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Flanking sequence of disease-resistant transgenic soybean event B5B9013-4 exogenous insertion element and application thereof

A technology of genetically modified soybeans and exogenous insertion, applied in the field of plant biology, can solve problems such as undiscovered disease-resistant genetically modified soybeans, and achieve effective supervision and management

Active Publication Date: 2018-07-03
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] According to the analysis of existing patents and documents, no articles or patent reports related to the flanking sequence of the exogenous insert fragment of the disease-resistant transgenic soybean event B5B9013-4 have been found.

Method used

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  • Flanking sequence of disease-resistant transgenic soybean event B5B9013-4 exogenous insertion element and application thereof
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  • Flanking sequence of disease-resistant transgenic soybean event B5B9013-4 exogenous insertion element and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Analysis of the insertion site of the exogenous fragment of the transgenic soybean event B5B9013-4

[0039] 1. Genomic DNA extraction of transgenic soybean B5B9013-4

[0040] (1) Genomic DNA extraction: Take 1-2 g of young soybean leaves, grind them into powder with liquid nitrogen, and put them into a 50 mL centrifuge tube. Add 5mL extract solution A (100mmol / L Tris-HCl, pH8.0, 0.35mol / L sorbitol, 5mmol / L EDTA, pH8.0, 1% 2-mercaptoethanol), 3.5mL extract solution B (50mmol / L L Tris-HCl, pH8.0, 4.0mol / LNaCl, 1.8% CTAB, 25mmol / L EDTA, pH8.0), 0.3mL 30% sodium lauroyl sarcosinate and 2% PVP-360, incubated at 55°C 60-90 minutes, shaking gently several times during the period. Take out the centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), shake it upside down for 15 minutes, and then centrifuge at room temperature for 10 minutes (13000rpm). Aspirate the supernatant, add 2 / 3 volume of pre-cooled isopropanol mixed with 1 / 10 volume of sup...

Embodiment 2

[0045] Example 2. Analysis of the left and right border flanking sequences of the transgenic soybean event B5B9013-4 exogenous insert

[0046]According to the exogenous insertion sequence of the transgenic soybean event B5B9013-4 and the upstream and downstream sequences of the insertion site in the soybean reference genome, PCR detection primers were designed. B5B9013-4 insertion site upstream sequence amplification primers are B5B9013LB-F1 (5'-CCCTCACTCCATTTGTCCTCT-3') and B5B9013LB-R1 (5'-TCCCACATACTTCCTCCCTCT-3'); B5B9013-4 insertion site downstream sequence amplification primers are B5B9013RB-F1 (5'-TACTTCCTCCCTCTTCAGCACC-3') and B5B9013RB-R1 (5'-GGTAGCGATGTGGACCTTAGCCTT-3').

[0047] Using the B5B9013-4 genomic DNA as a template, PCR amplification was performed using the above primers. The PCR reaction system (25uL) is: 10×PCR buffer 2.5uL, 10mmol / L dNTPs 0.5uL, 5U / uL Taq enzyme 0.5uL, sample DNA 1.0uL, 10umol / L forward primer 0.5uL, 10umol / L reverse Primer 0.5uL, ddH ...

Embodiment 3

[0049] Example 3. Specific PCR detection of transgenic soybean event B5B9013-4

[0050] According to the left border flanking sequence (as shown in SEQ-2) and the right border flanking sequence (as shown in SEQ-3) of the exogenous insert fragment of the transgenic soybean event B5B9013-4, specific detection primers were designed respectively. In the left border flanking sequence-specific detection primer combination, one of the primers is a forward primer designed based on the sequence of SEQ-2 No. 1-1071, as shown in SEQ-4; the other primer is based on SEQ-2 No. 1072- The reverse primer designed for the 1401 site sequence is shown in SEQ-5. In the right border flanking sequence-specific detection primer combination, one of the primers is a forward primer designed based on the sequence of the 1-323 positions of SEQ-3, as shown in SEQ-6; the other primer is based on the 324- The reverse primer designed for the sequence at position 845 is shown in SEQ-7.

[0051] The DNA sampl...

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Abstract

The invention provides a flanking sequence of a disease-resistant transgenic soybean event B5B9013-4 exogenous insertion element and application thereof, belonging to the field of plant biotechnology.Specifically, the invention relates to the left-boundary and right-boundary flanking sequences of broad-spectrum mosaic virus-resistant transgenic soybean event B5B9013-4 exogenous insertion element,and application thereof. The left-boundary flanking sequence of the transgenic soybean event B5B9013-4 exogenous insertion element is shown by SEQ-2, and the right-boundary flanking sequence is shownby SEQ-3. The flanking sequence of the disease-resistant transgenic soybean event B5B9013-4 exogenous insertion element can be adopted as a target DNA sequence to establish a specific detection method for the transgenic event. The flanking sequence of the exogenous insertion element and the detection method provided by the invention are applicable to the specific detection of the transgenic soybean events including parent and derivative strain or variety as well as products thereof such as plants, tissues, seeds and products.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a broad-spectrum anti-mosaic virus transgenic soybean event B5B9013-4 exogenous insertion fragment flanking sequence and application thereof. Background technique [0002] Potyvirus is the largest genus of plant viruses, including about 200 confirmed and tentative species. This type of virus can infect various plants such as Solanaceae, Chenopodiaceae, Fabaceae, Cucurbitaceae, etc., and cause serious yield loss. Soybean mosaic virus (SMV), bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV) all belong to the Potatovirus genus. All three viruses can be transmitted through seed poisoning or aphids, and cause symptoms such as mosaic, leaf curling, and plant dwarfing (Gao et al. 2015; Yang et al. 2014). SMV is a major viral disease affecting soybean production, and it is also one of the most important diseases in major soybean producing areas in my country. SMV can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13
Inventor 杨向东董英山牛陆李启云杨静邢国杰贺红利郭东全钱雪燕姚瑶
Owner JILIN ACAD OF AGRI SCI
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