Identification and characterisation of three genes determining embryogenesis by means of T-DNA mutagenesis in Arabidopsis thaliana.
| Authors | |
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| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Abstract Book of the 15th International Conference on Arabidopsis Research, July 11-14, Berlin, Germany. |
| MU Faculty or unit | |
| Citation | |
| Field | Genetics and molecular biology |
| Keywords | Arabidopsis thaliana; T-DNA |
| Description | One of the possible approaches to a plant gene function study in model plant Arabidopsis thaliana leads across T-DNA mutagenesis. Collection of 2500 T-DNA lines have been obtained by A. thaliana transformation with Agrobacterium tumefaciens containing plasmid pPCVRN4 with HPT selectable gene (Koncz et al. 1994). Screening of individual lines revealed three different mutations with defects in immature seed development (marked 1265, 1293, 1321) which have been characterised and analysed in detail. One T-DNA insert was determined in each line and the mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Morphological studies of embryo development were performed by clearing in chloral hydrate. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development. In 1265 mutation defect in globular stage was observed. It was associated with abnormal pattern of cell division connected with shoot apical meristem formation during embryogenesis. In 1293 mutation defect in cotyledon formation was observed and in 1321 mutation defect was connected with late embryogenesis, seed maturation and storage products formation. Cosegregation of T-DNA with the mutation was confirmed only in the 1265 line. The inverse PCR method was applied for isolation of flanking plant DNA. The obtained fragment of DNA was sequenced and subjected to BLAST and The Arabidopsis Information Resource database search, which revealed T-DNA insertion in At1g53330 gene coding pentatricopeptide repeat (PPR) containing protein. The hypothetical function of this gene is connected with transporter activity and probably cell - cell signalling. The other mutations, 1293 and 1321, which did not cosegregated with T-DNA, were subjected to genetic mapping for perspective isolation of the affected genes by map-based cloning strategy. DNA markers, SSR and CAPS, linked to the mutated gene were used to delimit the region containing the gene of interest. 1321 mutation was located on chromosome 2 in tight linkage with marker nga 1126 and 1293 mutation was linked with marker nga 63 on chromosome 1. The next work will be aimed at fine mapping enabling candidate gene choice and the isolation of DNA sequence that underlines the phenotype of interest. |
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