大麦表皮细胞中一种改良的单细胞转基因技术-粒子枪法

Shiwei Bai; Cheng  Chang; Xinyun  Han; Qian-Hua  Shen

doi:10.21769/BioProtoc.690

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Peer-reviewed

Modified Single-Cell Transient Gene Expression Assay in Barley Epidermal Cells

大麦表皮细胞中一种改良的单细胞转基因技术-粒子枪法

SB Shiwei Bai

CC Cheng Chang

XH Xinyun Han

QS Qian-Hua Shen email

发布: 2013年05月05日第3卷第9期 DOI: 10.21769/BioProtoc.690 浏览次数: 9966

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Jun 2012

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Abstract

Transient gene expression via biolistic particle delivery is a widely used technique for gene functional analysis in plants. In this protocol we describe a modified single-cell transient expression assay through transformation with a particle inflow gun of the model PDS-1000/He system (Bio-Rad). This assay was originally optimized for analyzing cell death activity and disease resistance function of the barley MLA (mildew locus A) disease resistance proteins against the powdery mildew fungus, which can be further adopted for other purposes for other types of plant proteins and in some other plant species, including Arabidopsis thaliana.

Keywords: Transient gene expression (瞬时基因表达)

Single epidermal cell (单个表皮细胞)

Particle bombardment (粒子轰击)

DNA coating (DNA涂层)

Barley, wheat (大麦、小麦)

Materials and Reagents

Barley (Hordeum vulgare L.) plants, 1 week old seedlings
Powdery mildew strain(s), fresh conidiospores as inoculum
Benzimidazol (Genview, catalog number: 51-17-2 )
Agar (Japan, plant cell culture tested)
CaCl₂(Sigma-Aldrich, catalog number: C7902 )
X-gluc (Inalco, catalog number: 1758-0600 )
Coomassie Brilliant Blue R-250 (Amresco, catalog number: 0472 )
Ethanol (Beijing Chemical Works)
Glycerol (Beijing Chemical Works)
Methanol (Beijing Chemical Works)
Lactic acid (Beijing Chemical Works)
Plasmid
Reporter DNA
K₃Fe [CN₆] (Sinopharm Chemical Reagent, catalog number: 10016718 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Spermidine (Sigma-Aldrich, catalog number: S-4139 ) (see Recipes)
GUS staining solution (see Recipes)
Destaining solution (see Recipes)
Benzimidazol plates (see Recipes)
Coomassie blue solution (see Recipes)

Equipment

PDS-1000/He delivery system (Bio-Rad Laboratones)
Macrocarrier (Bio-Rad Laboratones, catalog number: 1652335 )
Rupture disc (900 psi) (Bio-Rad Laboratones, catalog number: 1652328 )
Centrifuges (Eppendorf, catalog number: 5424 )
Fluorescence microscope (Carl Zeiss, Axio Scope. A1 )
pH meter (Mettler Toledo, FE20K)
Gold microcarrier: 1.0 μm in diameter (Bio-Rad Laboratones,catalog number: 165-2263 )

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Bai, S., Chang, C., Han, X. and Shen, Q. (2013). Modified Single-Cell Transient Gene Expression Assay in Barley Epidermal Cells. Bio-protocol 3(9): e690. DOI: 10.21769/BioProtoc.690.
Bai, S., Liu, J., Chang, C., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., Takken, F. L., Schulze-Lefert, P. and Shen, Q. H. (2012). Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance. PLoS Pathog 8(6): e1002752.