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Anti-glyphosate EPSP (enolpyruvyl shikimate phosphate) synthase GmEPSPS-2 as well as encoding gene and application thereof

A coding and amino acid technology, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as low expression efficiency

Inactive Publication Date: 2012-11-14
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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  • Anti-glyphosate EPSP (enolpyruvyl shikimate phosphate) synthase GmEPSPS-2 as well as encoding gene and application thereof
  • Anti-glyphosate EPSP (enolpyruvyl shikimate phosphate) synthase GmEPSPS-2 as well as encoding gene and application thereof
  • Anti-glyphosate EPSP (enolpyruvyl shikimate phosphate) synthase GmEPSPS-2 as well as encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, the modification synthesis of GmEPSPS-2 gene

[0074] According to the mutant gene of 5-enolpyruvate-3-phosphate synthase (EPSPS) derived from Pseudomonas fluorescens, the original gene ORF sequence 5 was deleted Two base sequences of 147 bp at the 'end and 531 bp at the 3' end, leaving only a base sequence of 654 bp in the middle core domain sequence, which is spliced ​​with a base sequence of 636 bp from the EPSPS gene of Agrobacterium tumefaciens CP4; In the case of ensuring that the amino acid sequence of the transformed EPSP synthetase protein remains unchanged, base substitutions are performed with plant-favored codons to initially obtain a modified DNA sequence; the presence of DNA sequences that cause unstable transcription in plants is eliminated Rich in AT sequences and commonly used restriction enzyme sites (BamH I, EcoR I, Hind III, Nco I, Xho I, Xba I, etc.), and then corrected and eliminated by replacing codons; and added at the 5' end Maize...

Embodiment 2

[0077] Embodiment 2, the construction of anti-glyphosate fusion gene plant expression vector

[0078] Artificially synthesize the GmEPSPS-2 gene shown in Sequence 2 in the sequence listing, double-enzyme-cut the GmEPSPS-2 gene with Xba I and Sac I respectively, and simultaneously double-enzyme-cut the plant expression vectors CPB and CUB with Xba I and Sac I respectively, The digested GmEPSPS-2 fragment and the backbone fragments of the CPB vector and the CUB vector were recovered with a gel extraction and purification kit. The GmEPSPS-2 fragment was ligated with the backbone fragments of the CPB vector and the CUB vector respectively to obtain the target plasmid. Transfer the target plasmid into Escherichia coli, screen for resistance, pick positive single clones, carry out liquid culture of positive single clones, extract positive clone plasmids for Xba I and Sac I double enzyme digestion identification, and initially determine the correctness after enzyme digestion identifi...

Embodiment 3

[0081] Embodiment 3, glyphosate herbicide tolerance test

[0082] Preparation of M9 medium:

[0083] (1) First prepare 1M MgSO 4 : Weigh MgSO 4 ·7H 2 Add 2.46g of O to 10mL of double distilled water to dissolve, autoclave for later use;

[0084] (2) Prepare 1M CaCl 2 : CaCl 2 ·6H 2 O 2.191g was dissolved in 10mL of double-distilled water, and autoclaved for later use;

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

[0086] Na 2 PO 4 ·7H 2 O 12.8g

[0087] K H 2 PO 4 3.0g

[0088] NaCl 0.5g

[0089] NH 4 Cl 1.0g

[0090] Add 200ml of double distilled water to dissolve, and sterilize at 121°C 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 of double distilled water, and sterilized by filtering through a 0.22 micron filter;

[0093] (5) Preparation of M9 medium by aseptic operation

[009...

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Abstract

The invention discloses an anti-glyphosate protein as well as an encoding gene and application of the anti-glyphosate protein. The protein, provided by the invention, is the following (a), (b) or (c): (a) the protein consists of amino acid sequences shown at 56th-532th position of the sequence 1 in a sequence table; (b) the protein consists of amino acid sequences shown by the sequence 1 in a sequence table; and (c) the protein with glyphosate resistance is derived from the sequence 1 and subjects the amino acid sequences of (a) or (b) to substitution and / or deletion and / or addition of one or more amino acid residues. The GmEPSPS-2 protein and the encoding gene of the GmEPSPS-2 protein, provided by the invention, have remarkable resistance effect on glyphosate phytocide; experiments prove that GmEPSPS-2 transgenic tobaccos and GmEPSPS-2 transgenic corns can grow normally under the action of 0.15% glyphosate, while growth of the non-transgenic tobaccos or corns are obviously inhibited, leaves of the non-transgenic tobaccos or corns become yellow and wither, which shows that GmEPSPS-2 can be stably and efficiently expressed in plants, and is further used for producing anti-glyphosate transgenic plants.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, relates to a glyphosate-resistant EPSP synthetase and its encoding gene and its application, in particular to an artificially modified and synthesized glyphosate-resistant EPSP synthetase and its encoding gene GmEPSPS-2, and contains The expression carrier of the gene and its application in the development of herbicide-resistant transgenic plants. Background technique [0002] Glyphosate is a systemic conduction non-selective, broad-spectrum herbicide with stable physical and chemical properties, high efficiency, low toxicity, low residue, easy to be decomposed by microorganisms, and does not damage the soil environment. It has been widely used in agriculture In production, it has become the largest pesticide variety in the world. The relevant mechanism of action is that glyphosate is an analogue of phosphoenolpyruvate (PEP), so glyphosate, shikimate-3-phosphate, and EPSP synthetase are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 谢树章李新海雷开荣林清翁建峰杨小艳
Owner CHONGQING ACAD OF AGRI SCI
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