DNA insert from MIR162 vector 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
contains: (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 pNOV1300.
- 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
DNA insert from MON89034 vector PV-ZMIR245:
Maize line MON89034 expresses two Bt-toxins encoded by Bacillus
thuringiensis cry1A.105 and cry2Ab2.
Transcription of cry1A.105 begins at the Cauliflower
Mosaic Virus (CaMV) Enhanced 35S promoter and finishes at the wheat
(Triticum aestivum) wheat heat shock protein 17.3
terminator. The transcript initially includes (5' to 3'): wheat 5'
untranslated leader from the chlorophyll a/b-binding protein,
Oryza sativa (rice) actin 1 intron and Cry1A.105.
The wheat 5' untranslated leader sequence and the rice intron
enhance the expression of cry1A.105.
Transcription of cry2Ab2 commences from the Figwort Mosaic Virus
(FMV) 35S promoter and terminates at the Agrobacterium
tumefaciens nopaline synthase (nos) terminator. The
transcript initially includes (5' to 3'): maize heat shock protein
70 (hsp70) intron, maize transit peptide and first intron
from the small subunit of Ribulose-1,5-bisphosphate
carboxylase/oxygenase and cry2Ab32. The hsp70
regulates and enhances gene expression, while the transit peptide
targets Cry2Ab2 to the chloroplast.
- The viral promoters are expected to be constitutively active and
promote high levels of transcription.
- The coding sequence of cry2Ab2 was codon-optimized for
expression within plant systems.
- A second T-DNA insertion (containing CaMV 35S promoter,
Escherichia coli neomycin phosphotransferase and A.
tumefaciens nos terminator) was initially inserted into
the genome for kanamycin selection during transformation. However,
once transformants were regenerated, the selectable marker was bred
out of the parental line using convention breeding
- Southern blot analyses indicated a single copy of the
cry1A.105 and the cry2Ab2 cassettes. No backbone
plasmid DNA or nptII sequences were detected. PCR and DNA
sequence analyses provided the complete DNA sequence of the insert
and confirmed the organization of the elements within the insert.
Furthermore, sequence analysis indicated that MON 89034 no longer
has the duplicated enhancer elements compared to the original e35S
promoter in PV-ZMIR245, possibly due to a recombination event that
resulted in its deletion.
DNA insert from GA21 vector pDPG434
Transcription of Zea mays modified
5-enolpyruvylshikimate-3-phosphate synthase (mepsps)
commences from the Oryza sativa (rice) actin 1 promoter
and terminates at the Agrobacterium tumefaciens nopaline
synthase terminator. The transcribed elements (from 5' to 3') are
expected to be as follows: first intron of rice actin 1, a
synthetic transit peptide and mepsps. Transcription of
mepsps is expected to occur constitutively due to the rice
actin promoter. Gene expression is additionally enhanced by the
rice actin intron. Post-translation, the optimized transit peptide
targets mEPSPS to the chloroplasts.
- The coding sequence of mepsps was obtained through
site-directed mutagenesis to create a modified version of the
native enzyme to confer glyphosate tolerance with similar enzymatic
- The Rice Actin 1 promoter contains a portion of the first intron
of the Actin 1 and thus corresponds to the 5' end of the
- The optimized transit peptide was derived from maize and
sunflower (Helianthus sp.) ribulose 1,5 -bisphosphate
carboxylase oxygenase sequences.
- Southern blot analysis indicated that an insert containing three
complete tandem copies of the insert and one incomplete copy were
inserted into the parental genome. The incomplete copy contains
rice actin promoter, the optimized transit peptide and a truncated
mepsps sequence without the nos 3' untranslated
region (as uncovered by sequence analysis).
- Sequencing analysis indicated a truncated rice actin promoter in
the 5' end of the insertion event, only containing 148 bp of the
- The modified maize expresses only the full-length mEPSPS protein.