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Flanking sequence of disease-resistant transgenic soybean event B5B9104-3 exogenous insertion element and application thereof

A transgenic soybean and exogenous insertion technology, applied in the field of plant biology, can solve problems such as disease-resistant transgenic soybeans that have not yet been found

Inactive 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 B5B9104-3 have been found so far

Method used

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  • Flanking sequence of disease-resistant transgenic soybean event B5B9104-3 exogenous insertion element and application thereof
  • Flanking sequence of disease-resistant transgenic soybean event B5B9104-3 exogenous insertion element and application thereof
  • Flanking sequence of disease-resistant transgenic soybean event B5B9104-3 exogenous insertion element and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Analysis of insertion site of foreign fragment of transgenic soybean event B5B9104-3

[0039] 1. Extraction of genomic DNA from genetically modified soybean B5B9104-3

[0040] (1) Genomic DNA extraction: Take 1-2g of young soybean leaves, grind it to powder with liquid nitrogen, and put it into a 50mL centrifuge tube. Add 5mL extract A (100mmol / L Tris-HCl, pH8.0, 0.35mol / L sorbitol, 5mmol / L EDTA, pH8.0, 1% 2-mercaptoethanol), 3.5mL extract B (50mmol / L Tris-HCl, pH 8.0, 4.0 mol / L NaCl, 1.8% CTAB, 25 mmol / L EDTA, pH 8.0), 0.3 mL of 30% sodium lauroyl sarcosinate and 2% PVP-360, incubated at 55°C Shake gently several times during 60 to 90 minutes. Take out the centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1), turn it upside down and gently shake for 15 minutes, and then centrifuge at room temperature for 10 minutes (13000 rpm). Aspirate the supernatant, add 2 / 3 volume of pre-cooled isopropanol mixed with 1 / 10 volume of supernatant sodium...

Embodiment 2

[0045] Example 2. Analysis of left and right border flanking sequence of transgenic soybean event B5B9104-3 exogenous insert

[0046] According to the transgenic soybean event B5B9104-3 exogenous insertion sequence and the upstream and downstream sequences of the insertion site in the soybean reference genome, PCR detection primers were designed. B5B9104-3 insertion site upstream sequence amplification primers are B5B9104LB-F1 (5'-GCATTATGTTTGAGGGAGACAAGC-3') and B5B9104LB-R1 (5'-AAGAGGGAGGAAGTATGTGGGAG-3'); B5B9104-3 insertion site downstream sequence amplification primers are B5B9104RB-F1 (5'-CTTTCTTCTGAGTTACATCTTTGTCTG-3') and B5B9104RB-R1 (5'-ATACACAAATGGAGGCTACAACG-3').

[0047] Using B5B9104-3 genomic DNA as a template, PCR amplification was performed using the above primers. PCR reaction system (25uL): 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 2 O 19.5uL. The PCR r...

Embodiment 3

[0049] Example 3. Specific PCR detection of transgenic soybean event B5B9104-3

[0050] According to the left border flanking sequence (shown in SEQ-2) and the right border flanking sequence (shown in SEQ-3) of the foreign insert of transgenic soybean event B5B9104-3, 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 positions 1-480 of SEQ-2, as shown in SEQ-4; the other primer is based on SEQ-2, 481- The reverse primer designed for the 1200 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 positions 1-582 of SEQ-3, as shown in SEQ-6; the other primer is based on SEQ-3, 583- The reverse primer designed for the 1294 site sequence is shown in SEQ-7.

[0051] DNA samples from the roots, stems, leaves,...

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Abstract

The invention provides a flanking sequence of a disease-resistant transgenic soybean event B5B9104-3 exogenous insertion element and application thereof, belonging to the field of plant biotechnology.Specifically, the invention relates to the left- and right-boundary flanking sequences of broad-spectrum mosaic virus-resistant transgenic soybean event B5B9104-3 exogenous insertion element, and application thereof. The left-boundary flanking sequence of the transgenic soybean event B5B9104-3 exogenous insertion element is shown by SEQ-2, and the right-boundary flanking sequence is shown by SEQ-3. The flanking sequence of the disease-resistant transgenic soybean event B5B9104-3 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 present invention relates to the field of plant biotechnology, in particular to a broad-spectrum anti-mosaic virus transgenic soybean event B5B9104-3 exogenous insert flanking sequence and its application. Background technique [0002] Potato Y virus (Potyvirus) is the largest genus of plant viruses, including about 200 confirmed and tentative species. This type of virus can infect a variety of plants such as Solanaceae, Chenopodiaceae, Leguminosae, Cucurbitaceae, and cause serious yield losses. Soybean mosaic virus (SMV), bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV) all belong to the potato Y virus 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 countr...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13
Inventor 牛陆杨向东董英山邢国杰杨静贺红利郭东全钱雪燕姚瑶
Owner JILIN ACAD OF AGRI SCI
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