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Maize event dp-098140-6 and compositions and methods for the identification and/or detection thereof

An event, plant technology, applied in the field of molecular biology, can solve the problem of transgene insertion affecting endogenous gene expression and so on

Inactive Publication Date: 2010-02-03
PIONEER HI BRED INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was also observed that transgene insertion may affect the expression of endogenous genes

Method used

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  • Maize event dp-098140-6 and compositions and methods for the identification and/or detection thereof
  • Maize event dp-098140-6 and compositions and methods for the identification and/or detection thereof
  • Maize event dp-098140-6 and compositions and methods for the identification and/or detection thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0602] Example 1: Sequence table of inserts and flanking edges of corn event DP-098140-6 Sign

[0603] The modification was performed by inserting the glyphosate acetyltransferase gene (glyat4621) and the maize acetolactate synthase gene (zm-hra) derived from Bacillus licheniformis into maize (Zea mays) in a modified form. The vector used for genetic modification is the plasmid of Agrobacterium tumefaciens strain LBA4404, which eliminates the pathogenicity by removing its natural T-DNA. By using the plasmid PHP24279 ( figure 1 The Agrobacterium-mediated transformation of) obtained corn event DP-098140-6. The T-DNA of plasmid PHP24279 contains two expression cassettes which will be described further below.

[0604] The immature embryos of maize were aseptically removed from the developing caryopsis and treated with the Agrobacterium tumefaciens strain LBA4404 containing GLYAT4621 and ZM-HRA expression cassette. After the embryo and Agrobacterium are co-cultured in a solid medium...

Embodiment 2

[0637] Example 2. Characterization of corn event DP-098140-6 by Southern blotting

[0638] The DNA inserted into DP-098140-6 (hereinafter referred to as "98140") corn was characterized by Southern blot analysis. Table 8 summarizes the results from various Southern blot analyses. The method used is described below. Genomic DNA was extracted from freeze-dried leaf tissue sampled from 98140 corn and non-genetically modified control plants. The genomic DNA was digested with restriction enzymes and separated by size on an agarose gel. Run a molecular weight marker next to the sample to estimate the size. The DNA fragments separated on the agarose gel are depurinated, denatured and neutralized in situ; then transferred to a nylon membrane. After transfer to the membrane, the DNA is bound to the membrane by UV cross-linking. The fragments homologous to the glyat4621 and zm-hra genes were generated from the plasmid PHP24279 by PCR, separated on an agarose gel by size, cut, and puri...

Embodiment 3

[0650] Example 3: Expression of insert

[0651] The expression of GLYAT4621 and Zm-HRA protein was evaluated in leaf tissue collected at the V5 stage of the growth of cultivated plants in the greenhouse. For each sample, four fresh leaf perforations were collected and ground in extraction buffer using GenoGrinder (Spex Certiprep). Bio-Rad protein analysis was used to determine the total extractable protein (TEP), which is based on the Bradford dye binding method. The sample extract was diluted in sample extraction buffer for ELISA analysis.

[0652] GLYAT4621 and ZM-HRA ELISA utilize a "sandwich" model to quantify specific target proteins in plant tissue extracts. In these analyses, standards (triplicate wells) and samples (duplicate wells) are incubated in a fixed plate that has been pre-coated with antibodies specific for the protein of interest. After one hour of incubation, unbound material was washed away from the plate and the bound protein was incubated with different pr...

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Abstract

Compositions and methods related to transgenic glyphosate / ALS inhibitor-tolerant maize plants are provided. Specifically, the present invention provides maize plants having a DP-098140-6 event which imparts tolerance to glyphosate and at least one ALS-inhibiting herbicide. The maize plant harboring the DP-098140-6 event at the recited chromosomal location comprises genomic / transgene junctions having at least the polynucleotide sequence of SEQ ID NO:5 and / or 6. The characterization of the genomic insertion site of the DP-098140-6 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the maize DP-098140-6 events are provided.

Description

Invention field [0001] The invention belongs to the field of molecular biology. More specifically, the present invention relates to multiple herbicide tolerance conferred by the joint expression of a sequence conferring tolerance to glyphosate and a sequence conferring tolerance to one or more ALS inhibitor chemicals. Background of the invention [0002] It is known that the expression of exogenous genes in plants is affected by their position in the plant genome, possibly due to the proximity of chromatin structure (for example, heterochromatin) or transcriptional regulatory elements (for example, enhancers) to the integration site Sex (Weising et al. (1988) Ann. Rev. Genet 22:421-477, 1988). At the same time, the presence of transgenes at different locations in the genome affects the entire phenotype of the plant in different ways. For this reason, it is often necessary to screen a large number of events to identify events that are characterized by optimized expression of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor T·K·奇库安J·W·德里C·B·黑兹尔N·L·亨德森D·洪德里德D·哈奇N·J·利森斯D·刘B·F·麦卡钦W·梅尔K·A·皮普尔斯D·W·绍恩德斯N·N·韦伯J·K·杨G·-Y·钟C·X·钟
Owner PIONEER HI BRED INT INC
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