Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
|
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 ...
|
|
A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)
|
Author:
Date:
2017-06-05
[Abstract] The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along with insect survival analysis, is reported. This protocol allowed the distinction between virulent A. nidulans strains (such as TNO2A3), which induced high larval mortality rates, to those in which gene deletion was accompanied by reduced pathogenicity such as ∆gcsA and ∆sdeA (Fernandes et al., 2016).
[摘要] 作为一种有效的异源宿主,越来越多的蜡蛾已经出现了一种有效的异源宿主,用于研究真菌发病机制和有希望的抗真菌药物的功效(Mylonakis等人,2005; Li& et al。2013)。这里,描述了一种描述构巢曲霉感染的方法。 mellonella 幼虫与昆虫存活分析一起报道。该协议允许区分有毒的 A。造成高幼虫死亡率的构巢组织菌株(如TNO2A3)与基因缺失伴随着致病性降低的病例如ΔGCA和ΔSAA(Fernandes 等人,2016)。
背景 -G。 mellonella 是一种廉价的模型,易于处理,其先天免疫反应与哺乳动物免疫系统分享功能相似性。此外,感染真菌突变菌株的幼虫和小鼠表现出相似的存活率(Brennan等人,2002)。因此,幼虫构成了一种方便的动物宿主,用于在真菌发病机理分析中代替脊椎动物的使用。尽管昆虫模型具有所有优点,但只有少数报告显示了G中曲霉菌感染的作用。蜡螟。该协议描述了一种有效的方法,用于分析G中的构巢曲霉发病机制。 mellonella 幼虫。
|
|
Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants
|
Author:
Date:
2017-03-20
[Abstract] Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen’s virulence/pathogenicity and its interaction with hop plants. In this protocol, we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.
[摘要] 苜蓿轮枝菌是一种土壤传播的植物病原体,通过根感染其宿主。它传播在植物的木质部,并通过分泌不同的毒力因子引起枯萎病症状。 Hop(umulus lupulus)是V主要的主机。非苜蓿,因此它被用作研究这种植物病原真菌的示范植物。啤酒植物的人工感染和疾病评分是研究病原体的毒力/致病性及其与啤酒花植物的相互作用的先决条件。在该方案中,我们描述了用于进行V的致病性测定的根浸渍接种方法。非苜蓿植物。
背景 轮枝孢属 spp。感染400多种不同的宿主植物,每个物种都有自己的宿主范围。主要的主持人。非苜蓿是跳。然而,跳跃具有用作致病性测定的测试植物的几个缺点;例如,它是一种多年生植物,需要经历休眠阶段。因此,从春季到夏末,植物只能用于几年的致病性测定。 Hop品种在温室中作为软木切片繁殖繁殖,或作为从砧木的休眠扦插。种子是通过交叉雌性和雄性植物获得的,并且仅用于育种目的。根浸渍接种方法已被广泛应用于轮枝孢属菌株的致病性测定。在其他植物宿主上,例如,番茄(Fradin等人,2009),N。 (Klosterman等人,2011)和拟南芥拟南芥(Ellendorff等人,2009)。
|
|