Novel epsp synthase genes conferring herbicide resistance

A technology of EPSP synthase and herbicide, applied in the direction of genetic engineering, enzyme, transferase, etc.

Inactive Publication Date: 2009-07-22
ATHENIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the EPSP synthases of certain bacteria are highly tolerant to glyphosate

Method used

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  • Novel epsp synthase genes conferring herbicide resistance
  • Novel epsp synthase genes conferring herbicide resistance
  • Novel epsp synthase genes conferring herbicide resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] The separation of embodiment 1.ATX8909

[0097] ATX8909 was isolated by plating soil samples on HEPES mineral salt medium (HMSM) containing glyphosate as the sole phosphorus source. Because HMSM does not contain aromatic amino acids, strains must be resistant to glyphosate in order to grow on this medium.

[0098] 2 grams of soil were resuspended in approximately 10 ml of water, vortexed for 15 seconds, and allowed to settle for 15 minutes. A 10 μl loop of this suspension was added to 3 ml HMSM (pH 7.0) supplemented with 10 mM glyphosate. HMSM contains (per liter): 10g glucose, 2gNH 4 SO 4 , 9.53g HEPES, 1.0ml 0.8M MgSO 4 , 1.0ml 0.1M CaCl 2 , 1.0ml trace element solution (in 100ml of 1000x solution: 0.1g FeSO 4 ·7H 2 O, 0.5mg CuSO 4 ·5H 2 O, 1.0 mg H 3 BO 3 , 1.0mg MnSO 4 ·5H 2O, 7.0mg ZnSO 4 ·7H 2 O, 1.0mg MoO 3 , 4.0 g KCl). The culture was grown for 4 days at 28°C in a shaking incubator before 20 μl was used to inoculate 2.5 ml of fresh HMSM conta...

Embodiment 2

[0102] Example 2. Cloning of glyphosate-resistant EPSP synthase

[0103] Genomic DNA was extracted from the strains described in Table 1, and the resulting DNA was partially digested with restriction enzyme Sau3A1, thereby generating a DNA fragment of about 5 kilobases in size. These DNA molecules were size selected on agarose gels, purified, and ligated into LAMBDA predigested with BamHI in the carrier arm. The ligated arms are then packaged into phage particles, and phage titers are determined as known in the art. The resulting library was amplified by methods known in the art to generate 3 x 10 7 -3 x 10 8 Library titer in PFU / mL. For each independent library, phage from the amplified library were then co-transfected into E. coli (XL1 Blue MRF') together with the M13 helper phage to allow extensive excision of the library as infectious circular ssDNA , as known in the art (Short et al. (1988) Nucleic Acids Research 16:7583-7600). Following centrifugation of the co-...

Embodiment 3

[0107] Example 3. DNA and protein sequence of EPSP synthase

[0108] For each of the clones in Table 2, the DNA sequence of the glyphosate-resistant EPSP synthase was determined by methods well known in the art.

[0109] grg25. The DNA sequence of grg25 is provided herein as SEQ ID NO:1. The predicted translation product of grg25 (GRG25) is provided herein as SEQ ID NO:2.

[0110] grg26. The DNA sequence of grg26 is provided herein as SEQ ID NO:3. The predicted translation product of grg26 (GRG26) is provided herein as SEQ ID NO:4.

[0111] grg27. The DNA sequence of grg27 is provided herein as SEQ ID NO:5. The predicted translation product of grg27 (GRG27) is provided herein as SEQ ID NO:6.

[0112] grg28. The DNA sequence of grg28 is provided herein as SEQ ID NO:7. The predicted translation product of grg28 (GRG28) is provided herein as SEQ ID NO:8.

[0113] grg29. The DNA sequence of grg29 is provided herein as SEQ ID NO:9. The predicted translation product of...

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Abstract

Compositions and methods for conferring herbicide resistance or tolerance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a polypeptide that confers resistance or tolerance to glyphosate herbicides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated nucleic acid molecules corresponding to glyphosate resistant nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, or 14 or the nucleotide sequence set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 28, 29, 30, 31, 32, 33, or 34.

Description

field of invention [0001] The present invention provides a novel gene encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers herbicide resistance. These genes can be used in plant biology, crop breeding and plant cell culture. Background of the invention [0002] N-phosphonomethylglycine, commonly known as glyphosate, is an important agronomic compound. Glyphosate inhibits the enzyme that converts phosphoenolpyruvate (PEP) and 3-phosphoshikimate to 5-enolpyruvyl-3-phosphoshikimate. Inhibition of this enzyme (5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as "EPSP synthase") inhibits aromatase by shutting down the shikimate pathway. Biosynthesis of family amino acids kills plant cells. [0003] Because glyphosate herbicides inhibit the biosynthesis of aromatic amino acids, they not only kill plant cells but are also toxic to bacterial cells. Glyphosate inhibits the EPSP synthase of many bacteria and is therefore toxic to these bacteria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10
CPCC12N15/8275C12N9/1092
Inventor B·卡尔B·范德伯格D·J·汤姆索C·皮特斯
Owner ATHENIX
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