| 15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice
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Author:
Date:
2016-08-20
[Abstract] 15N is a nonradioactive heavy isotope of nitrogen, widely used for biochemical and physiological research in plants. For instance, 15N-KNO3 was used as the nitrogen source in plants in order to investigate nitrate uptake activity and transport from roots to shoots (Lin et al., 2008). Here, we describe a detailed pipeline used for labeling living rice (Oryza sativa) plants with 15N-KNO3 and determination of net nitrate uptake and transport activity, and this protocol was proved to be valid in Arabidopsis and rice (Lin et al., 2008; Hu et al., 2015).
[摘要] N是氮的非放射性重同位素，广泛用于植物的生化和生理研究。 例如，使用N-KNO 3作为植物中的氮源，以研究硝酸盐吸收活性和从根到芽的转运（Lin等人 。， 2008）。 在这里，我们描述了用于标记具有 N-KNO 3的活水稻（>稻）植物和测定硝酸盐硝酸盐吸收的详细管道 和转运活性，并且该方案在拟南芥（Arabidopsis）和水稻中被证明是有效的（Lin等人，2008; Hu等人， 2015）。
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| Expression, Purification and Enzymatic Assay of Plant Histone Deacetylases
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Author:
Date:
2016-04-05
[Abstract] Histone deacetylases (HDACs) catalyzing the removal of acetyl groups from lysine residues of histone and non-histone proteins play vital roles in regulation of gene transcription. In plants, HDACs can be grouped into three families, including RPD3-type, SIR2-type and plant specific HD2-type HDACs. Here we describe a method to determine plant HDAC enzymatic activity. This protocol includes expression, purification and enzymatic activity assay of recombinant plant HDACs expressed in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).
[摘要] 组蛋白脱乙酰酶（HDAC）催化从组蛋白和非组蛋白蛋白的赖氨酸残基去除乙酰基在调节基因转录中起重要作用。 在植物中，HDAC可以分为三个家族，包括RPD3型，SIR2型和植物特异性HD2型HDAC。 在这里我们描述了一种确定植物HDAC酶活性的方法。 该方案包括在大肠杆菌（大肠杆菌）和拟南芥（）中表达的重组植物HDAC的表达，纯化和酶活性测定 >拟南芥）。
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| Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)
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Author:
Date:
2013-11-20
[Abstract] RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic ...
[摘要] RNA结合蛋白（RBP）在包括前mRNA剪接，多聚腺苷酸化，mRNA稳定性，mRNA定位和翻译的多个步骤在调节转录后水平的基因表达中起关键作用。 RBP主要通过结合新生或成熟转录物中的特定序列元件来调节这些过程。植物中有几百个RBP，但其中大多数的目标是未知的。已经开发了各种实验方法来鉴定RBP的目标。这些包括RNA免疫沉淀（RIP），UV交联和免疫沉淀（CLIP）和CLIP的许多变体（例如PAR-CLIP，iCLIP）。这些方法取决于使用针对该RBP的抗体与特异性RBP结合的RNA的免疫沉淀。电泳迁移率变动分析（EMSA），也称为凝胶移位分析，已被用于分析蛋白质 - 核酸相互作用。它是一种简单而强大的方法来分析蛋白质-RNA/DNA相互作用。在RNA EMSA中，通过在蛋白质存在下比较RNA的迁移来可视化RNA-蛋白质复合物。通常，在RNA EMSA中，使用特异性RNA序列来分析其与蛋白质的相互作用。将具有荧光标记的体外转录的32 P标记的或化学合成的RNA与或不与目标蛋白一起温育，然后将反应混合物在天然聚丙烯酰胺凝胶电泳上运行。 RNA-蛋白复合物与游离RNA相比缓慢迁移，其可以使用成像系统可视化。除了测试RBP与RNA的结合之外，EMSA还用于绘制参与相互作用的RNA和/或蛋白质中的区域。此外，还可以使用EMSA定量结合亲和力。
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