Name: analysis and specification of starch granule size distributions
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这项工作得到了合作农业研究中心和农业与人文科学学院综合粮食安全研究中心、草原景观A&M大学的部分支持。我们感谢华天的技术支持。

1. 淀粉样品的准备
<ol><li>按照既定程序（例如，土豆15、甜薯28、小麦粒13、29、玉米仁30等），从各种植物物种的淀粉积累组织中准备两个（或三个）克级的复制淀粉样品。</li>
	<li>用丙酮或甲苯彻底清洗淀粉样品 3-4 倍，以最大限度地减少颗粒聚合并完全干燥。 注：使用每次制剂产生超过1克淀?...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+starch+granule+size+distribution+between+hard+and+soft+wheat+cultivars+in+Eastern+China.">Comparison of starch granule size distribution between hard and soft wheat cultivars in Eastern China.</a> Agricultural Sciences China. 7 (8), 907-914 (2008).</li><li>Park, S. H., Wilson, J. D., Seabourn, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+granule+size+distribution+of+hard+red+winter+and+hard+red+spring+wheat:+Its+effects+on+mixing+and+breadmaking+quality.">Starch granule size distribution of hard red winter and hard red spring wheat: Its effects on mixing and breadmaking quality.</a> Journal of Cereal Science. 49 (1), 98-105 (2009).</li><li>Limpert, E., Stahel, W. A., Abbt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Log-normal+distributions+across+the+sciences:+keys+and+clues.">Log-normal distributions across the sciences: keys and clues.</a> Bioscience. 51 (5), 341-352 (2001).</li><li>Gao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-preserving+lognormal+volume-size+distributions+of+starch+granules+in+developing+sweetpotatoes+and+modulation+of+their+scale+parameters+by+a+starch+synthase+II+(SSII).">Self-preserving lognormal volume-size distributions of starch granules in developing sweetpotatoes and modulation of their scale parameters by a starch synthase II (SSII).</a> Acta Physiologiae Plantarum. 38 (11), 259 (2016).</li><li>Wattebled, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STA11,+a+Chlamydomonas+reinhardtii+locus+required+for+normal+starch+granule+biogenesis,+encodes+disproportionating+enzyme.+Further+evidence+for+a+function+of+alpha-1,4+glucanotransferases+during+starch+granule+biosynthesis+in+green+algae.">STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis, encodes disproportionating enzyme. Further evidence for a function of alpha-1,4 glucanotransferases during starch granule biosynthesis in green algae.</a> Plant Physiology. 132 (1), 137-145 (2003).</li><li>Ji, Y., Seetharaman, K., White, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+Small-Scale+Corn-Starch+Extraction+Method+for+Use+in+the+Laboratory.">Optimizing a Small-Scale Corn-Starch Extraction Method for Use in the Laboratory.</a> Cereal Chemistry. 81 (1), 55-58 (2004).</li><li>Halloy, S., Whigham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lognormal+as+universal+descriptor+of+unconstrained+complex+systems:+a+unifying+theory+for+complexity.">The lognormal as universal descriptor of unconstrained complex systems: a unifying theory for complexity.</a> Proceedings of the 7th Asia-Pacific Complex Systems Conference. , 309-320 (2004).</li><li>Furusawa, C., Suzuki, T., Kashiwagi, A., Yomo, T., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquity+of+log-normal+distributions+in+intra-cellular+reaction+dynamics.">Ubiquity of log-normal distributions in intra-cellular reaction dynamics.</a> Biophysics (Nagoya-shi). 1, 25-31 (2005).</li></ol>

概述的程序解决了淀粉颗粒大小分析的几种现有方法中的一些关键问题， 包括 3D 颗粒的不当 1D 或 2D 尺寸、由于颗粒形状不均匀而导致尺寸测量失真、由于颗粒样本大小有限而重复性差和统计有效性可疑、颗粒大小不准确或不当（特别是使用平均尺寸）的颗粒大小存在颗粒形状和非正常大小分布。它使用 ESZ 技术测量淀粉颗粒的 3D 大小（体积），对颗粒形状无响应。从具有非常大颗粒样本大小（...

淀粉颗粒是植物光合作用和储存组织中的两种主要储备同质聚合物、线性或稀疏分支淀粉样蛋白和高度分支的淀粉样蛋白，与一些小成分（包括脂质和蛋白质）有序地包装在一起的物理结构。来自各种植物物种的淀粉颗粒呈现出许多三维（3D）形状（参考1、2），包括球体、椭圆体、多壳体、血小板、立方体、立方体和不规则的管状物。即使是来自同一植物物种的同一组织或不同组织的组织，也可能有一组不同发生频率的形状。换句话说，来自植物物种的淀粉颗粒可能有一个特征的统计形状分布，而不是一个特定的形状。非均匀和非球形颗粒形状使得难以正确测量和定义淀粉颗粒大小。此外，来自植物物种同一组织的淀粉颗粒具有不同比例的大小，即表现出特征大小分布。这种大小分布使淀粉颗粒大小的分析和描述进一步复杂化。
淀粉颗粒尺寸已使用几类颗粒大小技术进行分析（在参考3中回顾），包括显微镜、沉积/星体场流分馏（Sd/StFFF）、激光衍射和电传感区（ESZ）。然而，这些技术并不同样适合在颗粒形状和大小分布存在的情况下确定淀粉颗粒大小。显微镜，包括光，共生和扫描电子显微镜，是优秀的形态学研究4，5，6，7，结构8，9和发展10，11淀粉颗粒，但很难定义其大小分布由于一些固有的缺陷。光学显微镜数据（IAOM）的微小颗粒图像或软件辅助图像分析的直接测量，这些数据已用于确定来自几个物种的淀粉颗粒大小， 包括玉米12，小麦13，14，土豆15和大麦16，只能测量1D（通常最大长度）或2D（表面积）大小非常有限的数量（数万至几千）淀粉颗粒图像。考虑到淀粉单位重量的巨大颗粒数（假设所有 10 μm 球体的密度为1.5 g/cm3），固有的受技术约束的小颗粒采样尺寸在统计学上很少具有代表性，因此可能导致结果的可重复性差。Sd/StFFF技术可能具有高速和分辨率，淀粉颗粒17的窄尺寸部分，但很少使用，可能是因为它的准确性可能受到损害，不同的形状和淀粉颗粒密度的严重影响。激光衍射技术是应用最广泛的技术，已应用于淀粉颗粒大小分析的所有主要作物品种3，14，16。虽然该技术有许多优点，但它实际上并不适合在颗粒形状分布的情况下确定淀粉颗粒大小。大多数并发激光衍射仪器依靠米光散射理论18来制造均匀的球形粒子，而修改后的Mie理论18依赖于其他一些均匀形状的粒子。因此，该技术本身对颗粒形状非常敏感，甚至不完全适合某些均匀性19的形状，更不用说淀粉颗粒具有一组不同比例的形状了。ESZ 技术测量电场扰动与通过孔径的粒子体积成正比。它以高分辨率提供颗粒体积大小、数量和体积分布信息等。由于ESZ技术独立于粒子的任何光学特性，包括颜色、形状、成分或折射指数，结果非常可重复，因此特别适合确定具有一组形状的淀粉颗粒的大小分布。
淀粉颗粒大小也通过使用许多参数来定义。它们通常用平均直径简单描述，在某些情况下，平均直径是2D图像12、20或等值球直径3平均最大长度的算术手段。在其他情况下，颗粒大小分布使用大小范围21，22，分布平均体积或平均直径（球面等价物，按数字、体积或表面积加权）来指定，假设正常分布 14、23、24、25、26。这些描述的淀粉颗粒大小从各种分析是一个完全不同的性质，并没有严格的可比性。如果直接比较来自不同物种的淀粉颗粒的这些"大小"，甚至同一物种的相同组织，可能会非常具有误导性。此外，在大多数研究中，测量分布宽度（即大小差）的标准偏差σ（或图形标准偏差σg）的公差（或形状）参数被忽略。
为了解决淀粉颗粒大小分析面临的上述关键问题，我们概述了使用 ESZ 技术对淀粉样品颗粒大小分布进行可重复且统计上有效的确定的程序，并使用采用的两参数倍增形式27正确指定已确定的颗粒日体大小分布，提高了准确性和可比性。为了验证和演示，我们使用该程序对甜品淀粉样品进行了复制颗粒大小分析，并使用其图形几何手段 <img alt="Equation 1" src="/files/ftp_upload/61586/61586eq1.jpg"> 和乘法标准偏差s* 以 <img alt="Equation 1" src="/files/ftp_upload/61586/61586eq1.jpg"> x/（乘以除法） s* 形式指定日志异常差量百分比体积等价球直径分布。

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			<td height="21" style="height:21px;width:261px;">Analytical beaker</td>
			<td style="width:211px;">Beckman Coulter Life Sciences</td>
			<td style="width:171px;">A35595</td>
			<td style="width:359px;">Smart-Technology (ST) beaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aperture tube, 100 &#181;m</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>A36394</td>
			<td>For the MS4E, , 1000 &#181;m</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable transfer pipettor,</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td style="width:171px;">13-711-9AM</td>
			<td style="width:359px;">Other disposable transfer pipettors with similar orifice can also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Fisherbrand Conical Polypropylene Centrifuge Tubes, 50 ml</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td>05-539-13</td>
			<td>Any other similar types of tubes can be used.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:261px;">Glass beakers, 150 to 250 ml</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td>02-540K</td>
			<td style="width:359px;">These beakers are used to contain methanol for washing the aperture tube and stirer between runs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">LiCl</td>
			<td style="width:211px;">Fisher Chemical</td>
			<td>L121-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol</td>
			<td>Fisher Chemical</td>
			<td>A412-500</td>
			<td>Buy in bulk as the analysis uses a large quantity of methanol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mettler Toledo ML-T Precision Balances</td>
			<td>Mettler Toledo</td>
			<td style="width:171px;">30243412</td>
			<td style="width:359px;">Any other precision balance with a readablity 0.01 g to 1 mg will work.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Multisizer 4e Coulter Counter</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>B23005</td>
			<td style="width:359px;">The old model, Multisizer 3 can also be used with slight adjustment of parameters. The 4e model comes with a 100 &#956;m aperture tube. Other aperture tubes of different diameter can be purchased separately from the company.</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;">Ultrasonic processor UP50H</td>
			<td>Hielscher Ultrasound Technology</td>
			<td>UP50H</td>
			<td style="width:359px;">Other laborator sonicator having a low-power (&#60;50 Watt) output can be also used. Both MS1 and MS2 sonotrodes for the particular sonicator can be used to disperse starch granules in 5 ml methanol. Always use the lowest setting first, 20% amplitude and 0.1 or 0.2 cycle, and raise the setting if aggregates persist in suspension.</td>
		</tr>
	</tbody>
</table>

analysis and specification of starch granule size distributions

来自所有植物来源的淀粉由颗粒组成，其大小和形状具有不同的发生频率，即显示大小和形状分布。 使用几种类型的颗粒大小技术确定的淀粉颗粒大小数据通常是有问题的，因为一些不可逾越的系统错误导致的可重复性差或缺乏统计意义，包括对颗粒形状的敏感性和颗粒样本大小的限制。我们概述了使用电传感区技术可重复和统计有效确定淀粉颗粒大小分布的程序，以及使用采用的两参数乘法来指定已确定的颗粒日志正常大小分布的程序，提高了准确性和可比性。它适用于克级淀粉样品的所有颗粒大小分析，因此，可以促进淀粉颗粒大小如何由淀粉生物合成装置和机制成型的研究：以及它们如何影响淀粉用于食品和工业用途的特性和功能。通过使用概述的程序对甜品淀粉样品的颗粒大小分布进行复制分析，可以呈现具有代表性的结果。我们进一步讨论了该过程的几个关键技术方面，特别是颗粒日志正常大小分布的倍增规格，以及克服颗粒聚合频繁孔径堵塞的一些技术手段。

为了验证该过程，并证明已确定颗粒大小分布的可重复性，我们对甜品淀粉样品进行了复制大小分析。我们准备复制（S1和S2）淀粉样品从田间种植的甜味品的育种线SC1149-19在类似的发育年龄使用先前描述的程序28。从每个淀粉提取物中，抽取两个0.5克的aliquot（a和b），悬浮在5 mL的甲醇中，用几脉冲低能超声波进行声波，以分解聚合物。两对淀粉-甲醇悬架中的每一对都经过滴样?...

Watch this Scientific Journal Video about Analysis and Specification of Starch Granule Size Distributions at JoVE.com

Analysis and Specification of Starch Granule Size Distributions

此处介绍了淀粉颗粒大小分布的可重复和统计有效确定过程，以及使用双参数乘法指定已确定的颗粒日志正常大小分布。它适用于植物和食品科学研究的克级淀粉样品的所有颗粒大小分析。

淀粉颗粒大小分布的分析和规格

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...

该技术可重复和统计有效地确定淀粉颗粒大小，并提供统计分布颗粒大小的正确描述。该技术确定淀粉颗粒体积，独立于其形状，并使用统计上有效的颗粒样本大小进行大小分布，并提供颗粒大小分布的改进规格。这种方法适用于植物和食品科学研究，涉及淀粉颗粒大小，除了任何其他淀粉应用，需要颗粒大小信息。在开始分析之前，在500毫升甲醇中溶解25克氯化锂，并选择一个直径范围覆盖淀粉样品已知颗粒大小范围的孔径管进行分析。要设置标准操作方法，请启动分析器软件并在主菜单中单击 SOP 并创建 SOM 向导并选择适当的设置，然后单击编辑 SOM 并选择适当的设置。要在 SOP 下选择首选项文件，请单击创建首选项向导并选择适当的首选项设置，然后单击创建 SOP 向导，输入描述和作者，并选择 SOM 和首选文件以创建和保存 SOP。要设置分析仪进行分析，请打开分析仪并验证分析器软件中的现成状态。将电解质罐中填充以前制备的电解质，并在必要时清空废罐。根据用户手册中的指南安装并固定所选的孔径管，并按锁定释放夹解锁检测平台。手动将平台降低到底部，并将包含 100 毫升电解质的分析烧杯放在平台上。将搅拌器移到搅拌位置，手动将平台提升到自锁的上部位置，将孔径管和搅拌器浸入电解质中。单击底部仪表工具栏，自动用电解质填充系统，然后单击冲洗以自动冲洗系统，然后单击 SOP 并加载 SOM 以加载标准操作方法，单击示例并输入样本信息以输入运行中的示例信息。要准备淀粉甲醇样品尺寸悬架，从两到三个复制淀粉提取物中各重一到两个0.5克样品，并在50毫升锥形离心管中加入每份五毫升甲醇。使用超声波处理器的几次低强度超声波脉冲来完全分散淀粉颗粒，并使用一次性转移移液器将第一个淀粉甲醇悬浮物的约200微升应用到烧杯中不断搅拌的100毫升氯化锂甲醇电解质上，然后关闭样品舱门，点击预览开始预览运行。在状态面板中，验证动态显示的浓度条是否为绿色，并显示悬架的 5% 到 8% 名义浓度范围。单击"停止"以停止预览，然后单击"开始"以开始分析。一旦大小颗粒的总计数（在运行中显示与状态面板上的运行时间一起显示）达到 SOM 控制模式设置的总计数，分析仪将自动完成运行。要使用相同的淀粉电解质悬架执行技术重复运行，请单击底部工具栏上的启动或重复。在分析结束时，清空烧杯，用甲醇冲洗，并补充100毫升新鲜电解质溶液，用于分析下一个样品。如果使用 SOM 来控制运行，则使用创建首选项向导根据需要选择首选项设置，以便查看、打印和统计分析结果。要在单个图形上覆盖多个运行的结果，请在覆盖窗口中选择要叠加的结果。单击"添加"将文件移动到选定的文件框，然后单击"确定"，在单个图形上叠加所选结果。要将文件添加到打开的叠加，请单击运行文件并打开覆盖以访问覆盖窗口，选择所需的文件，然后单击以添加。单击文件、文件工具和平均值以打开平均窗口，导航到并选择文件框中的多个预期结果文件。单击"添加"将文件移动到选定的文件框，然后单击"确定"以平均所选结果以显示在单个图形中。要在平均分布中包含额外的结果文件，请单击运行文件并打开并添加到平均值以打开添加到平均窗口并添加感兴趣的文件。新的平均值将显示在运行窗口中的图形上。要获取指定平均分布的图形几何平均值和标准偏差，请单击计算和平均统计以打开统计摘要窗口以显示数据的手段和标准偏差。表的第三行显示用于指定平均分布的图形几何平均值和标准偏差。第二栏显示平均分布的统计数据。在这里，显示了代表性红薯淀粉样品的四个复制大小分析及其平均分布的微分体积百分比等效球径分布。在这个数字中，可以观察到四个复制大小分析的平均累积数和体积百分比大小分布，这些分析是平均差量百分比大小分布的转换视图。比较这些淀粉颗粒样本的累计数量和体积百分比，发现体积较小的等效球径颗粒占总计数的百分比远远大于总体积。重要的是要尽量减少淀粉样品和检测悬浮物中的淀粉颗粒聚合物，并达到淀粉电解质悬浮物5%至8%名义浓度范围。还可进行淀粉样品物理、化学和热特性的分析，以及将颗粒大小与源植物中的淀粉合成特性联系起来。

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Extraction and Analysis of Taiwanese Green Propolis

台湾绿蜂胶的提取与分析

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...