Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves
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Author:
Date:
2021-02-05
[Abstract] Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. ...
[摘要] [摘要]脂质代谢由发生在不同亚细胞区室的反应网络组成。同位素标记是跟踪代谢物的转化和运动的好方法,而不会干扰整个细胞的新陈代谢。发TTY酸,膜脂和存储的构建块的三酰基甘油,在质体中合成的。脂肪酸合成的直接前体是乙酰辅酶A。外源乙酸盐可快速掺入叶片和分离的质体中的脂肪酸中,因为它可以通过细胞膜自由扩散,进入质体,然后迅速代谢成乙酰辅酶A。 因此,同位素标记的乙酸盐通常用作研究植物和其他生物中脂肪酸合成和复杂脂质代谢的示踪剂。同位素标记的基本原理及其最新技术进展已得到综述(Allen等,2015)。本协议描述了使用 的14 C标记的乙酸,以确定的脂肪酸合成和降解速率和跟踪的代谢甘油脂中的叶子。该方法通常被称为醋酸酯脉冲追踪标记法,已被广泛用于探查脂质代谢的各个方面(Allen等,2015),包括自噬在膜脂质更新中的作用(Fan等,2015)。,2019)和脂质与淀粉代谢途径之间的相互作用(Yu et al。,2018)。
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Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa
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Author:
Date:
2021-01-05
[Abstract] Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H2O2) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H2O2 solution of different concentrations; 1% H2O2 solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it ...
[摘要] [摘要]大麻种子的发芽对种植者和研究者都是重要的过程。许多生物技术应用需要可靠的无菌方法来发芽种子。该协议概述了使用过氧化氢(H 2 O 2 )溶液作为液体发芽介质的苜蓿种子的发芽过程。在这种PROT ocol,所有三个步骤,包括种子消毒,发芽,和幼苗发育中进行了一个ħ 2 ö 2种不同浓度的溶液; 1%H 2 O 2 解决方案显示出最快,最有效的发芽。该协议还显示了对于具有较低生存能力的非常老的大麻种子的高发芽效率。总的来说,与水控制相比,该协议证明了更好的发芽能力,并降低了污染的风险,使其适合组织培养和其他敏感应用。
[背景技术]大麻,否则称为大麻或大麻,是其中男性/女性性别由异型染色体(X和Y)确定的年主要雌雄异株的开花植物(德特等人,2020)。大麻种植有多种农业用途。几乎所有大麻植物的部分都被使用,种子用作食物,茎用作纤维,花/叶用作药物。花会产生大麻素和芳香化合物的混合物,这些化合物因其治疗和娱乐作用而受到重视(Chandra等人,2017)。大麻植物通过插条或通过种子发芽而无性繁殖。种子萌发是研究人员,育种非常重要,而且种植者的喜爱,尤其是从优良品种的种子可能是非常昂贵的和有价值的。此外,较老的种子发芽率可能降低,而细菌和真菌污染会危害发芽,特别是在种子发芽进行组织培养繁殖时。为了解决这些问题,我们已经开发了一种快速,无菌,使用1%的过氧化氢(H,高效的种子萌发协议2 ...
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K+ Release Assay and K+ Measurement in Oocyte Assay
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Author:
Date:
2020-11-05
[Abstract] The Xenopus oocyte is a powerful system for the exogenous expression and functional characterization of plant membrane transport proteins. Until now, a number of potassium transporters and channels have been identified in oocytes expression system by the two-electrode voltage clamp technology. It is difficult to characterize K+/H+ anti-transporters, especially, electroneutral transporter. The K+ efflux assay system enables easy, fast, large-scale measurement of the transporters activity without two-electrode voltage clamp technology. This protocol describes a technique to measure the efflux activity of potassium transporter in oocytes expressing system.
[摘要] [摘要]爪蟾卵母细胞是针对外源表达和植物膜转运蛋白的功能表征一个强大的系统。迄今为止,通过两电极电压钳技术已经在卵母细胞表达系统中发现了许多钾转运蛋白和钾通道。很难表征K + / H +反转运蛋白,尤其是电中性转运蛋白。K +外排测定系统无需两电极电压钳技术即可轻松,快速,大规模地测量转运蛋白的活性。该协议描述了一种测量卵母细胞表达系统中钾转运蛋白外排活性的技术。
[背景[非洲爪蟾卵母细胞中积累了酶,蛋白质和细胞器,这些都可以被利用来生产大量,正确翻译后修饰,正确定点的外来蛋白质(Miller and Zhou,2000)。因此,卵母细胞通过使用两电极电压钳技术提供了一种有效的表达系统,以功能上表征植物膜蛋白。在卵母细胞中表达的第一个植物钾转运蛋白是K +通道(Cao等,1992)。此后,大部分钾通道和转运已经在卵母细胞的研究(Schachtman等人,1994; V é RY等人,1994; Wang和吴,2013年)。但是一些K + / H +反转运蛋白是电中性的,两电极电压钳技术无法表征这些转运蛋白。为了克服这个问题,我们采用了一种新的方法来确定这些转运蛋白的活性,方法是测量孵育前后培养基中钾含量的变化。细胞内ķ +浓度为约70 - 150mM的(韦伯,1999年)。由表达的K +转运蛋白介导的K +外排可引起细胞内和细胞外K ...
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