Genetic elements from PV-ZMIR245
Two insecticidal protein expression cassettes were inserted into
the genome. Bacillus thuringiensis cry1A.105 is
transcribed from the Cauliflower mosaic virus 35S enhanced
promoter an terminates at the Triticum aestivum (wheat)
heat shock protein 17.3 terminator. The transcript is expected to
contain a wheat 5' untranslated region of the chlorophyll
a/b-binding protein (cab) and an Oryza sativa
actin 1 intron before (5') the Cry1A.105 coding sequence.
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.
Transcription of both gene cassettes is expected to occur at high
levels.
Note:
- The cry2Ab2 coding sequence was modified for optimal
expression in plants.
- Southern 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), 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.
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.
Note:
- 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
records.
|
