印记基因的DNA甲基化的测定拟南芥胚乳
Summary
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>拟南芥</em>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。
Abstract
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。
Protocol
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。…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
Supplies
- Dissecting Microscope
- Scissors
- Fine Tip Forceps
- Jewelry Tag
- Plant Stakes
- String or Twist-Ties
- 4″ X 2″ X 8″ Polyethylene Bags
- 3″ X 1″ X 1.0 mm Microscope Slides
- Liquid Nitrogen
- Liquid Nitrogen Containers
- Heat Block
- PCR Tubes
- Thermocycler
- Microcentrifuge Tubes
- Microcentrifuge
- Gel Electrophoresis facility
- Arabidopsis thaliana Columbia-0 Plants
- Arabidopsis thaliana Landsberg erecta Plants
Reagents
- 70% Ethanol
- 95% Ethanol
- 100% Ethanol
- 0.3 M Sorbitol and 5 mM MES-pH 5.7
- Cetyltrimethyl Ammonium Bromide (CTAB)
- 100% Ethanol
- Cholorform
- Restriction Enzymes
- 3 M NaOH
- 6.3 M NaOH
- 6.24 M Urea/ 4 M Sodium Bisulfite
- Sterile distilled H2O
- 10 mM Hydroquinone
- Wizard DNA Clean-Up System (Promega)
- 10 M NH4OAc
- 20 μg/ μL tRNA
- TE buffer
- The TOPO TA Cloning Kit (Invitrogen)
Riferimenti
- Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990-2997 (1994).
- 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. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature. 452, 215-219 (2008).
- Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA. 89, 1827-1831 (1992).
- 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. DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell. 124, 495-506 (2006).
- Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. Accurate sodium bisulfite sequencing in plants. Epigenetics. 5, 47-49 (2010).
- Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. Genome-wide demethylation of Arabidopsis endosperm. Science. 324, 1451-1454 (2009).
- Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. Ectopic hypermethylation of flower-specific genes in Arabidopsis. Curr. Biol. 10, 179-186 (2000).
- Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. Science. 303, 521-523 (2004).
- Lister, R., O’Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 133, 523-536 (2008).
- 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. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 462, 315-322 (2009).
- Paulin, R., Grigg, G. W., Davey, M. W., Piper, A. A. Urea improves efficiency of bisulfite-mediated sequencing of 5′- methylcytosine in genomic DNA. Nucl. Acids Res. 26, 5009-5010 (1998).
- Rogers, S. O., &, B. e. n. d. i. c. h., J, A. Extraction of DNA from plant tissues. Plant Molecular Biology Manual. A6, 1-10 (1988).
- Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. M. Early Flower Development in Arabídopsis. Plant Cell. 2, 755-767 (1990).
- Xiao, W., Gehring, M., Choi, Y., Margossian, L., Pu, H., Harada, J. J., Goldberg, R. B., Pennell, R. I., Fischer, R. L. Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase. Dev. Cell. 5, 891-901 (2003).
Tags
DNA MethylationImprinted GenesArabidopsis EndospermEpigenetic MechanismsDNA MethyltransferasesLoss Of DNA MethylationImprintingMaternal And Paternal AllelesReproduction DevelopmentFlowering PlantsDouble Fertilization EventMEDEA GeneFWA GeneDNA Methylation And DemethylationImprinting StatusMethylation PatternIsolate EndospermGenetic Cross
Citazione di questo articolo
Rea, M., Chen, M., Luan, S., Bhangu, D., Braud, M., Xiao, W. Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm. J. Vis. Exp. (47), e2327, doi:10.3791/2327 (2011).