The modified maize contains four gene cassettes: Escherichia coli phosphomannose isomerase (pmi), Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat), Diabrotica virgifera virgifera smooth septate junction protein 1 double stranded RNA (DvSSJ1-dsRNA) and Pseudomonas chlororaphis insecticidal protein ipd072Aa (ipd072Aa).

Transcription of pmi is under control of a Zea mays (maize) ubiquitin promoter and a Solanum tuberosum proteinase inhibitor II gene terminator. The  transcript is also expected to initially include a 5' untranslated region (5' UTR) and intron 1 of the maize ubiquitin at the 5' end of the pmi coding sequence. These sequences are not expected to be translated, but enhance transcription. A maize 19-kDA zein terminator is also present after the proteinase inhibitor II terminator and acts to prevent transcriptional interference between gene cassettes.

Transcription of pat is under control of an Oryza sativa (rice) actin 1 promoter and a Cauliflower mosaic virus (CaMV) terminator. A rice actin 1 intron is included at the 5' end of the pat to enhance expression of the coding sequence. Sorghum bicolor ubiquitin and gamma-kafarin terminators are also present after the CaMV terminator, which acts to prevent transcriptional interference between gene cassettes.

Transcription of the RNA interference (RNAi) cassette (DvSSJ1-dsRNA) is under control of a maize ubiquitin promoter and maize 27-kDA gamma zein terminator. From 5' to 3', the transcript is expected to contain: maize 5' UTR, maize ubiquitin intron 1, (sense) synthetic all-stop codon sequence, (sense) DvSSJ1, (sense) mini-stop codon sequence, maize alcohol dehydrogenase 1 intron, (anti-sense) mini-stop codon sequence, (anti-sense) DvSSJ1 and (anti-sense) synthetic all-stop codon sequence. The maize ubiquitin 5' UTR and intron promote high levels of transcription. After transcription, the sense and anti-sense regions base pair to form a double stranded structure (hairpin RNA; hpRNA) with the alcohol dehydrogenase sequence acting as a connecting loop. Translation is not expected to occur for the hpRNA because of RNAi processing within the host plant (see Additional information section below) and the all-stop and mini-stop synthetic sequences, which would terminate translation before any protein is produced. An Arabidopsis thaliana ubiquitin 14 and a maize In2-1 terminator are also present after the 27-kDA gamma zein terminator to prevent transcriptional interference between gene cassettes.

Transcription of the ipd072Aa is under control of a Banana streak virus promoter and an Arabidopsis thaliana putative mannose-binding protein superfamily terminator. A maize predicted calmodulin 5 gene intron was included at the 5' end of the ipd072Aa coding sequence to enhance expression.


Note:
- DP23211 maize was created through two subsequent transformations:
-- First transformation: particle bombardment of PHP56614 with two additional plasmids (PHP21139 and PHP31729). PHP21139 and PHP31729 were not integrated, but transiently express WUS and ODP2 proteins to improve regeneration of maize plants
-- Second transformation: Agrobacterium-mediated transformation with PHP74643,  subsequently resulting in site-specific recombination
- For information on the backbone sequences or removed genetic elements, kindly refer to the attached documents. Non-integrated or removed sequences include: WUS, ODP2, I-CreI, NPTII, DsRED2 and FLP.
- Next generation sequences (Southern-by-sequencing) confirmed the plants contained a single insertion, the intended genetic elements and no unintended insertions (such as antibiotic resistance from plasmid backbones)
- In silico analysis indicated that the 210 basepair region of the DvSSJ1 targeted by the RNAi construct is specific to corn rootworms in the Diabrotica genus, Chrysomelidae family and Coleoptera order.