Identification of novel genes expressed in Brassica napus during leaf senescence and in response to oxidative stress

Senescence is a genetically regulated oxidative process that involves a general degradation of cellular structures and enzymes and the mobilization of the products of degradation to other parts of the plant. The cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism) analysis has been used under stringent PCR conditions afforded by ligation of adapters to restriction fragments, and the use of specific primer sets. It was of interest to analyse the proportion of senescence-enhanced genes in Brassica napus (CV Falcon) that do respond to different stress signals and to determine whether other known stress response genes are also expressed during leaf senescence or in response to treatments that increase ROS (Reactive Oxygen Species). The cDNA-AFLP technique uses the standard AFLP protocol on a cDNA template. Total RNA was extracted from mature leaves treated with Ascorbic Acid (As-A), silver nitrate (AgNO3), UV-B irradiation, combined treatment (As-A/AgN03), (As-A/UV-B) as well as untreated control and senescence stage3 (S3). The first and double-stranded cDNAs were synthesised by PCR amplification. The result shows the expression pattern for 30 genes that were identified on the original cDNA-AFLP gel. Each gene that was identified as a band on the cDNA-AFLP gel and successfully cloned was characterised by both sequencing and expression analysis using hybridisation technique. The clone6 has high similarity to a DNA-binding protein that acts as a transcription factor and can be an important component of signalling pathways. This gene exhibits a senescence-enhanced expression and is highly induced by UV-B but not by AgNO3. The northern hybridisation results for this gene in different organs showed high expression in flowers, pod and senescing leaves and no expression in green leaves and roots. The combined treatments had large effect on the expression of clone9. The expression pattern for this gene showed high transcript level in roots and flowers, low level in early senescing leaves and no expression in pod, mature leaves and late senescing leaves.