Designing, Construction and Functional Analysis of Two New Plant Expression Vectors (pBI121GUS-9 and pBI1213+4) with Improved Cloning Sites

Most popular binary vectors used in plant transformation via Agrobacterium have limeted common restriction recognition sites due to large vector size which has caused difficulties in cloning of target genes. To overcome this problem, new plant expression vectors including pBI1213 with three additional recognition sites downstream of gus gene and pBI121GUS-9 with nine recognition sites between CaMV 35S and Nos terminator (by using two primers designed upstream and downstream of Nos terminator in a PCR reaction) were constructed and identified. To analyze the efficiency of new vectors both in cloning and transformation steps, gus reporter gene was cloned and the new recombinant constructs were transferred to model plant Nicotiana tabbacum L. cv. Samsun via Agrobacterium tumefaciens (LBA4404). DNA was extracted from putative transgenic seedlings, which were regenerated on selective media and analyzed by PCR method. GUS assay confirmed the expression of the transgene in leaves of transgenic seedlings. After verification of the pBI1213 and pBI121GUS-9 in transformation and expression of the reporter gene, these vectors can be used for transformation of strategic plants with commercial transgenes.