EPSP synthase of variable halomonas high resistance glyphosate and its encoding sequence

An EPSP synthase and coding sequence technology, applied in the direction of anti-enzyme immunoglobulins, enzymes, bacterial peptides, etc., can solve the problem of no glyphosate-resistant gene patent, and no glyphosate-resistant gene crops have entered the commercialization stage. reports, etc.

Active Publication Date: 2005-09-07
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] my country has made some progress in the research of transgenic glyphosate-resistant crops, but there is no report on the commercialization of transgenic glyphosate-resistant crops
At the same time, due to the ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Cloning of DNA fragments highly resistant to glufosinate

[0071] 1. Collection of soil samples in extremely polluted environments with glufosinate

[0072] In the natural environment, especially in the soil of areas where glyphosate and other herbicides have been used for a long time, there are a wide variety of bacterial strains that can tolerate glyphosate or other herbicides. Samples were collected from the soil (the open distribution point of the glyphosate production plant of Hebei Qifeng Chemical Co., Ltd.) that had been polluted by about 50% glufosinate for more than ten years.

[0073] 2. Isolation and identification of Halomonas mutans from extreme glufosinate-contaminated soil samples

[0074] The sample was inoculated at the final concentration of glufosinate (100mmol / L, 200mmol / L, 300mmol / L, 350mmol / L, 400mmol / L, 450mmol / L, 500mmol / L, 550mmol / L, 600mmol / L, 650mmol / L, 700mmol / L, 800mmol / L, 900mmol / L, 1000mmol / L) MOPs liquid culture medium, 30 ℃...

Embodiment 2

[0085] Example 2: Sequence analysis of highly glufosinate-resistant DNA fragments and functional verification of EPSP synthase

[0086] 1. Sequence analysis of DNA fragments highly resistant to glufosinate

[0087] The full nucleotide sequence of the highly glufosinate-resistant DNA fragment subcloned in Example 1 was determined. Analysis results show that it has a complete reading frame, encoding EPSP synthase whose sequence is shown in SEQ ID NO: 1, and encoding EPSP synthase (SEQ ID NO: 2) of 449 amino acids including stop codon.

[0088] Comparing the subcloned highly glufosinate-resistant coding sequence with the reported EPSP synthase coding gene (aroA), there is almost no homology at the nucleotide level.

[0089] The results of amino acid sequence homology analysis showed that the amino acid homology homology with known Class I EPSP synthases (derived from Escherichia coli, Salmonella typhimurium, etc.) was not high. The amino acid homology of synthases was 26.15% an...

Embodiment 3

[0094] Embodiment 3: the artificial synthesis of the EPSP synthase gene of high resistance to glufosinate

[0095]According to the determined nucleotide sequence containing the 1350bp coding region, first divide 8 segments into 150-200bp single-stranded oligonucleotide fragments with cohesive ends according to the positive and secondary strand sequences respectively. Anneal the 8 complementary single-stranded oligonucleotide fragments in one-to-one correspondence between the positive strand and the auxiliary strand respectively to form 8 double-stranded oligonucleotide fragments with sticky ends. The mixed double-stranded oligonucleotide fragments are catalyzed by T4 DNA ligase and assembled into a complete EPSP synthase gene. As verified by sequencing, the synthesized DNA fragment contains the nucleotide sequence of positions 1-1347 in SEQ ID NO: 1, and both ends of the synthetic gene contain XbaI and SacI sites.

[0096] The above-mentioned artificially synthesized 5' and 3...

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PUM

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Abstract

The invention discloses a novel 5-enol pyruvyl shikimate3-phosphate (EPSP), the polynucleotide for encoding the EPSP and the method for producing the EPSP through recombination technology. The invention also discloses the use of the polynucleotide for encoding the EPSP.

Description

technical field [0001] The invention belongs to the field of biology. Specifically, the invention relates to cloning 5-enolpyruvylshikimate-3- Phosphate synthase (EPSP synthase) gene and its protein product. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. The polypeptide of the present invention is an enzyme with resistance to glyphosate. Background technique [0002] Glyphosate (Glyphosate) is a systemic conduction type, broad-spectrum herbicide, and its mechanism of action is to inhibit the activity of 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS) in plants and interfere with the aroma in plants. biosynthesis of amino acids, leading to plant death. [0003] Using the aroA mutant produced by chemical mutagenesis, the drug resistance mechanism study confirmed that the aroA gene is the gene encoding the glyphosate target EPSP synthase. In recent years, the sharp increase in glyphosate production in countries ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C07K16/40C12N5/04C12N9/00C12N15/52C12N15/64C12N15/82
Inventor 林敏刘柱
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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