Construction of plant expression vector pCAMBIA1301-hs83 of hs83 gene

An agrobacterium binary vector carrying single chain variable fragment(scFv) hs83 gene was successfully constructed based on pCAMBIA1301.Initially,based on the squence information,two primers with appropriate restriction sites were redesigned to facilitate the insertion of the aim gene to replace the GUS second exon in pCAMBIA1301.Secondly,the aim gene was cloned with PCR amplification,and liagated into pMD-18 T-vetor for sequencing analysis.Thirdly,double enzymes digestions were carried out to plasimids of pCAMBIA1301 and T-hs83 to release two desired fragments,which were reclaimed separately and liagated,the recon was transformed into Agrobacterium tumefaciens EHA105 with the Freeze-Thaw Method after enzymes digestion analysis,the transformation was analysised by PCR.