| Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
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Author:
Date:
2017-08-05
[Abstract] The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate ...
[摘要] 植物内膜系统对蛋白质和脂质的合成，修饰和分泌起着至关重要的作用。 从经典观点来看，只有编码分泌蛋白质的mRNA才能通过协同翻译方式靶向内质网（ER）进行翻译，然而最近，这一模型已经被大量的细胞溶质mRNA也可能与 ER，并且一些类别的小RNA在ER上富集。 这些结果表明ER的功能超出了目前的知识。 在微粒体上大规模鉴定RNA和蛋白质对于显示ER功能至关重要，研究将由下一代测序技术提升。 该协议提供了从植物组织中分离细胞质，微粒体，游离多聚体（FP）和膜结合多聚体（MBP）的技术工作流程。 分离的级分适用于mRNA，小RNA和蛋白质的基因组广谱分析。
【背景】植物内膜系统对于细胞壁形成，脂质生物合成，蛋白质合成，修饰，折叠和贩运非常重要。根据共翻译易位模型，分泌蛋白N末端的信号肽由细胞溶质多核糖体合成，然后由ER上的信号识别粒子识别，其余蛋白质部分随后在ER上合成。根据该模型，只有编码分泌蛋白的mRNA可以被带到ER进行翻译（Peter和Johnson，1994）。然而，从哺乳动物和植物细胞ER（Lerner等人，2003; de ...
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| Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves
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Author:
Date:
2017-01-20
[Abstract] Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases of sugarcane. Dissemination of X. albilineans is mainly ensured by contaminated harvesting tools and infected stalk cuttings. However, some strains of this pathogen are transmitted by aerial means and are able to survive as epiphytes on the sugarcane phyllosphere before entering the leaves and causing disease. Here we present a protocol to estimate the capacity of attachment of X. albilineans to sugarcane leaves. Tissue-cultured ...
[摘要] 甘蔗（蔗糖种类的种间杂交种）是一种经济上重要的作物，其在世界范围内提供70％的生食糖生产，并在一些国家对生物乙醇和电力生产作出贡献。由细菌植物病原体Xanthomonas albilineans引起的叶片烫伤是甘蔗的主要疾病之一。传播X。 albilineans主要由受污染的收获工具和感染的茎秆确保。然而，这种病原体的一些菌株通过空中装置传播，并且能够在进入叶子并引起疾病之前作为甘蔗叶片上的附生植物生存。在这里，我们提出一个协议来估计X的附件容量。白皮书到甘蔗叶。将组织培养的甘蔗小植物浸入X的细菌悬浮液中。白皮书和X附件。通过两种方法确定了白皮书：叶片印记（半定量方法）和叶洗/匀浆（定量方法）。这些方法是评估甘蔗叶烫伤病原体菌株/突变体致病性的重要手段。
背景 管理X之间相互作用的机制。 albilineans 和它的宿主植物（甘蔗）不是公知的。 Albicidin是由白念珠菌产生的植物毒素，是已被证明在这种病原体的致病性中发挥作用的唯一分子因子（Birch，2001）。然而，X的致病性。白皮书不完全依赖于albicidin。缺乏Albicidin的突变体仍然能够有效地定殖甘蔗茎并产生疾病症状（Birch，2001; ...
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| Cycloheximide Assays to Measure Protein Degradation in vivo in Plants
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Author:
Date:
2016-09-05
[Abstract] The half-life of a protein is a characteristic property, and can be modulated by post-translational modifications, changes in subcellular localization, and/or interaction with other proteins or ligands. As one determinant of its steady-state level, a protein’s degradation represents an important distinguishing attribute relevant to its biological function. Because protein longevity cannot be elucidated from bioinformatics analyses, it must be determined empirically. Here we describe two approaches for in vivo half-life determination in plants: 1. pooled-seedling degradation assays monitoring either tagged versions of the protein (luciferase fusions or other epitope tags) or following the endogenous protein; 2. single-seedling degradation assays using luciferase fusion proteins. ...
[摘要] 蛋白质的半衰期是特征性质，并且可以通过翻译后修饰，亚细胞定位的改变和/或与其他蛋白质或配体的相互作用来调节。 作为其稳态水平的一个决定因素，蛋白质的降解代表与其生物学功能相关的重要的区别属性。 由于蛋白质的寿命不能从生物信息学分析中阐明，必须根据经验确定。 这里我们描述了在植物中体内半衰期测定的两种方法：1.监测蛋白质（萤光素酶融合物或其它表位标签）的标记形式或遵循内源蛋白质的混合 - 幼苗降解测定; 2.使用荧光素酶融合蛋白的单胚芽降解测定。 这些方法的优点是它们的简单性和低成本。
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