Herbicide tolerant, insect resistant cotton

The modified cotton was the result of crossing modified parental lines to achieve multiple herbicide tolerance and insect resistance. For Lepidopteran resistance, the modified cotton expresses Bacillus thuringiensis Cry1Ac, Cry1F and Vegetative insecticidal protein 3A in the aerial parts of the plant. For herbicide tolerance, the modified cotton expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase and Streptomyces hygroscopicus phosphinothricin N-acetyltransferase for tolerance to glufosinate, as well as Delftia acidovorans aryloxyalkanoate dioxygenase for tolerance to 2,4-dichlorophenoxyacetic acid. Additionally, an Escherichia coli hygromycin B phosphotransferase cassette is also present for hygromycin selection during transformation and breeding.

pAGM281; pMYC3006; pCOT-1; pDAB4468

DNA insert from 281-24-236 vector pAGM281
The DNA insertion expresses Bacillus thuringiensis var. aizawaicry1F and  Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat).

Transcription of pat is under control of a Zea mays ubiquitin promoter and an Agrobacterium tumefaciens open reading frame 25 (ORF25) terminator. The transcript is expected to initially contain a Z. mays ubiquitin intron 1 at the 5' end. The intron enhances the expression of pat.

Transcription of cry1F is under control of a synthetic 4ocs∆Mas2' promoter and the same ORF25 terminator as the pat expression cassette. The cassette is in the reverse orientation to utilize the same terminator.

Note:
- Transcription is expected to occur at elevated and constitutive levels due to the promoters.
- The coding sequences of pat and cry1F have been optimized for expression in plants
- Cry1F is a chimeric, full-length δ-endotoxin comprised of the core toxin of Cry1F and nontoxic portions of B. thuringiensis Cry1Ca3 and Cry1Ab1 proteins, which form the C-terminal end of the protein and are removed during the activation of the Cry1F protein.

DNA insert from 3006-210-23 vector pMYC3006
The insertion expresses Bacillus thuringiensis cry1Ac and Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat).

Transcription of pat is under control of a synthetic 4ocs∆Mas2' promoter and an Agrobacterium tumefaciens open reading frame 25 (ORF25) terminator. Transcription of cry1Ac is under control of a Zea mays ubiquitin promoter and the same ORF25 terminator as the pat expression cassette.

Note:
- The ORF25 terminator serves as the terminator for both gene expression cassettes. The cry1Ac cassette is in the reverse orientation to utilize the same terminator.
- The coding sequences of cry1Ac and pat have been optimized for expression in plant cells.
- Transcription is expected to occur at elevated and constitutive levels due to the promoters.
- Southern blot analysis demonstrated that a single, intact DNA insertion was present in the parental genome. The analysis also indicated that no vector backbone sequences were integrated during transformation.

DNA insert from COT102 vector pCOT-1
The DNA insertion expresses Escherichia coli hygromycin B phosphotransferase (hph) and Bacillus thuringiensis vegetative insecticidal protein 3A.

Transcription of hph is under control of the Arabidopsis thaliana ubiquitin 3 promoter and the Agrobacterium tumefaciens nopaline synthase (nos) terminator. The gene cassette is present in the counterclockwise orientation.

The expression of Bacillus thuringiensis vegetative insecticidal protein 3A (vip3A) is under transcriptional control of the A. thaliana actin 2 promoter and the nos terminator.

Note:
- The coding sequence of the vip3A was altered for optimal expression in plant cells.
- Transcription is expected to occur at elevated and constitutive levels due to the promoters.
- Southern blot analysis indicated the incorporation of a single copy of the transgenes without the integration of the vector backbone sequences.
- Southern blot analysis confirmed the expression of the proteins.

DNA insert from 81910 vector DAB4468
The DNA insertion expresses Delftia acidovorans aryloxyalkanoate dioxygenase (aad-12) and Streptomyces hygroscopicus phosphinothricin N-acetyltransferase (pat).

Transcription of aad-12 is under control of the Arabidopsis thaliana polyubiquitin 10 promoter and the Agrobacterium tumefaciens open reading frame 23 3' untranslated region. Transcription of pat is under control of the Cassava Vein Mosaic Virus promoter and the A. tumefaciens ORF1 3' untranslated region.

Note:
- Transcription is expected to occur at elevated and constitutive levels due to the promoters.
- Southern blot analysis indicated a single, intact insertion of aad-12 and pat into the parental genome without the integration of the vector backbone sequences.