Cloning And Function Characterization Of WRI1 And LACS Gene In Brassica Napus

Brassica napus is one of the world-wild oil crop plants, which is an important source of vegetable oil. The improvement on Brassica napus varieties is a critical strategy for oil production. Oil is usually in the form of triglycerides. Arabidopsis WRI1 gene codes an AP2/EREBP transcription factors, and plays a key role in the oil accumulation devrived from carbohydrate during seed development. In addition, LACS encoding long-chain acyl-coenzyme A synthetase is involved in long-chain fatty acid synthesis and fatty acid beta-oxidation, which plays important roles in seed formation and seedlings development in Arabidopsis. The present study is undertaken to clone an Arabidopsis WRI1 homologous gene, and several LACSs including BnLACSl,2,4,5,8,9 from Brassica napus for functional characterization via overexpression in Canola Westar. The main results of this study as follows:1. The full length cDNA of BnWRIl, BnLACSl,2,4,5,8 and 9 genes were isolated from Brassica napus and were ligased into plant expression vector. In addition, the specific sequences of BnLACSl, BnLACS2, and BnLACS9 fragments were also respectively cloned into the RNA interference vector.2. BnWRIl, BnLACSl,2,4,5,8,9 were ectopically overexpressed in Westar variety through agrobacteria mediated transformation. The expression levels of these genes in canola were verified by semi-quantitative PCR or Real time PCR, and most transgenic lines are overexpressed plants.3. The subcellular observation showed that BnWRIl is localized in nuclear visualized by GFP fused protein under laser confocal microscopy. In addition, the expression profiling showed that BnWRI is expressed in all tissues tested with highest level in siliques and roots.4. Overexpression of BnWRI1 resulted in increase the expression levels of several genes such as PKp2, MAT, KASI, GPDHcl, GPAT9, and LPAAT2 involved in fatty acid synthesis and lipid metabolism pathways.5. The overexpression of BnWRI1 and BnLACS5 enhanced oil accumulation in leaves and seeds as reveal by GC analysis. However, there were not significantly different among BnLACSl,2,4,8,9 overexpression plants and wild type.6. The proteins of BnLACS1,4,5,8,9 were also ectopically expressed in E.Coli cells for further enzyme activity study in the future.