Real-time PCR Analysis of PAMP-induced Marker Gene Expression in Nicotiana benthamiana
本塞姆氏烟草中PAMP诱导标记基因表达的实时定量PCR分析
发布: 2018年10月05日第8卷第19期 DOI: 10.21769/BioProtoc.3031 浏览次数: 9511
评审: Wende Liu Satyabrata NandaAnonymous reviewer(s)
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Feb 2018
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Abstract
Perception of pathogen-associated molecular patterns (PAMPs) often triggers various innate immune responses in plants. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. Here we described a protocol to monitor the relative expression level of marker genes in Nicotiana benthamiana upon treatment with PAMPs. The procedure includes leaf treatment using PAMPs, total RNA isolation, cDNA synthesis, quantitative real-time PCR and data analysis. This protocol is applicable to monitor marker gene expression triggered by different PAMPs in N. benthamiana.
Keywords: PAMPs (PAMPs)
PTI (PTI)
Real-time PCR (实时定量PCR)
Nicotiana benthamiana (烟草本塞姆氏)
Defense-related marker gene (防御相关标签基因)
Background
Pathogen-associated molecular patterns, namely PAMPs, are a class of molecules derived from pathogens and are relatively conserved across microorganisms. Multiple PAMPs such as flg22 and XEG1 (Felix et al., 1999; Ma et al., 2015), have been characterized that can be detected by plant cell surface localized pattern-recognition receptors (PRRs) and thereby induce PAMP-triggered immunity (Couto and Zipfel, 2016). One of the predominant PAMP-triggered responses is the activation of defense-related maker genes (Navarro et al., 2004; Zipfel et al., 2006). Nicotiana benthamiana has been used extensively as a model plants and is sensitive to multiple PAMPs. In N. benthamiana, the marker genes, such as NbCYP71D20, NbACRE31 and NbWRKY22, were previously found that are rapidly activated upon PAMP treatment (Heese et al., 2007; Segonzac et al., 2011; Wang et al., 2018). Here, we describe a detailed protocol for checking the PAMP-triggered marker gene expression in N. benthamiana. The relative gene expression was also determined in parallel using a negative control to exclude the background noise.
Materials and Reagents
- MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4312063 )
- 1,000 µl pipette tips (Corning, Axygen®, catalog number: TF-1000-R-S )
- 200 µl pipette tips (Corning, Axygen®, catalog number: TF-200-R-S )
- 10 µl pipette tips (Corning, Axygen®, catalog number: TF-300-R-S )
- 1 ml needless syringe (BD, catalog number: 309659 )
- 1.5 ml RNase-free tube (Corning, Axygen®, catalog number: MCT-150-C )
- Axygen® 0.2 ml Polypropylene PCR Tube Strips (8-Tubes/Strip) (Corning, Axygen®, catalog number: PCR-0208-C )
- MicroAmpTM Optical 96-Well Reaction Plate with Barcode (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4306737 )
- MicroAmpTM Optical Adhesive Film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4360954 )
- Tissue paper
- Healthy 5-6 weeks N. benthamiana plants (see Figure 1)
- Any PAMPs of interest and corresponding control solution
- Liquid nitrogen
- β-mercaptoethanol (Solarbio, catalog number: M8210 )
- E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, catalog number: R6834-01 )
- DNase/RNase-free ddH2O (Solarbio, catalog number: R1600 )
- PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Clontech, catalog number: RR047A )
- TB GreenTM Premix Ex TaqTM (Tli RNase H Plus) (Takara Bio, Clontech, catalog number: RR420A )
- 1 mM flg22 stock solution (Gene Script, RP19986) (see Recipes)
- 1 mM XEG1 stock solution (see Recipes)
- 70% ethanol (see Recipes)
Equipment
- Mortar and pestle
- Growth chamber
- NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000 )
- Vortexer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0246)
- Cold Centrifuge (Eppendorf, model: 5424 R , catalog number: 5404000014)
- Pipettes 100-1,000 µl (Eppendorf, catalog number: 3120000062 )
- Pipettes 10-100 µl (Eppendorf, catalog number: 3120000046 )
- PCR Thermal Cyclers (Thermo Fisher Scientific, Applied BiosystemsTM, model: 2720, catalog number: ED000651 )
- MicroAmpTM Adhesive Film Applicator (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4333183 )
- MPS 1000 Mini PCR Plate Spinner (Labnet International, catalog number: C1000 )
- ABI 7500 fast real-time PCR system (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4351107 )
Software
- Applied Biosystems Sequence Detection Software v1.4.0
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Liu, F., Xu, Y., Wang, Y. and Wang, Y. (2018). Real-time PCR Analysis of PAMP-induced Marker Gene Expression in Nicotiana benthamiana. Bio-protocol 8(19): e3031. DOI: 10.21769/BioProtoc.3031.
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分类
植物科学 > 植物分子生物学 > RNA > 转录
植物科学 > 植物生理学 > 生物胁迫
分子生物学 > RNA > RNA 提取
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