High oleic acid transgenic soybean event eb8072 foreign insert fragment flanking sequence and its application

A technology of transgenic soybeans and flanking sequences, 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: 2021-09-21
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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  • High oleic acid transgenic soybean event eb8072 foreign insert fragment flanking sequence and its application
  • High oleic acid transgenic soybean event eb8072 foreign insert fragment flanking sequence and its application
  • High oleic acid transgenic soybean event eb8072 foreign insert fragment flanking sequence and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Insertion site analysis of transgenic soybean events EB8072 exogenous fragment

[0038] 1. GM Soy EB8072 genome DNA extraction

[0039] (1) Genomic DNA extraction: Take 1-2 g of soybean young blades, liquid nitrogen to the powder, and load 50 ml of centrifuge tube. 5 ml of extract A (100 mmol / L Tris-HCl, pH 8.0, 0.35 mol / L), 5 mmol / l EDTA, pH 8.0, 1% 2-mercaptoethanol), 3.5ml extract B (50 mmol / L Tris-HCl, pH 8.0, 4.0 mol / l NaCl, 1.8% CTAb, 25mol / L EDTA, pH 8.0), 0.3ml 30% lauryl sodium sodium and 2% PVP-360, 55 ° C incubation 60 ~ 90 minutes, during the period. Take the centrifuge tube, add an equal volume of chloroform: isopentyl alcohol (24: 1), shake it for 15 minutes, then centrifuged at room temperature for 10 minutes (13000 rpm). The supernatant was absorbed, and 2 / 3 volume pre-cooling mixed isopropanol was mixed with 1 / 10 volume of sodium acetate, and 4 ° C was centrifuged for 20 minutes. Abandoned, rinsed with cold 75% ethanol. After drying D...

Embodiment 2

[0044] Example 2. Transgenic Soybean Event EB8072 Left, Right Boundary Side Wings Sequence Analysis

[0045] PCR detection primers are designed in the soybean reference genome based on the transgenic soybean event EB8072 exogenous insertion fragment sequence and insert sites in the soybean reference genome. EB8072 Insert sites upstream sequence amplification primers are EB8072LB-F1 (5'-AcccattattagagcctTGAGG-3 ') and EB8072LB-R1 (5'-TTCCCACAACATACGAGCCG-3'); EB8072 Insert sites downstream sequence amplification primers are EB8072RB-F1 5'-acgTGGGTTTCTGGCAGCTGG-3 ') and EB8072RB-R1 (5'-gcctaaggggTaaaggattgagTAGGTAAAAGGATTTGAGTAG-3').

[0046] The PCR amplification was performed by the above primers by the EB8072 genome DNA. The PCR reaction system (25 ul) is: 10 × PCR buffer 2.5 ul, 10 mmol / l DNTPS 0.5 ul, 5u / ul Taq enzyme 0.5 ul, sample DNA 1.0 ul, 10 umol / L forward primer 0.5 ul, 10 umol / L reverse Primers 0.5 ul, DDH 2 O 19.5UL. The PCR reaction conditions were: 95 ° C 5 m...

Embodiment 3

[0048] Example 3. Transgenic Soybean Event EB8072 Specific PCR Detection

[0049] The specific detection primers are separately design depending on the left boundary side sequence of the transgenic soybean event EB8072 exogenous insert fragment (e.g., SEQ-2) and the right boundary side wing sequence (as shown in SEQ-3). Among the left boundary side sequence specific detection primer combination, one primer is a forward primer designed in accordance with the SEQ-2 1-500 site sequence, such as SEQ-4; another primer is based on SEQ-2 501- The reverse primer designed by 1046-site sequence, as shown in SEQ-5. The right boundary side sequence specific detection primer combination, one of the primers is a forward primer designed in accordance with the SEQ-3 1-422 site sequence, such as SEQ-6; another primer is based on SEQ-3 No. 423- The reverse primers designed by 1022 sites are shown in SEQ-7.

[0050] The transgenic soybean plants were extracted from EB 8072, stems, leaves, flowers an...

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Abstract

The invention provides a high oleic acid transgenic soybean event EB8072 exogenous insertion fragment flanking sequence and application thereof, belonging to the field of plant biotechnology. Specifically, it relates to a high oleic acid transgenic soybean event EB8072 exogenous insert fragment left and right border flanking sequences and its application. The left border flanking sequence of the transgenic soybean event EB8072 exogenous insert fragment disclosed by the present invention is shown in SEQ-2, and the right border flanking sequence is shown in SEQ-3. The flanking sequence of the exogenous insertion fragment of the transgenic soybean event EB8072 disclosed in the present invention can be used as a target DNA sequence to establish a specific detection method for the transgenic event. The flanking sequence of the exogenous insert fragment and the detection method provided by the present invention are suitable for the specific detection of the transgenic soybean event including parents, derivative lines or varieties, and its products including plants, tissues, seeds and products.

Description

Technical field [0001] The present invention relates to the field of plant biological techniques, and in particular, to a high oleic acid transgenic soybean event EB8072 exogenous insertion fragment side wing sequence and its application thereof. Background technique [0002] Oleic acid is a class of unsaturated fatty acids existing in a variety of oil crops such as soybeans, rapeseed, peanuts, etc. Studies have shown that oleic acid has important nutrients and health functions. Oleic acid can reduce the content of cholesterol in human blood, reduce its role in blood vessels, softened blood, prevent arteriosclerosis. The nutrient community generally refers to the "safety fatty acid", and the oleic acid content is an important symbol of evaluating the quality of edible oil. Further, since the degree of fatty acid is low, the high proportion of oleic acid can effectively improve the stability of the edible oil. Soy seed oleic acid content is generally 20 to 25%, while the ratio of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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