Gene Expression Profiling of Microspore Embryogenesis in Brassica napus
Isolated microspores from selected cultivars of Brassica napus readily form embryos in culture after mild heat stress treatments (32°C for 1–3 days). Transcript profiling methods were used to identify differentially-expressed genes as well as shifts in metabolism during the early stages of microspore embryogen-esis. Approximately 20,000 expressed sequence tags (ESTs) from cDNA libraries representing freshly-isolated microspores (0 hours) and microspores cultured for 3, 5 or 7 days under embryogenesis-inducing conditions were prepared. In silico analyses of ESTs and semi-quantitative and real time reverse transcription-polymerase chain reaction (RT-PCR) based profiling identified differentially-regulated gene clusters and 16 genes that could be used as specific markers for microspore embryogenesis. These molecular marker genes also were expressed during zygotic embryogenesis, underscoring the common developmental path ways that function during zygotic and gametic embryogenesis. Future studies will focus on characterization of embryogenesis-related genes and development of fluorescently-labeled gene/protein probes to precisely mark and isolate early stages of microspore embryogenesis.
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