EPSP synthase gene derived from ochrobactrum anthropi and application thereof

A technology of EPSP synthase and Bacillus pallidus, applied in the field of microbial genetic engineering, can solve problems such as biosafety, and achieve the effect of strong affinity and high glyphosate resistance

Inactive Publication Date: 2012-11-07
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

EPSP synthetase is a key enzyme in the biosynthesis of aromatic amino acids (including tryptophan, tyrosine, phenylalanine, etc.) in plants and microorganisms. security issues

Method used

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  • EPSP synthase gene derived from ochrobactrum anthropi and application thereof
  • EPSP synthase gene derived from ochrobactrum anthropi and application thereof
  • EPSP synthase gene derived from ochrobactrum anthropi and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of DNA Fragment Highly Tolerant to Glyphosate

[0042] 1. Collection of soil samples in paddy fields where glyphosate is frequently used

[0043] Soil samples were collected from orchards that had been used at least four times a year and had been used continuously for more than ten years.

[0044] 2. Screening of glyphosate-resistant strains

[0045] Weigh 1g of the orchard soil sample frequently used by glyphosate, add 1ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.9% (w / v) 1ml of sodium chloride solution, shake and mix at 5000 rpm, let it stand on ice for 10 minutes, draw 150 μl of the solution, spread it and culture it in LB solid medium containing 60 mM glyphosate for 24 hours. Inoculate the grown single colony into a test tube with 1.6ml of LB liquid medium, culture at 28°C for 48h, then draw 150μl of the culture solution and apply it again to the LB solid medi...

Embodiment 2

[0064] Sequence analysis of DNA fragments highly tolerant to glyphosate and functional verification of EPSP synthase

[0065] 1. Sequence analysis of DNA fragments highly tolerant to glyphosate

[0066] The high-tolerance glyphosate plasmid pAroA-O.anthropi screened in Example 1, that is, the complete sequence of the EPSP synthase gene of the present invention, was subjected to DNA sequencing by step-by-step sequencing method. The analysis results show that the inserted fragment has a size of 3100bp, which contains a reading frame of 1353, and its sequence is shown in SEQID NO 1. The polynucleotide sequence it contains has a full length of 1353 bases and encodes 451 amino acids. Its amino acid sequence is shown in SEQ ID NO 2.

[0067]Amino acid sequence homology analysis results show: the amino acid sequence of AroA-O.anthropi has 82.89% homology with the EPSP synthase from Agrobacterium CP4 of type II (see figure 2 ), and less than 30% homology with type I EPSP synthase fr...

Embodiment 3

[0070] Artificial synthesis of EPSP synthase gene with high tolerance to glyphosate

[0071] The above-mentioned highly glyphosate-tolerant EPSP synthase gene was cloned by gene synthesis method [Nucleic Acids Research, 2004, 32, e98]. The designed primers are as follows:

[0072] 1. AroA-1: Tm=54, 60mer

[0073] ATG, TCC, CAT, TCT, GCA, TCC, CCG, AAA, CCA, GCA, ACC, GCC, CGC, CGG, TCG, GAG, GCA, CTT, ACG, GGC

[0074] 2. AroA-2: Tm=54, 60mer

[0075] ACG, ATG, CGA, GAT, GGA, TTT, GTC, ACC, CGG, AAT, GCG, AAT, TTC, GCC, CGT, AAG, TGC, CTC, CGA, GCG

[0076] 3. AroA-3: Tm=54, 60mer

[0077] GAC, AAA, TCC, ATC, TCG, CAT, CGT, TCC, TTC, ATG, TTC, GGC, GGC, CTT, GCA, TCG, GGT, GAA, ACC, CGC

[0078] 4. AroA-4: Tm=54, 60mer

[0079] GGT, ATT, GAT, GAC, GTC, TTC, GCC, TTC, CAG, AAG, GCC, GGT, GAT, GCG, GGT, TTC, ACC, CGA, TGC, AAG

[0080] 5. AroA-5: Tm=54, 60mer

[0081] GGC, GAA, GAC, GTC, ATC, AAT, ACC, GGT, CGC, GCC, ATG, CAG, GCC, ATG, GGC, GCG, AAA, ATC, CGC, AAG

[0...

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PUM

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Abstract

The invention discloses an EPSP synthase gene derived from ochrobactrum anthropi and application thereof. The EPSP synthase gene contains 1353 basic groups and 451 coded amino acids, the nucleotide sequence of the EPSP synthase gene is as shown in SEQ ID NO1, and the amino acid sequence of the coded proteins is as shown in SEQ ID NO2. The EPSP synthase gene derived from ochrobactrum anthropi disclosed by the invention is synthesized by a manual method, has high homology with the reported EPSP synthase gene derived from agrobacterium tumfaciens, has higher glyphosate tolerance, and can be usedfor culturing genetically modified crops.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to an EPSP synthase gene derived from human paleobacterium and its application. Background technique [0002] Glyphosate is an excellent broad-spectrum killing and systemic herbicide, widely used in orchards, rubber gardens, non-cultivated land, and no-tillage land for pre-sowing or post-sowing treatment of corn, soybeans, and cotton, as well as post-emergence directional treatment . Since it was registered in the United States in 1974, it has been registered in more than 100 countries around the world, becoming one of the most widely used herbicides in the world. But the herbicide is also a non-selective herbicide, which also has a killing effect on crops. In order to use glyphosate in agricultural production, crops must be bred to be glyphosate-resistant or degradable. [0003] The toxic action mechanism of glyphosate (N-phosphonomethyl-glycine, glyphosat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12R1/01C12R1/19
Inventor 田永生姚泉洪彭日荷熊爱生高峰付晓燕赵伟金晓芬朱波
Owner SHANGHAI ACAD OF AGRI SCI
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