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 NK603, vector PV-ZMGT32:
The plant expression plasmid vector, PV-ZMGT32 contains two adjacent plant gene expression cassettes each containing a single copy of the Agrobacterium tumefaciens strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (epsps). In the first expression cassette (5' end), the epsps gene is under transcriptional control of an Oryza sativa (rice) Actin 1 promoter and the A. tumefaciens nopaline synthase (nos) terminator. During transcription, a rice Actin 1 intron and an Arabidopsis thaliana chloroplast transit peptide 2 are included upstream (5') of the epsps coding sequence. The rice intron enhances EPSPS expression and the transit peptide targets EPSPS to the chloroplasts of the plant cells. The second epsps cassette is under control of the Cauliflower Mosaic Virus 35S enhanced promoter and the nos terminator. Similarly, transcription additionally includes a maize heat shock protein 70 intron and an A. thaliana chloroplast transit peptide 2. The heat shock protein intron also enhances expression of epsps.

Note:
The parental line (NK603) has one insertion site containing both epsps gene cassettes. No vector backbone (neomycin phosphotransferase and origin of replication) sequences were detected.


For additional information on this LMO, please refer to the records of the parental LMOs.