Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves
大麦幼苗叶的病毒诱导基因沉默（VIGS）
发布: 2015年06月20日第5卷第12期 DOI: 10.21769/BioProtoc.1506 浏览次数: 13189
评审: Samik BhattacharyaPablo Bolanos-VillegasSollapura J. Vishwanath
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参见作者原研究论文
The authors used this protocol in:
Sep 2014
Abstract
Virus induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterisation. This method exploits a dsRNA-mediated antiviral defence mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid time frame as compared to the generation of stable transformants. Here we describe a simple method for silencing a target gene in barley seedling leaves using vectors based on the Barley Stripe Mosaic Virus (BSMV).
Materials and Reagents
- Bacterial strain: Escherichia coli strain DH5 α
- Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 11304-011 )
- pGEM-T easy cloning kit (Promega Corporation, catalog number: A1380 )
- QIAGEN QIAprep Centrifuge Miniprep Kit (QIAGEN, catalog number: 27106 )
- Recombinant Taq DNA polymerase (Life Technologies, InvitrogenTM, catalog number: 10342-020 )
- Plasmid vectors: pα42 (pα), pβ42.sp1 (pβ), pSL038-1 (pγ), pSL038-PDS (pγ-PDS).
Note: See Scofield and Brandt (2012) for the plasmid maps (provided by Prof. Steven R. Scofield, https://link.springer.com/protocol/10.1007/978-1-61779-882-5_7/figures/1). - Luria-Bertani (LB) liquid broth (Sigma-Aldrich L3152) and solid media containing 1.2% (w/v) agar (OXOID, catalog number: LP0013 )
- Ampicillin (100 mg/l working concentration) (Sigma-Aldrich, catalog number: A0166-5G )
- Restriction enzymes:
PacI (New England Biolabs, catalog number: R0547S )
MluI (New England Biolabs, catalog number: R0198S )
SpeI (New England Biolabs, catalog number: R0133S ) - Ethanol (Sigma-Aldrich, catalog number: 459844 )
- sodium acetate (Sigma-Aldrich, catalog number: S2889 )
- mMessage mMachine T7 in vitro transcription kit (Ambion Sigma-Aldrich, catalog number: AM1344 )
- John Innes compost No 2 (Westland Horticulture)
- Glycine (Sigma-Aldrich, catalog number: 410225 )
- K2HPO4 dibasic (Sigma-Aldrich, catalog number: P3786 )
- Sodium pyrophosphate decahydrate (Sigma-Aldrich, catalog number: 221368 )
- Bentonite (Aldrich, catalog number: 28,523-4 )
- Celite (Fluka, catalog number: 22141 )
- 5x GP buffer (see Recipes)
- FES buffer (see Recipes)
Equipment
- Plant growth chambers (24 °C, 16/8 photoperiod and 55% humidity) (CambridgeHOK containment glasshouse)
- Sterile culture tubes (Falcon, catalog number: 352057 )
- Centrifuge tubes (SARSTEDT AG, catalog number: 72.695.500 )
- Filter paper (Whatman, catalog number: 1001-090 )
- Cooled centrifuge (Eppendorf)
- Shaking incubators for cultures (New Brunswick Scientific)
- Gel apparatus (Helixx Mupid-exU) and image system (Fusion Fx vilber lourmat)
- Thermal cycler (MJ Research PTC200)
- Gel electrophoresis chamber (Helixx Mupid-exU)
- Autoclave (Priorclave)
Software
- Primer3 software (version 0.4.0; http://frodo.wi.mit.edu/primer3/)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Gunupuru, L. R., Ali, S. S., Doohan, F. M. and Scofield, S. R. (2015). Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves. Bio-protocol 5(12): e1506. DOI: 10.21769/BioProtoc.1506.
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分类
植物科学 > 植物分子生物学 > RNA
植物科学 > 植物免疫 > 病害生物测定
分子生物学 > RNA > RNA 干扰
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