DNA insert from PV-ZMAP1043
Transcription of Agrobacterium tumefaciens
5-enolpyruvylshikimate-3-phosphate synthase (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, an Arabidopsis thaliana N-terminal chloroplast
transit peptide sequence for chloroplast targeting of the protein
and 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).
Note:
- 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 epsps coding sequence is the codon optimized coding
sequence of the aroA gene from Agrobacterium sp.
strain CP4 encoding EPSPS.
DNA insert from PV-ZMIR245
Two insecticidal protein expression cassettes were inserted into
the genome. Bacillus thuringiensis cry1A.105 expression is
under the control of the CaMV 35S enhanced promoter, which first
transcribes wheat (Triticum aestivum) 5' untranslated
region of the chlorophyll a/b-binding protein (cab) and a
rice actin 1 intron before transcribing cry1A.105.
Transcription terminates at the wheat heat shock protein 17.3
terminator. Expression of the B. thuringiensis cry2Ab2
starts at the Figwort mosaic virus (FMV) promoter, which
transcribes the Zea mays heat shock protein 70
(hsp70), then the Z. mays transit peptide and the
cry2Ab2 coding sequence, before terminating at the
nos terminator.
Note:
- The Cry2Ab2 coding sequence was modified for optimal expression
in plants.
- South blot analysis confirmed that single insertions of both
cry2Ab2 and cry1A.105, as well as no vector
backbone were present and in the parent.
- A deletion removed the duplicated enhancer elements compared to
the original CaMV e35S promoter in PV-ZMIR245.
- The selectable marker, nphII, cassette was bred out of
the parental line and became not associated with this
transformation event.
DNA insert from PV-ZMBK07
A partial insert containing Bacillus thuringiensis cry1Ab
was inserted into the parental maize genome. Transcription is
directed from the Cauliflower Mosaic Virus 35S enhanced promoter.
The transcript contains a Zea mays heat shock protein 70
(ZmHsp70) intron and the coding sequence of
cry1Ab. ZmHsp70 enhances expression of
cry1Ab.
Note:
- The coding sequence of cry1Ab has been codon optimized
for expression in plants. The codon optimization did not result in
any changes to the amino acid sequence relative to the native
sequence.
- Southern blot analysis indicated that a single partial insert is
found within the parental genome.
- Southern blot analysis did not detect the presence of the
Escherichia coli neomycin phosphotransferase II gene nor
any DNA from plasmid PVZMGT10 (containing genes for glyphosate
tolerance - cp4 epsps).
- ELISA protein analysis and feeding assays indicated expression of
Cry1Ab.
DNA insert from pNOV1300
In the parental MIR162 maize, a variant of the native B.
thuringiensis vegetative insecticidal protein 3Aa
(vip3Aa20), named vip3Aa19, which has codon
changes that result in a single M129I amino acid substitution
was inserted into the transformation cassette. During the
transformation process an additional DNA mutation resulted in a
K284Q amino acid substitution. This final form was designated the
name Vip3Aa20. 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. A second expression cassette, containing the E.
coli phosphomannose isomerase gene, 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 gene (nos)
terminator.
Note:
- Southern blot analyses demonstrated that the T-DNA insert
contains: i) single copies of a vip3Aa20 gene and a
pmi gene; ii) two copies of the ZmUbiInt promoter; iii)
one copy of the nos terminator; and iv) no backbone
sequences from transformation plasmid pNOV1300.
DNA insert from PV-ZMHT507801
The Streptomyces viridochromogenes phosphinothricin
N-acetyltransferase (pat) gene is under the control of the
Andropogon gerardii ubiquitin promoter and the
Oryza sativa alpha-amylase/trypsin inhibitor terminator.
The transcript includes the A. gerardii 5' untranslated
leader sequence and an intron from ubiquitin before (5') coding
sequence of pat.
The Stenotrophomonas maltophilia dicamba monooxygenase
(dmo) gene is under control of the Peanut chlorotic
streak caulimovirus (PC1SV) full-length transcript promoter
and the Triticum aestivum (wheat) heat shock protein 17
terminator. The transcript produced contains a wheat chlorophyll
a/b-binding 5' untranslated leader sequence (for improved gene
expression), an O. sativa actin 1 untranslated region and
intron (for improved gene expression), the untranslated and
targeting region of Petunia hybrida chloroplast transit
peptide 4 (for chloroplast targeting of the protein) and
dmo.
Note:
- Originally, the plasmid vector contained two T-DNA elements that
were inserted during the initial transformation event: one
containing the dmo and pat expression cassettes,
and a second containing an Agrobacterium tumefaciens
5-enolpyruvylshikimate-3-phosphate synthase (cp4-epsps)
expression cassette. The cp4-epsps expression cassette is
regulated by the O sativa actin 1 promoter and 5′
untranslated leader, O. sativa intron, the Arabidopsis
thaliana chloroplast targeting peptide 2 targeting sequence,
and the A. tumefaciens nopaline synthase 3′
untranslated region. Subsequent traditional breeding, segregation,
selection, and screening were used to isolate those plants that
contain the dmo and pat expression cassettes
(T-DNA I) and do not contain the cp4-epsps expression
cassette (T-DNA II).
- Molecular characterization of MON87419 indicated that a single
copy of T-DNA I was integrated into the maize genome at a single
intact locus that includes all expected elements within the insert,
with the exception of incomplete Right and Left Border sequences.
These analyses also showed no PV-ZMHT507801 backbone elements or
T-DNA II sequences were present in the event.
DNA insert from PV-ZMIR10871:
The MON87411 genome contains an RNA interference (RNAi) cassette
targeting Diabrotica virgifera virgifera, a Bacillus
thuringiensis Cry3Bb1 cassette and an Agrobacterium
tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase
(epsps) cassette.
Transcription of the RNAi cassette commences from the
Cauliflower mosaic virus 35S enhanced promoter and
terminates at the Pisum sativum ribulose bisphosphate
carboxylase small chain 2 terminator. The transcript initially
contains a Zea mays heat shock protein 70 intron, which
contributes to enhanced expression in vegetative tissues of the
plant, and two partial coding sequences of the D. virgifera
virgifera Snf7p gene, which encodes the SNF7 subunit of the
ESCRT-III complex. The two Snf7p sequences are in an inverted
orientation, separated by a 150 nucleotide intervening sequence,
which allows base pairing between the inverted sequences and
hairpin RNA formation post-transcription, which then triggers an
RNAi response. Due to RNAi processing, small interfering RNA
molecules (roughly 21-23 nucleotides in length) will be produced
and thus no translation into protein will occur from this cassette.
Transcription of the cry3Bb1 is under control of the
Z. mays physical impedance induced protein promoter and
Triticum aestivum (wheat) heat shock protein 17.3
terminator. The transcript also contains a wheat 5' untranslated
leader from chlorophyll a/b-binding protein and Oryza
sativa actin 1 intron for enhanced expression of the
transgene. Expression of epsps is under control of an
O. sativa alpha tubulin promoter and terminator. The
transcript additionally contains Arabidopsis thaliana
chloroplast targeting peptide 2 to sequester the protein to the
chloroplast.
Note:
- Sequencing, PCR and bioinformatic analyses indicate that a
single, intact insertions of the three gene cassettes occurred in
the parental line.
- No plasmid backbone was detected.
For more information, kindly refer to the parental line
records.
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