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.
Note:
- 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
techniques.
- 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 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.
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