Application of Arabidopis thaliana transcription factor at3g46080 gene
A transcription factor, technology of Arabidopsis thaliana, applied in the field of genetic engineering, can solve problems such as secondary pollution and ecological hazards, and achieve the effect of enhancing resistance
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Examples
Embodiment 1
[0018] Example 1 Obtaining of Arabidopsis transcription factor at3g46080 gene sequence and construction of plant expression vector
[0019] Log in to the Arabidopsis information resource database website: http: / / www.arabidopsis.org / servlets / TairObject? type=sequence&id=30040, find the sequence of transcription factor at3g46080 gene. Design PCR amplification primers according to the CDS of the at3g46080 gene,
[0020] at3g46080-F:5'-ATGGTTGCGAGAAGTGAGGAA-3'
[0021] at3g46080-R:5'-TGAAGAAAATCGTTCCCAACTC-3';
[0022] The wild-type Arabidopsis Col-0 was used as material, and the leaves of Arabidopsis thaliana grown for 3 weeks under short-day sunlight were used as materials. Total RNA was extracted by TRIzol method, and cDNA was obtained by reverse transcription. The reaction steps are as follows: Grind the leaves of Arabidopsis thaliana under the condition of liquid nitrogen. After the liquid nitrogen is completely volatilized, divide the sample into a centrifuge tube contain...
Embodiment 2
[0025] The acquisition of embodiment 2 transgenic Arabidopsis plants
[0026] (1) Cultivation of plants:
[0027] Arabidopsis thaliana seeds were surface-sterilized with 75% ethanol for 30 seconds, then rinsed once with sterile water, then surface-sterilized with 1% NaClO for 5-8 minutes, and rinsed three times with sterile water. After surface disinfection is completed, the treated seeds are planted on 1 / 2 MS medium plate. The well-seeded plates were sealed with parafilm and placed in the dark at 4°C. After 2-4 days, the plates were placed in a plant light incubator, and the light conditions were 12h light / 12h dark alternately. Eight days after the seeds germinated, the Arabidopsis seedlings were transferred to nutrient soil, grown under the same light conditions, and watered regularly. When the above-mentioned cultivated Arabidopsis grows to 6-8 weeks, the mature fruit pods are cut off for transformation.
[0028] (2) Arabidopsis transformation:
[0029] Transformation ...
Embodiment 3
[0033] Example 3 Validation of PCR detection of fusion gene at3g46080-flag insertion into genome in transgenic Arabidopsis thaliana.
[0034] (1) Extraction of genomic DNA
[0035] Take 50 mg of normal growing Arabidopsis leaves to be tested and add 400 μL plant genomic DNA extraction solution after grinding. The components of the extract are 200mM Tris-Cl pH7.4, 250mM NaCl, 25mM EDTA, 1% SDS. After vortexing and mixing, centrifuge at high speed for 5 minutes, absorb the supernatant and add an equal volume of isopropanol, and precipitate at room temperature for 30 minutes. Discard the supernatant after high-speed centrifugation for 5 minutes, wash the pellet twice with 70% ethanol, and dissolve the pellet in 20 μL of nuclease-free ddH 2 O.
[0036](2) Utilize the specific primer of PGWB11 carrier to carry out PCR amplification
[0037] PCR was performed using the above-mentioned extracted genomic DNA as a template, and the system included 4 μL of 5* buffer, 1 μL of upstrea...
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Description
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Application Information
- IPC
- C07K19/00; C12N15/29; C12N15/62; C12N15/82; A01H5/00; A01H6/20
- CPC
- C07K14/415; C12N15/8281; C07K2319/43
- Inventors
- 潘巧娜; 崔北米