- Why is a cell wall gene catalogue and macroarray useful in maize ?|
- What does MAIZEWALL contain ?
- How was MAIZEWALL built ?
- Sample description
- Cell wall macroarray construction and hybridization
Why is a cell wall gene catalogue and macroarray useful in maize ?
The plant cell wall provides structural support and defines cell size and shape, thereby playing a critical role in determining cell function. Primary cell walls are laid down in all plant cells and are mainly composed of cellulose, hemicellulose, pectin and protein. The structure and composition differ greatly among species, and especially between dicots and monocots. Dicots and non-commelinoid monocots possess type I cell walls, whereas type II cell walls are found in commelinoid monocotyledons which include maize and rice.
For example, the primary wall in maize contains less pectins and proteins and significant amounts of hydroxycinnamic acids. During the differentiation of specialized cell types such as fibers and xylem tracheary elements (TEs), in addition to primary walls, secondary walls are subsequently deposited to insure additional mechanical strength and solute conduction. Although much information is available for specific genes involved in dicot cell wall formation, nearly nothing is known in monocots, and in particular in maize. For this reason, we constructed a centralized cell wall database in maize that could be extremely useful for the scientific community working on cell walls and/or maize.
What does MAIZEWALL contain ?MAIZEWALL contains a bioinformatic analysis and gene expression data repertory of cell wall biosynthesis and assembly in maize.
- 735 maize sequences classified into :
- 174 gene families classified into :
- 18 functional cell wall-related categories
How was MAIZEWALL built ?Schematic view
Homology-based approach from a Zinnia elegans EST subtractive library enriched in genes expressed during secondary wall formation
Transcriptome analysis has been performed on roots, stem and leaves at the 4-5 leaf stage and internodes at silking. Lignin biochemistry and cytological studies have also been performed on these samples (Guillaumie et al.).
Macroarray construction and hybridizationSchematic view
In order to design a macroarray that guarantees signal specificity and sensitivity, a pilot experiment was carried out on a set of 3 O-methyltransferase genes selected for their known expression profiles. The experiment was designed to optimize the following parameters: 1/ the quantity of cDNA spotted on the membrane, 2/ the source of probe synthesis (total RNA vs polyA RNA), 3/ probe type (reverse-transcribed cDNA vs. random priming), 4/ the length of the Gene Specific Tag (GST) spotted, and 5/ the relative proportion of 3’UTR and coding sequence to insure hybridization specificity within a multigene family. This experiment allowed us to define the following technical parameters for the macroarray as follows : 150-250bp PCR products corresponding to the 3’UTR for each gene was spotted at a concentration of 0.25µg/µl (100nanolitres/spot); the cDNA probe was generated from reverse transcription of total RNA.