Transkriptionsanalyse einzelner isolierter Zellen zur Identifizierung von Genen, die während wichtiger Schritte der Entwicklung des weiblichen Gametophyten der Modelpflanze Arabidopsis eine Rolle spielen
Final Report Abstract
The life cycle of flowering plants alternates between a diploid sporophytic and two morphologically distinct haploid gametophytic phases. Transition between the sporophytic phase and the gametophytic phase is marked by the acquisition of the meiotic cell fate by a subset of cells within the ovule. Megasporogenesis involves meiosis of a megaspore mother cell (MMC) to give rise to the functional megaspore (FMC), and megagametogenesis is the formation of the female gametophyte (embryo sac) harboring the two gametes (the egg cell and the bi-nucleate central cell) from the functional megaspore. The goal of project is to identify genes expressed in single cells important for megasporogenesis and megagametogenesis in Arabidopsis thaliana by tanscriptome analysis of the MMC and the FMC, respectively. It was foreseen to adapt a microgenomics approach for this purpose, involving isolation of the individual cells by laser microdissection (LCM), RNA isolation and amplification and GeneCHIP hybridization. This method has been optimized before for reproductive cells (Vijverberg, Wüst, and Grossniklaus). During the fellowship I worked on adapting the method to the cell types of interest and encountered unexpected difficulties concerning (i) visibility of cells of interest, (ii) RNA integrity after isolation, and (iii) linear amplification. This resulted in higher time requirements than expected for several steps of the work proposed. (i) One problem was that the functional megaspore could not be distinguished from the surrounding sporophytic tissue with the optics available for LCM. Therefore subsequent work was focused on the MMC. As contaminations of the samples by surrounding tissues could not be completely excluded, MMC cells and samples of surrounding tissue (WR) were collected independently. Four arrays each of MMC samples and of samples from surrounding material (WR) were hybridized. Low level data analysis indicate a reasonably good data quality. Detailed data analysis, confirmation of expression and analysis of T-DNA insertion lines is initiated and currently ongoing. We are planning to write a scientific publication about the findings from this project. We anticipate that the data set generated during the fellowship will significantly contribute to our understanding of the molecular mechanism controlling embryo sac development and plant reproduction.
