Herbicide tolerance, insect 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. For Coleoptera resistance, the maize expresses B. thuringiensis Cry34Ab1 and Cry35Ab1. In addition to the insecticidal proteins, the maize also expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase for glufosinate tolerance and Sphingobium herbicidovorans aryloxyalkanoate dioxygenase for tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate acetyl coenzyme A carboxylase inhibitors.

PV-ZMIR245; PHP17662; 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 59122 vector PHP17662:
Transcription of Bacillus thuringiensis cry34Ab1 starts at Zea mays ubiquitin gene promoter and terminates at the Solanum tuberosum proteinase inhibitor II gene terminator. Transcription of B. thuringiensis cry35Ab1 commences from the (Triticum aestivum (wheat) peroxidase gene promoter and stops at another S. tuberosum proteinase inhibitor II gene terminator.

Note:
- The coding sequence of cry34Ab1 and cry35Ab1  has been adapted to the codon usage in maize as to achieve optimal expression in planta.
- The cry34Ab1 and cry35Ab1 were cloned from B. thuringiensis strain PS149B1.
Sequence analysis of 59122 done by the European Food Safety Authority indicated that this LMO contains one complete copy of the T-DNA of PHP17662 without internal rearrangements. All three gene cassettes, cry34Ab1, cry35Ab1 and pat, are intact within the transgenic event. The DNA sequences of the genes in 59122 are identical to those in the original plasmid except for two nucleotide differences in the wheat peroxidise promoter. At the 5' T-DNA end a deletion of 22 bp is observed and at the 3' T-DNA end a deletion of 25 bp is observed. The absence of vector backbone in maize 59122 was also demonstrated.

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.