Transgenic maize event LP007-1 and detection method thereof

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

Pending Publication Date: 2021-05-28
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • 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

Method used

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  • Transgenic maize event LP007-1 and detection method thereof
  • Transgenic maize event LP007-1 and detection method thereof
  • Transgenic maize event LP007-1 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0132]Example 1 Cloning and Transformation

[0133]1.1, carrier clone

[0134]Construct recombinant expression vector PLP007 using standard gene cloning technology (egfigure 2Indicated). The carrier PLP007 contains four series-in-line transgenic expression cassettes, and the first expression cassette is operatively coupled to corn thermal hindengene HSP70 protein intron (IzmHsp70) by mzi-gammousia virus 35S promoter (PRFMV). , Operability to corn green body transport peptide 2 (SPZMCTP2), operably linked to Cry2ab protein (ccry2ab) of Bacillus, Soviet, CRY2AB, operability, is operably linked to the transcription of carnoid enzyme The terminator (TNOS) is constituted; the second expression cassette is repeated by a series-containing tandem, which is repeated by the enhancer region, operability to the insect resistant gene VIP3AA of the Supreme Bacillus. CVIP3AA), operably linked to the 9th intron of the corn phosphate alkoxone pyruvic carboxygenase gene, operably linked to the 35S RNA sequ...

Example Embodiment

[0141]Example 2 Transgenic corn event LP007-1 was detected by TAQMAN

[0142]The leaves of the genetic corn event LP007-1 were taken from about 100 mg as a sample, and the genomic DNA was extracted with Qiagen's DneasyPlantmaxi Kit, and the copy number of Cry1Ab, Cry2ab, VIP3AA, and EPSPs were detected by TAQMan probe fluorescence quantitative PCR method. At the same time, the detection analysis was performed in accordance with the above method. The experiment has three repeats and averages.

[0143]The specific method is as follows:

[0144]Step 11, the blade of the gene corn event LP007-1 is taken, and the liquid nitrogen is used in a mortar into a homogenate, each sample takes three repetitions;

[0145]Step 12, extract the genomic DNA of the above sample using QIAGEN's DNEASY Plant Mini Kit, and the specific method refers to its product specification;

[0146]Step 13, the genomic DNA concentration of the above sample was measured by NanoDrop 2000 (Thermo Scientific);

[0147]Step 14, the genomic ...

Example Embodiment

[0171]Example 3 Transgenic corn event lp007-1 detection

[0172]3.1, genome DNA extraction

[0173]DNA extraction CTAB (cetyltrimethyla bromine) method in accordance with conventional CTAB: Take 2 grams of young turfrachnic corn incident LP007-1 leaves after the liquid nitrogen, add 0.5ml of temperature 65 Preheating DNA extraction of CTabbuffer [20 g / l CTAb, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA (ethylenediamine tetracetic acid)], using NaOH to adjust pH to 8.0, fully mixed, at a temperature of 65 ° C 90min; 0.5 times volume phenol, 0.5 times volume chloroform, reverse mix; 12000 rpm (revolutions per minute) at the rotational speed 10 min; absorb the supernatant, add 1x volume isopropyl alcohol, gently shake the center tube, at temperature -20 ° C for 30 min; 12000 rpm rotation, then centrifuge for 10 min; collected DNA to the tube; discard the supernatant, washed with 0.5 mL of volume concentration of 70% ethanol, 12000 rpm to centrifuge; vacuum Ultra-net is blown; DNA precipitation...

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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 LP007-1, a representative sample of the seed comprising said event having been preserved with preservation number CCTCC NO: P202014. The transgenic maize event LP007-1 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 LP007-1.

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 LP007-1 which is resistant to insects and tolerates glyphosate herbicide application and a method for detecting transgenic corn LP007- 1 nucleic acid sequence and method. Background technique [0002] Maize (Zea mays L.) is a major food crop in many parts of the world. Biotechnology has been applied to corn 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, especially gl...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895A01G13/00A01G22/20A23L7/10A23L29/30A23L7/109
CPCC12Q1/6895A01G13/00A01G22/20A23L7/198A23L29/30A23L7/109C12Q2600/13A23V2002/00A23V2250/5118Y02A40/146
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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