Name: determination of dna methylation of imprinted genes in arabidopsis endosperm
Uploaded: 2011-01-28T18:00:00
Description: Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

作者感谢维持拟南芥女士珍妮弗M.洛梅尔和塔拉北路Rognan。这项工作是从美国圣路易斯大学和美国国立卫生赠款1R15GM086846 - 01和3R15GM086846 - 01S1 W.小的启动资金支持。

一，遗传隧道 1。 Emasculating的母本为了区分使用的DNA序列多态性，两个不同生态型，如哥伦比亚0（COL - 0）和LER，将作为女性和男性的父母选择的母亲和父亲的等位基因。植物应年轻和健康。一个可以阉割母本，用解剖显微镜，放大镜遮阳帽，或肉眼。找到阶段- 12花（史密斯等人，1990年）和删除任何鲜花或角果上面和下面他们的基地花梗用剪刀裁剪。...

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. 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+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Lister, R., Pelizzola, M., Dowen, R. H., Hawkins, R. D., Hon, G., Tonti-Filippini, J., Nery, J. R., Lee, L., Ye, Z., Ngo, Q. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462, 315-322 (2009).</li><li>Paulin, R., Grigg, G. W., Davey, M. W., Piper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea+improves+efficiency+of+bisulfite-mediated+sequencing+of+5'-+methylcytosine+in+genomic+DNA.">Urea improves efficiency of bisulfite-mediated sequencing of 5'- methylcytosine in genomic DNA.</a> Nucl. Acids Res. 26, 5009-5010 (1998).</li><li>Rogers, S. O., &amp;amp, B. e. n. d. i. c. h., J, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+plant+tissues.">Extraction of DNA from plant tissues.</a> Plant Molecular Biology Manual. A6, 1-10 (1988).</li><li>Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Flower+Development+in+Arab&#237;dopsis.">Early Flower Development in Arab&#237;dopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Xiao, W., Gehring, M., Choi, Y., Margossian, L., Pu, H., Harada, J. J., Goldberg, R. B., Pennell, R. I., Fischer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+MEA+Polycomb+gene+is+controlled+by+antagonism+between+MET1+methyltransferase+and+DME+glycosylase.">Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase.</a> Dev. Cell. 5, 891-901 (2003).</li></ol>

这是比较容易分离胚胎胚乳和种皮，从种皮中分离出来的胚乳，在胚胎发育的早期或中期鱼雷阶段，特别是对种子，但它是单调乏味的。由于种皮不仅有助于某些基因，极少量的组织，例如，MEA和荧光增白剂 ，我们不从种皮中分离出来的胚乳。这意味着，我们可以从胚乳和种皮组织混合物中分离的RNA，检查一个基因的母亲和父亲的等位基因表达，比较，并确定该基因的印迹状态的表...

用品<ul><li>解剖显微镜</li><li>剪刀</li><li>精尖钳</li><li>珠宝标签</li><li>植物锦标</li><li> String或扭曲的关系</li><li> 4“× 2”× 8“聚乙烯袋</li><li> 3“X 1”X 1.0毫米显微镜载玻片</li><li>液氮</li><li>液氮容器</li><li>加热块</li><li> PCR管</li><li>热循环</li><li>离心管</li><li>离心</li><li>凝胶电泳设施</li><li>哥伦比亚0 拟南芥植物</li><li> 拟南芥兰茨贝格万寿菊植物</li></ul>试剂<ul><li> 70％的乙醇</li><li> 95％的乙醇</li><li> 100％的乙醇</li><li>山梨醇0.3米和5毫米的MES，pH值5.7 </li><li>十六烷基三甲基铵（CTAB） </li><li> 100％的乙醇</li><li> Cholorform </li><li>限制性内切酶</li><li> 3 M氢氧化钠</li><li> 6.3米氢氧化钠</li><li> 6.24 M尿素/ 4米的亚硫酸氢钠</li><li>无菌蒸馏水H 2 O </li><li> 10 mM的对苯二酚</li><li>向导DNA清理系统（Promega公司） </li><li> 10米NH 4醋酸</li><li> 20微克/微升的tRNA </li><li> TE缓冲液</li><li> TOPO TA克隆试剂盒（Invitrogen公司） </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

拟南芥是一种优秀的模式生物研究表观遗传机制。其中一个原因是DNA甲基丧失功能的空突变是可行的，从而提供了一个系统来研究如何在基因组DNA甲基化的损失，影响经济增长和发展。印指母系和父系等位基因表达差，在哺乳类动物和植物再生产的发展中起着重要作用。 DNA甲基化是决定是否印迹基因的母亲或父亲的等位基因表达或沉默的关键。在开花植物中，有一个再生产的双受精事件：一个精子细胞受精的卵细胞，形成与中央细胞上升到胚乳的胚胎和精子第二保险丝。胚乳是印迹发生在植物的组织。美狄亚，一个SET域polycomb的组基因和荧光增白剂，一种转录因子调节开花，胚乳和其表达的印迹显示前两个基因是由DNA甲基化和去甲基化控制植物。为了确定印记的基因和胚乳中的甲基化模式的状态，我们需要能够先隔离胚乳。由于种子是在拟南芥中的微小的，但它仍然具有挑战性，隔离拟南芥胚乳，并检查其甲基化。在此视频协议中，我们报告如何进行遗传交叉，隔离胚乳组织，并确定由亚硫酸氢钠测序的甲基化状态。

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

印记是在植物和哺乳动物繁殖的现象。 DNA甲基化起着重要作用的印迹机制。隔离胚乳和确定的印记基因的甲基化状态<em&gt;拟南芥</em&gt;可以是困难的。在这个协议中，我们介绍了如何隔离胚乳，并确定由亚硫酸氢钠测序的甲基化。

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Research

JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

科研

生物学

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...