Construction of Cry1Ac Plasmid Vector and Its Transformation Into Agrobacterium Tumefaciens

Introducing cry genes into rice genome is reported able to produce rice plant resistant to stemborer. DNA sequence encodes cry1Ac gene has been inserted into pGEM4Z, but this construct does not have a selectable marker gene for selection of transformed plant cells. The research aims were to construct a plasmid vector expressing a cry1Ac gene that has a transformation selectable gene and to transform it into Agrobacterium tumefaciens. Materials used were pAY560325 binary plasmid vector, pGEM4Z-cry1Ac vector, Escherichia coli strain DH5-α and A. tumefaciens strain LBA4404 competent cells. The methods consisted of plasmid DNA digestion using HindIII and EcoRI, electrophoresis, DNA (backbone and insert) dissection from the gel, purification, and ligation using T4 DNA ligase. Transformation of ligated DNA into E. coli by heat shock followed by cell plating onto selection medium, colony cultured, DNA isolation, and identification using restriction enzymes. Reconfirmation was done by cutting using restriction enzyme and PCR using F3 and R3, cry1Ac gene specific primers. Research result were DNA fragments of 3.8 kb ubiquitin::cry1Ac insert and pAY560325, the backbone vector, that after ligated and transformed into E. coli produced colonies. One of ten colonies containing plasmid DNA was evidently confirmed and named pAY560325-cry1Ac. Subsequently, it was transformed into A. tumefaciens by electrophoration method. Plasmid DNA was isolated from Agrobacterium that after digested with HindIII and EcoRI produced DNA fragments of 9.44 kb (pAY560325) and 3.814 kb (ubiquitin::cry1Ac). While by PCR, plasmid produced DNA fragment of about 711 bp. Thus, cry1Ac plasmid vector (pAY560325-cry1Ac) was successfully constructed and transformed into A. tumefaciens and is ready to be transformed into rice genome.