Cry3A gene obtained from Bacillus thuringiensis. The gene was
modified for enhanced expression in maize and such that the amino
acid sequence of the synthetic version of Cry3A is the same as the
native protein, except for the modified serine-protease recognition
site.
The pmi gene encodes the enzyme phosphomannose isomerase (PMI) that
allows the plants to utilise mannose as a carbon source and is used
as a selectable marker.
Truncations at the left and right border junctions of the T-DNA
insert as well as nucleotide changes were identified relative to
the intended DNA sequence. These substitutions have not resulted in
any apparent functional change in PMI as expressed in MIR604.
Southern hybridization data provide confirmatory evidence to
support the Taqman PCR analysis that Corn MIR604 contains a single
copy of mcry3A gene and the pmi gene.
Corn MIR604 contains a single copy of the MTL and ZmUbilnt
promoters, without any vector backbone sequences present in pZM26.
Sequence analysis revealed that truncations and deletions occurred
but have no effect on the efficacy of the T-DNA insert. Three base
pair changes were noted; one occurred in the regulatory region and
does not encode for a protein; two other base pair changes occurred
within the coding region but these amino acid changes did not
result in any apparent functional change in the new insert.
As expected for the mcry3A, pmi, MTL and ZmUbilnt probes the Kpn1
digest resulted in a single hybridization band demonstrating that a
single copy of each element is present in Corn MIR604.
Additionally, for the full length backbone probe, lack of
hybridization demonstrates the absence of any pZM26 vector backbone
sequences being incorporated into corn MIR604 during the
transformation process.
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