Virus-induced Gene Silencing (VIGS) in Phalaenopsis Orchids
蝴蝶兰的病毒诱导基因沉默（VIGS）
发布: 2014年12月20日第4卷第24期 DOI: 10.21769/BioProtoc.1359 浏览次数: 14114
评审: Tie LiuAnonymous reviewer(s)
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The authors used this protocol in:
May 2014
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Abstract
This is a protocol to produce stable silencing efficacy and efficiency for VIGS using CymMV as a silencing vector for floral functional genomics in Phalaenopsis orchids. This protocol is established based on a method created by Lu et al. (2007), and then modified by Hsieh et al. (2013a; 2013b), Lu et al. (2012) successfully engineered a cloning vector (pCymMV-Gateway) in that the target gene fragment is simple to insert and can be manipulated with high efficiency. The silencing vector is inoculated into plants by Agro-inoculation by using Agrobacterium tumefaciens (A. tumefaciens) strain EHA105. Agro-infiltration of leaves for use in VIGS study of orchid flowers is a time saver and produces less damage to flower buds.
Materials and Reagents
- Virus-free Phalaenopsis plants
- TOP10 Escherichia coli (E. coli) competent cells
- Agrobacterium tumefaciens strain EHA105
- Gel/PCR DNA fragments extraction kit (Geneaid Biotech, catalog number: DF100 )
- Gateway® BP-ClonaseTM II enzyme mix (Life Technologies, InvitrogenTM, catalog number: 11789-020 )
- Selective plate (LB plate with 50 µg/ml Kanamycin)
- High-speed plasmid mini kit (Geneaid Biotech, catalog number: PD100 )
- Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
- Murashige and Skoog salt (Sigma-Aldrich, catalog number: M5524 )
- Tryptone (US Biological, catalog number: 12855 )
- Yeast extract (Affymetrix, catalog number: 23547 )
- NaCl (Sigma-Aldrich, catalog number: S1446 )
- Tris-HCl (US Biological, catalog number: 22676 )
- CaCl2 (Inter-County Mechanical Corp., catalog number: 03-11250 )
- Glycerol (J.T.Baker®, catalog number: 2136-01 )
- Luria Broth medium (LB medium) (see Recipes)
- Proteinase K solution (see Recipes)
- Murishige and Skoog medium (MS medium) salt (see Recipes)
Equipment
- GeneAmp PCR system 9700 (Life Technologies, Applied Biosystems®)
- Laminar flow cabinet
- BioChrom Libra S50 UV/Vis Spectrophotometer (Biochrom)
- Incubator shaker
- Electroporation machine (Kaneka Corporation, Eurogentec)
- Electroporation cuvettes (EquiBio, catalog number: ECU-102 )
- Centrifuges for MiniSpin Eppendorf and 50 ml conical centrifuge tubes
- 1-ml syringe with a needle
- Water bath
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hsieh, M., Pan, Z., Yeh, H. and Chen, H. (2014). Virus-induced Gene Silencing (VIGS) in Phalaenopsis Orchids. Bio-protocol 4(24): e1359. DOI: 10.21769/BioProtoc.1359.
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分类
植物科学 > 植物分子生物学 > RNA > RNA 干扰
植物科学 > 植物分子生物学 > RNA > 转录
分子生物学 > RNA > RNA 干扰
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