Re-construction of a plant tranformation vector for expression in plants

The construction of a binary vector for gene expression in plants using Agrobacleriummediated plant transformation is important to achieve high beneficial vector system. In this study, the T-DNA region of pBIl2l and pGPTV/-SRN4/AGS was to be PCR amplified in order to be subcloned into pUC 19 vector. The knowledge of complete sequence of pBIl2l (Accession number: AF485783) was used in primer designed for TDNA amplification. However, the T-DNA was failed to amplifY. The problem was mainly due to the big size of the T-DNA region. Therefore, alcR gene and GUS gene located in the T -DNA was removed by using restriction enzymes to reduce the size of the I -DNA region. The ligation mixture was eventually transformed into E. coli strain XLIBlue. White colonies were observed indicates successful cloning. However, PCR amplification still failed as the reduced size of the I-DNA was still big (approximately 8 kbp)