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Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof

A glyphosate-resistant, coding technology that can be used in applications, genetic engineering, plant gene improvement, etc., and can solve problems such as low expression efficiency

Inactive Publication Date: 2012-11-07
CHONGQING ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese invention patent 03826892.2 discloses a glyphosate-resistant gene (EPSPS gene) derived from Pseudomonas fluorescens, but because it is derived from bacteria, the codon preference of bacteria is quite different from that of plants. The EPSPS gene from bacteria directly transforms plants, and its expression efficiency is low. Therefore, it is necessary in this field to artificially modify the glyphosate-resistant gene from microorganisms to ensure that the glyphosate-resistant gene can be efficiently and stably expressed in plants.

Method used

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  • Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof
  • Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof
  • Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Modification and synthesis of GmEPSPS gene

[0074] Based on the 5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) mutant gene derived from Pseudomonas fluorescens, the original ORF sequence of the gene was removed5 The two base sequences of 147bp at the'end and 531bp at the 3'end, leaving only a base sequence of 654bp in the middle core domain sequence, and a base sequence of 591bp from the scallion white EPSPS gene are spliced; When the composition of the amino acid sequence of the EPSP synthetase protein remains unchanged, base substitutions are made with plant-preferred codons to initially obtain a modified DNA sequence; exclude the presence of AT-rich sequences in the DNA sequence that cause plant transcription instability and Commonly used restriction sites (BamH I, EcoR I, Hind III, Nco I, Xho I, Xba I, etc.), then correct and eliminate them by replacing codons; and add rice ACT1-intron sequence at the 5'end (Positions 7-460 of sequence 2), petunia CTP4...

Embodiment 2

[0077] Example 2. Construction of plant expression vector for glyphosate-resistant fusion gene

[0078] The GmEPSPS gene shown in sequence 2 in the sequence table was artificially synthesized, and the GmEPSPS gene was double-cut with Xba I and Sac I, and the plant expression vectors CPB and CUB were double-cut with Xba I and Sac I, and recovered by gel The purification kit was used to recover the digested GmEPSPS fragments, as well as the backbone fragments of CPB vector and CUB vector. The GmEPSPS fragment was ligated with the backbone fragments of the CPB vector and the CUB vector to obtain the target plasmid. Transform the target plasmid into Escherichia coli, select for resistance screening, pick the positive single clone, culture the positive clone in liquid, extract the positive clone plasmid for double enzyme digestion with Xba I and Sac I, and it will be preliminarily judged to be correct after digestion The recombinant plasmid samples were sent for sequencing.

[0079] S...

Embodiment 3

[0081] Embodiment 3, glyphosate herbicide tolerance experiment

[0082] The preparation of M9 medium:

[0083] (1) Prepare 1M MgSO first 4 :Weigh MgSO 4 ·7H 2 O 2.46g add 10mL double distilled water to dissolve, autoclave sterilize for later use;

[0084] (2) Prepare 1M CaCl 2 : CaCl 2 ·6H 2 O 2.191g add 10mL double distilled water to dissolve, autoclave for later use;

[0085] (3) Prepare 5×M9 salt solution again:

[0086] Na 2 PO 4 ·7H 2 O 12.8g

[0087] KH 2 PO 4 3.0g

[0088] NaCl 0.5g

[0089] NH 4 Cl 1.0g

[0090] Add 200ml of double distilled water to dissolve, sterilize at 121℃ for 15 minutes,

[0091] Remarks: The above three samples are prepared separately, bottled separately, and can be sent to high pressure together.

[0092] (4) Prepare 20% glucose solution: 4g glucose is dissolved in 20ml double distilled water, filtered and sterilized by 0.22 micron filter;

[0093] (5) Aseptic operation to prepare M9 medium

[0094] 5×M9 salt solution 200ml

[0095] 1M of MgSO 4 2ml

[0096] 20%...

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Abstract

The invention discloses a glyphosate resistant protein, and a coding gene and application thereof. The protein provided by the invention is (a) or (b) or (c) as follows: (a) a protein composed of an amino acid sequence shown as 73rd-504th site of a sequence 1 in the sequence table; (b) a protein composed of the amino acid sequence shown in the sequence 1 in the sequence table; and (c) a protein, derived from the sequence 1 and with glyphosate resistance, obtained by substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence of (a) or (b). The GmEPSPS protein and the coding gene thereof provided by the present invention have significant resistance effect on a glyphosate herbicide. Experiments prove that GmEPSPS transgenic tobacco and GmEPSPS transgenic maize can still grow normally under 0.15% of glyphosate, but non-transgenic tobacco and maize are obviously suppressed in growth, and have leaves yellowing and withered. It shows that GmEPSPS can stably and efficiently express in plants, and be further used in production of glyphosate resistant transgenic plants.

Description

Technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a glyphosate-resistant EPSP synthase and its coding gene and application, and particularly relates to an artificially modified and synthesized glyphosate-resistant EPSP synthase and its coding gene GmEPSPS, and containing the gene The expression vector and its application in the development of herbicide-resistant transgenic plants. Background technique [0002] Glyphosate is a systemic, non-selective, broad-spectrum biocidal herbicide with stable physical and chemical properties, high efficiency, low toxicity, low residue, easy to be decomposed by microorganisms, and no damage to the soil environment. It has been widely used in agriculture In production, it has become the largest pesticide variety in the world. The related mechanism of action is that glyphosate is an analog of phosphoenolpyruvate (PEP), so glyphosate, shikimate-3-phosphate, and EPSP synthase are easily combined...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 雷开荣李新海谢树章翁建峰林清杨小艳
Owner CHONGQING ACAD OF AGRI SCI
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