M. Zangi, Z. Amini-Bayat, P. Ehsani, H. Ofoghi
Health Biotechnology and Biopharma, 2019, 3(1): 19-28.

Nowadays the dual binary vector system pGreen/pSoup is used for Agrobacterium transformation. The pGreen vector uses the pSa replication locus composed of the pSa ori and the pSa replicase gene (repA). The repA gene is resident on a pSoup and provides replication functions in Trans for pGreen. Therefore, the present study was a preliminary attempt to investigate how pgR107 binary vector replicates without subsidiary plasmid pSoup, in disarmed Agrobacterium LBA4404. So, human Nerve Growth Factor gene was cloned in a binary pgR107 vector based on pGreen. Recombinant pgR107-hNGF plasmid was introduced in competent A. tumefaciens strain LBA4404 using a freeze-thaw technique. Transformed colonies containing pgR107-hNGF grown on selective medium was approved by colony PCR using hNGF specific primers. Examination of A. tumefaciens LBA4404 genome for presence of pSa Rep (RepA) gene was done via basic local alignment search tool (BLAST). There was no homology observed between RepA sequence and A. tumefaciens strain LBA4404 genome. Our findings show although A. tumefaciens LBA4404 does not contain pSoup plasmid, binary vector could interestingly replicate in such strain. But, the mechanism of replication is unknown and being to be investigated.

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