一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量
Summary
程序性细胞死亡，DNA梯状或TUNEL法检测，如哺乳动物系统常用的检测往往难以重现植物。 GUS报告制度相结合，我们提出一个快速的，植物的瞬态检测，分析特定基因的潜在死亡属性。
Abstract
我们已经开发出一种新的瞬态植物表达系统，同时表达报告基因β-葡萄糖醛酸酶（GUS）的细胞死亡与公认的积极或消极的监管机构，。在这个系统中，N benthamiana叶渗透与35S驱动的表达，其中包含要分析的基因磁带，GUS载体pCAMBIA 2301使用车辆农杆菌菌株LBA4404。由于活细胞所需的GUS基因表达发生，GUS活性的损失预计时，这个标记基因与细胞死亡的积极监管机构的合作表示。同样，增加的GUS活性是观察时使用抗凋亡基因相比，矢量控制。如下图所示，我们已经成功地在我们的实验室中使用这个系统来分析亲和抗死亡的球员。这些措施包括植物抗凋亡的Bcl – 2的相关athanoGene（袋）的家庭，以及，如BAX已知的哺乳动物的细胞死亡诱导，。此外，我们已经用这个系统来分析具体截断内蛋白质死亡的功能，可提供线索，这些蛋白质可能的翻译后修饰/激活。在这里，我们提出一种快速，敏感的植物为基础的方法，如在初步调查的特定基因的死亡功能第一步。
Protocol
benthamiana烟草植物生长温度控制在25 ° C时生长室。使用3-6周老植物叶片完全展开的健康。 提示：通过使用新兴的叶子获得更好的成果 1。农杆菌介导的瞬态渗透协议： 第1天农杆菌介导的基因（S）来检测细胞死亡和载体，含有适当的载体（应变农杆菌）的甘油库存数的LB /利福平（25微克/毫升）/卡那霉素（100微克/毫升）?…
Discussion
<p class="jove_content"它往往是很难使用在哺乳动物系统中常见的植物细胞凋亡检测技术。 GUS报告制度相结合，我们目前的植物为基础的，敏感的方法检测和分析细胞死亡的球员。这种方法需要利用简单的事实，活细胞都需要GUS基因表达发生。为了确保测试结果有意义和可重复性，它是窝藏GUS卡带和要检测的基因，文化渗透在同等比例的关键。这些比率应保持比较时，额外的潜在细胞死亡的球员。如上所示，我们已经?…
Divulgazioni
The authors have nothing to disclose.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Rifampicin | VWR | IC19549001 | 25mg/mL stock in DMSO |
| 4′-hydroxy-3′,5′-dimethoxyacetophenone (Acetosyringone) | VWR | TCD2666 | 0.981 g/mL in DMSO (1M stock) |
| 2-(4-morpholino)ethanesulfonic acid monohydrate (MES) | VWR | EM-6110 | 1.92 g/L in water for making 1 L of infiltration media |
| Bacto-tryptone | Fisher | BP1421-2 | 10g/L in water for making 1 L of LB medium |
| Bacto-yeast extract | VWR | EM1.03753.0500 | 5g/L in water for making 1 L of LB medium |
| Sodium chloride | VWR | EM-7710 | 10g/L in water for making 1 L of LB medium |
| Sodium phosphate monobasic monohydrate | VWR | MK-7868-12 | 2.5g/L in water for making 50mM of buffer NaPi |
| Sodium phosphate dibasic heptahydrate | VWR | EMD-SX0715-1 | 5g/L in water for making 50mM of buffer NaPi |
| Magnesium sulfate heptahydrate | VWR | EM-MX0070-1 | 2.5 g/L in water for making 1 L of infiltration media |
| N-Lauroylsarcosine | VWR | TCL0151-500G | 0.1% v/v in GUS buffer |
| 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium salt (X-gluc) | Gold Biotechnology | G1281C | 1mg/100uL in methanol 100% |
| 4-Methylumbelliferyl-μ-D-glucuronide hydrate (MUG) | Sigma | M5664 | 2 mM in 100 uL of GUS extraction buffer |
| Potassium ferrocyanide trihydrate | VWR | EM-PX1460-1 | 100mM stock for X-gluc substrate solution |
| β-Methylumbelliferone (MU) | Sigma-Aldrich | M1381 | For MU standard curve use GUS extraction buffer |
| Sodium carbonate | VWR | EM-SX0395-11 | 0.2 M in water for making GUS stop buffer |
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Tags
A β-glucuronidase (GUS)Transient Plant Expression SystemReporter GenePositive Regulators Of Cell DeathN. Benthamiana LeavesCo-infiltration35S Driven Expression CassetteGene AnalysisGUS Vector PCAMBIA 2301Agrobacterium Strain LBA4404Live CellsMarker GeneAnti-apoptotic GenesPro- And Anti-death PlayersBcl-2 Associated AthanoGene (BAG) FamilyMammalian Inducers Of Cell DeathBAXPost-translational Modification/activation Of ProteinsRapid And Sensitive Plant Based Method
Citazione di questo articolo
Kabbage, M., Ek-Ramos, M., Dickman, M. A β-glucuronidase (GUS) Based Cell Death Assay. J. Vis. Exp. (51), e2680, doi:10.3791/2680 (2011).