Nucleic acid sequence and detection method for detecting maize plant dbn9968
A DBN9968, nucleic acid sequence technology, applied in the field of detecting the nucleic acid sequence of corn plant DBN9968 and its detection, capable of solving problems such as differences and inconsistent expression patterns
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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 the leaves of the transgenic corn event DBN9968 were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasy PlantMaxi Kit, and the copy numbers of Cry1Ab and EPSPS were detected by the Taqman probe fluorescent quantitative PCR method. At the same time, wild-type maize plants were used as controls, 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 get 3 repetitions 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 gen...
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 CTABBuffer (20g / L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix well, and extract at 65°C for 90min ; Add 0.5 times the volume of phenol, 0.5 times the volume of chloroform, mix upside down; centrifuge at 12000 rpm (revolutions per minute) for 10 min; absorb the supernatant, add 2 times the volume of absolute ethanol, shake the centrifuge tube gently, Centrifuge at 12000rpm for 10min; collect the DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1mL of 70% ethanol; centrifuge at 12000rpm for 5min; Air...
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