Flanking sequences of foreign insert fragment of high oleic acid transgenic soybean event e2d9050 and its application

A technology of transgenic soybeans and flanking sequences, which is applied in the field of plant biology and can solve undiscovered problems

Active Publication Date: 2022-02-11
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The analysis of existing patents and literature has found no articles or patent reports related to the flanking sequence of the foreign insert fragment of the high oleic acid transgenic soybean event E2D9050

Method used

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  • Flanking sequences of foreign insert fragment of high oleic acid transgenic soybean event e2d9050 and its application
  • Flanking sequences of foreign insert fragment of high oleic acid transgenic soybean event e2d9050 and its application
  • Flanking sequences of foreign insert fragment of high oleic acid transgenic soybean event e2d9050 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Analysis of the insertion site of the foreign fragment of the transgenic soybean event E2D9050

[0038] 1. Genomic DNA extraction of transgenic soybean E2D9050

[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 foreign insert fragment of the transgenic soybean event E2D9050

[0045] According to the exogenous insertion sequence of the transgenic soybean event E2D9050 and the upstream and downstream sequences of the insertion site in the soybean reference genome, PCR detection primers were designed. The primers for the upstream sequence amplification of the E2D9050 insertion site are E2D9050LB-F1: (5'-CGAATGAATAGGAGCGAAGAAGGA-3') and E2D9050LB-R1 (5'-GCACCATCGTCAACCACTACATC-3'); the downstream sequence amplification primers for the E2D9050 insertion site are E2D9050RB-F1 (5'-AATTTCCCCGATCGTTCAAACATTTGG-3') and E2D9050RB-R1 (5'-TGAGGTCTGTATCTTCTCCATTGACG-3').

[0046] Using the E2D9050 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...

Embodiment 3

[0048] Example 3. Specific PCR detection of transgenic soybean event E2D9050

[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 E2D9050, 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 1-521 positions of SEQ-2, as shown in SEQ-4; the other primer is based on the 522- The reverse primer designed for the sequence at position 966 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 1-376 positions of SEQ-3, as shown in SEQ-6; the other primer is based on the 377th- The reverse primer designed for the sequence at position 988 is shown in S...

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Abstract

The invention provides a high oleic acid transgenic soybean event E2D9050 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 E2D9050 exogenous insert fragment left and right border flanking sequences and its application. The left border flanking sequence of the transgenic soybean event E2D9050 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 E2D9050 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 invention relates to the field of plant biotechnology, in particular to a high oleic acid transgenic soybean event E2D9050 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 soy...

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 仲晓芳杨向东杨静邢国杰牛陆贺红利郭东全钱雪燕姚瑶
Owner JILIN ACAD OF AGRI SCI
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