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An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function.
T Koprek1, D McElroy, J Louwerse
1Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. tkoprek@nature.berkeley.edu
The Plant Journal : for Cell and Molecular Biology
|November 9, 2000
Summary
Researchers developed a novel gene tagging system in barley using maize Activator (Ac) transposase and Dissociation (Ds) elements. This system enables efficient gene isolation and characterization by facilitating targeted gene transposition and identifying mutant phenotypes for functional studies.
Area of Science:
- Plant Molecular Biology
- Genetics
- Biotechnology
Background:
- Developing functional gene isolation methods is crucial for understanding plant genetics.
- Transposon-based systems offer powerful tools for genetic manipulation and gene discovery.
- Barley (Hordeum vulgare) transformation and gene characterization require efficient and reliable methodologies.
Purpose of the Study:
- To establish a function-based gene isolation and characterization method in barley.
- To develop and validate a maize Activator/Dissociation (Ac/Ds) transposon system for barley.
- To assess the efficiency and utility of the Ac/Ds system for gene tagging and mutant phenotype analysis.
Main Methods:
- Stable transformation of barley with plasmids encoding maize Activator (Ac) transposase and Dissociation (Ds) elements.
- In vitro assays using a Ds-interrupted uidA reporter gene to assess Ds excision frequency.
- Crosses between transformed barley lines to analyze somatic and germinal transposition in F1 and F2 generations.
- Analysis of F3 progeny to determine reinsertion patterns and co-segregation of transposed Ds elements with mutant phenotypes.
Main Results:
- High-frequency Ds excision was demonstrated in vitro using a uidA reporter system.
- Significant Ds transposition (up to 47%) was observed in F2 progeny derived from crosses.
- Analysis of F3 plants revealed that transposed Ds elements reinserted into both linked (75%) and unlinked (25%) locations.
- Two mutant phenotypes were identified where transposed Ds elements co-segregated with the phenotype, indicating successful gene tagging.
Conclusions:
- The developed Ac/Ds transposon system is functional for stable transformation in barley.
- The system facilitates targeted gene tagging and functional gene characterization through transposition.
- The co-segregation of transposed Ds elements with mutant phenotypes highlights its utility for identifying and isolating genes underlying specific traits.
