Maize with increased production of the amino acid lycine through
introduction of the cordapA gene from Corynebacterium glutamicum
and regulated by a promoter from globulin 1 (Glb1) gene from Zea
mays, rice actin gene intron (rAct1) and Z. mays chloroplast
transit peptide sequence for DHDPS and a Glb1 gene 3' terminator
from a non-translated region from Z. mays. The DNA used in the
microprojectile bombardment also comprised a nptII cassette as a
selectable marker which was flanked by loxP recombination sites.
The progeny were screened for plants that did not contain the nptII
gene cassette, but contained the cordapA gene cassette. The Cre
recombinase protein gene, which was also integrated into the maize
genome, was segregated away from the cordapA gene through
subsequent breeding.
Information on the inserted DNA sequences:
The linear DNA fragment used in the transformation included two
expression cassettes, each with a single copy of a gene: cordapA,
and nptII. The 3′ nontranslated region of the globulin 1 gene
contains the polyadenylation signal.The nptII gene cassette confers
the paromomycin resistance that permits the selection of cells
containing the expression cassette.
Vector information
The plant expression plasmid vector, PV-ZMPQ76, contains three
expression cassettes, the cordapA sequence encoding a
lysine-insensitive cDHDPS protein, nptII, a selectable marker
flanked by loxP recombination sites and amp, a selectable marker
encoding ampicillin resistance which, along with nptII was used for
selection in E.coli. The 5.9 Kb fragment of this plasmid used for
transformation contained only the cordapA and nptII cassettes and
in the final product only the cordapA cassette was present. This
was accomplished by crossing plants positive for cordapA with a
maize line engineered to express the Cre recombinase protein.
The Cre recombinase in the resulting hybrid initiated the excision
of the DNA fragment containing the nptII cassette at the loxP
sites.
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