Nucleic acid sequence for detecting soybean plant DBN8002 and detection method thereof

A DBN8002, nucleic acid sequence technology, applied in the fields of plant molecular biology and transgenic crop breeding

Active Publication Date: 2020-07-10
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event

Method used

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  • Nucleic acid sequence for detecting soybean plant DBN8002 and detection method thereof
  • Nucleic acid sequence for detecting soybean plant DBN8002 and detection method thereof
  • Nucleic acid sequence for detecting soybean plant DBN8002 and detection method thereof

Examples

Experimental program
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Effect test

no. 1 example

[0152] The first embodiment, cloning and transformation

[0153] 1.1. Vector cloning

[0154] The recombinant expression vector pDBN4006 was constructed using standard gene cloning techniques (such as figure 2 shown). The vector pDBN4006 contains two tandem transgene expression cassettes, the first expression cassette is operably linked to the mVip3Aa gene ( CN103509808 B), and is operably linked to the transcription terminator (tNos) of nopaline synthase; the second expression cassette is composed of cauliflower mosaic virus promoter (pr35S), operably linked to Streptomyces The glufosinate-resistant phosphinothricin N-acetyltransferase gene (cPAT) is operably linked to the transcription terminator (t35S) of cauliflower mosaic virus.

[0155]The vector pDBN4006 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) with liquid nitrogen method, and 4-[hydroxyl (methyl)phosphono]-DL-homoalanine Acid was used as a selectable marker to select ...

no. 2 example

[0163] The second embodiment, detection of transgenic soybean event DBN8002 with TaqMan

[0164] About 100 mg of the leaves of the transgenic soybean event DBN8002 were taken as a sample, and the genomic DNA was extracted with a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy numbers of the mVip3Aa gene and the PAT gene were detected by the Taqman probe fluorescence quantitative PCR method. At the same time, the wild-type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

[0165] The specific method is as follows:

[0166] Step 1. Take 100 mg of leaves of the transgenic soybean event DBN8002, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

[0167] Step 2, using a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen) to extract the genomic DNA of the above sample...

no. 3 example

[0185] The third embodiment, analysis of the insertion site of the transgenic soybean event DBN8002

[0186] 3.1. Genomic DNA extraction

[0187]The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2g of young leaves of the transgenic soybean event DBN8002 and grind them into powder in liquid nitrogen, add 0.5mL of DNA extraction CTAB buffer (20g / L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix well, and extract at 65°C for 90min ; Add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, mix upside down; centrifuge at 12,000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, shake the centrifuge tube gently, and store at 4°C Let stand for 30min; centrifuge at 12000rpm for 10min; collect DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1mL of 70% ...

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Abstract

The invention relates to a nucleic acid sequence for detecting soybean plant DBN8002 and a detection method thereof. The nucleic acid sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, and / or SEQ ID NO: 2 or a complementary sequence thereof. The soybean plant DBN8002 has better resistance to lepidoptera insects, has better tolerance to glufosinate-ammonium herbicides, and has noinfluence on the yield, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of the transgenic soybean event DBN8002.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research. Specifically, the present invention relates to insect-resistant and glufosinate-ammonium herbicide-tolerant transgenic soybean event DBN8002 and a nucleic acid sequence for detecting whether a specific transgenic soybean event DBN8002 is contained in a biological sample and a detection method thereof. Background technique [0002] Soybean (Glycine max) is one of the five major crops in the world. Biotechnology has been applied to soybean to improve its agronomic traits and quality. Herbicide tolerance is an important agronomic trait in soybean production, especially tolerance to glyphosate herbicides, such as the successful soybean events GTS40-3-2 and MON89788, which have been widely planted in major soybean planting areas such as the United States. to plant. Another important agronomic trait ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11C12N15/82A01H5/00A01H6/54A01H1/02A01G22/40A23J7/00A23D9/00A23L11/00A23C20/02A23L11/45
CPCC12Q1/6895C12N15/8286C12N15/8277A01H1/02A01G22/40A23J7/00A23D9/00A23L11/07A23L11/01A23C20/025C12Q2600/13A23L11/03A23J3/16C12N15/8274C12Q2600/156Y02A40/146C12Q1/6837C12N15/8275A01H5/00A01H6/542A01H5/10
Inventor 韩超于彩虹谢香庭王登元杨淑靖崔广东康越景鲍晓明
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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