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.