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High-glyphosate-tolerance EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase), and coding gene and application thereof

An EPSP synthase and gene technology, which is applied in the field of biology, can solve the problems of decreased glyphosate inhibition constant, decreased binding ability, decreased PEP affinity, etc.

Active Publication Date: 2014-01-01
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high structural similarity between PEP and glyphosate, the inhibition constant to glyphosate also decreases while the affinity between EPSPS and PEP is increased; on the other hand, the binding ability of EPSPS to glyphosate decreases while It is often accompanied by a decrease in the affinity for PEP (Zhou, M., Xu, H., Wei, X., et al. (2006). Identification of a glyphosate-resistant mutant of rice5-enolpyruvylshikimate3-phosphate synthase using a directed evolution strategy. Plant Physiol 140, 184-195)

Method used

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  • High-glyphosate-tolerance EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase), and coding gene and application thereof
  • High-glyphosate-tolerance EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase), and coding gene and application thereof
  • High-glyphosate-tolerance EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase), and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1 Screening and identification of resistant strains

[0109] Soil samples heavily polluted by glyphosate were serially diluted with liquid M9 medium, and the dilution factor was 10 6 The soil suspension was spread on the M9 solid medium containing 300mM glyphosate, and cultured at 37°C. Since the M9 medium is auxotrophic, the bacteria that can survive and grow are glyphosate-resistant strains. Use liquid M9 medium containing 300mM glyphosate to verify the glyphosate resistance of the screened strains again, and use plasmids pACYC-HTG7, pACYC-AM79, pACYC-A1501, pACYC-RD, pACYC-G2 to transform the recombinant of ER2799 strains as control (Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lu W, Wang J, Lin M, Wang G: A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE2012,7(6):e38718). Such as figure 1 As shown, 1, 2, 3, 4, and 5 respectively represent recombinant strains containing ...

Embodiment 2

[0118] Example 2 Cloning of the gene encoding P818EPSPS (i.e. the gene encoding EPSP synthase, abbreviated as aroA818)

[0119] By analyzing the relevant EPSPS coding gene sequences, designing universal primers or degenerate primers according to the conserved sequences, and using thermal asymmetric PCR (Tail-PCR) method, the EPSPS coding genes were cloned.

[0120] The nucleotide sequence and amino acid sequence of EPSPS coding genes derived from microorganisms with known sequences were compared to analyze their sequence conservation at two levels. According to the principle that bases with slightly more occurrences are selected, the reverse primer was designed as an anchor primer, P818-universal-R: 5'-GTAATACGACTCACTATAGGCATGGCGATGCGATGATC-3' (SEQ ID NO.5); the forward primer was designed to merge Primer, P818-random-F: 5'-GTAATACGACTCACTATAGGAADMGNCC DWTDRR-3' (SEQ ID NO.6), the sequence of the T7 promoter is added to the 5' end of the primer to facilitate the next experimen...

Embodiment 3

[0125] Example 3 Similarity between P818EPSP synthase and other known glyphosate-resistant EPSP synthases

[0126] The EPSPS encoding gene aroA818 of Pseudomonas P818 has a full length of 1323bp and encodes 440 amino acids. Collect typical partial EPSPS sequences with a certain research basis and use the Neighbor-Joining algorithm to construct a phylogenetic tree (MEGA4.0), with a bootstrap value of 1000 times.

[0127] Phylogenetic analysis showed that AroA818 belongs to type II EPSPS, which has the closest homologous relationship with the EPSPS of Pseudomonas stutzeri A1501 ( image 3 ).

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Abstract

The invention provides a novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase) and a coding gene thereof. The EPSP synthase is (1) a protein composed of amino acid sequence disclosed as SEQ ID NO.2, or (2) a (1)-derived protein with glyphosate tolerance and EPSP synthase activity obtained by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence disclosed as SEQ ID NO.2. The invention also provides a preparation method of the EPSP synthase, and application of the EPSP synthase and coding gene thereof. After the EPSP synthase coding gene is transplanted and expressed into a plant, the obtained transgenic plant has higher tolerance to glyphosate.

Description

technical field [0001] The invention belongs to the field of biology, and relates to a microbial-sourced 5-enolpyruvylshikimate-3-phosphate synthase (5-enolpyruvylshikimate-3-phosphate Synthase, EPSP synthase), its coding gene and its ability to cultivate resistant Application of glyphosate in plants. Background technique [0002] Glyphosate was first discovered and synthesized by John E Franz of Monsanto in the early 1970s. Glyphosate has excellent characteristics such as high efficiency, low toxicity, fast degradation in the environment, and broad-spectrum killing, and the preparation process is simple. Due to the outstanding characteristics of glyphosate, it has rapidly developed into a herbicide with the largest production and sales volume and the most widely used area in agricultural production in the past few decades. [0003] The mechanism of action of glyphosate is mainly to competitively inhibit the EPSPS that catalyzes PEP and S3P to generate EPSP in the shikimat...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12N15/84A01H5/00C07K16/40C12Q1/68C12R1/38
CPCC07K16/40C12N9/1092C12N15/8275C12Y205/01019
Inventor 王国英曹高燚刘允军
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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