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Composition and method for conferring and/or enhancing tolerance against herbicides by using variants of ppo

An identity, amino acid technology, applied in the field of compositions and methods using PPO variants to confer and/or enhance tolerance to herbicides, capable of solving problems such as pollution

Pending Publication Date: 2020-09-29
FARMHANNONG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the case of large-scale cultivation of algae in bioreactors or in open or closed ponds, it may occur due to undesired competing organisms (for example, undesired algae, fungi, rotifers or zooplankton) Pollution

Method used

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  • Composition and method for conferring and/or enhancing tolerance against herbicides by using variants of ppo
  • Composition and method for conferring and/or enhancing tolerance against herbicides by using variants of ppo
  • Composition and method for conferring and/or enhancing tolerance against herbicides by using variants of ppo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1. Verification of herbicide tolerance of ApPPO1 and MxPPO isolated from prokaryotes

[0188] The PPO gene sequences were obtained from the Genebank database of two strains of Auxenochlorella protothecoides and Myxococcus xanthus, respectively. For encoding a PPO protein from oleaginous microalgae (ApPPO1; SEQ ID NO:1), the PPO gene designated ApPPO1 was isolated from oleaginous microalgae and optimized to have the nucleic acid sequence of SEQ ID NO:7. For encoding the PPO protein from M. xanthus (MxPPO; SEQ ID NO:3), the PPO gene designated MxPPO was isolated from M. xanthus and optimized to have the nucleic acid sequence of SEQ ID NO:8. To obtain herbicide-binding structures of PPO proteins, herbicides including Tifenacil, Saflufenacil, Flufenacil, and Sulfentrazone were used, and PPO proteins including ApPPO1 and MxPPO were used. ApPPO1 was constructed from the structure of CyPPO10 (PPO protein derived from Synechococcus elongatus BP-1; SEQ ID NO:5) using ...

Embodiment 2

[0192] Example 2. Construction of PPO variants

[0193] In order to enhance the PPO-inhibiting herbicide tolerance to ApPPO1 and MxPPO, the mutations at the herbicide-interacting positions obtained in Example 1 were introduced, respectively. Each PPO gene was codon optimized and synthesized (Cosmogenetech Co., Ltd.) for efficient herbicide tolerance testing using PPO-deficient E. coli strain BT3.

[0194] The detailed experimental procedure is as follows:

[0195] Using the primers listed in Table 2, PCR was performed under the following conditions to amplify the PPO gene.

[0196] PCR reaction mix

[0197] Template (synthetic DNA of ApPPO1 and MxPPO) 1 μl

[0198] 5 μl of 10X buffer

[0199] dNTP mix (10 mM each) 1 μl

[0200] Forward primer (10 μM) 1 μl

[0201] Reverse primer (10μM) 1μl

[0202] DDW 40μl

[0203] Pfu-X (Solgent, 2.5 units / μl) 1 μl

[0204] 50 μl total

[0205] [Table 1] PCR reaction conditions

[0206]

[0207] [Table 2] List of primers for ...

Embodiment 3

[0229] Example 3. Verification of PPO-inhibiting herbicide tolerance of PPO variants (tested in E. coli)

[0230] The mutated CyPPO gene obtained in Example 2 was transformed into a BT3 (ΔPPO) strain lacking PPO activity, and cultured with a PPO-inhibiting herbicide in LB medium, thereby checking whether the growth of the transformed BT3 was not inhibited.

[0231] The BT3(ΔPPO) strain was provided by Hokkaido University (Japan), which is a hemG-type PPO-deficient and kanamycin-resistant Escherichia coli strain (see "Watanabe N, Che FS, Iwano M, Takayama S, Yoshida S , Isogai A. Dual targeting of spinach protoporphyrinogen IX oxidase II tomitochondria and chloroplasts by alternative use of two in-frame initiationcodons), J.Biol.Chem.276(23):20474-20481, 2001; Che FS, Watanabe N, Iwano M, Inokuchi H, Takayama S, Yoshida S, Isogai A. Protoporphyrinogen IX oxidase in spinach chloroplasts Molecular Characterization and Subcellular Localization of Protoporphyrinogen IX oxidase i...

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PUM

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Abstract

Provided is a technology for conferring more enhanced tolerance of plants and / or algae against herbicides and / or more greatly enhancing tolerance by using amino acid variants of protoporphyrinogen IXoxidases derived from microorganisms.

Description

technical field [0001] PPO variants of protoporphyrinogen IX oxidase are provided for use in conferring and / or enhancing herbicide tolerance in plants and / or algae. Background technique [0002] The porphyrin biosynthetic pathway is used to synthesize chlorophyll and heme, which play crucial roles in the metabolism of plants, and it occurs in chloroplasts. In this pathway, protoporphyrinogen IX oxidase (hereinafter referred to as PPO; EC: 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. After protoporphyrinogen IX is oxidized to protoporphyrin IX, protoporphyrin IX combines with magnesium to synthesize chlorophyll through magnesium chelatase, or combines with iron through Fe chelatase to synthesize heme. [0003] Therefore, when PPO activity is inhibited, the synthesis of chlorophyll and heme is inhibited, and the substrate protoporphyrinogen IX leaves the normal porphyrin biosynthetic pathway, resulting in the rapid export of protoporphyrinog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N1/12C12N15/79C12N15/82A01N63/50
CPCC12N5/10C12N15/11C12N15/79C12N9/001C12Y103/03004C12N15/8274Y02A40/80A01H1/123C12N1/12A01N63/50C07K14/195
Inventor 成淳基尹俊善洪明基安荣玉禹主龙韩允贞朴重赫
Owner FARMHANNONG CO LTD
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