Cloning And Analysis Of The BAN-Homologous DNA Fragment And The Primary Study Of Floral-Dip Method In Oilseed Rape Brassica Napus

The objectives of this study are to clone the BAN fragment of Brassica napus and to apply the floral-dip in the transformation of oilseed rape to establish a convenient method for oilseed rape breeding. BAN is DFR-like gene in chalcone synthesis pathway functioned as a negative regulator, and has been well studied in Arabidopsis thaliana. Two primers, designed according to the conserved regions of BAN gene in Arabidopsis thaliana, were used to amplify the BAN homologous fragments from the genomic DNA of Brassica napus, B. chinensisl, B. juncea, A. thaliana and other cultural plants of Cruciferae. The very similar PCR fragments were obtained from all the amplifications, which implicated that BAN may be a conserved gene existing widely in the genomes of Cruciferae. PCR fragments were cloned and confirmed by restriction enzyme digestion. The clones inserted with BAN fragments of B. napus and A. thaliana were sequenced and compared with the sequences in GenBank. The results showed that the BAN fragment of A. thaliana had the same sequence (779bp) as reported. The BAN fragment of B. napus was 780bp and its extron had 90% of homology with those of A. thaliana BAN (90%). The sequences of intron, however, had only 74% of homology with those of A. thaliana BAN. In addition, amino acid sequence of oilseed rape BAN had 78% of homology and 87% of similarity with those of A. thaliana and had various homologies (55-64%) with many NADPH-dependent reductases of other plants.In the study of floral-dip method, flowering inflorescences of oilseed rape growing in field are immersed in floral-dip solution resuspended with culturedAgrobacterium tumefaciens LBA4404 or EHA105, resided binary plasmid pBI121. To investigate the best period of transformation, different flowering stages including before, after and pollination were chosen to do floral-dip. In another experiment the stigma is deliberately removed from the ovary in each flower and the ovary is then inoculated into the floral-dip solution with A. tumefaciens in order to find if it is useful for the bacterial infection and transformation. From total 16,660 dipped inflorescences, l,268g matured seeds were harvested. In kanamycin-resistent experiment the surface-sterilized non-treated seeds were placed on MS-medium with various kanamycin concentrations. The seedlings were then growing on the medium for 20 days. The primary results show that the tolerable concentration of kanamycin to the tested seedlings is up to 15ug/ml and the suggested concentration for selection of resistant plant is 25ug/ml...