谱仪。然而，这种方法缺乏一个类似于紫外线方法20的适当标准。后来，NIRS（近红外光谱）和NMR（核磁共振光谱）估计了木质素含量。虽然这些方法有缺点，但它们不会改变木质素的化学结构，保持其纯度20。
重度克拉森法是木质茎木质素估计的直接和最可靠的分析方法。重力木质素估计的基础是非木质化合物的水解/溶解和重力21不溶性木质素的收集。在这种方法中，通过浓缩H2SO4对生物量进行水解，提取木质素残留物20、22。。这种方法估计的木质素含量称为酸不溶性木质素或克拉森木质素。克拉森方法的应用取决于植物种类、组织类型和细胞壁类型。单宁、多糖和蛋白质等非木质素成分的可变量存在，导致酸不溶性/可溶性木质素含量的估计存在成比例差异。因此，只建议采用克拉森法，用于木质素对木质茎等高木质物质含量生物量的估计。溶解性方法，如乙酰溴化物（AcBr）、酸不溶性木质素和硫二甘醇酸（TGA），是估计各种植物生物质来源木质素含量的最常用方法。Kim等人建立了两种通过溶解提取木质素的方法。第一种方法通过溶解纤维素和半纤维素提取木质素作为不溶性残留物，而第二种方法将木质素分离在可溶性部分，将纤维素和血细胞素作为不溶性残留物24。
在木质素估计中采用的类似方法基于溶解性是硫二甲酸 （TGA） 和乙酰溴化物 （AcBr） 方法25.TGA和乙酰溴化物方法均通过测量溶解木质素在280纳米的吸收量来估计木质素含量：然而，AcBr方法在木质素溶解过程中降解木兰，并显示木质素含量26的虚假增加。硫二甲酸酯 （TGA） 方法是更可靠的方法，因为它取决于与木质素的苯甲酸酯醇组与 TGA 的具体结合。TGA结合木质素是在酸性条件下使用HCl沉淀的，木质素的吸收量估计为280nm27。TGA 方法具有结构修饰较少、木质素估计可溶性、非木质素成分干扰少以及与 TGA 特定结合导致木质素精确估计等额外优点。
这种TGA方法是根据用于木质素含量估算的植物生物质样品的种类进行修改的。在这里，我们修改和调整了快速TGA方法的稻草27棉花组织估计木质素含量。简言之，干粉植物样品经过蛋白质溶解缓冲和甲醇提取，以去除蛋白质和酒精可溶性成分。酒精不溶性残留物在酸性条件下用TGA和沉淀木质素处理。使用商业竹木质素生成木质素标准曲线，并获得回归线（y = mx+c）。"x"值使用280纳米的木质素平均吸收值，而"m"和"c"值则从回归线输入，以计算棉花植物生物质样品中未知的木质素浓度。该方法分为五个阶段：1）植物样品的制备：2） 用水和甲醇清洗样品：3） 用TGA和酸处理颗粒，沉淀木质素：4） 木质素降水：和 5） 样品的标准曲线制备和木质素含量估算。前两个阶段主要侧重于植物材料制备，其次是水、PSB（蛋白质溶解缓冲器）和甲醇提取，以获得酒精不溶性材料。然后，它与TGA（硫二甘油酸）和HCl一起在第三阶段形成木质素复合物。最后，HCl被用来沉淀木质素，木质素溶解在氢氧化钠中，以测量其吸收量在280纳米28。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BioSpectrophotometer kinetic</td>
			<td>Eppendorf kinetic</td>
			<td>6136000010</td>
			<td>For measuring absorbance at 280 nm</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>For centrifuging&#160; samples</td>
		</tr>
		<tr>
			<td>Commercial bamboo lignin</td>
			<td>Aldrich</td>
			<td>1002171289</td>
			<td>Used in the preparation of the standard curve</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Fischer Scientific</td>
			<td>16690382</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>VWR</td>
			<td>734-0448</td>
			<td>Containers for solutions</td>
		</tr>
		<tr>
			<td>Freezer mill</td>
			<td>Spex Sample Prep</td>
			<td>68-701-15</td>
			<td>For fine grinding of plant tissue samples</td>
		</tr>
		<tr>
			<td>Heat block/ Thermal mixer</td>
			<td>Eppendorf</td>
			<td>13527550</td>
			<td>For temperature controlled steps during lignin extraction</td>
		</tr>
		<tr>
			<td>Hotplate stirrer</td>
			<td>Walter</td>
			<td>WP1007-HS</td>
			<td>Used for preparation of solutions</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCL)</td>
			<td>Sigma</td>
			<td>221677</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisherbrand</td>
			<td>150152633</td>
			<td>For thorough drying of plant tissue samples</td>
		</tr>
		<tr>
			<td>Measuring scale</td>
			<td>Mettler toledo</td>
			<td>30243386</td>
			<td>For measuring plant tissue weight, standards and microfuge tubes</td>
		</tr>
		<tr>
			<td>Methanol (100 %)</td>
			<td>Fischer Scientific</td>
			<td>67-56-1</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Microfuge tubes (2 mL)</td>
			<td>Microcentrifuge</td>
			<td>Z628034-500EA</td>
			<td>Containers for extraction of lignin</td>
		</tr>
		<tr>
			<td>Plant biomass gerinder</td>
			<td>Hanchen</td>
			<td>Amazon</td>
			<td>Used for crushing dried samples</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher Scientific</td>
			<td>AE150</td>
			<td>Measuring pH for solutions prepared for lignin extraction</td>
		</tr>
		<tr>
			<td>Temperature controlled incubator/oven</td>
			<td>Fisher Scientific</td>
			<td>15-015-2633</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Thioglycolic acid (TGA)</td>
			<td>Sigma Aldrich</td>
			<td>68-11-1</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Vacuum dryer</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>Used for drying samples</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Eppendorf</td>
			<td>3340001</td>
			<td>For proper mixing of samples</td>
		</tr>
	</tbody>
</table>

estimation of plant biomass lignin content using thioglycolic acid (tga)

木质素是一种天然聚合物，是地球上仅次于纤维素的第二富足聚合物。木质素主要沉积在植物二级细胞壁中，是一种芳香异质体，主要由三种具有重要工业意义的单酚组成。木质素在植物生长发育中起着重要作用，在动物饲料、木材和工业木质素产品的质量方面具有重要的保护作用。准确估计木质素含量对于基本了解木质素生物合成和生物质的工业应用都至关重要。硫二甘油酸（TGA）方法是估算植物生物量中木质素总含量的高度可靠的方法。这种方法通过与木质素的苯酒精群形成硫化物来估计木质素的含量，这种醇组在碱性条件下是可溶性的，在酸性条件下不溶性。木质素总含量使用商业竹木质素生成的标准曲线进行估算。

比较了两条不同的棉花实验线，以弥补不同组织中木质素含量的差异。每个样本提取的木质素含量测量为280纳米，并记录其各自的吸收值。将每个生物复制品的平均吸收值与木质素标准曲线的回归线（表2，图3C）进行比较。回归线，y = mx + c，用于计算提取实验线、样本1和样品2的未知木质素含量。平均OD值的结果被替换在"x"中，而"m"和"c"值则从木质素标准曲线的...

Watch this Scientific Journal Video about Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA at JoVE.com

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

在这里，我们提出了一个修改后的TGA方法，用于估计草本植物生物质中的木质素含量。这种方法通过与木质素形成特定的硫化物键来估计木质素含量，并且比克拉森方法具有优势，因为它需要相对较小的木质素含量估计样本。

使用硫二甘酸（TGA）估计植物生物质木质素含量

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

利格宁是一种复杂的异质体，由三种单酚组成。它是地球上仅次于纤维素的第二大富合物。在这里，我们演示了硫化物酸方法，这是估计木质素含量的最可靠方法。这种方法基于木质素通过硫酸结合硫二甲酸的原则。此外，我们把该协议分为五个阶段。在第一阶段，我们准备植物材料。在第二阶段，我们分离酒精不溶性提取物。在第三阶段，我们使用硫化物酸和酸性条件沉淀木质素。在第四阶段，我们使用工业竹木质素准备木质素标准曲线。最后，在第五阶段，我们使用第四阶段准备的标准曲线估计木质素含量。收集植物材料，翻转锅根，在水中彻底清洗，空气干燥两天，将它们转移到标记容器中，并在49摄氏度的孵化器中孵化一周至10天。使用剪刀，组织进一步切成1毫米至3毫米大小的碎片。或者生物质研磨机用于粉碎植物组织。使用根组织 打开生物质研磨机将碎粉收集到预先标记的容器中。同样，重复茎组织和叶组织。碎粉被装进研磨花瓶，然后放入冰柜磨坊，以10 CP的速度运行冷冻机三个周期。称20毫克的地面组织粉末，并转移到预制的2毫升管。在实验室笔记本中用组织粉末和组织粉做空管和管子的笔记。将带盖子在 60 摄氏度开启的管子孵化一小时。孵化后，在15，000 RPM的每个样品涡离心机中加入1.8毫升水，10分钟。丢弃超自然添加1.8毫升甲醇。在15，000 RPM孵化离心机10分钟后，在预热的60摄氏度区块中涡流和孵育20分钟。再重复一次此步骤。离心后丢弃超自然剂，在真空干燥机中干燥颗粒两到三个小时，或直到托盘完全干燥。测量并记录实验室笔记本中每个管子的重量，以估算末尾的木质素含量。还包括积极控制工业竹木质在此点开始从0.5毫克到4毫克三脚架。在每个样品漩涡中加入1毫升3种普通盐酸，以及100微升甘油酸，在80摄氏度下孵育3小时。3小时后孵化将它们转移到机架上，并让它冷却10分钟。然后离心机在 15，000 RPM 10 分钟。离心后，解决方案的颜色看起来像这样。丢弃超自然人。加入1毫升水。漩涡离心机在 15，000 RPM 10 分钟。丢弃超自然人。加入1毫升1个正常氢氧化钠。涡流和孵化在37摄氏度摇床过夜。经过一夜孵化离心机，管子在15，000 RPM下10分钟。将超自然新鲜预标记的 2 毫升管转移到超自然人中，加入 200 微升浓缩盐酸。如此处所示，通过温和的反转混合它们。在冰箱中以4摄氏度的气温进行孵化。在 15，000 RPM 的孵育离心机后，10 分钟。丢弃超自然人。加入1毫升氢氧化钠。在室温下在摇床中旋转和孵化10至15分钟。使用光谱仪收集吸收读数280纳米，将2微升氢氧化钠作为空白，因为沉淀的木质素溶解在氢氧化钠中。将空白归零。同样，重复的样品。使用每个样本中的两个微升器来收集实验室笔记本中的吸收读数。每个样本需要三个读数，用于三个技术复制。在第一列中，我们有示例编号。在第二列中，我们有生物复制，其中R1、R2、R3代表一条实验线的三个生物复制品。在第三列中，我们平均吸收读数为280纳米。我们使用公式 y=mx-c 计算了第四列中的木质素含量，其中 x 是平均 OD .m 和 c 值是从编写的标准曲线的回归线绘制的。第五列是甲醇和水洗涤后的重量。因此，这是用于估计毫克中的木质素含量的方式。因此，我们将第四列获得的价值除以第五列，以获得第六列中每毫克木质素含量的价值。并且这个值乘以1000，以获得每克细胞壁的重量。木质素含量的百分比是通过将这个值除以1000和乘以100来计算的。最后，我们通过平均每个实验线的三个生物复制品的木质素来获得平均木质素，这些生物复制品将以条形图的形式与另一条实验线的平均值进行比较，以了解木质素含量的差异。我们也可以应用学生测试来测试他们的重要性。在A列中，我们有商业竹木质素，然后是TGA之前服用的竹木质素的重量，以及它们各自在280纳米的吸收读数。表 A 中的这些值用于在 B 列中绘制一个分散的图形，从而提供带有 m 和 c 值的回归线。在C中，我们比较了使用标准曲线获得的值，工业竹木质素实验线一、二以条形图的形式进行了比较，结果显示它们之间存在差异。

Research

JoVE Journal

Biochemistry

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Determination of the Glycogen Content in Cyanobacteria

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

分光光度法测定蓝藻Synechocystis中的蛋白含量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

使用稀有的科顿富标记识别氨基酸过度生产者

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...