Herbicide tolerant, Lepidoptera resistant maize

The modified maize was produced by crossing modified parental lines to result in a line with herbicide tolerance and insect resistance. For Lepidoptera resistance, the maize expresses Bacillus thuringiensis Cry1A.105 and Cry2Ab2. In addition to the insecticidal proteins, the maize also expresses Sphingobium herbicidovorans aryloxyalkanoate dioxygenase for tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate acetyl coenzyme A carboxylase inhibitors.

PV-ZMIR245 and pDAS1740

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 DAS40278 vector pDAS1740:
The LMO was generated using the Whiskers-mediated transformation method. Sphingobium herbicidovorans aryloxyalkanoate dioxygenase-1 (aad-1)  is under the control of Zea mays ubiquitin gene promoter and Z. mays root preferential cationic peroxidase terminator. The aad-1 coding sequence was optimized for expression in the plant.

Note:
- Southern blot analysis indicated that a single complete copy of the transformation cassette was stably integrated into the host genome at a single locus
- No integration of the vector backbone occurred.