| Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers
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Author:
Date:
2021-01-05
[Abstract] Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to ...
[摘要] [摘要]植物基因敲低是研究基因型与表型关系，提高作物对病害的抵抗力以及实现植物分子高效生物合成的有用方法。小干扰RNA（siRNA）介导的基因沉默是在植物中实现基因敲低的最常见方法之一。传统上，通过将siRNA序列编码到DNA载体中，将siRNA传递到完整的植物细胞中，然后通过病毒和/或细菌方法传递。在这个协议中，我们提供的siRNA分子的替代直接递送方法为完整的植物细胞的高效瞬时根Ë击倒在模型的烟草植物，烟草本塞姆氏烟草，叶子。我们的方法使用一维碳基纳米材料，单壁碳纳米管（SWNTs）来传递siRNA，而不依赖于病毒/细菌的传递。我们方法的独特优势在于：i ）不需要对siRNA序列进行DNA编码； ii）与非生物方法相比，这种非生物方法可在更广泛的植物物种中起作用，并且iii）使用非生物递送时，调节并发症更少方法，其中基因沉默是瞬时的，而无需对植物基因组进行永久性修饰。
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[背景技术[ 0002 ]在1990年代初，植物研究人员研究矮牵牛花的着色发现了通过RNA干扰（RNAi）引起的基因沉默（Van der ...
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| Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
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Author:
Date:
2020-03-05
[Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.
[摘要] [摘要 ] 超氧化物歧化酶（SOD能）充当主防御针对反应性氧物质（ROS）通过转换的超氧阴离子自由基（O 2 - ）为分子氧（O 2 ）和过氧化氢（H 2 ? 2 ）。这种酶的家庭成员包括CuZnSODs ，MnSODs ，FeSODs 和NiSODs ，这取决于是需要适当的活动辅助因子的性质。大多数真核生物，包括酵母，都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。
[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。：这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ，MnSODs ，FeSODs ，和NiSODs 。在酿酒酵母中，有两个S OD ：CuZn-Sod1p和Mn-Sod2p（Abreu和Cabelli ...
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| Preparation and Purification of Proteins Secreted from Phytophthora sojae
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Author:
Date:
2018-10-20
[Abstract] Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions.
[摘要] 大豆根和茎腐病的致病因子大豆疫霉（Phytophthora sojae）是造成大豆生产的巨大经济损失的原因。 P上。 sojae >秘密各种效应器重新编程宿主免疫力。植物质外体空间是植物 - 病原体相互作用的主要战场。在这里，我们描述了从 P中纯化和分离分泌蛋白的方案。 sojae >，包括来自 P的分泌蛋白的沉淀。 sojae >培养滤液，分泌蛋白的色谱纯化和质谱分析蛋白质。通过该协议，可以更容易地鉴定 Phytophthora >中潜在的质外体效应物，并有助于我们对植物 - 微生物相互作用的理解。
【背景】从 Phytophthora >物种中分泌的蛋白质的纯化对于理解 Phytophthora >发病机理至关重要。过去，V8果汁和植物（番茄和利马豆）果汁培养基已用于培养 Phytophthora >，培养物过滤后用于分析 Phytophthora >分泌的蛋白质。这些方案的缺点是培养基含有大量的植物蛋白，它们代表了大部分检测到的蛋白质。在该方案中，我们使用了不含任何蛋白质的合成液体培养基。该培养基显着降低了 Phytophthora >培养滤液的背景。此外，利用凝胶过滤脱盐和筛分柱代替离子交换柱，可以有效和大规模纯化 Phytophthora >分泌的蛋白质。
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