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Transgenic maize event LP007-4 and detection method thereof

A technology for transgenic corn and corn events, applied in the field of molecular biology

Active Publication Date: 2021-05-25
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 LP007-4 and detection method thereof
  • Transgenic maize event LP007-4 and detection method thereof
  • Transgenic maize event LP007-4 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1 cloning and transformation

[0133] 1.1. Vector cloning

[0134] Use standard gene cloning techniques to construct recombinant expression vector pLP007 (such as figure 2 shown). The vector pLP007 contains 4 transgene expression cassettes in series, the first expression cassette is operably linked to the maize heat shock protein gene HSP70 intein (iZmHSP70) by the Scrophulariaceae Mosaic Virus 35s promoter (prFMV) , operably linked to the maize chloroplast transit peptide 2 (spZmCTP2), operably linked to the insect-resistant Cry2Ab protein (cCry2Ab) of Bacillus thuringiensis, operably linked to the transcription of nopaline synthase terminator (tNos); the second expression cassette consists of a tandem repeat maize ubiquitin gene promoter Ubi (prZmUbi) containing an enhancer region, operably linked to the insect resistance gene Vip3Aa of Bacillus thuringiensis ( cVip3Aa) is operably connected to the 9th intron of maize phosphoenolpyruvate carboxykinase g...

Embodiment 2

[0141] Example 2 Detection of transgenic maize event LP007-4 with TaqMan

[0142] About 100 mg of leaves of transgenic maize event LP007-4 were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasyPlantMaxi Kit, and the copy numbers of cry1Ab, cry2Ab, vip3Aa and epsps 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.

[0143] The specific method is as follows:

[0144] Step 11, take 100 mg of leaves of transgenic corn event LP007-4, grind into a homogenate with liquid nitrogen in a mortar, and take 3 repetitions for each sample;

[0145] Step 12, use the DNeasy Plant Mini Kit of Qiagen to extract the genomic DNA of the above sample, the specific method is with reference to its product manual;

[0146] Step 13, measure the genomic DNA...

Embodiment 3

[0171] Example 3 Detection of transgenic corn event LP007-4

[0172] 3.1. Genomic DNA extraction

[0173] The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2 grams of tender leaves of the transgenic corn event LP007-4, grind 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, and mix evenly by inverting; centrifuge at 12000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 1 times the volume of isopropanol, shake the centrifuge tube gently, and keep at temperature Let stand at -20°C for 30 minutes; centrifuge at 12000rpm for 10min; collect DNA to the bottom of the tube; discard the sup...

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Abstract

The present invention provides 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 LP007-4, a representative sample of the seed comprising said event having been preserved with preservation number CCTCC NO: P202017. The transgenic maize event LP007-4 of the invention is resistant to ingestion impairment of lepidoptera pests and tolerant to phytotoxic effects of agricultural herbicides containing glyphosate. The corn plant with double characters has the following advantages: economic loss caused by lepidoptera pests is avoided; an agricultural herbicide containing glyphosate can be applied to corn crops; the corn yield is not reduced; and the breeding efficiency is enhanced, and the molecular marker can be used for tracking transgenic insertion fragments in a breeding population and a progeny thereof. Meanwhile, the detection method provided by the invention can be used for rapidly, accurately and stably identifying the existence of the plant material derived from the transgenic maize event LP007-4.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for transgenic plants and products thereof, in particular to a transgenic corn event LP007-4 which is resistant to insects and tolerates the application of glyphosate herbicides and a method for detecting transgenic corn LP007- 4 nucleic acid sequences and methods. Background technique [0002] Maize (Zea mays L.) is a major food crop in many parts of the world. Biotechnology has been applied to maize to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in maize production, especially resistance to Lepidoptera insects (such as corn borer, cotton bollworm, fall armyworm, armyworm, etc.). The resistance of maize to lepidopteran insects can be obtained by expressing the resistance gene of lepidopteran insects in maize plants through transgenic methods. Another important agronomic trait is herbicide tolerance, es...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11A01G13/00A01G22/20
CPCC12Q1/6895A01G13/00A01G22/20C12Q2600/13Y02A40/146
Inventor 吕玉平刘枫孙宇赵丽媛李涛张原贺志豪李斌李琪卢娟易金麒韩雨颖
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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