Herbicide tolerant, Insect resistant maize

The modified maize was produced through the cross breeding of two modified parental lines for herbicide tolerance and insect resistance. For Lepidoptera resistance, the maize expresses Bacillus thuringiensis Vegetative insecticidal protein 3Aa20. For herbicide tolerance, the maize expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase, which encodes a variant of the endogenous enzyme involved in aromatic amino acid synthesis and confers glyphosate tolerance in female and vegetative tissues. The tissue specificity additionally allows for glyphosate-treated plants to serve as a female parent in the production of hybrid lines. The modified maize also contains a selectable, Escherichia coli phosphomannose isomerase, for mannose selection during parental transformation.

PV-ZMAP1043; pNOV1300

Genetic elements from PV-ZMAP1043
Transcription of 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) from Agrobacterium tumefaciens commences from the Cauliflower mosaic virus (CaMV) enhanced 35S promoter and ends at the A. tumefaciens nopaline synthase (nos) gene terminator. The transcript contains a Zea mays heat shock protein 70 (Hsp70) intron, Arabidopsis thaliana N-terminal chloroplast transit peptide sequence, and cp4 epsps. The CaMV enhanced 35S promoter-hsp70 combination promotes gene expression in female and vegetative tissues, but not in male reproductive tissues (pollen microspores and tapetum).

Note:
- Southern blot analyses indicate that a single copy of the T-DNA was inserted at a single site in the parental maize genome and no plasmid vector backbone sequences were detected to have been integrated. DNA sequencing analyses further indicated that the expected T-DNA sequences were integrated.
-The cp4 epsps coding sequence is the codon optimized coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding CP4 EPSPS.

Genetic elements from pNOV1300
In the parental MIR162 maize, a variant of the native B. thuringiensis vegetative insecticidal protein 3Aa (vip3Aa20), named vip3Aa19, which has codon changes that result in a single  M129I amino acid substitution was inserted into the transformation cassette. During the transformation process an additional DNA mutation resulted in a K284Q amino acid substitution. This final form was designated the name Vip3Aa20. 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.

A second expression cassette, containing the E. coli phosphomannose isomerase gene, 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 gene (nos) terminator.

Note:
- Southern blot analyses demonstrated that the T-DNA insert contains: i) single copies of a vip3Aa20 gene and a pmi gene; ii) two copies of the ZmUbiInt promoter; iii) one copy of the nos terminator; and iv) no backbone sequences from transformation plasmid pNOV1300.