Recombinant plasmid for detecting components in transgenic plant and application thereof

A technology for transgenic plants and recombinant plasmids is applied to the detection of recombinant plasmids of transgenic plant components and its application field, which can solve the problems of lack and inability to make accurate judgments, and achieve the effects of simple method, small workload and easy acquisition.

Inactive Publication Date: 2012-03-28
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing commercialized transgenic products contain CaMV35S promoter and / or NOS terminator, and there are reports on the detection of transgenic plants and their products targeting the CaMV35S promoter and NOS terminator, but the CaMV35S promoter and NOS terminator The detection of seeds is mainly used for the detection of products with known product ingredients and genetically modified background. For products with unknown product ingredients and genetically modified background (especially processed products), it is impossible to make accurate judgments due to the lack of general standards or plasmid standard molecules.

Method used

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  • Recombinant plasmid for detecting components in transgenic plant and application thereof
  • Recombinant plasmid for detecting components in transgenic plant and application thereof
  • Recombinant plasmid for detecting components in transgenic plant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045] Example 1: Construction of recombinant plasmid pMD-RBCL-CaMV35S-NOS

[0046] 1. Experimental materials

[0047] Genetically modified soybean GTS40-3-2, purchased from European IRMM company.

[0048] 2. Reagents

[0049] PMDl8-T carrier, T 4 DNA ligase and buffer, 2×premix Taq DNA polymerase and buffer DL2000 Marker were purchased from Bao Bioengineering (Dalian) Co., Ltd.; PCR product recovery kit and plasmid extraction kit were purchased from OMEGA Biotechnology Co., Ltd.; CTAB , Tris, EDTA and other biochemical reagents were purchased from Guangzhou Xueyou Biotechnology Co., Ltd.; DH5a competent strains were stored in our laboratory; primers were synthesized by Shanghai Yingwei Jieji Bioengineering Service Co., Ltd.

[0050] 3. Main equipment

[0051] C1000 PCR instrument (Bio-Rad Laboratories, Inc.)

[0052] Gel-type gel imaging system (Bio-Rad Laboratories, Inc.)

[0053] Biophotometer plus UV spectrophotometer (Eppendorf, Inc.)

[0054] Other instruments include: constant tempe...

example 2

[0103] Example 2: Application of pMD-RBCL-CaMV35S-NOS in multiplex PCR kit

[0104] 1. Composition of multiplex PCR kit

[0105] (1) 2×Premix Taq DNA polymerase, containing Taq DNA polymerase, PCR reaction buffer and dNTP;

[0106] (2) Multiplex PCR amplification primers, as shown in the following table:

[0107]

[0108] Note: The primer names in the above table are referred to as abbreviations (continued to be used in Table 8 below), and the corresponding relationship between them and the primers in the kit of the present invention is shown in the sequence numbers in the table.

[0109] (3) The positive control DNA is the recombinant plasmid pMD-RBCL-CaMV35S-NOS prepared in Example 1;

[0110] (4) Ultra-pure water.

[0111] 2. Effect experiment of multiplex PCR kit

[0112] (1) Experimental samples

[0113] 1. Genetically modified soybean GTS40-3-2;

[0114] 2. Non-GMO soybeans;

[0115] 3. Other genetically modified materials: genetically modified rice TT51.1, genetically modified rape RT7...

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Abstract

The invention relates to a recombinant plasmid for detecting components in a transgenic plant, the plasmid comprises a vector and a gene fragment, and the plasmid is characterized in that the gene fragment is formed by fusion of the specific sequence of chloroplast RBCL genes, the specific sequence of a cauliflower mosaic virus 35S (CaMV35S) promoter and the specific sequence of an agrobacterium tumefaciens (NOS: nopaline synthase) terminator, wherein the specific sequence of the chloroplast RBCL genes is as shown in SEQ ID No. 1 (sequence identity number 1); the specific sequence of the CaMV35S promoter is as shown in SEQ ID No. 2; and the specific sequence of the NOS terminator is as shown in SEQ ID No. 3. The recombinant plasmid and a multiplex PCR (polymerase chain reaction) kit matched with the recombinant plasmid can be used for multi-target-point qualitative detection of the transgenic plants and products thereof and have the advantages of being convenient and fast.

Description

Technical field [0001] The present invention relates to an assay or test method containing enzymes or microorganisms, in particular to a recombinant plasmid containing chloroplast RBCL (ribulose, 1.5-bisphosphate carboxylose large subunit, ribulose 1,5-bisphosphate carboxylose large subunit) gene. Background technique [0002] With the rapid development of genetically modified organisms (GMO), its safety has aroused fierce debate and widespread concern around the world. In order to protect consumers' right to know and choose, many countries and regions in the world have formulated corresponding laws and regulations to strengthen the management of import and export of genetically modified plants and their products, which provides technical support for genetically modified organisms for safety supervision. Inspection work brings greater challenges. Nucleic acid-based genetic modification detection technology is an important method for genetic modification detection. At present, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/68
Inventor 肖维威刘唐书周琳华马文丽
Owner SOUTHERN MEDICAL UNIVERSITY
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