| Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds
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Author:
Date:
2018-07-05
[Abstract] Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation ...
[摘要] 植物液泡是植物细胞中最大的隔室，占细胞体积的80％以上。各种蛋白质，糖，色素和其他代谢物存储在这些细胞器中（Paris et al。，1996; Olbrich et al。，2007）。花产生多种特殊代谢物，其中一些是该器官特有的，如授粉综合征的成分， ie ，气味挥发物和黄酮类化合物（Hoballah et al。， 2007; Cna'ani et al。，2015）。为了研究存储在花液泡中的化合物，必须将该隔室与细胞的其余部分分开。为了能够分离液泡，首先通过将刺穿的花冠与纤维素酶和macrozyme酶一起孵育来产生原生质体。在过滤和几个离心步骤后，通过显微镜观察显示原生质体与碎片和受损/破裂的原生质体分离。裂解浓缩的原生质体，并通过Ficoll梯度离心提取液泡。 Vacuoles用于隔离代谢物的定量GC-MS分析。这种方法使我们能够将空泡识别为糖基化挥发性苯丙酸类的亚细胞聚集位点，并假设共轭气味化合物在通向顶空的途径中被隔离（Cna'ani et al。，2017） 。
【背景】植物空泡占植物细胞中细胞体积的80％。这些细胞器对植物生长和发育至关重要，在整个植物的生命中具有不同的功能。 ...
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| Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment
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Author:
Date:
2016-11-05
[Abstract] This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4+ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH4+ as the sole N source presented an increase of ...
[摘要] 该方案可用于抑制多胺，特别是腐胺在作为单独N源生长的番茄植物中的生物合成。通常，多胺是与生理过程相关的带正电荷的小代谢物，包括器官发生，胚胎发生，花起始和发育，叶衰老，花粉管生长，果实发育和成熟，并参与对非生物和生物胁迫的应答（Tiburcio等， et al 。，2014）。通过鸟氨酸脱羧酶（ODC）或精氨酸脱羧酶（ADC）将鸟氨酸或精氨酸脱羧，从氨基酸合成多胺（Walters，2003）。用NH 4+作为唯一N源生长的番茄植物在叶中表现出腐胺含量的增加（Fernández-Crespo等人，2015） ）。为了评估腐胺积累的重要性，将腐胺合成抑制剂DL-α-（二氟甲基）精氨酸（DFMA）和DL-α-（二氟甲基）鸟氨酸（DFMO）分别用作ADC和ODC酶的不可逆抑制剂Fallon和Phillips，1988），其目的是减少NH 4营养物诱导的细胞腐胺积累。在样品收集之前的一周内，将含有2mM DFMA和5mM DFMO的抑制剂溶液直接施用到每个罐中。
[背景] 应用程序（应用程序） 的抑制剂DFMA和DFMO通常在MS培养基和体外试验中进行（Perez-Amador等人，2002; Stes等人， 。，2011）。然而，我们需要测试这些抑制剂在体内的有效性以维持自然生长条件的目的。 Moschou 等。 ...
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| Affymetrix Genome-wide Human SNP Assay
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Author:
Date:
2016-05-20
[Abstract] To assess genomic variation, it is possible to identify the single nucleotide polymorphisms (SNP) which an individual carries. Using the Affymetrix Genome-wide Human SNP Assay, it is possible to assess 906,600 SNPs on a single array. This protocol, the next iteration of the GeneChip Mapping 500K array set, is based directly on the manufacturers’ protocol and shows steps which are highly similar to that which is found here: http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf.
[摘要] 为了评估基因组变异，可以鉴定个体携带的单核苷酸多态性（SNP）。 使用Affymetrix全基因组人类SNP测定，可以在单个阵列上评估906,600个SNP。 这个协议是GeneChip Mapping 500K数组集的下一个迭代，它直接基于制造商的协议，并显示了与这里非常相似的步骤：http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf 。
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