The promoter region of this LMO consists a duplicated enhancer
region from the cauliflower mosaic virus 35S promoter combined with
the promoter of the act1 gene, that encodes Actin 1, from Oryza
sativa.
In this modified organism, maize-specific splicing of the ATHB17
transcript results in a truncated protein, ATHB17Δ113, which is
missing the first 113 N-terminal amino acids that are expressed in
Arabidopsis thaliana. The ATHB17∆113 protein retains the
ability to form homo- and hetero-dimers and bind to target DNA
sequences like the full-length protein, however, it is unable to
function as a transcriptional repressor because the protein lacks a
functional repression domain.
The plasmid backbone also contained cp4 epsps and aadA expression
cassettes. The cp4 epsps expression cassette is regulated by the
act1 promoter from Oryza sativa, the act1 intron from
Oryza sativa, the CTP2 targeting sequence from Arabidopsis
thaliana, and the nos 3′ untranslated region from
Agrobacterium tumefaciens. The aadA expression cassette is
regulated by the bacterial promoter, and 3' untranslated region of
an aminoglycosidemodifying enzyme, 3''(9)-O-nucleotidyltransferase
from the transposon Tn7.
During transformation both the T-DNA and the cp4 epsps expression
cassette were inserted into the maize genome. Subsequently,
traditional breeding, segregation, selection and screening were
used to isolate those plants that contain the ATHB17 expression
cassette and not the cp4 epsps and aadA expression cassettes.
Based on sequencing, PCR and bioinformatic analysis, hte modified
LMO contains a single copy of the ATHB17 expression cassette that
is stably integrated at a single locus.
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