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Standard plasmid molecule for transgenic maize Mon810 detection and construction method thereof

A technology of transgenic corn and standard plasmids, which is applied in biochemical equipment and methods, introduction of foreign genetic material using vectors, and determination/inspection of microorganisms, etc. and strain-specific detection to achieve the effect of solving the lack of positive reference materials

Inactive Publication Date: 2012-06-13
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the plasmid standard molecules reported in the literature for the detection of transgenic maize, there are pMu15, pMD-ZM, p3SNTM-9 and ERM-AD413 related to transgenic maize Mon810, but some of them can only be used for detection of construction specificity and screening specificity Some can only be used to detect strain specificity, and cannot meet the requirements of a standard substance for screening specific detection, gene specific detection, construction specific detection and strain specific detection

Method used

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  • Standard plasmid molecule for transgenic maize Mon810 detection and construction method thereof
  • Standard plasmid molecule for transgenic maize Mon810 detection and construction method thereof
  • Standard plasmid molecule for transgenic maize Mon810 detection and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: the construction of standard molecule

[0036] 1. PCR primer sequence design for constructing standard plasmid molecule pMHZ

[0037] According to the gene sequence of the transgenic maize Mon810 exogenous insertion vector and the adjacent region of the maize genome published in GenBank (accession number AF434709), the sequence information of the main components of the exogenous vector (accession number AY326434) and the maize internal standard gene ZSSIIb The sequence information (accession number AF019297), using the software Primer 5.0 to design three pairs of specific primers (as shown in Table 1), are:

[0038] MT-F, its sequence is shown in Seq ID No: 4, and the sequence 3-8 base pairs are Bam HI restriction site,

[0039] MT-R, its sequence is shown in Seq ID No:5; MT-F is located on the maize genome, and MT-R is located at the exogenous insertion vector of transgenic maize Mon810 CaMV35S On the promoter sequence, it is used to amplify the line...

Embodiment 2

[0084] Example 2: Application of constructed plasmid standard molecules in actual detection

[0085] 1. Experimental materials

[0086] Genetically modified maize Mon810.

[0087] Regular corn varieties.

[0088] Other genetically improved plants: soybean GTS 40-3-2, Shengmian No. 1, corn BT11, corn Mon863, rice SK-2.

[0089] 2. Experimental method and process

[0090] 1. Genomic DNA extraction and detection

[0091] 1) Extraction of plant genomic DNA

[0092] a. Take an appropriate amount of corn sample, add it to a mortar, grind it into powder in the presence of liquid nitrogen, weigh about 200 mg of the ground sample and transfer it to a 2ml centrifuge tube;

[0093] b. Add 1mL 65°C preheated extraction solution (20mM EDTA, 2% CTAB, 100mM Tris-HCl pH 8.0, 1.4mol / L NaCl, 1% PVP), mix gently, and keep warm in 65°C water bath for 30min. Shake and mix occasionally;

[0094] c. Add an equal volume of phenol / chloroform solution (24:1) into the tube, mix well by turning it...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a standard plasmid molecule for genetically modified maize strain Mon810 detection and a construction method thereof. The standard plasmid molecule comprises a strain specific sequence, a construction specific sequence and a maize internal standard gene zSSIIb specific segment of genetically modified maize strain Mon810; by analyzing an exogenous insertion vector border sequence of the genetically modified maize strain Mon810 and a sequence of a joining region of two elements Hsp70 and cryIA (b), strain specific and construction specific PCR (Polymerase Chain Reaction) primers are designed, amplification is performed to obtain the strain specific segment and the construction specific segment of the genetically modified maize strain Mon810 and the maize internal standard gene specific sequence; and an artificial recombinant plasmid molecule pMHZ is obtained by constructing the sequences and the segment into the plasmid molecule through a molecular cloning method. A positive standard sample of the genetically modified maize strain Mon810 can be fully substituted by the constructed standard plasmid molecule; and the plasmid molecule is used for quantitative PCR analysis and detection of the genetically modified maize strain Mon810 sample.

Description

technical field [0001] The invention relates to a standard plasmid molecule used for the detection of transgenic corn Mon810 and a construction method thereof, belonging to the technical field of biological detection. Background technique [0002] The emergence and application of genetic engineering technology has opened up a new era of development for agriculture, medicine and other fields. Since the first genetically modified tobacco crop came out in 1983, the development of genetic engineering technology has been changing with each passing day. New varieties of genetically modified crops have emerged continuously, and they have developed by leaps and bounds in just over 20 years. According to the latest data from the International Agricultural Biotechnology Application Agency (ISAAA), From 1996 to 2010, the total area of ​​GM crops in the world increased by 87 times, reaching 148 million hectares. At the same time, a large number of genetically improved agricultural prod...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63C12N15/66
Inventor 杨正友郭燕风田园赵凤春李文华
Owner SHANDONG AGRICULTURAL UNIVERSITY
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