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Gene aroA for encoding 5-enolpyruvyl-shikimate-3-phosphate synthase and application thereof

A technology of pyruvyl shikim and phosphate synthase, which can be used in application, genetic engineering, plant genetic improvement and other directions, and can solve problems such as low glyphosate resistance

Inactive Publication Date: 2012-11-07
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the defect of low glyphosate resistance of existing transgenic crops, the primary purpose of the present invention is to provide a gene aroA encoding 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) S001

Method used

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  • Gene aroA for encoding 5-enolpyruvyl-shikimate-3-phosphate synthase and application thereof
  • Gene aroA for encoding 5-enolpyruvyl-shikimate-3-phosphate synthase and application thereof
  • Gene aroA for encoding 5-enolpyruvyl-shikimate-3-phosphate synthase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The screening and isolation of highly glyphosate-resistant strains is to take soil samples from vegetable fields in Guangdong and use glyphosate as a screening marker to isolate and obtain Klebsiella pneumoniae kpS001 strains; specifically, the following steps are included:

[0022] 1. Isolation of Klebsiella pneumoniae highly resistant to glyphosate:

[0023] (1) Take 10 g of vegetable field soil samples in Guangdong, add them to 90 mL of 150 mmol / L glyphosate (original powder) basal salt medium (liquid), and culture them at 37 °C and 120 r / min for 72 h.

[0024] (2) Take 10mL of the culture solution from step (1) and add it to the basal salt medium (liquid) containing 200mmol / L glyphosate (original powder) for cultivation, and cultivate at 37°C and 120r / min for 72h; and so on, the previous Add 300, 350, 400mmol / L glyphosate (raw powder) to the basal salt medium (liquid) step by step in the round culture solution, and cultivate at 37°C and 120r / min for 72h.

[0025] (...

Embodiment 2

[0039] Cloning the gene aroA encoding Klebsiella pneumoniae EPSP synthetase, the present embodiment utilizes the method of homologous cloning to clone the gene aroA encoding EPSP synthetase from bacterial strain kpS001; Specifically comprises the following steps:

[0040] 1. Genome extraction method of Klebsiella pneumoniae kpS001 strain: Genome extraction is extracted with the Genome Extraction Kit of Tiangen Bacteria. The operation steps are basically the same as the instruction manual, with slight modifications as follows:

[0041] (1) Take 2 mL of bacterial culture solution, centrifuge at 12,000 rpm for 1 min, and suck up the supernatant as much as possible.

[0042] (2) Add 200 μL buffer GA to the cell pellet and shake until the cell is completely suspended.

[0043] (3) Add 20 μL proteinase K solution to the tube and mix well.

[0044] (4) Add 220 μL buffer GB, shake for 15 seconds, and place at 70°C for 10 minutes. After the solution becomes clear, briefly centrifuge t...

Embodiment 3

[0086] build aroA S001 The recombinant vector with the prokaryotic expression vector pProA comprises the following steps:

[0087] 1PCR amplification

[0088] According to the sequencing results of Example 2 (SEQ ID NO.3), the primers were redesigned as follows:

[0089] KpF1: 5-GAATTCATGGAATCCCTGACGTTACAAC-3

[0090] KpR1: 5-CTCGAGTCAGGCGAGGGTACTGATCCG-3

[0091] PCR reaction system:

[0092] 10*Taq buffer 3μL dNTPs (10mmol) 1μL kpF (10pmol) 1μL kpR (10pmol) 1μL dna 2μL rTaq 0.3μL wxya 2 o 21.7μL Total 30μL

[0093] The PCR reaction procedure is as follows:

[0094] 94℃ 5min

[0095]

[0096] 72°C 10min

[0097] 16℃Forever

[0098] After the PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered with the Tiangen DNA Product Rapid Purification and Recovery Kit, with a length of about 1300bp. Ligated to pMD18-T vector. The recombinant vector was transformed into Escher...

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Abstract

The invention discloses a gene aroAS001 for encoding 5-enolpyruvyl-shikimate-3-phosphate synthase, and an application of the gene, wherein the base sequence of the gene is represented by SEQ ID No.3; the gene is suitable for enhancing glyphosate resistance of crops; specifically, gene aroAS001, and an expression vector or a cloning vector containing gene aroAS001, or strains containing the expression vector or cloning vector are transferred in the crops. The gene provided by the invention is an excellent glyphosate resistant gene, the aroA gene defect type strains transferred with the gene can grow in a culture medium containing lower than 400mM glyphosate; compared with other known EPSP (enolpyruvyl-shikimate-3-phosphate) synthase genes, the gene provided by the invention has significant glyphosate resistance and brings about unexpected beneficial effects.

Description

technical field [0001] The present invention relates to a gene aroA encoding 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) and its application. The gene is cloned from a Klebsiella pneumoniae strain highly resistant to glyphosate The resulting, exhibited enhanced resistance to glyphosate. Background technique [0002] Glyphosate (Glyphosate) has been favored by the market since it was successfully developed by Monsanto in 1971 because of its stable physical and chemical properties, high efficiency, broad spectrum, low toxicity, low residue, easy decomposition and no damage to the soil environment. The world's largest selling plant protection product. As a result, glyphosate has also become the first choice for research on herbicide-resistant transgenic crops, and glyphosate-resistant transgenic crops are currently the most widely planted transgenic crops in the world. The cloning, expression and functional verification of glyphosate target enzyme EPSP synthetase (EP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54A01H5/00
Inventor 田兴山张纯冯莉吴丹丹张妤杨彩宏岳茂峰崔烨
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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