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Nucleic acid sequence for detecting corn plant DBN9968, and detection method thereof

A kind of DBN9968, nucleic acid sequence technology, applied in the nucleic acid sequence for detecting maize plant DBN9968 and its detection field

Active Publication Date: 2015-08-12
BEIJING DABEINONG BIOTECHNOLOGY 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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  • Nucleic acid sequence for detecting corn plant DBN9968, and detection method thereof
  • Nucleic acid sequence for detecting corn plant DBN9968, and detection method thereof
  • Nucleic acid sequence for detecting corn plant DBN9968, and detection method thereof

Examples

Experimental program
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Effect test

no. 1 example

[0180] The first embodiment, cloning and transformation

[0181] 1.1. Vector cloning

[0182] Use standard gene cloning techniques to construct recombinant expression vector DBN10124 (such as figure 2 shown). The vector DBN10124 contains two tandem transgene expression cassettes, the first expression cassette consists of a tandem repeat cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to a maize heat shock 70kDa protein containing (iZmHSP70), operably linked to the insect-resistant Cry1Ab protein (cCry1Ab) of Bacillus thuringiensis, and operably linked to the transcription terminator (tNos) of nopaline synthase; the second The expression cassette consists of the rice actin 1 promoter (prOsAct1), operably linked to the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), operably linked to the glyphosate-tolerant 5 -enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) and is operably linked to the cauliflower mosaic virus 35S ...

no. 2 example

[0191] The second embodiment, using TaqMan to detect the transgenic corn event DBN9968

[0192] About 100 mg of leaves of transgenic maize event DBN9968 was taken as a sample, and its genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy numbers of Cry1Ab 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.

[0193] The specific method is as follows:

[0194] Step 11, take 100 mg of leaves of the transgenic corn event DBN9968, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

[0195] 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;

[0196] Step 13, measure the genomic DNA co...

no. 3 example

[0213]The third embodiment, detection of transgenic corn event DBN9968

[0214] 3.1. Genomic DNA extraction

[0215] The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2 grams of tender leaves of the transgenic corn event DBN9968 and grind them into powder in liquid nitrogen, add 0.5mL and pre-cook at 65°C. Hot DNA extraction CTAB Buffer (20g / L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and extract at a temperature of 65°C 90 min; add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, and mix them upside down; centrifuge at 12,000 rpm (revolutions per minute) for 10 min; absorb the supernatant, add 2 times the volume of absolute ethanol, gently shake the centrifuge tube, and put it at a temperature of 4 Stand still at ℃ 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 invention relates to a nucleic acid sequence for detecting corn plant DBN9968, and a detection method thereof. The nucleic acid sequence of the corn plant comprises a nucleic acid sequence represented by SEQ ID NO:1 or a complementary sequence thereof, or a nucleic acid sequence represented by SEQ ID NO:2 or a complementary sequence thereof. The corn plant DBN9968 has good resistance to lepidopteran insects, has good tolerance to glyphosate herbicides and has no influences on the output, and the detection method can be used to accurately and rapidly identify whether a biological sample includes the DNA molecule of transgenic corn event DBN9968 or not.

Description

technical field [0001] The present invention relates to a nucleic acid sequence for detecting corn plant DBN9968 and a detection method thereof, in particular to a transgenic corn event DBN9968 which is resistant to insects and tolerant to glyphosate herbicide application and used for detecting in biological samples Whether it contains the nucleic acid sequence of the specific transgenic maize event DBN9968 and its detection 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, 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 tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N5/10A01N47/44A01N63/02A01P7/04A01P21/00A01G7/06A01G1/00C12N15/82C12N15/32C12N15/54A01H1/02A01H5/00
Inventor 丁德荣康越景张云珠刘海利庞洁王利君贾志伟黄金存郭函子王磊傅学乾周毅李风鲍晓明吕玉平张世平
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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