Nucleotide sequence and detection method for detecting corn plant DBN9981
A technology of DBN9981 and nucleic acid sequence, applied in the field of detection of nucleic acid sequence of corn plant DBN9981 and its detection
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no. 1 example
[0169] The first embodiment, cloning and transformation
[0170] 1.1. Vector cloning
[0171] 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
[0180] The second embodiment, detection of transgenic corn event DBN9981 with TaqMan
[0181] About 100 mg of leaves of transgenic maize event DBN9981 were 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.
[0182] The specific method is as follows:
[0183] Step 11, take 100 mg of leaves of the transgenic corn event DBN9981, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
[0184] 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;
[0185] Step 13, measure the genomic DNA con...
no. 3 example
[0205] The third embodiment, detection of transgenic corn event DBN9981
[0206] 3.1. Genomic DNA extraction
[0207]DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2 grams of tender leaves of the transgenic corn event DBN9981 and grind them into powder in liquid nitrogen, add 0.5mL to 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 supernat...
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