Herbicide tolerant, insect resistant maize

The modified maize (Zea mays) was produced through the cross breeding of three modified parental lines for herbicide tolerance and insect resistance. For Lepidoptera resistance, the maize expresses Bacillus thuringiensis vegetative insecticidal protein 3Aa20, Cry1A.105 and Cry2Ab2. For herbicide tolerance, the maize expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase, a variant of an endogenous enzyme, in female and vegetative tissues for glyphosate tolerance. The tissue specificity allows for glyphosate-treated plants to serve as the female parent in the production of further hybrid lines. The modified maize also contains a selectable, Escherichia coli phosphomannose isomerase, for mannose selection during parental transformation.

PV-ZMAP1043; PV-ZMIR245; 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 PV-ZMIR245
Two insecticidal protein expression cassettes were inserted into the genome. Bacillus thuringiensis cry1A.105 expression is under the control of the CaMV 35S enhanced promoter, which first transcribes wheat (Triticum aestivum) 5' untranslated region of the chlorophyll a/b-binding protein (cab) and a rice actin 1 intron before transcribing cry1A.105. Transcription terminates at the wheat heat shock protein 17.3 terminator. Expression of the B. thuringiensis cry2Ab2 starts at the Figwort mosaic virus (FMV) promoter, which transcribes the Zea mays heat shock protein 70 (hsp70), then the Z. mays transit peptide and the cry2Ab2 coding sequence, before terminating at the nos terminator.

Note:
- The Cry2Ab2 coding sequence was modified for optimal expression in plants.
- South blot analysis confirmed that single insertions of both cry2Ab2 and cry1A.105, as well as no vector backbone were present and in the parent.
- A deletion removed the duplicated enhancer elements compared to the original CaMV e35S promoter in PV-ZMIR245.
- The selectable marker, nphII, cassette was bred out of the parental line and became not associated with this transformation event.


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.