Differential Profiling And Functional Analysis Of Genes In Seeds And Silique Wall During Seed Filling Stage Of Brassica Napus

As the third largest source of vegetable oil, rapeseed(Brassica napus) is widely grown in the world and it plays an important role in the supply of both high quality edible oils and raw materials for industry and biodiesel. The seed of rapeseed is the most important harvesting organ and its quality and yield depends on how it develops.Additionally, the silique wall, encapsulating the developing seeds, not only protects the seed from pests and pathogens, but also serves as an photosynthetic organ providing assimilates and nutrient to the seeds. Thus the silique wall also plays an important role in the seed development, which naturally has an effect on the final yield of rapeseed. Genes involved in seed development have been reported frequently in Arabidopsis, while rarely in rapeseed. Although B. napus is closely related to Arabidopsis, the duplicated and rearranged nature of the B. napus genome precludes simple one-to-one relationships between the B. napus and A. thaliana genomes. Moreover, the regulation of seed development is far more complicated compared to that in Arabidopsis. To address these issues, two major works were conducted in this study: 1) Global transcriptional profiles in siliques of Brassica napus from 21-22 day after flower were analyzed using a Brassica95 K EST microarray. Differentially expressed genes in the SW and seeds were extracted for further analysis. 2) According to the microarray data, several genes that may have an effect on seed development were chose to clone and construct expression vectors. The expression vectors were then transformed into Brassica napus and Arabidopsis for gene function identification. The major results are as follows:1. In total, 17,459 m RNAs were found to be expressed in seeds or the SW, with3,278 preferentially expressed in the SW, 2,425 preferentially expressed in seeds and9,377 expressed in both organs. More genes preferentially expressed in the SW indicated that the SW plays an active role in silique development at the seed-filling stage.2. Metabolism pathways analyzed by Map Man software reflected that:(1) genes involved in photosynthesis and transport-related pathways were more actively transcripted in the SW, while those involved in lipid metabolism were more active in seeds during the seed filling stage. This was consistent with the role of the SW as the“source” and the seed as the “sink”, respectively;(2) genes involved in the secondary metabolisms were active in both seeds and the SW but showed an unequal distribution of gene expression between the two organs. For example, genes related to flavonoidmetabolism were highly expressed in seeds, while genes involved in the metabolism of phenylpropanoids and terpenes were enriched in the SW;(3) large numbers of transcription factors were identified to be differentially expressed between the SW and seeds, suggesting a complex pattern of transcriptional control in these two organs.3. Six candidate genes were cloned and used to construct sense and antisense expression vectors. Transgenic Brassica napus and Arabidopsis plants with these vectors were obtained. The analysis of gene expression level of transgenic plants revealed that all the trangenes were over-expressed in the transgenic lines obtained with high expression levels.4. Gene function analysis showed that:(1) the leaf development was affected in F28 K over-expression plants of Brassica napus but not in the plants of Arabidopsis. The leaves dysplasia may be the result of abnormal cell division;(2) during the germination stage, F11K3 over-expression Arabidopsis was more sensitive to Na Cl treatment than the wild type and the mutant showed an opposite performance, while their germination rates were nearly the same at different Na Cl concentrations. This indicated that F11K3 may affect the rate of Na+ entering the cells;(3) at the medium containing 0.3m M NO3-, F46over-expression Brassica napus had the same performance with the wild type; when the NO3- concentration increased to 3m M, the dry weight of the over-expression plants was significantly higher than the wild type. It suggested that F46 was a low-affinity nitrate transporter and can promote the nitrogen use efficiency of Brassica napus.