Genetic elements from PV-ZMAP1043
Transcription of Agrobacterium tumefaciens
5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps)
commences from the Cauliflower mosaic virus (CaMV)
enhanced 35S promoter and terminates at the A. tumefaciens
nopaline synthase (nos) terminator. The transcript
contains a Zea mays heat shock protein 70 (hsp70)
intron for enhanced gene expression, an Arabidopsis
thaliana N-terminal chloroplast transit peptide sequence for
chloroplast targeting of the protein and cp4 epsps.
The CaMV enhanced 35S promoter-hsp70 combination promotes
gene expression in female and vegetative tissues, but not in male
reproductive tissues (pollen microspores and tapetum).
- Southern blot analyses indicate that a single copy of the T-DNA
was inserted at a single site in the parental maize genome and no
plasmid vector backbone sequences were detected to have been
integrated. DNA sequencing analyses further indicated that the
expected T-DNA sequences were integrated.
-The cp4 epsps coding sequence is the codon optimized coding
sequence of the aroA gene from Agrobacterium sp.
strain CP4 encoding CP4 EPSPS.
Genetic elements from pNOV1300
In the parental MIR162 maize, a variant of the native B.
thuringiensis vegetative insecticidal protein 3Aa
(vip3Aa), termed vip3Aa20, was inserted into the
transformation cassette. Transcription of vip3Aa20
commences at the Z. mays ubiquitin gene promoter and then
transcribes vip3Aa20 followed by intron 9 of Z.
mays phosphoenolpyruvate carboxylase, before terminating at
the CaMV 35S terminator. The intron enhances expression of the
A second expression cassette, containing E. coli
phosphomannose isomerase (pmi), was also inserted into the
parental genome. The gene is under the control of another ubiquitin
promoter and transcription terminates at the Agrobacterium
tumefaciens nopaline synthase (nos) terminator.
- Southern blot analyses demonstrated that the T-DNA insert
(i) single copies of vip3Aa20 and pmi gene;
(ii) two copies of the maize ubiquitin promoter;
(iii) one copy of the nos terminator; and
(iv) no backbone sequences from transformation plasmid
- Vip3Aa20 is a variant of the native Vip3Aa, which has codon
changes that result in M129I (methionine to isoleucine at position
129) and K284Q (lysine to glutamine at position 284) amino acid
Genetic elements from PV-ZMGT32
The plant expression plasmid vector, PV-ZMGT32 contains two
adjacent plant gene expression cassettes each containing a single
copy of the cp4 epsps. In the first (5' end) expression
cassette, the cp4 epsps gene is under the transcriptional
regulation of an Oryza sativa actin promoter and a
nos terminator. An O. sativa actin intron is also
present in the transcript for enhanced expression of the coding
sequence. The second cassette consists of another cp4
epsps gene regulated by an CaMV enhanced 35S promoter
(containing a duplicated enhancer region) and a nos
terminator. Similarly, an intron from the maize heat shock protein
70 (hsp70) was included for enhancing expression of the
coding sequence. Both promoters of the gene cassettes are expected
to promoter high levels of transcription.
- The parental NK603 line contained a single, intact insertion
containing both cp4 epsps gene cassettes.
- Due to restriction digest prior to particle bombardment, the
vector backbone, containing E. coli neomycin
phosphotransferase II and origin of replication, were not
incorporated into the parental genome.
For more information, kindly refer to the parental line