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
transgene.
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.
Note:
- 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
substitutions.
DNA insert from MIR604 vector pZM26
The parental plant contains two expression cassettes: (i) modified
Cry3a (mCry3a) originally from Bacillus
thuringiensis and (ii) phosphomannose isomerase (pmi)
from Escherichia coli.
Expression mCry3a is under control of a Zea mays
metallothionein-like gene promoter and an Agrobacterium
tumefaciens nopaline synthase (nos) terminator.
Transcription of pmi is under the control of Z.
mays ubiquitin gene promoter and an A. tumefaciens
nos terminator. The transcript initially also contains an
intron from Z. mays ubiquitin-1 to enhance gene
expression.
Note:
- mCry3a was originally obtained from the native
cry3a gene, but was modified to enhance gene expression in
maize. The synthetic version of the protein (mCry3a) contains the
same amino acid sequences of the native version, except for the
modified serine-protease recognition site.
- The following changes in the pmi occurred: the valine at
position 61 has been substituted by alanine (V61A) and glutamine at
position 210 has been substituted by histidine (Q210H). Please note
no apparent change of function occurred.
- Southern blot and qPCR analysis indicated that a single insertion
of both expression cassettes occurred and there was no integration
of the vector backbone.
DNA insert from 5307 vector pSYN12274
The DNA insert contains two gene cassettes for an engineered
chimeric protein eCry3.1Ab and an Escherichia coli
phosphomannose isomerase (pmi).
Transcription of eCry3.1Ab is under control of a Cestrum
Yellow Leaf Curling Virus promoter and an Agrobacterium
tumefaciens nopaline synthase (nos) terminator.
Transcription of pmi is under control of a Zea
mays ubiquitin gene promoter and a nos terminator.
The promoter contains the first intron of the ubiquitin gene, which
will be initially included in the mRNA before splicing and for
enhancing expression of pmi. Transcription is expected to
be constitutive under both promoters and result in elevated levels
of transgene expression.
Note:
- eCry3.1Ab is a result of a fusion of the 5′ end (Domain I, Domain
II and 15 amino acids of Domain III) of a modified Cry3A gene
(mcry3A) and the 3′ end (Domain III and Variable Region 6) of a
synthetic Cry1Ab gene. The sequences were sourced from Bacillus
thuringiesis.
- Southern blot analysis indicated that the parental line contains
a single insertion of the vector and there was no integration of
the vector backbone.
- Sequencing analysis indicated that the right and left T-DNA
borders were truncated.
The modified maize event was a result of cross-breeding modified
parental lines and demonstrates herbicide tolerance and insect
resistance. For Lepidoptera resistance, the maize expresses
Bacillus thuringiensis Cry1Ab, Vegetative insecticidal
protein 3Aa20, and Cry1F. For Coleoptera resistance, the maize
expresses B. thuringiensis mCry3A. In addition to the
insecticidal proteins, the maize also expresses Streptomyces
viridochromogenes phosphinothricin N-acetyltransferase for
glufosinate tolerance and a modified native Zea mays
5-enolpyruvylshikimate-3-phosphate synthase for tolerance to
glyphosate. A selectable marker, Escherichia coli
phosphomannose isomerase, is also present and was used during
the transformation of the parental line using mannose
selection.
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.
Note:
- 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
function.
- 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
gene.
- 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
promoter region.
- The modified maize expresses only the full-length mEPSPS protein.
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