DNA insert from MON89034 vector PV-ZMIR245:
Maize line MON89034 expresses two Bt-toxins encoded by the
Bacillus thuringiensis genes cry1A.105 and cry2Ab2.
Transcription of cry1A.105 begins are the Cauliflower Mosaic Virus
(CaMV) 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 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 Rubsico and cry2Ab32. The Hsp70 regulates and enhances
gene expression, while the transit peptide targets cr2Ab2 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 MON88017 vector PV-ZMIR39
Maize line 88017 contains A. tumefaciens
5-enolpyruvylshikimate-3-phosphate (epsps) and B.
thuringiensis cry3Bb1.
Transcription of epsps starts from the rice Actin 1 promoter and
terminates at the A. tumefaciens nos terminator. The
transcript initially includes (5' to 3'): a rice Actin 1 intron for
enhanced gene expression, Arabidopsis thaliana chloroplast
transit peptide 2 for chloroplast targeting of the EPSPS protein
and epsps.
Transcription of the cry3Bb1 commences from the CaMV 35S enhanced
promoter and terminates at the wheat heat shock protein 17.3
terminator. The transcript initially includes (5' to 3'): wheat 5'
untranslated leader from chlorophyll a/b-binding, rice Actin 1
intron and cry3Bb1. The wheat untranslated leader and the rice
actin intron regulate and enhance expression of the downstream
cry3Bb1 element.
Note:
- The wild-type cry3Bb1 coding sequence was modified to encode six
specific amino acid substitutions, resulting in the synthetic
cry3Bb1 coding sequence present in the vector. The differences at
the six positions are: 2A (insertion), H232R, S312L, N314T, E318K,
Q349R.
- Molecular analyses of MON 88017 confirmed that single copies of
the cp4 epsps and cry3Bb1 genes are integrated at a single locus in
the corn genome with all expression elements intact and no plasmid
bacterial backbone present. Plasmid PV-ZMIR39 contains the left and
right transfer DNA (T-DNA) border sequences that facilitate
transformation.
For additional information on this LMO, please refer to the
records of the parental LMOs.
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