Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato
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Author:
Date:
2021-03-05
[Abstract] Targeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor-related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. ...
[摘要] [摘要]靶向代谢麦克风是评估作物育种研究的有用方法。抗氧化剂和与风味有关的性状受到越来越多的关注,被认为是番茄育种中的品质性状。本研究提供了色谱方法来研究番茄中的抗氧化剂(类胡萝卜素,维生素C,维生素E,酚类化合物和谷胱甘肽)和与风味相关的特征(糖和有机酸)。应用了两种不同的提取方法(针对极性和非极性实体)来分离目标化合物。由于不需要进一步纯化,因此这项工作中开发的提取方法既省时又经济。使用RP C18色谱柱,通过RP-C18色谱柱在适当波长下对每种化合物进行类胡萝卜素,维生素C,谷胱甘肽和酚酸的分析。用RP C18和NH2色谱柱通过HPLC分析维生素E和糖,并分别通过FLD和RI检测器进行检测。此外,MSTFA衍生化后,使用Rtx 5DA色谱柱使用GC-FID分析有机酸。结果,开发了定量重要植物代谢物的灵敏分析方法,并在本文中进行了描述。这些方法不仅适用于番茄,但都还表征其他物种的香味相关的和抗氧化的化合物。因此,这些协议可用于指导作物育种的选择。
[背景]代谢组学是其受到关注的其潜在的援助作物育种研究施加生化途径。在番茄育种中,由于消费者对提高作物品质的偏好,抗氧化剂和与风味有关的代谢产物都受到了越来越多的关注。人类无法自己合成抗氧化剂分子; ...
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Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila
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Author:
Date:
2021-02-20
[Abstract] Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating ...
[摘要] [摘要]功能丧失的研究为基因或基因产物在不同细胞过程中的参与提供了重要启示。短发夹RNA(shRNA)介导的RNA干扰(RNAi)是一种经典而直接的技术,经常用于敲低基因以评估其功能。也可以通过siRNA,microRNA或CRISPR-Cas9方法实现类似的基因表达扰动。在果蝇遗传学中,UAS-GAL4系统用于表达RNAi,并使遍在和组织特异性的基因敲除成为可能。UAS-GAL4系统借鉴了酿酒酵母的遗传成分,因此排除了系统意外表达的可能性。特别地,该技术使用靶标特异性shRNA,并且其表达受上游激活序列(UAS)支配。由特定启动子调节的GAL4受控表达可以普遍或以组织特异性方式驱动干扰RNA的表达。通过RNA分离和半定量RT-PCR反应,然后进行琼脂糖凝胶电泳来测量敲低效率。我们还采用了免疫染色程序来评估击倒效率。
RNAi为研究人员提供了降低基因产物水平(相当于亚同型条件)并研究结果的选择。基于UAS-GAL4的RNAi方法提供了基因表达的时空调节,还有助于推断早期发育阶段所需的基因功能。
[背景]果蝇果蝇(果蝇)是在研究实验室经常使用的一种通用模式生物。果蝇易于处理,繁殖和维护。而且,精心制作却寿命短,繁殖力高的果蝇具有更多的优势。果蝇遗传学工具的易用性有助于发展对基因功能的全面了解。由于果蝇基因中有60%与人类基因同源,并且具有前面提到的其他优点,因此果蝇是研究体内基因功能的显而易见的模型生物。 ...
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Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
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Author:
Date:
2020-06-20
[Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique faces two main disadvantages: it is not a direct measure of the kinase activity and it is a time-consuming approach. However, since PKA can be readily obtained from sperm extracts, in vitro assays such as the “radioactive assay” can be performed using the native enzyme. Unlike western-blot, the radioactive assay is a straightforward technique to evaluate PKA activity by quantification of incorporated 32P into a peptidic ...
[摘要] [Abstract]为了获得受精潜能,哺乳动物的精子必须经历一个被称为电容的过程,这个过程依赖于蛋白激酶A(PKA)的早期激活。通常,在全细胞实验中,通过分析其底物在western-blot中的磷酸化状态来评估PKA的活性。这种技术面临着两个主要的缺点:它不是对激酶活性的直接测量,而且是一种耗时的方法。然而,由于PKA可以很容易地从精子提取物中获得,体外检测如"放射性检测"可以使用原生酶进行。与western-blot不同的是,放射性测定法是一种直接的技术,通过将整合的32P定量到肽类底物中来评价PKA的活性。这种方法很容易允许分析PKA的不同激动剂或拮抗剂。由于小鼠精子是可溶性PKA的丰富来源,这种测定法可以进行简单的分馏,使PKA既可用于体外测试药物对PKA活性的影响,也可用于跟踪PKA活性在增容开始时的变化。
[Background] ...
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