水稻原生质体制备和转化 Protocol

​ — edited by Roc_Yan 20221113

Abstract

Rice (Oryza sativa) is an important cereal crop and a model monocot plant for biology research. The reliable system of foreign DNA transformation and expression is a valuable strategy for basic research and molecular breeding application in rice. The Agrobacterium tumefaciens-mediated foreign DNA transformation system was a powerful tool for genetic research. However, it needs a long period to obtain the stable transformants for further analysis and the transformation rate limits in some organism. Protoplasts are plant cells without a cell wall, and it is much easier for foreign DNA transformation and expression. It has been widely applied in transient expression. Here, we describe a simple method for efficient protoplast isolation and transfection in rice.

植物非生物胁迫信号（Zhu. 2016）

生物胁迫(如病原体感染和食草动物的啃食)
非生物胁迫(例如干旱、高温、冷害、营养匮乏、盐害以及土壤中铝、砷、镉等有毒金属毒害)。
干旱、盐害以及温度胁迫是影响植物的地理分布、限制农作物产量,并威胁粮食安全的主要环境因子。
干旱造成的初级胁迫信号是高渗透压胁迫,通常被简称为渗透胁迫。这是由于典型的低渗透压条件对植物细胞来说并不是问题。盐胁迫同时对细胞造成渗透胁迫和离子毒害。干旱胁迫和盐胁迫有各自独立和一些共同的信号转导机制。干旱和盐胁迫的一个重要特征是渗透胁迫信号能够引起植物激素脱落酸(ABA)的累积，进而引起植物的适应性反应。

感知：感受蛋白的功能冗余,即一个感受蛋白的缺失并不导致胁迫应答的显著变化,可能是科学家面临的主要困难。另外一种情形是,某个感受器可能是植物生长必需的,而功能缺失突变体将不会存活,致使无法对其深入研究。此外,很难从技术上证明一种蛋白质或其他大分子是物理信号(如渗透压的变化、离子浓度或温度)的感受器。

Regulation of Ubiquitination Is Central to the Phosphate Starvation Response ^Review

泛素化在植物 PSR，Pi acquisition, and utilization efficiency 中具有重要的调节作用。
ER 相关的自噬受到磷酸盐饥饿刺激，从而调节根尖的局部磷感知。
不同磷酸盐供给条件下，磷酸盐转运体的内部分选和降解取决于泛素化。
泛素化和 SUMO化反应能够调节 PSR 途径中的转录因子和其他功能元件。

Background

• PSR
  • Plants modulate their root system architecture by developing shorter primary roots and increasing lateral root length and density to increase the root surface area.
  • Plants improve Pi uptake and translocation by regulating the expression and activity of Pi transporters, such as PHOSPHATE TRANSPORTER 1s (PHT1s) and PHOSPHATE 1 (PHO1).
  • Plants secrete organic acids and enzymes, such as phosphatases, to activate Pi in the soil and increase their association with mycorrhiza to enhance Pi uptake.
  • PSR modulates plant metabolism pathways, such as anthocyanin accumulation and Pi remobilization from senescing organs, via enzymes such as phosphatases.
• PTMs, including ubiquitination and SUMOylation, show a different and more precise mechanism.
  • Attachment of the small molecule ubiquitin (Ub) could regulate the activity, longevity, and/or localization of intracellular molecules.
  • Ub is often conjugated to the lysine residue of target substrates or another Ub to form polyubiquitin chains via the E1-E2-E3 cascade.
  • Ubiquitin Code: different Ub linkage sites and types determine the distinct fates of target proteins.
  • Ub can be reversibly erased by deubiquitinating enzymes (DUBs), which specifically function in different processes.
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端午节快乐！昨天入学体检，身高185cm，体重59kg。病理性偏瘦：少熬夜，饮食规律，多运动。

组蛋白 H3 上主要的四种甲基化修饰

H3K4 Methylation

Plant H3K4 methylation occurs almost exclusively in gene-coding regions.

@ H3K4me1 helping prevent cryptic transcription from the gene body and regulating transcription of overlapping genes, and playing an antagonistic role with H3K9me2. It’s also required for a subset of DNA methylation.

拟南芥和水稻中Pi吸收和分配

涉及蛋白：PHT1，PHT2，PHT3，PHT4，PHT5(VPT1)，SPX-MFS，PHO1，phosphate translocators

背景

  磷是植物生长发育所必需的大量营养元素之一，但由于其在土壤溶液中的溶解度和迁移性较差，以及磷与有机化合物的结合性，磷也是较难获取的元素之一。

常见组蛋白修饰及其生物学功能

• 组蛋白修饰对染色质结构和基因表达具有重要的作用和影响，了解目前已经报道的组蛋白修饰对基因表达影响，能够对基因表达相关课题研究提供很好的帮助。
• 以下是一些常见的组蛋白修饰：

原核蛋白提取纯化

His & GST Tag

Buffer Preparation

• 细菌裂解：1x PBS；1mM PMSF

• Beads Washing Buffer：1x PBS

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背景：

• 一、表观遗传学

  主要涉及 DNA 甲基化对基因表达的抑制、组蛋白修饰对基因表达的调控、small RNA 对靶基因的干扰以及 ATP 依赖的染色质重塑等表观遗传调控过程。

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