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Method and primer pair for detecting transgenic G2-aroA gene herbicide-resisting corn G1105E-823C

A G1105E-823C, herbicide-resistant technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., and can solve problems such as low expression levels

Active Publication Date: 2015-06-24
BEIJING ORIGIN SEED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the glyphosate-resistant gene G2-aroA was discovered in 2004, it can be highly expressed in the host Pseudomonas fluorescens G2 and Escherichia coli, showing high resistance to glyphosate (Yichen Sun, Min Lin and Yiping Wang.Novel AroA with high tolerance to Glyposate, encoded by a gene of Pseudomonas putida4G-1isolated from an extremely polluted environment in China.Aplied and environmental microbiology.2005,71(8):4771-4776), but its in plants, especially Important food crops have not been utilized due to low expression levels, so it is urgent to study its application in plants, such as codon optimization, to accelerate its application to agricultural production

Method used

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  • Method and primer pair for detecting transgenic G2-aroA gene herbicide-resisting corn G1105E-823C
  • Method and primer pair for detecting transgenic G2-aroA gene herbicide-resisting corn G1105E-823C
  • Method and primer pair for detecting transgenic G2-aroA gene herbicide-resisting corn G1105E-823C

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1. Acquisition and Character Identification of G1105E-823C Transgenic G2-aroA Gene Herbicide-tolerant Corn G1105E-823C

[0038] 1. Acquisition of G2-aroA transgenic herbicide-tolerant corn G1105E-823C

[0039] According to the embodiment 2 of the Chinese patent (application number 201210107071.2, authorized announcement number CN102676553B) (the 36th paragraph to the 88th paragraph of the announcement text) applied by the team of the inventor of the present invention, several mG2-aroA were obtained Gene T 6 Generation of transgenic maize lines, one of which was designated as G1105E-823C.

[0040] 2. Identification of herbicide-tolerant maize G1105E-823C with transgenic G2-aroA gene

[0041] For several Ts obtained in step 1 and transferred into mG2-aroA gene 6 Identification of glyphosate resistance in transgenic maize lines. The specific operation is as follows:

[0042] 1. Experimental design: 5M row length, 3 row areas, 3 repetitions, density: 60×35cm. ...

Embodiment 2

[0048] Example 2. Preparation of a kit for detecting herbicide-tolerant corn G1105E-823C transgenic for G2-aroA

[0049] 1. Cloning of the flanking sequence of G2-aroA transgenic herbicide-tolerant maize G1105E-823C

[0050] The following two flanking sequences of the exogenously inserted gene in the transgenic G2-aroA herbicide-tolerant maize G1105E-823C were isolated by Genome walking method. Specifically refer to the instructions of the Genome walking kit (TaKaRa Code: D316) for operation.

[0051] 1. 5′ flanking sequence

[0052] Its nucleotide sequence is shown in sequence 5 in the sequence listing. The flanking sequence at the 5' end consists of two parts, the maize genome sequence fragment and the T-DNA RB sequence fragment on the transformation vector. Specifically, the 1-212th position of sequence 5 is the maize genome sequence fragment, and the 213-256th position is the T-DNA RB sequence fragment on the transformation vector.

[0053] 2, 3' flanking sequence

[...

Embodiment 3

[0072] Example 3. Specific detection of the kit for detecting the herbicide-tolerant corn G1105E-823C transgenic G2-aroA gene

[0073] Samples for the test: transgenic G2-aroA gene herbicide-tolerant corn G1105E-823C CGMCC No.9154, 9 other T 6 Generation of transgenic maize lines, and transformation recipient maize variety Zong 31.

[0074] The two specific primer pairs obtained in Example 2 (primer pair 1 and primer pair 2) and the internal reference primer pair were used to test each sample to verify the specificity of primer pair 1 and primer pair 2. Each sample adopts the same detection method, and the details of the package are as follows:

[0075] Genomic DNA was extracted from each test sample, and used as a template, two specific primer pairs (primer pair 1 and primer pair 2) obtained in Example 2 and an internal reference primer pair were used for PCR amplification respectively. The reaction system and reaction procedure adopted by the three primer pairs were the sa...

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Abstract

The invention discloses a method and a primer pair for detecting transgenic G2-aroA gene herbicide-resisting corn G1105E-823C. The primer pair is specifically a primer pair group for detection or auxiliary detection on whether corn to be detected is transgenic G2-aroA gene herbicide-resisting corn G1105E-823C or not; the primer pair consists of a primer pair 1 and a primer pair 2; the primer pair 1 consists of two single-chain DNA molecules as shown in a sequence 1 and a sequence 2 in a sequence list; the primer pair 2 consists of two single-chain DNA molecules as shown in a sequence 3 and a sequence 4 in the sequence list. Experiment shows that whether the corn to be detected is transgenic G2-aroA gene herbicide-resisting corn G1105E-823C or not can be detected through the primer pair 1 and the primer pair 2 by using a PCR method, and the method is high in accuracy rate, high in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method and a pair of primers for detecting the herbicide-resistant corn G1105E-823C of transgenic G2-aroA gene. Background technique [0002] Weeds are a serious hazard to crop production. Since modern weed control technology appeared in 1942, chemical herbicides have developed greatly. Glyphosate herbicide is a broad-spectrum, non-selective herbicide, which inhibits EPSPS (5-enolpyruvyl oxalate-3-phosphate synthase, which is an important pathway in the synthesis of aromatic amino acids in plants. enzyme) activity, blocks the biosynthesis of shikimate pathway in plants, strongly inhibits cell division, and has a strong inhibitory effect on many annual and perennial weeds. Since glyphosate is easily decomposed by microorganisms, has no residual toxicity in the soil, and is non-toxic to animals, it has been widely used since the successful development of Roundup in 1976. [0003] How...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 韩庚辰宋哲姜付坤张华
Owner BEIJING ORIGIN SEED TECH
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