蝴蝶兰的病毒诱导基因沉默（VIGS）

Ming-Hsien Hsieh; Zhao-Jun Pan; Hsin-Hung Yeh; Hong-Hwa Chen

doi:10.21769/BioProtoc.1359

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Peer-reviewed

Virus-induced Gene Silencing (VIGS) in Phalaenopsis Orchids

蝴蝶兰的病毒诱导基因沉默（VIGS）

Ming-Hsien Hsieh

ZP Zhao-Jun Pan

Hsin-Hung Yeh

Hong-Hwa Chen email

发布: 2014年12月20日第4卷第24期 DOI: 10.21769/BioProtoc.1359 浏览次数: 13205

评审: Tie LiuAnonymous reviewer(s)

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Cover of New Phytologist, featuring study using the protocol.

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-Clonase^TM II enzyme mix (Life Technologies, Invitrogen^TM, 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

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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.