| Drought is one of the most important abiotic stresses that dicreases plant growth and crop productivity. The annual loss in yield caused by drought stress is equivalent to the sum of all other environmental stresses caused worldwide. As an important oil crops, one of the five preponderant crops, oilseed annual production losses more than20%caused by drought, even reaching25%-32%sometimes under severe conditions. It is critical to study the drought-responsive mechanism at the molecular level so as to improve drought resistance in rapeseed using biotechniques.To explore the mechanisms of drought stress, a cDNA library, which were from the high tolerant varietiy Q2in Brassica napus, were constructed using suppression subtractive hybridization (SSH). By reverse northern hybridization, there were186positive clones selected. Three of them were randomly selected for further testing by Real-time PCR.Sequencing results of186clones showed that156ESTs represented different gene fragments. They were divided into11gene categories as follows:metabolism11.1%; Catalytic and antioxidant activity10.9%; signal transduction10%; transcription regulation6.4%; transporter facilitation3.8%; translation regulation2%; protein degradation1.3%; stress-induction1%; energy1%; unclassified protein36.4%and unknown function16.1%.Originally, most of them come from Arabidopsis thaliana, B. rape, B. oleracea, B. rapa, Lilium longiflorum and other plants. Morever, the functional analysis of the156ESTs, mainly involved in drought stress response of photosynthesis and chloroplast structure, stress resistance and defense, membrane and transporters, transcription regulation and signal transduction. Many drought resistant related ESTs were obtained, like chlorophyll a/b binding protein,1,5-bisphosphate carboxylase/oxygenase, peroxidase, phosphatidylinositol transfer protein, ABC transporters, transcription factor, serine/threonine kinase and GTP binding protein. These ESTs basically reflected gene expression profiling in B. napus under drought stress.RT-PCR was used to detect the expression models of the B and C genes (similar to peroxidase of of Arabidopsis thaliana), and one unknown EST(A gene). The results revealed that significant difference existed in the expression of A, B and C at the different timeponits after treated with drought stress. In the meantime, they all showed the highest expression at24h drought stress. To confirm the results, the fluorescent real-time quantitive PCR was used for further accurate quantification. Three ESTs took different trends, but showed the same highest expression at12-24h infected by drought stress. By comparison, the expression levels of B, C gene detection were lower at3h. Therefore, we can conclude that the time during12-24h after treatment was the key timepoint for B. napus to resist drought. |
