Name: plastoglobule lipid droplet isolation from plant leaf tissue and cyanobacteria
Uploaded: 2022-10-06T14:20:00
Description: Watch this Scientific Journal Video about Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria at JoVE.com

Lundquist实验室小组的研究得到了NSF（MCB-2034631）和美国农业部（MICL08607）对P.K.L.的资助。作者感谢Carrie Hiser博士（MSU）对蓝藻质体球分离方法开发的支持。

1.粗质体球分离
<ol><li>从非胁迫玉米叶片组织中提取粗质体球<ol><li>获得6株约3周龄且接近V5生长期的健康玉米幼苗，重约120克。</li>
		<li>剪掉茎基部的所有叶子，迅速将它们浸泡在冰浴中，然后运送到冷藏室。</li>
		<li>在绿色安全灯下工作，从冰浴中取出玉米叶，然后用剪刀将它们剪成小块（约 5 厘米 x 5 厘米）。</li>
		<li>在商用搅拌机中，在350 mL研磨缓冲液中轻轻但彻底研磨一半?...

<ol>
	<li>Lundquist, P. K., Shivaiah, K. K., Espinoza-Corral, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+throughout+the+evolutionary+tree.">Lipid droplets throughout the evolutionary tree.</a> Progress in Lipid Research. 78, 101029 (2020).</li><li>Lundquist, P. K., 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=The+functional+network+of+the+Arabidopsis+plastoglobule+proteome+based+on+quantitative+proteomics+and+genome-wide+coexpression+analysis.">The functional network of the Arabidopsis plastoglobule proteome based on quantitative proteomics and genome-wide coexpression analysis.</a> Plant Physiology. 158 (3), 1172-1192 (2012).</li><li>Ytterberg, A. J., Peltier, J. B., van Wijk, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+of+plastoglobules+in+chloroplasts+and+chromoplasts.+A+surprising+site+for+differential+accumulation+of+metabolic+enzymes.">Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes.</a> Plant Physiology. 140 (3), 984-997 (2006).</li><li>Lundquist, P. K., 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=Loss+of+plastoglobule+kinases+ABC1K1+and+ABC1K3+causes+conditional+degreening,+modified+prenyl-lipids,+and+recruitment+of+the+jasmonic+acid+pathway.">Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.</a> The Plant Cell. 25 (5), 1818-1839 (2013).</li><li>Vidi, P. A., 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=Tocopherol+cyclase+(VTE1)+localization+and+vitamin+E+accumulation+in+chloroplast+plastoglobule+lipoprotein+particles.">Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.</a> Journal of Biological Chemistry. 281 (16), 11225-11234 (2006).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+and+the+fine+structure+of+plastids.">Plastoglobuli and the fine structure of plastids.</a> Endeavour. 27 (102), 144-149 (1965).</li><li>Lichtenthaler, H. K., Peveling, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+in+different+types+of+plastids+from+Allium+cepa+L.">Plastoglobuli in different types of plastids from Allium cepa L.</a> Planta. 72 (1), 1-13 (1966).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+Plastoglobuli+von+Spinat,+ihre+Gr&#246;&#946;e,+Isolierung+und+Lipochinonzusammensetzung.">Die Plastoglobuli von Spinat, ihre Gr&#246;&#946;e, Isolierung und Lipochinonzusammensetzung.</a> Protoplasma. 68 (1-2), 65-77 (1969).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+and+lipoquinone+content+of+chloroplasts+from+Cereus+peruvianus+(L)+Mill.">Plastoglobuli and lipoquinone content of chloroplasts from Cereus peruvianus (L) Mill.</a> Planta. 87 (4), 304-310 (1969).</li><li>Simpson, D. J., Baqar, M. R., Lee, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromoplast+ultrastructure+of+Capsicum+carotenoid+mutants+I.+Ultrastructure+and+carotenoid+composition+of+a+new+mutant.">Chromoplast ultrastructure of Capsicum carotenoid mutants I. Ultrastructure and carotenoid composition of a new mutant.</a> Zeitschrift f&#252;r Pflanzenphysiologie. 83 (4), 293-308 (1977).</li><li>Hansmann, P., Sitte, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+molecular+structure+of+chromoplast+globules+of+Viola+tricolor.">Composition and molecular structure of chromoplast globules of Viola tricolor.</a> Plant Cell Reports. 1 (3), 111-114 (1982).</li><li>Steinmuller, D., Tevini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+function+of+plastoglobuli+:+I.+Isolation+and+purification+from+chloroplasts+and+chromoplasts.">Composition and function of plastoglobuli : I. Isolation and purification from chloroplasts and chromoplasts.</a> Planta. 163 (2), 201-207 (1985).</li><li>Kessler, F., Schnell, D., Blobel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+plastoglobules+isolated+from+pea+(Pisum+sativum+L.)+chloroplasts.">Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts.</a> Planta. 208 (1), 107-113 (1999).</li><li>Grennan, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobule+proteome.">Plastoglobule proteome.</a> Plant Physiology. 147 (2), 443-445 (2008).</li><li>Peramuna, A., Summers, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+occurrence+of+lipid+droplets+in+the+cyanobacterium+Nostoc+punctiforme.">Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme.</a> Archives of Microbiology. 196 (12), 881-890 (2014).</li><li>Austin, J. R., Frost, E., Vidi, P. A., Kessler, F., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobules+are+lipoprotein+subcompartments+of+the+chloroplast+that+are+permanently+coupled+to+thylakoid+membranes+and+contain+biosynthetic+enzymes.">Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes.</a> The Plant Cell. 18 (7), 1693-1703 (2006).</li><li>Eugeni Piller, L., Abraham, M., Dormann, P., Kessler, F., Besagni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+lipid+droplets+at+the+crossroads+of+prenylquinone+metabolism.">Plastid lipid droplets at the crossroads of prenylquinone metabolism.</a> Journal of Experimental Botany. 63 (4), 1609-1618 (2012).</li><li>Eugeni Piller, L., Glauser, G., Kessler, F., Besagni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+plastoglobules+in+metabolite+repair+in+the+tocopherol+redox+cycle.">Role of plastoglobules in metabolite repair in the tocopherol redox cycle.</a> Frontiers in Plant Science. 5, 298 (2014).</li><li>Xu, C., Fan, J., Shanklin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+functional+connections+between+cytoplasmic+and+chloroplast+triacylglycerol+storage.">Metabolic and functional connections between cytoplasmic and chloroplast triacylglycerol storage.</a> Progress in Lipid Research. 80, 101069 (2020).</li><li>Izquierdo, Y., Fernandez-Santos, R., Cascon, T., Castresana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplet+isolation+from+Arabidopsis+thaliana+leaves.">Lipid droplet isolation from Arabidopsis thaliana leaves.</a> Bio-Protocols. 10 (24), 3867 (2020).</li><li>Espinoza-Corral, R., Schwenkert, S., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+changes+of+Arabidopsis+thaliana+plastoglobules+facilitate+thylakoid+membrane+remodeling+under+high+light+stress.">Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress.</a> Plant Journal. 106 (6), 1571-1587 (2021).</li><li>Espinoza-Corral, R., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plastoglobule-localized+protein+AtABC1K6+is+a+Mn2+-dependent+kinase+necessary+for+timely+transition+to+reproductive+growth.">The plastoglobule-localized protein AtABC1K6 is a Mn2+-dependent kinase necessary for timely transition to reproductive growth.</a> Journal of Biological Chemistry. 298 (4), 101762 (2022).</li><li>Espinoza-Corral, R., Herrera-Tequia, A., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+topology+and+membrane+interaction+characteristics+of+plastoglobule-localized+AtFBN1a+and+AtLOX2.">Insights into topology and membrane interaction characteristics of plastoglobule-localized AtFBN1a and AtLOX2.</a> Plant Signalling &amp; Behavior. 16 (10), 1945213 (2021).</li><li>Besagni, C., Piller, L. E., Br&#233;h&#233;lin, C., Jarvis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Plastoglobules+from+Arabidopsis+Plastids+for+Proteomic+Analysis+and+Other+Studies.">Preparation of Plastoglobules from Arabidopsis Plastids for Proteomic Analysis and Other Studies.</a> Chloroplast Research in Arabidopsis. , 223-239 (2011).</li><li>Yang, H., Murphy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+preparation,+sucrose+density+gradients+and+two-phase+separation+fractionation+from+five-day-old+Arabidopsis+seedlings.">Membrane preparation, sucrose density gradients and two-phase separation fractionation from five-day-old Arabidopsis seedlings.</a> Bio-Protocols. 3 (24), 1014 (2022).</li><li>Kelekar, P., Wei, M., Yang, P., Pazour, G. J., King, 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=Isolation+and+Analysis+of+Radial+Spoke+Proteins.">Isolation and Analysis of Radial Spoke Proteins.</a> Cilia: Motors and Regulation. Methods in Cell Biology. 92, 181-196 (2009).</li><li>Chen, J. H., 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=Nuclear-encoded+synthesis+of+the+D1+subunit+of+photosystem+II+increases+photosynthetic+efficiency+and+crop+yield.">Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield.</a> Nature Plants. 6 (5), 570-580 (2020).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy+reveals+that+senescence+induces+in-situ+and+vacuolar+degradation+of+plastoglobules+in+aging+watermelon+leaves.">Ultramicroscopy reveals that senescence induces in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.</a> Micron. 80, 135-144 (2016).</li><li>Singh, D. K., Laremore, T. N., Smith, P. B., Maximova, S. N., McNellis, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+FIBRILLIN4+gene+expression+in+apple+decreases+plastoglobule+plastoquinone+content.">Knockdown of FIBRILLIN4 gene expression in apple decreases plastoglobule plastoquinone content.</a> PLoS One. 7 (10), 47547 (2012).</li><li>Singh, D. K., 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=FIBRILLIN4+is+required+for+plastoglobule+development+and+stress+resistance+in+apple+and+Arabidopsis.">FIBRILLIN4 is required for plastoglobule development and stress resistance in apple and Arabidopsis.</a> Plant Physiology. 154 (3), 1281-1293 (2010).</li><li>Zheng, X., 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=Gardenia+carotenoid+cleavage+dioxygenase+4a+is+an+efficient+tool+for+biotechnological+production+of+crocins+in+green+and+non-green+plant+tissues.">Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.</a> Plant Biotechnology Journal. , (2022).</li><li>Bligh, E. G., Dyer, W. 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为了最大限度地减少材料的生理/生化变化并保护某些光不稳定和热不稳定的异戊烯基脂质色素，这些色素是塑料球的丰富成分，在4°C下进行隔离并避光至关重要。如上所述，初始步骤是在冷室的安全灯下使用绿色发光灯泡进行的。在实验室中进行的后续步骤是在昏暗的灯光下，并使用冰或冷藏离心。出于类似的原因，加入新鲜蛋白酶抑制剂（如果有兴趣研究蛋白质的磷酸化调节，则包括磷酸酶?...

目前对质体球组成和功能的理解是通过详细的蛋白质组学和脂质组学研究出现的1，2，3，4，5。这种研究得到了快速有效的分离方法的极大帮助，该方法依赖于其非常低的密度，使用蔗糖梯度进行有效分离。从山毛榉树（Fagus sylvatica），苏格兰扫帚（Sarothamnus scoparius），洋葱（葱属cepa），菠菜（Spinacia oleracea），三色堇（中提琴三色），胡椒（辣椒）和豌豆（Pisum sativum）等物种中实现了质体球分离的初始方法6，7，8，9，10，11，12，13.Ytterberg等人后来提出了一种以更有效和更好的产量方式分离叶绿体质体球的更新方法3，14。虽然最初用于研究拟南芥叶叶绿体的质球，但我们已经成功地将这种更新的方法用于其他植物物种的健康叶组织，包括单子叶植物和双子叶植物，包括玉米（Zea mays）、番茄（Solanum lycopersicum）、爱情草（Eragrostis nindensis）、紫色假凤梨（Brachypodium distachyon）和野生烟草（Nicotiana benthamiana;未发表的结果）。此外，分离方法已成功适应蓝藻的质体球，包括集胞藻属PCC 6803和Anabaena sp. PCC 712015，以及复活植物E. nindensis的干燥叶组织。
健康叶组织的叶绿体质体球与类囊体膜物理连接16。尽管有这种物理连续性，两个叶绿体亚区室保持不同的脂质和蛋白质组成，尽管已经提出了两个区室之间脂质和蛋白质的调节交换2，4，17，18，19。事实上，最近提出了一个有趣的半融合模型，用于叶绿体和细胞质19之间中性脂质的运输。由于质体球和类囊体的物理连续性，健康叶组织的分离方法从收集颗粒粗类囊体制剂开始，随后对其进行超声处理以将质体球与类囊体分离，这与用于分离胞质脂滴的方法相反20.然后在蔗糖垫上进行超速离心，然后将低密度质体球向上漂浮通过蔗糖，有效地将它们与类囊体、细胞核和其他高密度材料分离。相比之下，蓝藻中的质体球以及干燥的叶组织中的质体球显然以自由漂浮的形式存在于体内。因此，它们的分离涉及直接漂浮在蔗糖梯度上。本文演示了从健康叶片组织中分离的方法，并进一步展示了可用于从干燥的叶组织或蓝藻培养物中分离质体球的两种变体，极大地扩展了可以研究塑料球的生理广度和进化背景。
分离的质体球随后可用于任意数量的下游分析，以研究分子特征。我们使用从拟南芥叶组织中分离出的质体球在不同环境条件或基因型下进行广泛的蛋白质组学和脂质组学分析，证明了蛋白质和脂质组成的选择性修饰以适应压力2，4，21，22。此外，已经进行了体外激酶测定，该测定已证明与分离的质体球相关的反式磷酸化活性22，已使用天然凝胶电泳21研究了蛋白质组分的寡聚状态，并且已经进行了蛋白酶剃须测定23。
这种方法的主要优点是程序的相对速度。根据我们的经验，下面概述的协议可以在大约4小时内完全完成。已经描述了从叶组织中分离质体球的替代方法，该方法允许同时分离其他叶绿体亚区室24。当需要或需要与其他叶绿体亚区室进行定量比较时，这种替代方法提供了一些明显的优势。然而，这种替代方法也更加繁琐，并且将从相当数量的叶组织中提供显着降低的分离质体球的产量。当以质体球为重点研究时，此处概述的方法论是最佳选择。尽管如此，在样品制备过程中可以收集总叶和粗类囊体等分试样，强烈建议这样做，以便有参考样品进行后续比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AEBSF</td>
			<td>Milipore Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Antipain.2HCl</td>
			<td>Bachem</td>
			<td>H-1765.0050BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Milipore Sigma</td>
			<td>A6106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>BDH</td>
			<td>BDH9242</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin</td>
			<td>Sigma Aldrich</td>
			<td>B8385</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-Glycerophosphate. 2Na5H2O</td>
			<td>EMD Millipore</td>
			<td>35675</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Proliant Biological</td>
			<td>68700</td>
			<td></td>
		</tr>
		<tr>
			<td>Chymostatin</td>
			<td>Sigma Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>Eragrostis nindensis</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>E-64</td>
			<td>Milipore Sigma</td>
			<td>E3132</td>
			<td></td>
		</tr>
		<tr>
			<td>French Pressure cell (model FA-079)</td>
			<td>SLM/Aminco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron shaking incubator</td>
			<td>Infors HT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-ramidon.2 Na</td>
			<td>Sigma Aldrich</td>
			<td>R7385</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Chemicals</td>
			<td>M16050</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced Cysteine</td>
			<td>MP Biochemicals</td>
			<td>101444</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma Aldrich</td>
			<td>S7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Ortho-vanadate</td>
			<td>Sigma Aldrich</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyrophosphate &#183; 10H2O</td>
			<td>Sigma Aldrich</td>
			<td>3850</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma Aldrich</td>
			<td>S3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylvania 15 W fluorescent Gro-Lux tube light bulb, 18&#34;</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Synechocystis sp. PCC 6803</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>Waring Blender (1.2 L)</td>
			<td>VWR</td>
			<td>58977-227</td>
			<td>Commercial blender</td>
		</tr>
		<tr>
			<td>Zea mays</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

plastoglobule lipid droplet isolation from plant leaf tissue and cyanobacteria

塑性球脂滴是植物叶绿体和蓝藻的动态亚区室。它们在光合作用物种中无处不在，被认为在快速变化的环境条件下在类囊体膜的适应和重塑中起着核心作用。分离高纯度质体球的能力极大地促进了通过蛋白质组、脂质组和其他方法进行的研究。使用高纯度和高产量的质体球，可以研究它们的脂质和蛋白质组成、酶活性和蛋白质拓扑以及其他可能的分子特征。本文提出了一种从植物叶片组织叶绿体中分离质体球的快速有效的方案，并介绍了从玉米叶、复活植物的干燥叶组织 Eragrostis nindensis 和蓝藻集 胞藻 中分离质球和相关脂滴结构的方法学变化 PCC 6803。分离依赖于这些富含脂质的颗粒的低密度，这有助于通过蔗糖密度浮选纯化它们。这种方法将在研究来自不同物种的质体球方面证明是有价值的。

完成协议的步骤1后，应该能够容易地看到相当数量的质体球/脂滴材料漂浮在蔗糖垫的顶层（或附近）（图1B-C）。在这个阶段也可以收集其他馏分。例如，类囊体将被沉淀，并可以用培养基R 0.2重新悬浮以进行后续分析。随后离心后，将在蔗糖梯度表面或附近获得纯化的质体球，如图1A，C所示。已经看到在某些情?...

Watch this Scientific Journal Video about Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria at JoVE.com

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

提出了一种快速有效的方案，用于分离与各种光合生物相关的质球脂滴。成功制备分离的质体球是蛋白质组学和脂质组学分析等详细分子研究之前的关键第一步。

从植物叶片组织和蓝藻中分离出塑体球脂滴

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...

分离高纯度的质体球对他们的研究至关重要。本文介绍了一种快速有效的分离方案，有助于其下游生化研究。这种方法的主要优点是程序的相对速度及其对众多植物物种和环境条件的适应性。在对类囊体悬浮液进行超声处理时必须特别小心，以确保有足够的力从类囊体中剥离质体球，并随后从蔗糖梯度中提取质体球。演示这一程序的将是我实验室的博士后研究员Elsinraju Devadasu博士和我实验室的博士生Febri Susanto。首先获得六种健康玉米的三周龄幼苗，具有V5生长期，重约120克。剪掉茎基部的所有叶子，并迅速将它们浸入冰浴中，然后将它们运送到冷藏室。在绿色安全灯下工作，从冰浴中取出玉米叶，用剪刀将它们剪成五乘五厘米的小块。在商用搅拌机上以低于7的水平在350毫升研磨缓冲液中轻轻彻底研磨一半的剪枝叶组织。启动和停止搅拌机五到六次，以确保所有叶子都被切掉。通过大漏斗上的一层 25 微米尼龙布过滤匀浆到四个 250 毫升离心瓶中。用剩余的夹住的叶组织重复混合步骤后，将滤液均匀地分配到四个瓶子之间。取出等分试样的叶滤液并将其放在一边，作为代表性的总叶样品储存。接下来，将滤液在 1， 840 G 和 4 摄氏度下离心六分钟。倒出所得的上清液，用刷子轻轻旋转，将沉淀重悬于含有0.2摩尔蔗糖的12毫升培养基R 0.2中。将颗粒重新悬浮在一个瓶子中后，将悬浮液添加到下一个瓶子中，然后重复重悬液以将悬浮液合并到一个瓶子中。将混合悬浮液分布在六个三毫升超速离心管之间，每个管中的最大体积达到 2.5 毫升。然后使用100%振幅的尖端超声仪对每个管进行四次超声处理，每次10秒。确保超声器喇叭浸没并远离液体表面，以防止起泡。在每轮中，在悬架内缓慢上下移动超声器喇叭。在交替使用四个试管的同时，在每次超声处理后将每个试管放回冰桶中，以使样品冷却。完成后，将超声处理的粗类囊体悬浮液在 150， 000 G 下在 4 摄氏度下离心 30 分钟。使用注射器和 22 号针头撇去垫子表面，并从蔗糖垫中收获产生的黄色和油性浮式粗质体球垫。从每个管中回收约500微升，并将它们放入2.0毫升的管中。在超声处理和释放质体球之前和之后收集粗类囊体等分试样。完成后，要么继续植物组织处理，要么将粗质体球储存在零下80摄氏度，稍后再纯化。在BG11培养基中生长50毫升集胞藻属PCC 6802培养物7至10天以达到固定期。使用分光光度计，将细胞密度调整为2.0纳米处的750光密度。为了去除多糖，离心50毫升培养物并除去上清液。在50毫升缓冲液A中重复洗涤细胞两次。将洗涤后的沉淀重悬于25毫升缓冲液A中，并使用1， 100磅平方英寸的法国压力池破碎细胞。在寒冷条件下重复该过程三次，直到裂解的颜色在白光下从绿色变为红色，蓝色，绿色。取出细胞匀浆的等分试样并将其放在一边，作为代表性的总细胞样品储存。将所得匀浆分布在八个三毫升超速离心管中，每个管中最多填充 2.5 毫升。用 400 微升培养基 R 覆盖该匀浆，产生阶梯度。在离心管之前，根据需要添加额外的培养基R来仔细平衡试管。类囊体和其他较重的细胞器，包括任何多羟基链烷酸小体，显示沉淀形成，而质体球在蔗糖梯度顶部或附近形成黄色油状垫。如前所述，用注射器收获所得的粗质体球漂浮垫，并将收获物放入两毫升管中。如有必要，用针尖从超速离心管壁的侧面刮下质球。要么继续蓝藻加工，要么将粗质体球储存在零下80摄氏度，稍后再纯化。为了使用植物组织处理方法收获纯质体球，使用500微升粗质体球，400微升培养基R 0.2和400微升培养基R制备前面描述的蔗糖梯度.离心后，将所得的纯质体球漂浮垫收获到两毫升管中。将纯质体球分装并在液氮中快速冷冻后，将它们储存在零下80摄氏度或将其冻干成干粉。为了从蓝藻中收获纯质体球，如前所述，使用 500 微升粗质体球、750 微升培养基 R 0.7 和 750 微升培养基 R 0.2 创建蔗糖梯度。离心后，将纯质体球收集在1.5毫升管中，并将纯质体球等分。完成后，将纯质体球等分试样在液氮中快速冷冻，并直接在零下80摄氏度下储存或冻干成干粉。在该方法中，可以看到大量的质体球或脂滴材料漂浮在蔗糖垫的顶层。随后离心后，在蔗糖梯度表面或附近获得纯化的质体球。成功分离质体球后，使用针对拟南芥原纤维蛋白1a和拟南芥光系统II亚基D1的抗体开发的用于纯度评估的免疫印迹显示，原纤维蛋白同系物主要与类囊体相关，而不是质体球。为确保高效、集中地提取浮动垫，请在完全浸没时将注射器针头孔放在缓冲液液表面的正下方，然后轻轻向上拉。分离的质体球样品适用于随后的蛋白质组学或脂质组学研究，并已用于证明不同环境条件下靶向蛋白质-脂质组成的变化。该技术促进了质体球的新研究，包括通过随后的生化研究发现相关的蛋白激酶活性和寡聚蛋白复合物的存在。

Research

JoVE Journal

Biology

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

LeafJ：ImageJ的插件半自动叶形状测量

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

科研

生物学

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

从食虫植物组织分离存活的多细胞腺的，猪笼草</i

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

植物表皮脂质聚酯单体的分离及成分分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

利用成熟的细胞隔离试剂盒快速分离脂滴器

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

蓝藻中完整藻胆体的分离与表征

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...