Herbicide tolerant maize

The maize has been produced through cross breeding of three parental lines to confer tolerance to multiple herbicides. For tolerance to glyphosate, the maize expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase, a variant of an endogenous gene, which prevents the herbicide interference of the shikimate pathway (aromatic amino acid biosynthesis). For tolerance to glufinosate, the modified maize expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase, which inactivates phosphinothricin (glufinosate) by transferring an acetyl group from acetyl-CoA. For diacamba tolerance, the maize expresses Stenotrophomonas maltophilia dicamba monooxygenase, which catalyzes the oxygen demethylation of diacamba.

PV-ZMAP1043; PV-ZMHT507801; PV-ZMGT32

DNA insert from PV-ZMAP1043
Transcription of  Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) commences from the Cauliflower mosaic virus (CaMV) enhanced 35S promoter and terminates at the A. tumefaciens nopaline synthase (nos) terminator. The transcript contains a Zea mays heat shock protein 70 (hsp70) intron, an Arabidopsis thaliana N-terminal chloroplast transit peptide sequence for chloroplast targeting of the protein 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.


DNA insert from PV-ZMHT507801
The Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat) gene is under the control of the Andropogon gerardii ubiquitin  promoter and the Oryza sativa alpha-amylase/trypsin inhibitor terminator. The transcript includes the A. gerardii 5' untranslated leader sequence and an intron from ubiquitin before (5') coding sequence of pat.

The Stenotrophomonas maltophilia dicamba monooxygenase (dmo) gene is under control of the Peanut chlorotic streak caulimovirus (PC1SV) full-length transcript promoter and the Triticum aestivum (wheat) heat shock protein 17 terminator. The transcript produced contains a wheat chlorophyll a/b-binding 5' untranslated leader sequence (for improved gene expression), an O. sativa actin 1 untranslated region and intron (for improved gene expression), the untranslated  and targeting region of Petunia hybrida chloroplast transit peptide 4 (for chloroplast targeting of the protein) and dmo.

Note:
- Originally, the plasmid vector contained two T-DNA elements that were inserted during the initial transformation event: one containing the dmo and pat expression cassettes, and a second containing an Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) expression cassette. The cp4-epsps expression cassette is regulated by the O sativa actin 1 promoter and 5′ untranslated leader, O. sativa intron, the Arabidopsis thaliana chloroplast targeting peptide 2 targeting sequence, and the A. tumefaciens nopaline synthase  3′ untranslated region. Subsequent traditional breeding, segregation, selection, and screening were used to isolate those plants that contain the dmo and pat expression cassettes (T-DNA I) and do not contain the cp4-epsps expression cassette (T-DNA II).
- Molecular characterization of MON87419 indicated that a single copy of T-DNA I was integrated into the maize genome at a single intact locus that includes all expected elements within the insert, with the exception of incomplete Right and Left Border sequences. These analyses also showed no PV-ZMHT507801 backbone elements or T-DNA II sequences were present in the event.


DNA insert from PV-ZMGT32
The plant expression plasmid vector, PV-ZMGT32 contains two adjacent plant gene expression cassettes each containing a single copy of the cp4 epsps. In the first (5' end) expression cassette, the cp4 epsps gene is under the transcriptional regulation of an Oryza sativa actin promoter and a nos terminator. An O. sativa actin intron is also present in the transcript for enhanced expression of the coding sequence. The second cassette consists of another cp4 epsps gene regulated by an CaMV enhanced 35S promoter (containing a duplicated enhancer region) and a nos terminator. Similarly, an intron from the maize heat shock protein 70 (hsp70) was included for enhancing expression of the coding sequence. Both promoters of the gene cassettes are expected to promoter high levels of transcription.

Note:
- The parental NK603 line contained a single, intact insertion containing both cp4 epsps gene cassettes.
- Due to restriction digest prior to particle bombardment, the vector backbone, containing E. coli neomycin phosphotransferase II and origin of replication, were not incorporated into the parental genome.

For more information, kindly refer to the parental line records.