Abstract
A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.
Highlights
A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays)
When abundant double-stranded RNA (dsRNA) are produced by a transgene, they are processed by a dicer-like protein into small interfering RNAs
Over 86,000 embryos were subjected to biolistic transformation by 106 different transgene constructs, resulting in nearly 3,000 transgenic events that were identified based on their ability to grow on culture media containing bialophos
Summary
A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). A functional genomics approach was pursued to study the role of 130 chromatin-related maize (Zea mays) genes in controlling a range of epigenetic phenomena including paramutation, epimutation, transgene silencing, DNA methylation, and imprinting. In Arabidopsis (Arabidopsis thaliana), lines bearing a single copy of an inverted repeat (IR) transgene silenced the endogenous target more efficiently than lines with multicopy insertions (Kerschen et al, 2004). This parameter, together with several others, required optimization for our large-scale study in maize
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