Agrobacterium-Mediated Transformation Of Sweet Protein Brazzein Gene In Fragaria×Ananassa Duch.’Akihime’

Strawberry (Fragaria ananassa Duch.), perennial herbaceous plant in the Rosaceae family, which is the major fruit crops and it is widely grown in the world, The higher demand has been put forward for the fruit quality under live level development. Modern genetic engineering improvements directly in the gene level, breaking the boundaries between species, it play an increasingly important role in nurturing disease resistance, insect resistance, herbicide resistance to stress and other strawberry varieties’development. In recent years, a lot of valuable genes have been transformed into strawberry. Sweet protein Brazzein has been transformed into many higher plants, but the expression showed differently. In our study, a vector based on the Brazzein expression property and former study was constructed. The Brazzein gene was used as the purpose gene, the transformation of strawberry by Agrobacterium-mediated was studied. The experiment research results were as follows:1. Brazzein gene, added the sequence of DFN signal peptide nucleotide, that was isolated from Pyrus pyrifolia Nakai ’Huanghua’ at the5’-end of the above gene, devised according to the preference codon of dicotyledonous plants and53amino acid residues was successfully synthesized as modified brazzein gene using overlapping PCR. Then a binary vector was successfully construcred which was named as PYH4215-bra with the modified gene. The vector PYH4215-bra harbouring β-glucuronidase (gusA) gene disrupted by a catalase intron as reporter, hygromycin phosphotransferase (hpt) gene as selectable marker for transformants and the modified brazzein gene in its T-DNA region was transformed into the disarmed Agrobacterium tumefaciens strain EHA105and used for strawberry transformation. All the mentioned genes were driven by the CaMV35S promoter.2. A genetic transformation system was studied with the ’Akihime’ leaf disc mediated by Agrobacterium tumefaciens. Effect of the bacterial concentration, infection time, co-culture time, AS concentration, pre-culture time, hygromycin concentration and carbenicilin concentration on genetic transformation were discussed, a transgenic system for’Akihime’ was established. The explants were inoculated with0.4-0.5of OD600for6and8min were appropriate for different bacterial concentration. Co-cultured for3d and transferred to the medium containing4mg/L hygromycin and200mg/L carbenicilin for14d in dark. Then the explants were cultured in light. It’s not good to add AS to activate bacterial, pre-culture didn’t improve the ’Akihime’ transformation rate.3. One hundred and twenty-four hygromycin-resistant plantlets were detected through X-glucuronide staining solution, and58plantlets were stained obviously, the positive rate was46.8%. PCR and RT-PCR analysis was carried out to confirm the brazzein gene was transferred into strawberry among13(3-glucuronidase positive plantlets, the clear brazzein gene fragment was amplified in8transgenic strawberry lines and these8transgenic strawberry lines indicated by number bra-1to bra-8.4. The ripened fruits of four transgenic lines and control line were taking as the materials, the fruits were tasted one line by one line, the soluble solids and the soluble sucrose were identified. The results showed that, in the four transgenic lines, the Bra-3fruits were tasted sweeter, and the soluble solids content was higher than other lines, and reached21.33, there was no significant differences among the soluble sucrose of the transgenic lines and control line.