Construction of Cry1Ac Plasmid Vector and Its Transformation into Agrobacterium tumefaciens

Sri Koerniati, Alifah R. Heritiera


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.


Construction of expression vector; cry1Ac gene; Agrobacterium tumefaciens; rice stemborer.

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