Loading...
You are viewing a DELETED record.
This record information is displayed for reference purpose only and should be not used.
This document has been updated. This is not the latest published version. Click here to view the latest version of the record.
Living Modified Organism (LMO)
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.
Borer-resistant, herbicide-tolerant maize
EN
DP915635
Yes
DP-915635-4
-
loading record details...Organization:Pioneer Hi-Bred International Inc. ()Private sector (business and industry)7100 NW 62nd Avenue PO Box 1000Johnston, Iowa
50131, United States of AmericaPhone: +1 515 535-3200,Fax:Email:Website: www.pioneer.com/,
The maize was modified through a site-specific transformation protocol for insect resistance and herbicide tolerance. For resistance to Western corn rootworm (Diabrotica virgifera), the maize expresses Ophioderma pendulum insecticidal protein IPD079Ea, which has a pore-forming mode of action against feeding larvae. The protein binds receptors in the insect's midgut which are different from the receptors that Bt toxins interact with, allowing the maize to overcome Bt-resistance in Coleoptera pests. For tolerance to glufosinate, the maize expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase, which inactivates the herbicidal compound through acetylation. In addition, the maize also contains an Escherichia coli phosphomannose isomerase cassette, which allows for modified plants to use mannose as a carbon source and thus is a selectable marker during transformation. The transformation protocol involved two steps to achieve a site specific integration into the maize genome. In the first step, four plasmids were introduced by microparticle bombardment, which introduced recombination sites at a specific location using transiently expressed CRISPR/Cas9. In the second step, Agrobacterium-mediated transformation was used to introduce the final gene cassettes through a recombination with sequences present in the first insertion site. More information regarding the two-step site-specific transformation is provided below.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
-
loading record details...BCH-ORGA-SCBD-246-6 Organism Zea mays (Maize, Corn, MAIZE)Crops
Maize variety PHR03
EN
PHP73878 and PHP83175
EN
- Agrobacterium-mediated DNA transfer
- Biolistic / Particle gun
- Gene editing (e.g. CRISPR-Cas, etc.)
0.048 kb
|
1.216 kb
|
0.311 kb
|
0.742 kb
|
1.682 kb
|
0.000 kb
|
0.552 kb
|
0.194 kb
|
0.034 kb
|
0.584 kb
|
0.464 kb
|
1.581 kb
|
1.581 kb
|
1.581 kb
|
0.911 kb
|
0.856 kb
|
1.440 kb
|
0.953 kb
|
0.460 kb
|
0.940 kb
|
0.048 kb
|
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
-
loading record details...BCH-GENE-SCBD-260877-1 Flippase recombinase recognition targets | Saccharomyces cerevisiae (Yeast, YEASX)Recognition sequence
-
BCH-GENE-SCBD-15003-7 Phosphomannose Isomerase gene | Escherichia coli (ECOLX)Protein coding sequence | Mannose tolerance,Selectable marker genes and reporter genes
-
BCH-GENE-SCBD-100367-4 Proteinase inhibitor II gene terminator | Solanum tuberosum (Potato, SOLTU)Terminator
-
loading record details...BCH-GENE-SCBD-116046-1 19-kDa zein gene terminator | Zea mays (Maize, Corn, MAIZE)Terminator
-
loading record details...BCH-GENE-SCBD-100364-5 Rice actin 1 gene promoter | Oryza sativa (Rice, ORYSA)Promoter
-
BCH-GENE-SCBD-100355-6 Rice actin 1, intron | Oryza sativa (Rice, ORYSA)Intron
-
loading record details...BCH-GENE-SCBD-15002-4 Phosphinothricin N-acetyltransferase gene | Streptomyces viridochromogenes (STRVR)Protein coding sequence | Resistance to herbicides (Glufosinate)
-
BCH-GENE-SCBD-100290-6 CaMV 35S terminator | Cauliflower mosaic virus (CaMV)Terminator
-
BCH-GENE-SCBD-103069-3 loxP recombination site | Bacteriophage P1 (Phage P1)recombination site
-
loading record details...BCH-GENE-SCBD-116047-2 Ubiquitin terminator | Sorghum bicolor (Sorghum)Terminator
-
BCH-GENE-SCBD-116062-1 Gamma kafarin terminator | Sorghum bicolor (Sorghum)Terminator
-
BCH-GENE-SCBD-260878-1 RCc3 enhancer | Sorghum bicolor (Sorghum)Enhancer
-
BCH-GENE-SCBD-116052-1 Predicted calmodulin 5 gene intron | Zea mays (Maize, Corn, MAIZE)Intron
-
BCH-GENE-SCBD-260880-1 PCOa promoter | Zea mays (Maize, Corn, MAIZE)Promoter
-
BCH-GENE-SCBD-260899-1 Insecticidal protein IPD079Ea | Ophioderma pendulum (Old World adder's-tongue, Daun rambu, OPHPE)Protein coding sequence | Resistance to diseases and pests (Insects, Coleoptera (beetles), Western corn rootworm (Diabrotica virgifera))
-
loading record details...BCH-GENE-SCBD-260900-2 Subtilisin-chymotrypsin inhibitor 1B terminator | Sorghum bicolor (Sorghum)Terminator
-
BCH-GENE-SCBD-116051-1 27-kDA gamma zein terminator | Zea mays (Maize, Corn, MAIZE)Terminator
-
loading record details...BCH-GENE-SCBD-105058-3 In2-1 Terminator | Zea mays (Maize, Corn, MAIZE)Terminator
The modified maize contains three gene cassettes: Escherichia coli phosphomannose isomerase (pmi); Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat) and Ophioderma pendulum insecticidal protein IPD079Ea (ipd079ea).
The pmi coding sequence was inserted near an endogenous promoter and thus is still expected to have sufficient activity for expression. Transcription is terminated by a Solanum tuberosum proteinase inhibitor II (pinII) terminator. A second terminator, maize 19-kDa zein gene terminator, was included to prevent transcription beyond the gene cassette (limits read through/'leaky' expression of the adjacent gene cassette).
The pat coding sequence is under control of an Oryza sativa actin promoter and Cauliflower mosaic virus 35S terminator. The promoter contains an intron from the rice actin gene for enhanced expression. Two additional terminators, Sorghum bicolor ubiquitin and gamma-kafarin, are included to isolate the gene cassette, preventing transcription beyond the gene cassette (limits read through/'leaky' expression of the adjacent gene cassette).
The ipd079ea coding sequence under control of a maize PCOa promoter and S. bicolor subtilisin-chymotrypsin inhibitor 1B terminator. The promoter is enhanced by S. bicolor RCc3 enhancers and promotes root-specific expression. An additional two terminators, maize 27-kDa gamma zein and ln2-1, were included to prevent transcription beyond the gene cassette.
Note:
- Sequencing analysis indicated that the maize contains a single, intact insertion of the expected sequences into chromosome 1 of the maize genome. The sequences were not rearranged or truncated. The analysis also indicated that PHP73878 and PHP83175 vector backbone sequences were absent.
- Sequencing analysis indicated that PHP70605, PHP21139 and PHP21875 were absent from the maize genome (also see below).
Transformation of the maize:
The transformation of the maize (site-specific integration) was performed in two steps to insert the transgene cassettes into chromosome 1 in a controlled manner.
- The first transformation (microparticle bombardment)
- PHP73878 (for integration into maize genome)
- Contains sequences for CRISPR/Cas9 mediated recombination, loxP site for Cre recombination, FRT1 site for FLP recombination, nptII, pinII terminator and FRT site.
- PHP70605 (facilitates recombination; not inserted into the maize genome)
- Contains T3 promoter, maize ubi promoter, nuclear locating signal from SV40, cas9 exon 1, intron 1 from potato LS1, cas9 exon 2, nuclear locating signal from A. tumefaciens virD2, pinII terminator, maize polIII U6 promoter, guide RNA (gRNA) for Cas9 and maize polIII U6
- PHP21139 (aids in regeneration during tissue culture; not inserted into genome)
- Contains maize Wuschel 2 (wus2)
- PHP21875 (aids in the regeneration of plants in vitro; not inserted into genome)
- Contains coding optimized ovule development protein 2 (odp2)
- Following microparticle bombardment, the gRNA and Cas9 are transiently expressed. The gRNA targets sequences on chromosome 1 (which are also present in PHP73878) to cause a site specific integration of PHP73878 into chromosome 1.
- Plants were regenerated using tissue culture and selected for using kanamycin (from the introduced E. coli neomycin phosphotransferase II cassette).
- PHP73878 (for integration into maize genome)
- The second transformation (Agrobacterium tumefaciens-mediated)
- PHP83175:
- Integrated into genome: pmi, pat and ipd079ea.
- Not integrated into genome (transiently expressed): wus2, odp2 and yeast flippase.
- Following introduction of the plasmid into host cells by Agrobacterium tumefaciens, expression of flippase directs a recombination between the FRT sites in PHP73878 and PHP83175, resulting in the replacement of the nptII cassette with pmi, pat and ipd079ea cassettes. Transient expression of wus2 and odp2 facilitate the regeneration and tissue culture of transformed plants.
- PHP83175:
For more information, kindly refer to the documents attached in the 'Additional information' section of this record.
EN
- Feed
- Food
- DP-915635-4 - EU Reference Laboratory for GM Food and Feed (EURL-GMFF) ( JRC ) [ English ]
EN
EN
- EUginius - DP915635 [ English ]
- Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion.pdf [ English ]
- Anses - BIOT2021SA0116.pdf [ French ]
- US20210381000A1 - Maize event DP-915635-4 and methods for detection thereof.pdf [ English ]
- コウチュウ目害虫抵抗性及び除草剤グルホシネート耐性トウモロコシ.pdf [ Japanese ]