Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence

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

Inactive Publication Date: 2012-07-25
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These results suggest that co-expression of Cry1Ac and Cry1Ie in transgenic maize may delay the development of resistance in the corn borer

Method used

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  • Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence
  • Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence
  • Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 The acquisition of insect-resistant transgenic corn IE034

[0037] 1. Construction of transformation vector p3301UbiIe

[0038] The Cry1Ie sequence was designed according to the principle of maize codon preference, as shown in SEQ ID NO.2, synthesized by Shanghai Bioengineering Co., Ltd., and connected to the vector pUC57 to construct the vector pUC57Ie. Plasmid pAHC17 was digested with PstI, the 2.0kb ubiquitin promoter fragment was recovered, and both ends were filled in with T4 DNA polymerase. At the same time, the plasmid pUC57Ie was digested with SalI, and the 4.9kb fragment was recovered and filled in. The two fragments were connected to construct the vector pUbi-cry1Ie, so that the ubiquitin promoter was connected to the front of the Cry1Ie gene. The vector pUbi-cry1Ie was digested with BamHI, the 6.9kb fragment was recovered and blunted, and then partially digested with HindIII, and the 4.2kb fragment was recovered. At the same time, the plasmid p33...

Embodiment 2

[0146] Example 2 Obtaining the flanking sequence at the 5' end of the exogenous gene integration site of insect-resistant transgenic maize IE034 by TAIL-PCR

[0147] 1. Extraction of total DNA from insect-resistant transgenic corn event IE034

[0148] For the method, refer to 5.1 in Example 1.

[0149] 2. Primer design

[0150] Design specific primers based on CaMV35S polyA terminator sequence

[0151] 35S-0: 5'-CGACAAGCTCGAGTTTCTCCATAATAAT-3'; (SEQ ID No.8)

[0152] 35S-1: 5'-ACGATGGACTCCAGTCCGGCCCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCA-3'; (SEQ ID No. 9)

[0153] 35S-2: 5'-GGGTTTCGCTCATGTGTTGAGCATA-3'; (SEQ ID No. 10)

[0154] Design TAIL-PCR random primers:

[0155] LAD1-1: 5'-ACGATGGACTCCAGAGCGGCCGC(T / A / C)N(A / G / C)NNNCCAC-3' (SEQ ID No. 11);

[0156] LAD1-2: 5'-ACGATGGACTCCAGAGCGGCCGC(G / C / A)(G / C / A)N(G / C / A)NNNCCAA' (SEQ ID No. 12);

[0157] Design nested primers

[0158] AC: 5'-ACGATGGACTCCAGAG-3' (SEQ ID No. 13)

[0159] 3. PCR reaction

[0160] The first round of ampl...

Embodiment 3

[0174] Example 3 Application of side sequence of insect-resistant transgenic maize IE034

[0175] Using the flanking sequence of insect-resistant transgenic maize IE034 and the CaMV35S polyA sequence in the foreign fragment, a pair of primers were designed for two different insertion sites, and a qualitative PCR identification method for IE034 event and its derivatives was established. The primer designed based on the maize genome at the 5' end of the integration site in one of the exogenous fragments is 5'-AGGGGCTCCTCTGTTGTTGTA-3' (SEQ ID No.4), and the primer designed based on the CaMV35SpolyA terminator sequence is 5'-TCGCTCATGTGTTGAGCATATAA-3' (SEQ ID No. 5).

[0176] The extraction of corn genomic DNA and the PCR reaction system followed the method in Example 1. The PCR reaction program was 95°C for 5min, 35 cycles (94°C for 30s, 58°C for 30s, 72°C for 1min), and 72°C for 7min. When using this specific primer for PCR amplification, there are no amplified bands in water,...

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Abstract

The invention belongs to the field of plant biotechnology, and particularly relates to a flanking sequence of a foreign insert segment in the maize genetic modification event IE034 and application of the flanking sequence. Testing by the Southern blot method show that an insertion side is present in the maize genetic modification event IE034, and a 5'-end flanking sequence of the foreign insert segment on the insertion site is showed as SEQ ID No.1. The invention provides a primer for detecting the flanking sequence, such as nucleotide sequences showed in SEQ ID No.4 and SEQ ID No.5. Testing of the flanking sequence of the foreign insert segment in the maize genetic modification event IE034 is applicable to detection of genetic modification IE034 of insect-resistant maize (including parents, hybrids F1 and offspring) and products of the insect-resistant maize ( including plants, tissues and seeds and products of these).

Description

technical field [0001] The present invention relates to the field of plant biotechnology, in particular to a side sequence of a transgenic corn IE034 exogenous insertion fragment and a PCR primer for detecting the sequence, and also relates to a method and a kit for detecting transgenic corn using the primer . Background technique [0002] Insect pests (especially corn borer) can cause severe yield loss in corn, and insect-resistant transgenic corn is one of the earliest research traits in the world. The insect-resistant gene is mainly derived from the Bt insecticidal protein of Bacillus thuringiensis, and its mechanism of action is to perforate the insect's gastrointestinal tract, so that the insect's metabolism is disordered and it dies. Monsanto Corporation of the United States is a leader in the development and application of transgenic insect-resistant corn. The insect-resistant transgenic corn MON810 and MON863 developed by it have been in commercial production for ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王国英刘允军张煜文刘艳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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