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Transgenic maize event LW2-2 and detection method thereof

A technology for transgenic maize and maize events, which is applied in the field of molecular biology to achieve the effects of excellent performance, enhanced breeding efficiency and high tolerance

Pending Publication Date: 2021-08-20
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event

Method used

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  • Transgenic maize event LW2-2 and detection method thereof
  • Transgenic maize event LW2-2 and detection method thereof
  • Transgenic maize event LW2-2 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Embodiment 1 cloning and transformation

[0165] 1.1. Vector cloning

[0166] Use standard gene cloning techniques to construct recombinant expression vector pLW2 (such as figure 2 shown). The vector pLW2 comprises two transgenic expression cassettes in series, the first expression cassette is operably linked to the coding sequence (spAtCTP2) of the Arabidopsis EPSPS chloroplast transit peptide by the rice actin 1 promoter (pOsAct1), which can operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) of the Agrobacterium sp. CP4 strain, operably linked to the transcription terminator of cauliflower mosaic virus 35s ( CaMv35S); the second expression cassette consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (p35S) containing an enhancer region operably linked to the glufosinate-tolerant phosphinothricin of Streptomyces N-acetyltransferase (cPAT) and is operably linked to cauliflower mosaic virus 35S termin...

Embodiment 2

[0174] Example 2 Detection of transgenic maize event LW2-2 with TaqMan

[0175] About 100mg of the leaves of the transgenic maize event LW2-2 were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasyPlantMaxiKit, and the copy numbers of the EPSPS gene and the PAT gene were detected by fluorescent quantitative PCR with Taqman probes. At the same time, wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

[0176] The specific method is as follows:

[0177] Step 11, take 100 mg of leaves of transgenic corn event LW2-2, grind into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

[0178] Step 12, using the DNeasy Plant Mini Kit of Qiagen to extract the genomic DNA of the above sample, the specific method refers to its product manual;

[0179] Step 13, measure the genomic DNA concentra...

Embodiment 3

[0196] Example 3 Detection of transgenic corn event LW2-2

[0197] 3.1. Genomic DNA extraction

[0198] The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: after taking 2 grams of tender leaves of the transgenic corn event LW2-2 and grinding them into powder in liquid nitrogen, add 0.5mL at a temperature of 65 ℃ preheated DNA extraction CTABBuffer (20g / L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and pump at a temperature of 65℃ Extract for 90 minutes; add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, mix upside down; centrifuge at 12,000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, and gently shake the centrifuge tube. Let stand at 4°C for 30 minutes; centrifuge at 12,000 rpm for 10 minutes; collect DNA to the bottom of the tube; discard the supernatant an...

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Abstract

The present application discloses a nucleic acid sequence comprising one or more selected from the group consisting of sequences SEQ ID NO: 1-7 and complementary sequences thereof, said nucleic acid sequence being derived from a plant, seed or cell comprising the corn event LW2-2, a representative sample of the seed comprising said event having been preserved with preservation number CCTCC NO: P202022; the invention further discloses a detection method for detecting the LW2-2 event. The transgenic maize event LW2-2 has good tolerance to glyphosate herbicide and glufosinate-ammonium herbicide, and the detection method can accurately and rapidly identify whether a biological sample contains the plant material of the transgenic maize event LW2-2.

Description

technical field [0001] This application relates to the technical field of molecular biology, and relates to detection methods for transgenic plants and their products, in particular to the transgenic corn event LW2-2 tolerant to glyphosate herbicide application and the nucleic acid sequence and method for detecting transgenic corn LW2-2 . Background technique [0002] N-phosphonomethylglycine, also known as glyphosate, is a systemic, chronic broad-spectrum herbicide. Glyphosate is a competitive inhibitor of phosphoenolpyruvate (PEP), the synthetic substrate of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and can inhibit the synthesis of PEP and 3-phosphoshikimate. The conversion of the two substrates to 5-enolpyruvylshikimic acid-3-phosphoshikimic acid under the catalysis of EPSPS blocks the synthetic pathway of shikimic acid, the precursor of aromatic amino acid synthesis, and interferes with the synthesis of proteins leading to plant and bacteria die. [0003] G...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 李晓娇贾志伟王强吕玉平刘枫赵晓娜孙宇董雷李胜兵赵丽媛李涛张原卢娟韩雨颖易金麒
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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