Genetic elements from PV-ZMAP1043
Transcription of 5-enolpyruvylshikimate-3-phosphate synthase
(cp4 epsps) from Agrobacterium tumefaciens
commences from the Cauliflower Mosaic Virus (CaMV) enhanced 35S
promoter and ends at the A. tumefaciens nopaline synthase
(nos) gene terminator. The transcript contains a Zea mays
heat shock protein 70 (Hsp70) intron, Arabidopsis thaliana
N-terminal chloroplast transit peptide sequence, and cp4
epsps.
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 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 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 gene 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.
Genetic elements from pNOV1300
In the parental MIR162 maize, a variant of the native B.
thuringiensis vegetative insecticidal protein 3Aa (Vip3Aa),
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 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.
Genetic elements from PV-ZMHT507801
The Streptomyces viridochromogenes phosphinothricin
N-acetyltransferase (PAT) gene is under the control of the
Andropogon gerardii ubiquitin (Ubq) gene promoter and the
O. sativa alpha-amylase/trypsin inhibitor terminator. The
transcript includes the A. gerardii 5' untranslated leader
sequence and an intron from Ubq before the PAT.
The Stenotrophomonas maltophilia Dicamba monooxygenase
(DMO) gene is under control of the peanut chlorotic streak
caulimovirus (PC1SV) Full-length transcipt (FLt) promoter and the
Triticum aestivum (wheat) heat shock protein 17
terminator. The transcript produced contains the wheat chlorophyll
a/b-binding 5' untranslated leader sequence (for improved gene
expression), the O. sativa Actin 1 untranslated region
(UTR) and intron (for improved gene expression), the targeting and
UTR of Petunia hybrida Chloroplast Transit Peptide 4, 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 a cp4 epsps expression cassette. The cp4 epsps
expression cassette is regulated by the Ract1 promoter from Oryza
sativa, the Ract1 5′ untranslated leader from Oryza sativa, the
Ract1 intron from Oryza sativa, the CTP2 targeting
sequence from Arabidopsis thaliana, and the nos 3′
untranslated region from Agrobacterium tumefaciens.
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 MON 87419 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.
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 regulation of the rice actin
promoter (P-Ract1) and the rice actin intron (I-Ract1). The second
cassette, which is fused to the 3' end of the first, consists of
the cp4epsps gene regulated by the CaMV enhanced 35S promoter
(e35S) and an intron from the corn heat shock protein 70 (HSP70).
Both expression cassettes incorporate the 3'untranslated region of
nos for signal polyadenylation.
Note:
- The vector also contains the nptII gene encoding kanamycin
resistance allowing selection of bacteria containing the plasmid,
and an origin of replication (ori) necessary for replicating the
plasmid in Escherichia coli. For the transformation
through particle bombardment, a fragment of the vector (obtained
after digestion with the restriction enzyme MluI) was used which
contains only the two cp4 epsps gene expression cassettes.
Therefore, the nptII gene and the origin of replication were not
inserted into the parental NK603.
- The parental NK603 contained one insertion site containing a
single copy of the linear DNA of PV-ZMGT32 used for transformation.
Both cp4 epsps gene cassettes within the single insert which are
intact.
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