Glufosinate tolerant, insect resistant maize

The modified maize event was a result of cross-breeding modified parental lines and demonstrates herbicide tolerance and insect resistance. For Lepidoptera resistance, the maize expresses Bacillus thuringiensis Cry1Ab, Vegetative insecticidal protein 3Aa20, and Cry1F. For Coleoptera resistance, the maize expresses B. thuringiensis mCry3A and eCry3.1Ab. In addition to the insecticidal proteins, the maize also expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase for glufosinate tolerance. A selectable marker, Escherichia coli phosphomannose isomerase, is also present and was used during transformation of the parental line using mannose selection.

pZO1502; pNOV1300; pZM26; PHI8999A; pSYN12274

DNA insert from Bt11 vector pZO1502
Transcription of the Bacillus thuringiensis cry1Ab gene is under control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the Agrobacterium tumefaciens nopaline synthase (nos) terminator. The transcript initially contains Zea mays alcohol dehydrogenase 1 intron 6, which enhance gene expression.

Transcription of the Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat) is under control of the CaMV 35S promoter and nos terminator. The transcript initially contains Zea mays alcohol dehydrogenase 1 intron 2 to enhance gene expression.

Note:
- The CaMV promoter associated with cry1Ab, was isolated from the CM1841 strain of CaMV using DdeI restriction digestion. However, the DdeI sites were converted into SacI sites.
- The CaMV promoter associated with pat was isolated from the Cabb-S strain of CaMV (AluI to DdeI fragment) and subsequently modified.
- The cry1Ab coding sequence encodes a truncated version corresponding to the N-terminal 615 amino acids of the full length Cry1Ab gene.
- The cloning of pat did not result in any amino acid sequence changes in the parental line.
- The nos terminator corresponds to the 423 to 678 basepairs of the nopaline synthase gene in A. tumefaciens.

DNA insert from MIR162 vector pNOV1300
In the parental MIR162 maize, a variant of the native B. thuringiensis vegetative insecticidal protein 3Aa (vip3Aa), termed vip3Aa20, was inserted into the transformation cassette. Transcription of vip3Aa20 commences at the Z. mays ubiquitin gene promoter and then transcribes vip3Aa20 followed by intron 9 of Z. mays phosphoenolpyruvate carboxylase, before terminating at the CaMV 35S terminator. The intron enhances expression of the transgene.

A second expression cassette, containing E. coli phosphomannose isomerase (pmi), was also inserted into the parental genome. The gene is under the control of another ubiquitin promoter and transcription terminates at the Agrobacterium tumefaciens nopaline synthase (nos) terminator.

Note:
- Southern blot analyses demonstrated that the T-DNA insert contains: (i) single copies of vip3Aa20 and pmi gene; (ii) two copies of the maize ubiquitin promoter; (iii) one copy of the nos terminator; and iv) no backbone sequences from transformation plasmid pNOV1300.
- Vip3Aa20 is a variant of the native Vip3Aa, which has codon changes that result in a M129I (methionine to isoleucine at position 129) and K284Q (lysine to glutamine at position 284) amino acid substitutions.

Genetic elements from pZM26
The parental plant contains two expression cassettes: (i) modified Cry3a (mCry3a) originally from Bacillus thuringiensis and (ii) phosphomannose isomerase (pmi) from Escherichia coli.

Expression mCry3a is under control of a Zea mays metallothionein-like gene promoter and an Agrobacterium tumefaciens nopaline synthase (nos) terminator. Transcription of pmi is under the control of Z. mays ubiquitin gene promoter and an A. tumefaciens nos terminator. The transcript initially also contains an intron from Z. mays ubiquitin-1 to enhance gene expression.

Note:
- mCry3a was originally obtained from the native cry3a gene, but was modified to enhance gene expression in maize. The synthetic version of the protein (mCry3a) contains the same amino acid sequences of the native version, except for the modified serine-protease recognition site.
- The following changes in the pmi occurred: the valine at position 61 has been substituted by alanine (V61A) and glutamine at position 210 has been substituted by histidine (Q210H). Please note no apparent change of function occurred.
- Southern blot and qPCR analysis indicated that a single insertion of both expression cassettes occurred and there was no integration of the vector backbone.

DNA insert from TC1507 vector PHI8999A
DNA fragment PHI8999A contains two adjacent plant gene expression cassettes for Bacillus thuringiensis cry1F and Streptomyces viridochromogenes pat.

Transcription of cry1F is directed by the promoter and first exon and intron of the maize (Zea mays) ubiquitin gene and terminates at the Agrobacterium tumefaciens ORF25 terminator.

Transcription of the pat gene 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 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 5307 vector pSYN12274
The DNA insert contains two gene cassettes for an engineered chimeric protein eCry3.1Ab and an Escherichia coli phosphomannose isomerase (pmi).

Transcription of eCry3.1Ab is under control of a Cestrum Yellow Leaf Curling Virus promoter and an Agrobacterium tumefaciens nopaline synthase (nos) terminator. Transcription of pmi is under control of a Zea mays ubiquitin gene promoter and a nos terminator. The promoter contains the first intron of the ubiquitin gene, which will be initially included in the mRNA before splicing and for enhancing expression of pmi. Transcription is expected to be constitutive under both promoters and result in elevated levels of transgene expression.

Note:
- eCry3.1Ab is a result of a fusion of the 5′ end (Domain I, Domain II and 15 amino acids of Domain III) of a modified Cry3A gene (mcry3A) and the 3′ end (Domain III and Variable Region 6) of a synthetic Cry1Ab gene. The sequences were sourced from Bacillus thuringiesis.
- Southern blot analysis indicated that the parental line contains a single insertion of the vector and there was no integration of the vector backbone.
- Sequencing analysis indicated that the right and left T-DNA borders were truncated.