A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

Yu Bai, Ning Han, Jinxia Wu, Yinong Yang, Junhui Wang, Muyuan Zhu, Hongwu Bian

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

Original languageEnglish (US)
Pages (from-to)211-219
Number of pages9
JournalPlant Cell, Tissue and Organ Culture
Volume119
Issue number1
DOIs
StatePublished - Sep 30 2014

Fingerprint

microRNA
protoplasts
barley
gene expression
genes
osmolarity
transfection
mannitol
quantitative analysis
proteins
viability
screening
stems
assays
methodology
tissues
leaves

All Science Journal Classification (ASJC) codes

  • Horticulture

Cite this

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abstract = "Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.",
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A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes. / Bai, Yu; Han, Ning; Wu, Jinxia; Yang, Yinong; Wang, Junhui; Zhu, Muyuan; Bian, Hongwu.

In: Plant Cell, Tissue and Organ Culture, Vol. 119, No. 1, 30.09.2014, p. 211-219.

Research output: Contribution to journalArticle

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AU - Zhu, Muyuan

AU - Bian, Hongwu

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