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Gene encoding acetolactic acid sy nthase gene

A technology of acetolactate synthase and gene, applied in the field of rate-limiting enzymes

Inactive Publication Date: 2004-04-07
KUMIAI CHEM IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mutant ALS genes targeting PC herbicide resistance and involving specific resistance to PC herbicides have not been reported

Method used

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  • Gene encoding acetolactic acid sy nthase gene
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  • Gene encoding acetolactic acid sy nthase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Generation of PC herbicide-resistant rice (Kinmaze) cultured cells

[0087] Rice seeds (variety; Kinmaze, scientific name: Oryza sativa var. Kinmaze) were dehulled. The seeds were soaked in 70% ethanol for 5 minutes and then in about 5% antiformin for 20 minutes, followed by several washes with sterile distilled water. Then, place the seed in table 1 with

[0088] Cultured statically on the medium with the indicated composition.

[0089] Inorganic salts (Murashige-Skoog cultured

base mixed brine)

1 package

Thiamine hydrochloride (0.1g / l)

1ml

Niacin (0.5g / l)

1ml

Pyridoxine hydrochloride (0.5g / l)

1ml

Glycine (2g / l)

1ml

Inositol (50g / l)

2ml

2,4-D (200ppm)

10ml

sucrose

30g

[0090] In the above medium composition, 2,4-D is a synthetic auxin. To prepare the medium, first, the medium having the above composition was placed in a 1 L...

Embodiment 2

[0098] Example 2: Herbicide susceptibility of partially purified ALS enzymes from resistant mutants

[0099] In this example, the mutant ALS protein was partially purified from the resistant mutant obtained in Example 1, and then the herbicide sensitivity of the resulting mutant ALS protein was examined. The mutant ALS protein was partially purified as follows.

[0100] First, 200 g or more of resistant mutants were prepared by a liquid culture method (without supplementing bispyribac), and the composition of the medium was the same as that shown in Table 1, but Gelrite was not included. Then, using a Hiscotron, buffer 1 [100 mM potassium phosphate buffer (pH 7.5) containing 20% ​​(v / v) glycerol, 0.5 mM thiamine pyrophosphate (TPP), 10 μM yellow adenine dinucleotide (FAD), 0.5mM MgCl 2 About 150 g of the resistant mutant was homogenized with polyvinylpolypyrrolidone in a volume 1 / 10 of the tissue volume. The homogenate was filtered through a nylon mesh and then centrifuge...

Embodiment 3

[0114] Embodiment 3: Cloning of mutant ALS gene

[0115] A probe for cloning a gene encoding a mutant ALS protein derived from a resistant mutant (mutant ALS gene) was prepared as follows. Part of the cDNA used as a probe in this example was derived from rice (Nippon-bare) showing high homology to the maize ALS gene.

[0116] (1) Determination of the partial cDNA nucleotide sequence derived from rice (Nippon-bare) showing high homology with the corn ALS gene

[0117] As part of the IneGenome Project (Ine Genome Project) conducted by the Society for Techno-inmovation of Agriculture, Forestry and Fisheries and National Institute of Agrobiological Sciences, the partial nucleotide sequence of rice (Nippon-bare) cDNA has been determined, and A partial nucleotide sequence database of cDNA has been established. A cDNA clone (Accession No. C72411) with a known nucleotide sequence of about 350 bp contained in this database shows a high degree of homology with the maize ALS gene. T...

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Abstract

The present invention provides a gene encoding ALS protein which shows extremely high level of resistance to PC herbicides. The gene of the present invention encodes the following protein (a) or (b): (a) a protein which comprises an amino acid sequence of SEQ ID NO: 1; (b) a protein which comprises an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution, deletion or addition of at least one or more amino acids, has resistance to a pyrimidinyl carboxy herbicide and has acetolactate synthase activity.

Description

field of invention [0001] The invention relates to a gene encoding acetolactate synthase, which is a rate-limiting enzyme in the biosynthesis pathway of branched-chain amino acids. Background of the invention [0002] Acetolactate synthase (hereinafter referred to as "ALS") is a rate-limiting enzyme in the biosynthetic pathway of branched-chain amino acids such as leucine, valine, and isoleucine, and is considered an essential enzyme for plant growth. In addition, ALS is known to exist in various higher plants. In addition, ALS is present in various microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Salmonella typhimurlum. [0003] Three types of ALS isozymes are known to exist in E. coli and S. typhimurium. Each of these isozymes is a hetero-oligomer composed of a large molecular weight catalytic subunit that controls the enzyme's catalytic activity and a small molecular weight regulatory subunit that functions as a regulatory pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/82
CPCC12Y202/01006C12N9/88C12N15/8278
Inventor 清水力中山礎永山孝三福田笃德田中喜之角康一郎
Owner KUMIAI CHEM IND CO LTD
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