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Method for producing hsl protein having improved catalytic activity for 2-oxoglutaric acid-dependently oxidizing 4-hppd inhibitor

A 4-HPPD, oxoglutaric acid technology, applied in chemical instruments and methods, biochemical equipment and methods, oxidoreductases, etc., can solve the problems of chlorophyll disintegration, withering, plant whitening, etc., to improve catalytic activity , the effect of increasing resistance

Active Publication Date: 2019-09-20
日本史迪士生物科学株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These herbicides are all agents (4-HPPD inhibitors) that inhibit the function of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), by inhibiting the function of this enzyme, thus as figure 1 As shown, indirectly inhibit the carotenoid synthesis system, causing the disintegration of chlorophyll, resulting in plant albinism and death

Method used

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  • Method for producing hsl protein having improved catalytic activity for 2-oxoglutaric acid-dependently oxidizing 4-hppd inhibitor
  • Method for producing hsl protein having improved catalytic activity for 2-oxoglutaric acid-dependently oxidizing 4-hppd inhibitor
  • Method for producing hsl protein having improved catalytic activity for 2-oxoglutaric acid-dependently oxidizing 4-hppd inhibitor

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Embodiment

[0122] Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.

[0123] The present inventors previously found that a gene possessed by rice (HIS1) and its homologous gene (HSL1 gene) contribute to resistance or sensitivity to 4-HPPD inhibitors. In addition, it has been clarified that by using such a gene, plants having improved resistance or sensitivity to 4-HPPD inhibitors can be produced, and it has been further found that a gene having high homology with the HIS1 gene of rice is found in barley, sorghum, and maize. etc. also exist (Patent Document 1).

[0124] In addition, the present inventors deduced that HIS1 and OsHSL1 are oxidases and 2-oxoglutarate-dependent dioxygenases (2 -oxoglutarate dependent dioxygenas, 2OGDs). 2OGD is a protein containing non-heme iron ions, and is a soluble protein localized in the cytoplasm in plants. 2OGD requires 2-oxoglutaric acid (2OG) and oxygen m...

Embodiment 2

[0138] Deduction of Amino Acid Residues Involved in 4-HPPD Inhibitor Catalytic Activity of HIS1 Protein

[0139] Therefore, based on this new knowledge, the present inventors hypothesized that a slight difference in the amino acid sequences of the HIS1 protein and the OsHSL1 protein contributes to the decomposing activity of the 4-HPPD inhibitor. Furthermore, in the HIS1 protein, amino acid residues involved in the degradation activity of the 4-HPPD inhibitor were estimated by the method shown below.

[0140] First, in order to predict the three-dimensional structure of the HIS1 protein, crystal structure analysis was attempted. However, the purified protein was extremely unstable and prone to insolubilization, so it was discarded.

[0141] Instead, the enzyme of Arabidopsis thaliana with the highest sequence similarity to HIS1 among the oxidases dependent on ferric ion and 2-oxoglutarate whose protein crystal structure has been resolved, that is, anthocyanin Synthase (Ant...

Embodiment 3

[0147] Production of OsHSL1 protein variants and evaluation of the 4-HPPD inhibitor decomposing activity of these variants

[0148] Therefore, in order to examine such a possibility, as to whether or not HIS1-type enzymatic activity can be imparted to the protein by appropriately substituting the amino acid residues different from the HIS1 protein in the OsHSL1 protein with the amino acid residues of HIS1, the following method was used: method for analysis.

[0149]

[0150] First, in order to replace any of the amino acid residues at positions 118, 140, 204, 229, and 298 of the OsHSL1 protein with amino acid residues of the HIS1 protein by site-directed mutagenesis, as exemplified below The primers for introducing mutations used in this method are designed in this way.

[0151] 1) Amino acid replacement of the valine residue at position 118 of OsHSL1 with an isoleucine residue (HSL1 V118I)

[0152] Primers for mutation introduction were designed in such a manner that th...

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Abstract

The purpose of the present invention is to provide: a method for producing an HSL protein having improved catalytic activity for 2-oxoglutaric acid-dependently oxidizing a 4-HPPD inhibitor; and a method for producing a plant body having improved resistance against a 4-HPPD inhibitor using the method for producing an HSL protein. It has been made clear that by displacing the 140th locus in the HSL protein by means of a basic amino acid, the catalytic activity for 2-oxoglutaric acid-dependently oxidizing a 4-HPPD inhibitor can be improved in the protein and, further, the activity for decomposing said inhibitor can be improved.

Description

technical field [0001] The present invention relates to a method for producing an HSL protein having improved catalytic activity for 2-oxoglutarate-dependent oxidation of 4-HPPD inhibitors. Furthermore, the present invention relates to a method for producing a plant having improved resistance to a 4-HPPD inhibitor using this method. In addition, the present invention also relates to a method for determining the resistance of a plant to a 4-HPPD inhibitor, and a method for breeding a plant having improved resistance to a 4-HPPD inhibitor using the method. Background technique [0002] Recently, herbicide components such as bicyclic sulcotrione, tefurfuryl ketone, sulcotrione, mesotrione, and tembotrione have been developed and put into practical use. These herbicides are all agents (4-HPPD inhibitors) that inhibit the function of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), by inhibiting the function of this enzyme, thus as figure 1 As shown, it indirectly inhibits the car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02A01H1/00C12N9/02C12N15/09C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12N15/8274C12N9/0071C12Y114/11C07K14/415
Inventor 户泽让武井里美大岛正弘广濑咲子川田元滋关野景介山崎明彦
Owner 日本史迪士生物科学株式会社
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