| Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for the Overexpression of The COI1 Receptor
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Author:
Date:
2021-01-05
[Abstract] Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities.
In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long ...
[摘要] [摘要]细胞悬浮培养已经研究了数十年，以生产天然分子。然而，产生稳定转化的细胞系的困难限制了它们用于可再现地和高产量地生产高价值化学品的用途。
在该协议中，设计并详细介绍了稳定转化和维持拟南芥细胞悬浮培养物的方法。直接用根癌农杆菌转化拟南芥细胞培养物，以过量表达可乐宁不敏感1（COI1）茉莉酸酯受体。转化后建立细胞培养物，并连续保持并测试COI1的过表达。该协议以前也曾用于沉默拟南芥过氧化物酶，并允许长期维持转化细胞。提供了在液体和固体培养基中培养维持的详细信息，以及蛋白质表达的证据来证实转化。
所描述的系统为合成生物学研究独立于发育控制的信号传递并获得生物技术和医学领域感兴趣的代谢物提供了强大的工具。
[背景]植物细胞悬浮培养为植物次生代谢产物的生产和去分化组织的研究提供了可行的替代方法（Wu和Ge，2004； Lee等，2010）。数十年来，人们对它们的使用兴趣不断提高，因为它们可以为人类使用的高价值生物技术产品提供连续生产系统。有各种各样的细胞培养物可产生大量具有商业和工业意义的次级代谢产物，例如花青素，甜菜碱，调味剂，甜菊苷等（Rao和Ravishankar，2002）。
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| Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection
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Author:
Date:
2018-01-05
[Abstract] Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and ...
[摘要] Plasmodesmata（PD）是包裹在相邻植物细胞的细胞壁中的纳米（〜20nm宽）膜衬里的孔。它们允许细胞交换从糖，激素，RNA到各种蛋白质等营养物质的所有类型的分子。不幸的是，它们也被植物病毒劫持，使它们从细胞间传播，然后在整个植物体系中传播。它们在植物生物学中的核心地位使得理解其生理机制，尤其是将其功能与其结构联系起来至关重要。在过去的50年中，电子显微镜观察家们已经观察到了这些现象，并试图用超微结构来表征它们。他们为已知的这些毛孔奠定了基础（Tilney等人，1991; Ding等人，1992; Oparka和Roberts，2001; Nicolas等人， et al。，2017a）。
尽管三维电子显微镜（3D-EM）爆炸，PD超微结构仍然不支持这种技术。第一个技术难点是在尽可能接近原生状态的情况下处理它们。其次，由于染色/固定试剂穿透率差，其体积增大，含水量高以及存在酸性液泡，植物样品显示自己难以加工。最重要的是，它们在细胞壁上的独特位置和它们的纳米尺寸使得难以方便地染色以便看到这些孔隙的内部运作。
这里我们详细描述Nicolas et al。（2017b）中使用的协议，对PD进行细节化处理，并生成高分辨率的X线断层图。
【背景】高压冻结（HPF）依赖于样品中存在的水的玻璃化。通过以足够高的冷冻速度（10 ...
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