Molecular Cloning And Preliminary Functional Analysis Of BnWRI1s From Brassica Napus

Five homologous genes of Arabidopsis WRI1 was cloned from Brassica napus cv tapidor silique by the method of PCR-based cDNA library-screening.Homologous analysis showed that the amino acid sequence deduced from five genes share high homology with the Arabidopsis WRI1 and contained two copies of the plant-specific AP2 /EREBP DNA-binding domain . BnWRI1b was found to be identical to BnWRI1 from GenBank(DQ370141).There must be a multigene family of BnWRI1s in Brassica napus.Because the amino acid sequence deduced from five genes have characteristic features of transcriptional factors that they all contained two copies of the plant-specific AP2 DNA-binding domain,the N-terminal region contained a sequence enriched in serine and threonine, and the C-terminal part of it was enriched with acidic amino acids.we speculated that WRI1 in Brassica napus functions as AP2/EREBP transcriptional factor.When the full- cDNA of five genes ligated with the vector pGBKT7 to construct pGBKT7/BnWRI1s recombinant plasmid in yeast two hybrid system, All showed transcriptional activation.And we also find that if BnWRI1a-ΔC truncated derivatives of BnWRI1a with deletions in the C-terminal acidic region failed to show transcriptional activation.These results suggest that BnWRI1s in Brassica napus also encode a putative transcription factor of the AP2/EREBP family and the C-terminal acidic region of BnWRI1a is required for transactivation. Experimental evidence for the regulation of BnWRI1a mRNA abundance was obtained by semi-RT-PCR.The expression of the BnWRI1a gene was tissue-specific with the highest abundance of mRNA in reproductive organs such as flowers and siliques with seeds, although BnWRI1a mRNA was also detected in stems,seedling and roots.The seed-specific overexpression vector pGE-N-WR11 was constructed under the control of the napin promoter and Brassica napus were transformed by floral dip method via Agrobacterium-mediated transformation procedure.Transgenic T0 seeds were obtained and the further work is carrying on.