Deliverables
cDNA microarray carrying dormancy related pine cDNAs and relevant conserved genes from other species
A dedicated cDNA microarray was produced carrying 1451 genes that were pre-selected for relevance to dormancy and/or hardiness. Additional genes were added to a total of 153 genes. These included fragments of genes that were differentially present in a PCR assay performed during the previous project (amplified fragment length polymorphisms: AFLP fragments), and some genes that were described in the plant literature to be associated with stress or dormancy. Additionally a number of control genes were spotted on the array to allow data normalisation. These included 4 different yeast genes that are known not to be present in most plants, to allow correction for background hybridisation, and different sized fragments of the firefly luciferase gene. The luciferase gene serves as a positive control during hybridisation experiments.
The array can be used to dissect biological processes occurring in pine. The primary use will be for stress related or bud development related processes. The array can be used for scientific purposes or for the selection of indicator genes that can serve as content for molecular diagnostic assays. To our knowledge this is the first dedicated array for pine seedling bud tissue.
Since no prior sequence or other molecular information was available for beech, two new subtracted beech libraries have been made and characterised for constructing the beech cDNA array. Subtraction resulted in libraries enriched for genes related to active growth or dormancy/cold hardiness, respectively. Both libraries had excellent properties to serve as starting point for a small beech array. 364 clones were selected to be spotted on the array. In making this selection we ensured that representatives from all major functional groups were present, including metabolism, energy, cell cycle and DNA processing, transcription, protein synthesis, protein destination, cellular communication/signal transduction mechanism, stress response, control of cellular organization and transport facilitation. In addition more than 50% of the spots represent 'unknowns'. Since not much sequence data is available form beech, many of the genes we isolated produced no reliable hit in the BLAST search. Supplemented with positive, negative and technical controls this resulted in a highly dedicated array of 384 spots.
The array can be used to dissect biological processes occurring in beech. The primary use will be for stress related or bud development related processes. The array can be used for scientific purposes or for the selection of indicator genes that can serve as content for molecular diagnostic assays. To our knowledge this is the first dedicated array for beech seedling bud tissue.
We selected 9 pine genes that are likely to be involved in pine cold hardiness development and developed RT-PCR primers to allow rapid and robust analysis of the quantitative expression levels of these genes. It concerns
- dehydrin Psdhn1
- dehydrin Psdhn2
- dehydrin family Psdhn3-8 (group-specific)
- dehydrin Psdhn5
- dehydrin Psdhn3/7 (double detection)
- a stress inducible protein
- a Tubulin
- Glucose-6-Phosphate Dehydrogenase
- Ribosomal 18S RNA.
We developed a reliable protocol that takes into account the difficulties of the tissue type (dormant buds). The protocol starts with a CTAB RNA isolation. Preparations were DNAseI and used for cDNA synthesis. Dilutions of this cDNA were used for Realtime PCR. Product formation was measured using the iCycler system (BIORAD Laboratories, The Netherlands). Relative changes in expression were calculated using the Gene Expression Macro (Version 1.1) supplied by BIORAD.
These primers can be used for quantitative analysis of stress related gene expression.
The can be used in scientific research.
In addition, they may serve as content for a molecular diagnostic assay for hardiness of pine seedlings
Transcriptional profiling of pine and beech samples grown in either field or controlled climate conditions was used to select two subsets of each 15 genes that can be used to molecularly characterize the samples from these and other experiments and locations. The selected genes represent different expression profiles in relation to the examined conditions, either increasing, decreasing or constant.
Based on their expression pattern, these groups of genes can be linked to physiological events that occur during acquisition of frost hardiness. The expression profile of the descriptive gene set provides a useful molecular signature of the various bud samples. All genes display a high relative change in expression over the season, a strong correlation to physiological parameters and a high absolute expression level. The genes that were selected in this manner are candidates for development into molecular markers for frost hardiness
We selected 4 beech genes that are likely to be involved in beech cold hardiness development and developed RT-PCR primers to allow rapid and robust analysis of the quantitative expression levels of these genes. It concerns
- ABA inducible protein
- alpha tubulin
- a gene with unknown function
- a protein kinase
These primers can be used for quantitative analysis of stress related gene expression.
The can be used in scientific research.
In addition, they may serve as content for a molecular diagnostic assay for hardiness of beech seedlings
We developed a reliable protocol that takes into account the difficulties of the tissue type (dormant buds). The protocol starts with a CTAB RNA isolation. Preparations were DNAseI and used for cDNA synthesis. Dilutions of this cDNA were used for Realtime PCR. Product formation was measured using the iCycler system (BIORAD Laboratories, The Netherlands). Relative changes in expression were calculated using the Gene Expression Macro (Version 1.1) supplied by BIORAD.
We will use these primers in follow up research projects and for the development of diagnostics tests
Based on the expression patterns of two dehydrin genes a test system was developed for assessment of hardiness of pine seedlings. The test is based on the principle of RT-PCR (cDNA synthesis followed by amplification of specific gene fragments) combined with immunodetection of the amplified gene fragments. For immunodetection the Nucleic Acid Lateral Flow Immuno Assay (NALFIA) was used. A mixture containing the amplified fragments is applied to the lateral flow system. At specific locations on the membrane antibodies are immobilized that capture specific labels attached to the amplified fragments. A second carbonated compound that binds to each amplified fragment is used to visualize the fragments on the membrane. Use of this assay system is simple, cheap and results are clear. The change in ratio of the signal intensity of both dehydrin genes indicates when the seedlings have become frost tolerant. a constitutively expressed gene serves as a technical control. Preliminary data, obtained from pine trees of Dutch origin, suggests that needle tissue can be used equally well as bud tissue. The use of gene expression as monitoring tool for seedling vitality is highly innovative.
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