Name: caffeine extraction, enzymatic activity and gene expression of caffeine synthase from plant cell suspensions
Uploaded: 2018-10-02T13:00:00
Description: Watch this Scientific Journal Video about Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions at JoVE.com

我们实验室的工作由委员会 Ciencia y Tecnología (CONACyT 219893) 的赠款提供给 SMTHS。这项研究还得到了 CONACyT 和 Sistema 全国 Investigadores (4422) 授予 RJPK (37938 号) 的奖学金的支持。作者感谢 CIATEJ 在撰写本手稿时使用了它的安装。特别感谢 Víctor 博士的所有建议在分子生物学科和 Valentín 门多萨, 国际金融公司, UNAM 的设施在拍摄这篇文章。

1. 咖啡因在咖啡细胞悬浮液中的提取。
<ol><li>使用C. 咖啡细胞悬浮液9。保持悬浮由双周亚文化在 Murashige 和 Skoog 培养基在 pH 值4.3 与恒定的 100 rpm 震动25°c 在连续的光之下 (8.3 瓦特/m2)。</li>
	<li>用11µm 孔滤纸和傅书礼漏斗在真空过滤下收获细胞。</li>
	<li>用鳞片将收集到的细胞的新鲜重量进行登记, 用铝箔包裹, 将它们冷冻在液氮中, 并保持在-80 摄氏度直到...

<ol>
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B., Diener, H. C., Robbins, M. S., Garas, S. Y., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+in+the+management+of+patients+with+headache.">Caffeine in the management of patients with headache.</a> Journal of Headache and Pain. 18 (1), 1-11 (2017).</li><li>De Mejia, E. G., Ramirez-Mares, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+caffeine+and+coffee+on+our+health.">Impact of caffeine and coffee on our health.</a> Trends in Endocrinology &amp; Metabolism. 25 (10), 489-492 (2014).</li><li>Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., Sano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+production+in+tobacco+plants+by+simultaneous+expression+of+three+coffee+N-methyltrasferases+and+its+potential+as+a+pest+repellant.">Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltrasferases and its potential as a pest repellant.</a> Plant Molecular Biology. 59 (2), 221-227 (2005).</li><li>Denoeud, F., Carretero-Paulet, L., Dereeper, A., Droc, G., Guyot, R., Pietrella, M., Aury, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coffee+genome+provides+insight+into+the+convergent+evolution+of+caffeine+biosynthesis.">The coffee genome provides insight into the convergent evolution of caffeine biosynthesis.</a> Science. 345 (6201), 1181-1184 (2014).</li><li>Kurata, H., Matsumura, S., Furusaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+irradiation+causes+physiological+and+metabolic+changes+for+purine+alkaloid+production+by+a+Coffea+arabica+cell+suspension+culture.">Light irradiation causes physiological and metabolic changes for purine alkaloid production by a Coffea arabica cell suspension culture.</a> Plant Science. 123 (1-2), 197-203 (1997).</li><li>Pech-K&#250;, R., Mu&#241;oz-S&#225;nchez, J. A., Monforte-Gonz&#225;lez, M., V&#225;zquez-Flota, F., Rodas-Junco, B. A., Gonz&#225;lez-Mendoza, V. M., Hern&#225;ndez-Sotomayor, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+aluminum+stress+and+caffeine+biosynthesis+in+suspension+cells+of+Coffea+arabica+L.">Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.</a> Journal of Inorganic Biochemistry. 181, 177-182 (2018).</li><li>Huang, R., O'Donnell, A. J., Barboline, J. J., Barkman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+of+caffeine+in+plants+by+co-option+of+exapted+ancestral+enzymes.">Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes.</a> Proceedings of the National Academy of Sciences. 113 (38), 10613-10618 (2016).</li><li>Mizuno, K., Okuda, A., Kato, M., Yoneyama, N., Tanaka, H., Ashihara, H., Fujimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+new+dual-functional+caffeine+synthase+gene+encoding+an+enzyme+for+the+conversion+of+7-methylxanthine+to+caffeine+from+coffee+(Coffea+arabica+L.).">Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.).</a> FEBS letters. 534 (1-3), 75-81 (2003).</li><li>Kato, M., Mizuno, K., Fujimura, T., Iwama, M., Irie, M., Crozier, A., Ashihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+caffeine+synthase+from+tea+leaves.">Purification and characterization of caffeine synthase from tea leaves.</a> Plant Physiology. 120 (2), 579-586 (1999).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Method. 25 (4), 402-408 (2001).</li><li>Kurata, H., Achioku, T., Furusaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light/dark+cycle+operation+with+an+hour-scale+period+enhances+caffeine+production+by+Coffea+arabica+cells.">The light/dark cycle operation with an hour-scale period enhances caffeine production by Coffea arabica cells.</a> Enzyme and Microbial Technology. 23 (7-8), 518-523 (1998).</li><li>Sartor, R. M., Mazzafera, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+formation+by+suspension+cultures+of+Coffea+dewevrei.">Caffeine formation by suspension cultures of Coffea dewevrei.</a> Brazilian Archives of Biology Technology. 43 (1), 1-9 (2000).</li><li>Koshiro, Y., Zheng, X. Q., Wang, M. L., Nagai, C., Ashihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+content+and+biosynthetic+activity+of+caffeine+and+trigonelline+during+growth+and+ripening+of+Coffea+arabica+and+Coffea+canephora+fruits.">Changes in content and biosynthetic activity of caffeine and trigonelline during growth and ripening of Coffea arabica and Coffea canephora fruits.</a> Plant Science. 171 (2), 242-250 (2006).</li><li>Schimpl, F. C., Kiyota, E., Mayer, J. L. S., de Carvalho Gon&#231;alves, J. F., da Silva, J. F., Mazzafera, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+biochemical+characterization+of+caffeine+synthase+and+purine+alkaloid+concentration+in+guarana+fruit.">Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.</a> Phytochemistry. 105, 25-36 (2014).</li><li>Perrois, C., Strickler, S. R., Mathieu, G., Lepelley, M., Bedon, L., Michaux, S., Privat, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+caffeine+metabolism+in+Coffea+arabica+(Arabica)+and+Coffea+canephora+(Robusta).">Differential regulation of caffeine metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta).</a> Planta. 241 (1), 179-191 (2015).</li></ol>

我们在这里提出了评价咖啡因含量, CS 活动和转录水平的最佳条件在体外植物组织培养, 如细胞悬浮的C. 阿拉伯咖啡。以前的报告已经证实, 在光照射下维持细胞和在培养基中存在可可碱, 是提高咖啡因含量的合适参数, 从而可以评价咖啡因分离方法。采用反相高效液相色谱法 (RP)。目前, 很少有报道使用一种简单而快速的方法和经济优势来研究细胞悬浮液中的咖啡因生物合成。在这里, ...

咖啡因是咖啡1属植物 biosynthesized 的次生代谢产物。这生物碱属于甲基黄嘌呤家庭并且被认为作为一个化工厂防御, 因为它可能采取行动反对病原体和草食动物的有害作用2,3。此外, 这种代谢物负责的刺激性能的咖啡饮料, 这是普遍消费全球4,5。由于其特性, 一些研究小组对咖啡因6,7的生物合成途径和分解代谢有兴趣。目前, 植物体外细胞/组织培养作为评价咖啡因积累的替代品, 在不同的生物和非生物学的战略8,9。
咖啡因的生物合成涉及 7-甲基黄嘌呤的水解释放从相应的核糖核苷, 其次是有序的n-methylations 在位置3和1。特定的s-甘基蛋氨酸 (SAM) 依赖 n-甲基 (NMT) 催化甲基化在位置 7, 而可可碱合酶 (TS) 和 CS 分别参与3和 1-methylations, 生产可可碱和咖啡因。对不同 NMTs 基因编码的研究, 使人们了解调节咖啡因生产的机制10,11。CS 具有n-甲基活性, 催化了咖啡因11生物合成途径的最后两个步骤。在咖啡树幼苗中, 光辐射可以增加 CS 的活性, 从而增加咖啡因的生物合成。最近, 我们发现, 在光照射下维持细胞悬浮液是评价产生非生物胁迫因子影响咖啡因生物合成途径的最佳条件.8。这些研究中获得的信息可能在代谢工程和系统生物学中的应用, 以最大限度地研究这种体外系统中的咖啡因生物合成途径。
鉴于获得合适的咖啡因生物合成模型的优点, 我们优化了咖啡因在细胞悬浮液中的提取条件. 还有可能制定一个有用的协议来研究酶活性, 以及评估咖啡咖啡因合酶 1 (CCS1) 编码这种酶的基因转录水平的方法学步骤。在此, 我们报告了一个协议, 以提取和量化的咖啡因在C.

<table border="0" cellpadding="0" cellspacing="0" style="width:883px;" width="883">
	<tbody>
		<tr>
			<td>Murashige &#38; Skoog Basal salt mixture</td>
			<td>PhytoTechnology Laboratories</td>
			<td>M524</td>
			<td>Packge Size: 50 L 
			Reagent (mg/L) 
			Ammonium Nitrate (1650) 
			Boric acid (6.2) 
			Calcium chloride, anhydrous (322.2) 
			Cobalt Chloride&#8226;H2O (0.025) 
			Cupric Sulfate&#8226;5H2O (0.025) 
			Na2EDTA&#8226;2H2O (37.26) 
			Ferrous Sulfate&#8226;7H2O (27.8) 
			Magnesium Sulfate, Anhydrous (180.7) 
			Manganese Sulfate&#8226;H2O (16.9) 
			Molybdic Acid (Sodium Salt)&#8226; 2H2O (0.25) 
			Potassium Iodide (0.83) 
			Potassium Nitrate (1900) 
			Potassium Phosphate, Monobasic (170) 
			Zinc Sulfate&#8226;7H2O (8.6) 
			Supplemented with 
			myo-inositol (100) 
			thiamine (10) 
			cysteine (25) 
			sucrose (30000) 
			2,4-dichlorophenoxyacetic acid (3) 
			6-benzylamine purine (1)</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>SIGMA</td>
			<td>C0750-5G</td>
			<td>STANDARD-5g</td>
		</tr>
		<tr>
			<td>Theobromine</td>
			<td>SIGMA</td>
			<td>T4500</td>
			<td>20 g</td>
		</tr>
		<tr>
			<td>CAMAG TLC Scanner-4</td>
			<td>CAMAG</td>
			<td>27.62</td>
			<td></td>
		</tr>
		<tr>
			<td>WinCATS Planar Chromatography Manager software</td>
			<td>CAMAG</td>
			<td>1.4.10</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Isoamyl alcohol (24:1)</td>
			<td>SIGMA</td>
			<td>C-0549</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Cyclohexane</td>
			<td>JALMEX</td>
			<td>C4375-13</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>J.T. BAKER</td>
			<td>900643</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T. BAKER</td>
			<td>9093-03</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>JALMEX</td>
			<td>C-4425-15</td>
			<td>3.5 L</td>
		</tr>
		<tr>
			<td>TLC silica gel 60 F254</td>
			<td>Merck</td>
			<td>1.05554.0001</td>
			<td>TLC plate</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>M6250</td>
			<td>SIGMA</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>(+)-sodium L- ascorbate</td>
			<td>A4034</td>
			<td>SIGMA</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>SIGMA</td>
			<td>T6066</td>
			<td>1 Kg</td>
		</tr>
		<tr>
			<td>Hydrochloric acid 36.5-38%</td>
			<td>J.T. Baker</td>
			<td>9535-05</td>
			<td>2.5 L</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo scientific</td>
			<td>232227</td>
			<td>Kit</td>
		</tr>
		<tr>
			<td>Methyl [3H]-S-adenosyl methionine</td>
			<td>Perkin Elmer</td>
			<td>NET155</td>
			<td>Specific activity of 15 Ci/mmol</td>
		</tr>
		<tr>
			<td>Liquid scintillation vials</td>
			<td>SIGMA</td>
			<td>Z253081</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic bath/circulator</td>
			<td>Cole Parmer</td>
			<td>60714</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifugue tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Tube of 1.5 mL</td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Heathrow Scientific</td>
			<td>HS23202A</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Centrifuge 5804</td>
			<td>Eppendorf</td>
			<td>5804 000925</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Thermolyne</td>
			<td>LR 5947</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcelain mortar</td>
			<td>Fisherbrand</td>
			<td>FB961B</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>Z274844</td>
			<td>Porosity medium</td>
		</tr>
		<tr>
			<td>Picofuge</td>
			<td>Stratagene</td>
			<td>400550</td>
			<td>2000 x g</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>AND</td>
			<td>HR-120</td>
			<td>Model HR-120</td>
		</tr>
		<tr>
			<td>Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel photodocumentation system</td>
			<td>Bio-Rad</td>
			<td>Chemic XRS</td>
			<td>Model Chemic XRS</td>
		</tr>
		<tr>
			<td>Compact UV lamp</td>
			<td>UVP</td>
			<td>95002112</td>
			<td>UVGL-25</td>
		</tr>
		<tr>
			<td>Scienceware HDPE Buchner funnel</td>
			<td>SIGMA</td>
			<td>2419907</td>
			<td>Type 37600 mixer</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Thermo scientific</td>
			<td>15596-018</td>
			<td>200 mL</td>
		</tr>
		<tr>
			<td>ReverdAid Reverse transcriptase</td>
			<td>Thermo scientific</td>
			<td>#EP0441</td>
			<td>10000 U</td>
		</tr>
		<tr>
			<td>Oligo (dT)18 primer</td>
			<td>Thermo scientific</td>
			<td>#S0131</td>
			<td>100 &#181;M</td>
		</tr>
		<tr>
			<td>DNase I, RNase-free</td>
			<td>Thermo scientific</td>
			<td>#EN0525</td>
			<td>1000 U</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Thermo scientific</td>
			<td>EN0525</td>
			<td>1.25 mL</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Thermo scientific</td>
			<td>EN0525</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Thermo scientific</td>
			<td>R0191</td>
			<td>R0191</td>
		</tr>
		<tr>
			<td>SYBR Green qPCR Master Mix (2X)</td>
			<td>Thermo scientific</td>
			<td>K0251</td>
			<td>For 200 reactions of 25 &#181;L</td>
		</tr>
		<tr>
			<td>PikoReal</td>
			<td>Thermo scientific</td>
			<td>2.2</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Phenol, pH 8.0, equilibrated, Molecular Biology Grade, Ultrapure</td>
			<td>USB</td>
			<td>J75829</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Karal</td>
			<td>2040</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>SIGMA</td>
			<td>64175</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate</td>
			<td>SIGMA</td>
			<td>D5758</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Lab Rotator</td>
			<td>LW Scientific</td>
			<td>Mod. LW210</td>
			<td></td>
		</tr>
	</tbody>
</table>

caffeine extraction, enzymatic activity and gene expression of caffeine synthase from plant cell suspensions

咖啡因 (13, 7-trimethylxanthine) 是一种嘌呤生物碱, 目前流行饮料, 如咖啡和茶。这种次生代谢产物被认为是一种化学防御, 因为它具有抗菌活性, 被认为是一种天然杀虫剂。咖啡因也可以产生负面的化感作用, 防止周围植物的生长。此外, 世界各地的人们使用咖啡因的镇痛和刺激作用。由于对咖啡因技术应用的兴趣, 这种化合物的生物合成途径的研究已经发展。这些研究主要集中于了解调节咖啡因生物合成的生物化学和分子机制。体外组织培养已成为研究这种生物合成途径的有用系统。本文将介绍一步一步的协议, 以量化咖啡因和测量的转录水平的基因 (CCS1) 编码咖啡因合成酶 (CS) 的细胞悬浮液, 以及它的活动。

根据图 1所示的方案, 通过对样品进行色谱分析, 对通过本工艺获得的咖啡因提取物进行了薄层定量分析。为了量化细胞提取物中咖啡因的含量, 使用了这种化合物的各种商业标准的曲线 (图 2A)。用 273 nm 的最大吸收峰值 (图 2B) 分析了咖啡因的吸光度模式, 在薄层色谱板上, 咖啡因的峰值分离显示出0.34 和0.39 ...

Watch this Scientific Journal Video about Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions at JoVE.com

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

本协议描述了一种高效的方法, 用于提取和定量的咖啡因在细胞悬浮液中的表达水平和评价咖啡因合酶酶活性的实验过程。编码这种酶的基因。

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

该方法有助于回答分析化学、生物化学和生物技术领域的关键问题。例如，咖啡因生物合成中发生了什么变化。这项技术的主要优点是，它可以应用于产生咖啡因的植物内模型。首先，在冻干细胞中加入10毫升丙酮，并在与涡流混合器混合30秒之前密封烧瓶。然后，在 100 rpm 时将样品与旋转器在室温下混合 5 小时。在此之后，用25微升丙酮重新悬浮样品。首先，将先前准备的咖啡因提取物的微升剂涂抹到硅胶板中。然后，在色谱室中开发TLC，其移动相为10毫升，即环丙烷丙酮。使用紧凑型紫外线灯在短波长紫外线中可视化咖啡因波段。在此之后，通过密度测定在273纳米量化咖啡因水平。使用滤纸，真空过滤以前准备的电池悬浮液。然后，使用瓷砂浆用液氮和RNA分离试剂对细胞样品进行检测，直到其均质化。接下来，将样品的500微升转移到无菌微离心管中。然后，加入300微升氯仿异丙胺醇和300微升平衡酚。将样品与涡流混合器混合，在20，000G下离心，在4摄氏度下15分钟。在此之后，将上相的300微升转移到一个干净的微型离心管中。然后，加入200微升异丙醇，在负20摄氏度下孵育管子一小时。在颗粒中加入一毫升乙醇。然后，在12，000G下将管离心10分钟，然后将液相 dec。接下来，在室温下干燥样品1.5小时。然后，用25微升DEPC处理水重新悬浮RNA提取物。首先，在无菌微离心管中加入两微克总RNA提取物。然后，添加一个10X反应缓冲液的微升和一个DNase微升。使用 DEPC 处理的水将最终反应量带到 10 微升。然后，将样品混合在2000G下，并离心机一分钟。接下来，在37摄氏度下孵育样品30分钟。在此之后，加入一微升50微摩尔二甲胺二胺四乙酸酯，以停止反应。在65摄氏度下孵育样品10分钟。然后，将100纳米的RNA涂抹在一个阿加罗斯凝胶上，然后运行凝胶。使用凝胶照片文档系统可视化 RNA 的完整性。首先，在微型离心管中加入2.5微克总RNA。然后，加入一个微升的寡氧脱氧米丁底剂，并将管子填充到13微升的最终体积中，用无核酸酶水。混合样品，以2000G离心1分钟。在65摄氏度下孵育样品5分钟。然后在四摄氏度的两分钟。接下来，在样品中加入4个5X反应缓冲液微升、2个dNTP混合物微升和1微升逆转录酶。然后，轻轻混合样品，在2000G下离心一分钟。在此之后，在45摄氏度下孵育样品50分钟，然后在70摄氏度下孵育10分钟。将 7.5 微升 2X Taq DNA 聚合酶和 0.1 微摩尔 RAX 添加到 PCR 微离心管中。然后，添加0.75微升每个前进和反向底因为：放大CCS1基因。在此之后，添加 600 纳米的 CDNA 模板。如文本协议所述，实时PCR预造放大反应。然后，使用 PCR 软件分析数据。称出一克蜂窝材料，用铝箔包装。将样品抽取后，将其转移到玻璃瓶中，并加入2.5毫升的萃取缓冲液。接下来，在20，000G下将样品离心20分钟，温度为4摄氏度。将 500 微升等分转化为低温小瓶。使用分光光度计，使用双辛辛酸测定，以牛血清白蛋白为标准，测量样品在562纳米的蛋白质浓度。首先，根据文本协议，在微型离心管中准备反应混合物。然后，将总体积增加至200微升，带100毫摩尔三升HCL。请记住，在制备与放射性物质进行反应混合物时，为了自己的安全，必须遵循适当的预防措施。将微型离心管放在冰上，加入一个可溶性分数的体积，含有7至9毫克蛋白质。然后，在30摄氏度下旋转混合物，孵育30分钟。之后，将样品在11，000G下离心5分钟。然后，小心地回收900微升的体积，将样品转移到闪烁的小瓶中。接下来，蒸发氯仿，在室温下完全干燥。在小瓶中加入五毫升的闪烁液。最后，使用闪烁计数器分析咖啡因中的放射性。在该协议中，通过TLC密度测定法，通过可见光谱，在273纳米的最大吸收量下，对咖啡因的吸收模式进行了分析。TLC板上咖啡因的分离曲线显示RF介于0.34和0.39之间。从悬浮细胞中提取的RNA表现出28、18和5s RRNA亚基的清晰分离，表明样品质量高。最后，从QPCR分析中获得熔融曲线，显示咖啡因合成酶基因的单一扩增产品。该技术开发后，为生物技术和生物化学领域的研究人员探索咖啡、茶和可可的二次代谢组织培养铺平了道路。不要忘记，使用放射性和分子病理学可能是极其危险的，在执行本程序时，应始终采取预防措施，如使用手套、护目镜和放射性物质专用设备。

Research

JoVE Journal

Biochemistry

Expression, Purification, and Antimicrobial Activity of S100A12

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

科研

生物化学

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

mRNA Interactome Capture from Plant Protoplasts

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

PCR 突变体、克隆、表达、快速蛋白质纯化协议和色氨酸合成酶野生类型和突变形式的结晶

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...