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Flanking sequences of exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application

A technology of EB8072 and transgenic soybeans, applied in the field of plant biology, can solve the problems of undiscovered EB8072 foreign insert fragment flanking sequence articles and patent reports, etc., and achieve the effect of effective supervision and management

Inactive Publication Date: 2018-08-07
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 high oleic acid transgenic soybean event EB8072 have been found so far

Method used

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  • Flanking sequences of exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application
  • Flanking sequences of exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application
  • Flanking sequences of exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Analysis of the insertion site of the exogenous fragment of the transgenic soybean event EB8072

[0038] 1. Genomic DNA extraction of transgenic soybean EB8072

[0039] (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 supernata...

Embodiment 2

[0044] Example 2. Analysis of the flanking sequences of the left and right borders of the exogenous insert in transgenic soybean event EB8072

[0045] PCR detection primers were designed according to the sequence of the exogenous insertion fragment of the transgenic soybean event EB8072 and the upstream and downstream sequences of the insertion site in the soybean reference genome. The primers for the upstream sequence amplification of the EB8072 insertion site are EB8072LB-F1 (5'-ACCACCATTATTAGAGCCTTGAGG-3') and EB8072LB-R1 (5'-TTCCACACAACATACGAGCCG-3'); the downstream sequence amplification primers for the EB8072 insertion site are EB8072RB-F1 ( 5'-ACGTGGGTTTCTGGCAGCTGG-3') and EB8072RB-R1 (5'-GCCTAAGGGTAAAGGGATTTGAGTAG-3').

[0046] Using EB8072 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 0.5uL, 10...

Embodiment 3

[0048] Example 3. Specific PCR detection of transgenic soybean event EB8072

[0049] According to the flanking sequence of the left border (as shown in SEQ-2) and the flanking sequence of the right border (as shown in SEQ-3) of the exogenous insert fragment of the transgenic soybean event EB8072, 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 according to the sequence of the first 1-500 positions of SEQ-2, as shown in SEQ-4; the other primer is based on the 501- The reverse primer designed for the sequence at position 1046 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 1-422 positions of SEQ-3, as shown in SEQ-6; the other primer is based on the 423- The reverse primer designed for the 1022 site sequence is shown in SEQ-7....

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Abstract

The invention provides flanking sequences of an exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application, belongs to the technical field of plant biotechnology, and particularly relates to left and right boundary flanking sequences of the exogenous insert of high-oleic-acid transgenic soybean event EB8072 and their application. The left and right boundary flankingsequences are shown as in SEQ-2 and SEQ-3. The flanking sequences may act as target DNA sequences to establish a transgenic event specific detection method. The boundary flanking sequences and the method are applicable to the specific detection for transgenic soybean events, including parents and derivative strains or varieties, as well as their articles of manufacture, including plants, tissues,seeds and products.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a high oleic acid transgenic soybean event EB8072 exogenous insertion fragment flanking sequence and application thereof. Background technique [0002] Oleic acid is a kind of unsaturated fatty acid widely present in the seeds of various oil crops (such as soybean, rapeseed, peanut, etc.). Studies have shown that oleic acid has important nutritional and health functions. Oleic acid can reduce the content of cholesterol in human blood, reduce its deposition on blood vessels, soften blood vessels, and prevent arteriosclerosis. In the field of nutrition, oleic acid is generally called "safe fatty acid", and the content of oleic acid is an important indicator for evaluating the quality of edible oil. In addition, due to the low degree of fatty acid unsaturation, a high proportion of oleic acid can effectively improve the stability of edible oil. The content of oleic acid in soyb...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13
Inventor 杨向东杨静仲晓芳牛陆贺红利邢国杰郭东全钱雪燕姚瑶
Owner JILIN ACAD OF AGRI SCI
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