Herbicide-tolerant, 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, Cry2Ab2 and Cry1F. For Coleoptera resistance, the maize expresses B. thuringiensis Cry3Bb1. In addition to the insecticidal proteins, the maize also expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase for glyphosate tolerance, Streptomyces viridochromogenes for glufosinate tolerance and Sphingobium herbicidovorans aryloxyalkanoate dioxygenase for tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate acetyl coenzyme A carboxylase inhibitors.

Please note: The development of this event has not been confirmed.

PV-ZMIR245; PHI8999A; PV-ZMIR39;  pDAS1740

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 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 Rubsico 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 TC1507 vector PHI8999A
DNA fragment PHI8999A contains two adjacent plant gene expression cassettes for the cry1F and pat genes. Transcription of the cry1F gene was directed by the promoter and first exon and intron of the maize ubiquitin gene and terminates at the Agrobacterium tumefaciens ORF25 terminator.
Transcription of the pat gene from Streptomyces viridochromogenes commences from the Cauliflower Mosaic Virus (CaMV) 35S promoter and ends at the CaMV 35S terminator.

Note:
- The coding sequence of both genes has been optimized to achieve a high level of expression in maize.
- The sequences of the complete cry1F and pat genes are identical to those in the original plasmid.
- the CRY1F protein includes the F604K (phenylalanine to lysine at position 604) amino acid substitution, which was introduced to create a specific restriction site for cloning purposes.

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 Oryza sativa (rice) Actin 1 promoter and terminates at the A. tumefaciens nos  terminator. The transcript initially includes (from 5' to 3'): rice actin 1 intron for enhanced gene expression, Arabidopsis thaliana chloroplast transit peptide 2 and epsps. The A. thaliana transit peptide 2 targets the EPSPS protein to the chloroplast of the plant's cell.

Transcription of cry3Bb1 commences from the Cauliflower Mosaic Virus (CaMV) 35S enhanced promoter and terminates at the wheat heat shock protein 17.3 terminator. The transcript initially includes (from 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 the following six specific amino acid substitutions, resulting in the synthetic cry3Bb1 coding sequence present in the vector:
-- 2A (alanine insertion at position 2)
-- H232R (histidine to arginine substitution at position 223)
-- S312L (serine to leucine substitution at position 312)
-- N314T (asparagine to threonine substitution at 314)
-- E318K (glutamic acid to lysine substitution at position 318)
-- Q349R (glutamine to arginine substitution at position 349).
- Molecular analyses of MON88017 confirmed that single copies of the 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.


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

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 segments from the vector backbone occurred.