Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof

A technology of transgenic soybean and exogenous insertion, applied in the field of plant biology

Inactive Publication Date: 2016-05-11
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few international studies on improving soybean Phytophthora root rot resistance by using transgenic technology

Method used

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  • Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof
  • Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof
  • Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Acquisition of disease-resistant transgenic soybean event B4J8049

[0036] 1. Construction of transformation vector pTF101-Gmubi3-hrpZm

[0037] Design of hrpZ based on soybean codon bias Psta The gene sequence is shown in SEQ-2, synthesized by Nanjing GenScript Biotechnology Co., Ltd., and connected to the cloning vector pUC-57 to construct the vector pUC57-hrpZm. The pUC57-Gmubi3 and the intermediate vector pTF101-35S were digested with PstI / XbaI respectively, the Gmubi3 digested fragment and the pTF101-35S digested large fragment were recovered from the gel, ligated with T4 DNA ligase, and the intermediate expression vector pTF101-Gmubi3 was constructed. On this basis, the pUC57-hrpZm plasmid and the intermediate vector pTF101-Gmubi3 were digested with XbaI / SacI, the hrpZm fragment and the pTF101-Gmubi3 fragment were recovered, and the plant expression vector pTF101-Gmubi3-hrpZm was constructed. figure 1 shown.

[0038] 2. Obtaining of transgenic soybea...

Embodiment 2

[0121] Example 2. Using TAIL-PCR to obtain the sequence flanking the right border of the transgenic soybean event B4J8049 insert

[0122] 1. Total DNA Extraction of Transgenic Soybean Event B4J8049

[0123] Refer to 4.1 in Example 1 for the specific method.

[0124] 2. Primer design

[0125] Design specific primers according to the sequence on the left side of the right border (RB) of the transformation vector pTF101-Gmubi3-hrpZmT-DNA region (located in the T-DNA region):

[0126] 101RB-0a: 5'-ACGTGGTTAAACAAATGCAGAAAATCGACGTCGTC-3'

101RB-1a: 5'-AGTAGACTGACAAATAAATTACCTGACAACATCGTTTCAC-3'

101RB-2a: 5'-CACAAAAAAGGGAGTGCATTTTCCAGGGC-3'

[0127] Design TAIL-PCR high degeneracy random primers:

[0128] LAD-1: 5'-ACGATGGACTCCAGAGCGGCCGC(G / C / A)N(G / C / A)NNNGGAA-3'

LAD-2: 5'-ACGATGGACTCCAGAGCGGCCGC(T / A / C)N(A / G / C)NNNCCAC-3'

[0129] Design nested primers:

[0130] Ac1: 5'-ACGATGGACTCCAGAG-3'

[0131] 3. PCR amplification reaction ...

Embodiment 3

[0148] Example 3 Application of Side Sequences of Disease-resistant Transgenic Soybean Event B4J8049

[0149] According to the side sequence of disease-resistant transgenic soybean event B4J8049 and the sequence on the left side of the right border of the foreign fragment (in the T-DNA region), design specific upstream primer B4J8049F: 5'-TTTCCCGCCTTCAGTTTAAACTATCAG-3' (SEQ-5); according to the soybean genome Sequence Design Specific downstream primer B4J8049R: 5'-GACGCCGTCAACAATGGTGAAC-3' (SEQ-6).

[0150] Soybean genomic DNA extraction and PCR reaction system follow the method in Example 1. PCR amplification program 95°C for 5 minutes; (94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1.5 minutes) 35 cycles; 72°C for 10 minutes. When this specific primer was used for PCR amplification, there was no amplified band in non-transgenic plants or plasmids, and only transgenic soybean B4J8049 plant leaf and seed DNA produced 1010bp specific amplified bands ( Figure 4 and Fig...

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Abstract

The invention belongs to the technical field of plant biology, and relates to a transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof. The provided anti-disease transgenic soybean event B4J8049 is identified as unit point insertion through the Southern hybrid, and the right boundary flanking sequence of the exogenous fragment of the insertion site is represented by SEQ-1. The specific primers, which are designed according to the flanking sequence, are represented by the SEQ-5 and SEQ-6, and a specific PCR detection method of the anti-disease transgenic soybean event B4J8049 is established. The provided detection primer and detection method are suitable for carrying out specific detection on the anti-disease transgenic soybean event B4J8049, which comprises parent, derivation species, and products (plants, tissue, seed, and product).

Description

technical field [0001] The invention belongs to the field of plant biology technology, and specifically relates to a side sequence of a disease-resistant transgenic soybean event B4J8049 exogenous insert fragment and PCR primers for detecting the sequence, and the present invention also relates to the use of the primers to conduct the transgenic soybean event B4J8049 Methods and kits for specific detection. Background technique [0002] Phytophthora root rot (PRR) is a soil-borne quarantine disease caused by Phytophthorasojae. The pathogen can infect and cause harm to soybeans throughout the entire growth period of soybeans, leading to early soybean seed rot, damping off, and middle and late root and stem rot. Soybean Phytophthora root rot occurs in all major soybean producing areas in the world, causing soybean yield losses of more than 1 billion US dollars every year, and is one of the devastating diseases that endanger soybean production. Since the pathogen was first is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
CPCC12N15/11C12N2310/10C12Q1/6895
Inventor 杨向东董英山李启云郭东全杜倩杨静邢国杰牛陆李海云钱雪燕姚瑶张原宇
Owner JILIN ACAD OF AGRI SCI
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