Non-destructive bioluminescence detection system for monitoring defense gene expression in tobacco BY-2 cells
Non-destructive bioluminescence detection system for monitoring defense gene expression in tobacco BY-2 cells
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用于监测烟草BY-2细胞防御基因表达的无损生物发光检测系统
DOI：
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
K. Hiratsuka
中科院分区：
文献类型：
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作者：
Y. Watakabe;Sachiko Ono;Tsuneyuki Tanaka;K. Hiratsuka
利用启动子-荧光素酶基因融合转化的烟草BY-2细胞，建立了一种非破坏性、灵敏的体内检测系统，用于监测高等植物细胞中防御基因的表达。从烟草by -2细胞基因组DNA中分离到烟草水杨酸诱导致病相关基因PR-1a的启动子片段，通过农杆菌介导的转化将其与荧光素酶报告基因融合并导入植物细胞。为了检测PR-1a启动子作为荧光素酶活性的表达，将转化的细胞与荧光素溶液混合，并使用常规发光计监测生物发光水平。由于by -2细胞PR-1a启动子表达水平相对较高，因此在对数相生长条件下，SA处理对荧光素酶活性的诱导作用几乎检测不到。然而，我们可以观察到在固定相下施用SA后荧光素酶活性的浓度依赖性诱导。苯并（1,2,3）噻二唑-7-碳硫酸s -甲酯（BTH）和甲基-2,6-二氯异烟酸（INA）处理也能显著提高转基因细胞的荧光素酶活性，且呈剂量依赖性。另一方面，用无活性SA类似物4-羟基苯甲酸（4-HBA）处理对荧光素酶活性没有影响。该检测系统的灵敏度高于先前报道的检测SA或BTH诱导的技术。这些结果表明，这种快速、廉价和通用的检测系统将有助于鉴定和表征高等植物细胞中能够诱导防御基因表达的化学物质。
Using tobacco BY-2 cells transformed with a promoter-luciferase gene fusion, we developed a non-destructive and sensitive in vivo assay system for monitoring defense gene expression in higher plant cells. A promoter fragment of a tobacco salicylic acid (SA) inducible pathogenesis-related gene, PR-1a isolated from the genomic DNA of tobacco BY-2 cells, was fused to the luciferase reporter gene and introduced into plant cells by Agrobacterium-mediated transformation. To detect the PR-1a promoter expression as a luciferase activity, transformed cells were mixed with luciferin solution and the bioluminescence levels were monitored in vivo using a conventional luminometer. Because the PR-1a promoter expression levels of the BY-2 cells are relatively high, the induction of the luciferase activities by the treatment with SA was barely detectable under log phase growth conditions. However, we could observe concentration dependent induction of the luciferase activities following SA application under stationary phase. Treatment with benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl-2,6-dichloroisonicotinic acid (INA) also resulted in a drastic increase in luciferase activities of transgenic cells in a dose dependent manner. On the other hand, treatment with an inactive SA analog 4-hydroxybenzoic acid (4-HBA) showed no influence on luciferase activities. The sensitivity of the assay system was higher than the previously reported techniques for the detection of induction by SA or BTH. These results indicate that this rapid, inexpensive and versatile assay system would be useful for the identification and characterization of chemicals capable of inducing defense gene expression in higher plant cells.
广谱抗病拟南芥突变体的分离和表征。
影响因子：
3.3
作者：
Maleck,Klaus;Neuenschwander,Urs;Cade,RebeccaM;Dietrich,RobertA;Dangl,JefferyL;Ryals,JohnA
通讯作者：
Ryals,JohnA