Luciferase-mediated assay to detect the PAMP-triggered gene expression in transgenic Nicotiana benthamiana.

Plant signaling & behavior Pub Date : 2024-12-31 Epub Date: 2024-11-25 DOI:10.1080/15592324.2024.2411918
Ola Barakat, Fushuang Zhang, Mengzhu Zeng, Yuanchao Wang
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Abstract

Luciferase is one of the bioluminescence-producing agents, which was widely used as a reporter enzyme for constructing bioassay systems to study gene expression with high accuracy and within a broad dynamic spectrum. Perception of pathogen associated molecular patterns (PAMPs) in plants often lead to significant transcriptional changes. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. In this study, we showed that the marker gene CYP71D20 was rapidly activated in Nicotiana benthamiana upon treatment with the bacterial PAMP flg22 and the Phytophthora elicitin INF1. In addition, we generated transgenic N. benthamiana using the luciferase as a reporter gene to analyze CYP71D20 gene expression upon PAMP treatment. The transgenic line carrying the luciferase gene driven by CYP71D20 promoter was treated with the bacterial PAMP flg22 or Phytophthora elicitin INF1. Transcriptional activation of CYP71D20 was measured by monitoring the luciferase activity. The results showed that the LUC activity was increased after treatment with different PAMPs, indicating that the CYP71D20-promotor luciferase assay can be used to study the PAMP-triggered gene expression in N. benthamiana.

以荧光素酶为介导的检测方法,用于检测转基因烟草中 PAMP 触发的基因表达。
荧光素酶是生物发光剂之一,被广泛用作构建生物测定系统的报告酶,以高精度和宽动态光谱研究基因表达。植物对病原体相关分子模式(PAMPs)的感知往往会导致显著的转录变化。防御相关基因的转录变化通常被用作检测 PAMP 触发的植物免疫反应的标记。在本研究中,我们发现在用细菌 PAMP flg22 和 Phytophthora elicitin INF1 处理烟草后,标记基因 CYP71D20 被迅速激活。此外,我们还利用荧光素酶作为报告基因生成了转基因烟草,以分析 PAMP 处理时 CYP71D20 基因的表达情况。将携带由 CYP71D20 启动子驱动的荧光素酶基因的转基因品系用细菌 PAMP flg22 或 Phytophthora elicitin INF1 处理。通过监测荧光素酶活性来测量 CYP71D20 的转录激活情况。结果表明,经不同的 PAMP 处理后,荧光素酶活性均有所提高,这表明 CYP71D20 启动子荧光素酶检测法可用于研究 PAMP 触发的 N. benthamiana 基因表达。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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