Construction of Plant Expression Vector with manA Gene

Objective:To construct the plant expression vector using manA gene as the selective marker.Method:The manA gene was amplified from the genomic DNA of Escherichia coli 'DH5α' by using specific primers containing XhoⅠenzyme site,the manA gene fragment was inserted into the plasmid pCAMBIA1301 to substitute the hpt gene,it was identified by enzyme digestion and PCR.Then,the Gateway Binary Vector(pGWCBF) was cut by XbaⅠ and HindⅢ to obtain the fragment including P35S-T35S-attR1-attR2-CmR-ccdB and inserted into the expression vector pCAMBIA1301.The donor vector with CBF gene was mixed with pCAMBIA1301-manA-GW by BP and LR reaction.Result:The recombinant vector pCAMBIA1301-manA-CBF was successfully constructed and verified by enzyme digestion.Conclusion:The vector can be transformed into Agrobacterium tumefaciens 'GV3101' by freeze thaw method and used in the grape genetic transformation,the following research could be done to obtain the cold tolerance transgenic plants with safe marker gene.