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Flanking sequence of stress-resistant transgenic soybean event WB1 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 that have not yet been discovered, and achieve the effect of 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

AI Technical Summary

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 WB1 exogenous insertion fragment of the retrograde soybean event have been found.

Method used

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  • Flanking sequence of stress-resistant transgenic soybean event WB1 exogenous insertion element and application thereof
  • Flanking sequence of stress-resistant transgenic soybean event WB1 exogenous insertion element and application thereof
  • Flanking sequence of stress-resistant transgenic soybean event WB1 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 foreign fragment of the transgenic soybean event WB1

[0039] 1. Genomic DNA extraction of transgenic soybean WB1

[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 supernatant sodiu...

Embodiment 2

[0045] Example 2. Analysis of the flanking sequences of the left and right borders of the WB1 exogenous insert in the transgenic soybean event

[0046] PCR detection primers were designed according to the sequence of the exogenous insertion fragment of the transgenic soybean event WB1 and the upstream and downstream sequences of the insertion site in the soybean reference genome. The primers for amplification of the upstream sequence of the WB1 insertion site were FA8015LB-F1 (5'-TTTGTCACTCCACCCATACCTGG-3') and FA8015LB-R1 (5'-CACCATCGTCAACCACTACATCG-3'); the primers for the amplification of the downstream sequence of the WB1 insertion site were FA8015RB-F1 ( 5'-TTTACGGCGAGTTCTGTTAGGTC-3') and FA8015RB-R1 (5'-TCATACACACCCTCTCACAC-3').

[0047] Using WB1 genomic DNA as a template, the above primers were used for PCR amplification. 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 0.5u...

Embodiment 3

[0049] Example 3. Transgenic soybean event WB1-specific PCR detection

[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 transgenic soybean event WB1, 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 the 1-401 site of SEQ-2, as shown in SEQ-4; the other primer is based on the 402- The reverse primer designed for the sequence at position 977 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 first 1-605 positions of SEQ-3, as shown in SEQ-6; the other primer is based on the 606th- The reverse primer designed for the sequence at position 1254 is shown in SEQ-7.

[0051] The DNA samples o...

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Abstract

The invention provides a flanking sequence of a stress-resistant transgenic soybean event WB1 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 stress-resistant transgenic soybean event WB1 exogenous insertion element, and application thereof. The left-boundary flanking sequence of the transgenic soybean event WB1 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 WB1 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 flanking sequence of an exogenous insertion fragment of WB1 resistant to a retrogenic soybean event and an application thereof. Background technique [0002] Soil salinization is one of the important factors affecting crop production, and its degree of harm is second only to drought. According to statistics, about 20% of the world's cultivated land is threatened by salt damage, accounting for about 7.6% of the world's land area. The total area of ​​saline-alkali land in my country is 27 million hm 2 saline-alkali land. In addition, due to climate change, improper irrigation methods, overgrazing and other reasons, 6.7×10 6 hm 2 of secondary salinized land. With the acceleration of the industrialization process, the quality of irrigation water continues to decline, and the rate of land salinization (including secondary salinization) is gradually accelerating. It is estima...

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 杨向东牛陆赵倩倩马瑞于志晶杜茜杜娟蔡琴安
Owner JILIN ACAD OF AGRI SCI
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