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EPSP synthase gene for Zymomonasmobilis glyphosate and application therefor

A glyphosate and gene technology, applied to bacteria, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc., can solve problems such as reports that no glyphosate-resistant crops have entered the commercialization stage.

Inactive Publication Date: 2007-04-25
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the glyphosate-resistant EPSP synthase derived from Zymomonas mobilis (Zymomonasmobilis) strains
[0005] 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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning of DNA fragments highly resistant to glyphosate

[0022] 1. Collection of soil samples in extremely glyphosate polluted environment

[0023] 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 activated sludge polluted by glyphosate for more than ten years (an open distribution point of a glyphosate production plant).

[0024] 2. Isolation and identification of Zymomonas mobilis from extremely glyphosate-contaminated soil samples

[0025] The sample was inoculated at the final concentration of glyphosate (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 medium, 30°C, 200r / min shaking flask shaking culture for 2d, dilute the bacterial s...

Embodiment 2

[0035] Example 2 Sequence analysis of highly glyphosate-resistant DNA fragments and functional verification of ZM-EPSP synthase

[0036] 1. Sequence analysis of DNA fragments highly resistant to glyphosate

[0037] The full nucleotide sequence of the highly glyphosate-resistant DNA fragment subcloned in Example 1 was determined. The analysis results show that it has a complete reading frame, encoding ZM-EPSP synthase, its sequence is shown in SEQ ID NO: 1, aroA encodes ZM-EPSP synthase with 453 amino acids including stop codon.

[0038]Comparing the subcloned highly glyphosate-resistant coding sequence with the reported EPSP synthase coding gene (aroA), the homology at the nucleotide level is very low. The amino acid sequence homology analysis results show that the amino acid homology with the known Class I EPSP synthase (derived from Escherichia coli, Salmonella typhimurium, etc.) is not high, and the amino acid homology with EPSP synthase derived from Escherichia coli and S...

Embodiment 3

[0042] Example 3 Artificial synthesis of highly glyphosate-resistant ZM-EPSP synthase gene

[0043] According to the completed nucleotide sequence containing the 1362 coding region, first divide 8 segments into single-stranded oligonucleotide fragments with a length of 150-200bp and sticky ends according to the positive and secondary strand sequences respectively. Anneal the 8 complementary single-stranded oligonucleotide fragments with 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 ZM-EPSP synthase gene. The synthetic DNA fragment contains the nucleotide sequence of positions 1-1347 in SEQ ID NO: 1, and the two ends of the synthetic gene contain XbaI and SacI sites.

[0044] The above-mentioned artificially synthesized 5' and 3' end restriction sites ...

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PUM

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Abstract

This invention involves a novel EPSP synthase and its applications. The novel EPSP synthase gene is screened out and introduced respectively into E. coli bacteria and the model plant tobacco, qualifying the engineering, genetic strain and transgenic tobacco with herbicide glyphosate tolerance.

Description

Technical field: [0001] The present invention relates to an EPSP synthase gene. The invention also relates to a plasmid containing the EPSP synthase gene and the application of the gene in preparing transgenic engineering bacteria and cultivating transgenic plants. Background technique: [0002] Glyphosate is a systemic, broad-spectrum herbicide, and its mechanism of action is to inhibit the activity of EPSP synthase (full name: 5-enolpyruvylshikimate-3-phosphate synthase) in plants. Interferes with the biosynthesis of aromatic amino acids in plants, leading to plant death (S.R.Padgette et al., in Herbicide-Resistent Crops: Agricultural, Environmental, Economic, Regulatory, and Technical Aspects, S.O.Duke, Ed. (CRC Press, Boca Raton, FL , 1996), pp.53-84). [0003] If the EPSP synthase gene in the plant is mutated to produce an EPSP synthase with low affinity to glyphosate, which cannot bind glyphosate well or even at all, then the plant has glyphosate resistance ability....

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/63C12N15/82C12N15/70C12N1/21
Inventor 陈明林敏顿宝庆张维平淑珍陆伟
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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