| Purification of Rubisco from Chlamydomonas reinhardtii
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Author:
Date:
2015-12-05
[Abstract] Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell ...
[摘要] 衣藻衣原体是叶绿体研究的模式生物体。 除了其他方便的特点，叶绿体基因组转化的可行性区分这种单细胞藻理想的操纵叶绿体基因表达瞄准生物技术目标，如改善生物燃料和生物质生产。 核酮糖1,5-二磷酸羧化酶/加氧酶（EC 4.1.1.39，Rubisco）是光合作用碳固定酶，其被认为对藻类培养物中的生物量累积是至关重要的。 Rubisco的野生型和定点突变体的纯化。 通常进行研究其催化性质并评估菌株的碳固定潜力。 在该方案中，通过细胞沉淀的超声处理提取Rubisco，并通过硫酸铵沉淀，蔗糖梯度离心（Goldwaithe和Bogorad，1975）和阴离子交换层析纯化。
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| Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea
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Author:
Date:
2015-12-05
[Abstract] Plant proteins can be targeted to intracellular (i.e., cytosol, vacuole, organelles etc.) or extracellular (i.e., cell walls, apoplast) compartments. Dual targeting is a key mechanism with important implications for plant metabolism, growth, development and defense etc. Harsh Hakea (Hakea prostrata R.Br.) is a perennial species and member of the Proteaceae family that thrives on extremely phosphate impoverished soils of southwestern Australia. Harsh Hakea is not a common model organism, but has been widely developed for physiological and molecular/biochemical studies of the endogenous adaptations of an ‘extremophile’ plant species to abiotic stress, including low phosphorus tolerance. Tissues of Harsh Hakea contain large amounts of compounds (e.g. ...
[摘要] 植物蛋白可以靶向细胞内（即胞质溶胶，液泡，细胞器等）或细胞外（即细胞壁，质外体）区室。双重靶向是对植物新陈代谢，生长，发育和防御等具有重要影响的关键机制。 Harsh Hakea（ Hakea prostrata R.Br.）是一种多年生物种和成员的泛革兰科家族，在澳大利亚西南部的极端磷酸盐贫困土壤上生长。 Harsh Hakea不是一种常见的模式生物，而是广泛开发用于"极端"植物物种对非生物胁迫（包括低磷耐性）的内源性适应的生理学和分子/生物化学研究。 Harsh Hakea的组织含有大量干扰可溶性蛋白质提取的化合物（例如，酚类化合物）。我们以前优化了来自Harsh Hakea蛋白质组织根的细胞内蛋白质的提取，将可溶性蛋白质产量提高至少10倍（Shane等人，2013）。在这里，我们描述的提取和细胞内分离从"松散绑定"细胞壁蛋白质在Harsh Hakea的协议。
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| In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
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Author:
Date:
2015-11-05
[Abstract] An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified ...
[摘要] 水生被子植物Vallisneria（Alismatales：Hydrocharitaceae）已经在一个世纪内用作研究动态细胞内运动的光调节的优良实验材料，包括叶绿体再分布和细胞质流（Senn，1908; Seitz，1987 ; Takagi，1997）。然而，对分子机制的理解落后，因为难以应用现代技术，如基因转化到这个植物。特别地，哪种类型的光感受器在这些有趣的反应中起作用长期以来一直是未解决的主题。最近，在水生植物中首次在Vallisneria中分离编码植物特异性蓝光受体光托品的基因（Sakai等人，2015）。光金蛋白首先被鉴定为拟南芥中下胚轴向往性的光感受器，并且现在已知调节许多反应，包括各种植物物种中的叶绿体光定位运动（Christie，2007）。光催化素主要位于质膜上，并且它们由蓝光诱导的自磷酸化是信号转导途径的关键步骤（Sakamoto和Briggs，2002; Kong等，2006; et al。，2013; Inoue et al。，2010）。在这里，我们描述了使用Vallisneria的原始微粒体和质膜富集级分的体外蛋白磷酸化测定的方案，其使得我们能够验证光催化素的存在并表征它们的自身磷酸化反应。在这些分析之后，Sakai等人（2015）提出 Vallisneria 光催化素介导高强度蓝光诱导的叶绿体回避反应。
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