本塞姆氏烟草中PAMP诱导标记基因表达的实时定量PCR分析

Fan Liu; Yuanpeng Xu; Yan Wang; Yuanchao Wang

doi:10.21769/BioProtoc.3031

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Peer-reviewed

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

FL Fan Liu

YX Yuanpeng Xu

Yan Wang

Yuanchao Wang email

发布: 2018年10月05日第8卷第19期 DOI: 10.21769/BioProtoc.3031 浏览次数: 8122

评审: Wende Liu Satyabrata NandaAnonymous reviewer(s)

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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

MicroAmp^TMSplash-Free 96-Well Base (Thermo Fisher Scientific, Applied Biosystems^TM, 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 )
MicroAmp^TM Optical 96-Well Reaction Plate with Barcode (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4306737 )
MicroAmp^TM Optical Adhesive Film (Thermo Fisher Scientific, Applied Biosystems^TM, 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 )
PrimeScript^TMRT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Clontech, catalog number: RR047A )
TB Green^TM Premix Ex Taq^TM (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
NanoDrop^TM1000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop^TM 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 Biosystems^TM, model: 2720, catalog number: ED000651 )
MicroAmp^TM Adhesive Film Applicator (Thermo Fisher Scientific, Applied Biosystems^TM, 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 Biosystems^TM, catalog number: 4351107 )

Software

Applied Biosystems Sequence Detection Software v1.4.0

Procedure

English

中文翻译

文章信息

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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.