Nucleic acid sequence and method for detecting herbicide-tolerant maize plant dbn9858
A DBN9858, nucleic acid sequence technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, horticultural methods, etc.
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no. 1 example
[0170] The first embodiment, cloning and transformation
[0171] 1.1. Vector cloning
[0172] Use standard gene cloning techniques to construct recombinant expression vector DBN10006 (such as figure 2 shown). The vector DBN10006 contains two tandem transgene expression cassettes, the first of which consists of a tandem repeat of the cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to the glufosinate-resistant gene of Streptomyces. Competent phosphinothricin N-acetyltransferase (cPAT) and operably linked to the cauliflower mosaic virus 35S terminator (t35S); the second expression cassette is initiated by rice actin 1 (prOsAct1), operably linked to the coding sequence of the Arabidopsis EPSPS chloroplast transit peptide (spAtCTP2), operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimic acid of the Agrobacterium sp. CP4 strain -3-phosphate synthase (cEPSPS), which is operably linked to the transcription terminator (tNos) ...
no. 2 example
[0181] The second embodiment, detection of transgenic corn event DBN9858 with TaqMan
[0182] About 100 mg of leaves of transgenic maize event DBN9858 were taken as a sample, and its genomic DNA was extracted with Qiagen's DNeasy PlantMaxi Kit, and the copy numbers of EPSPS gene and PAT gene were detected by Taqman probe fluorescence quantitative PCR method. 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.
[0183] The specific method is as follows:
[0184] Step 11, take 100 mg of leaves of the transgenic corn event DBN9858, grind it into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
[0185]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;
[0186] Step 13, measure the genomic ...
no. 3 example
[0203] The third embodiment, detection of transgenic corn event DBN9858
[0204] 3.1. Genomic DNA extraction
[0205] The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2 grams of tender leaves of the transgenic corn event DBN9858 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 thoroughly, and extract at 65°C for 90 minutes; add 0.5 times the volume of phenol, 0.5 times the volume of chloroform, mix upside down; centrifuge at 12000rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, gently shake the centrifuge tube, Let stand at 4°C for 30 minutes; centrifuge at 12,000 rpm for 10 minutes; collect DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1 mL o...
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