Promoter for early specific expression of plant anther and application of promoter
A technology of promoters and plant flowers, applied in the field of plant genetic engineering, can solve problems such as few and few, affecting male fertility, etc.
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Embodiment 1
[0023] Example 1. Cloning of AT4G14811 gene promoter fragment EASEpro:
[0024] The length of 1.409 kb upstream of the ATG site of the initiation codon of the Arabidopsis AT4G14811 gene was selected as the candidate promoter region, and the Arabidopsis col-0 genomic DNA was used as the template, and primers F1 and R1 (below) were used for amplification. Subsequently, the amplified PCR products were constructed into the pENTR intermediate vector by TOPO reaction, and the LR reaction was carried out with the pKGWFS7 and pK7FWG0 final vectors respectively to construct the analysis vectors EASEpro-GUS and EASEpro-GFP with the EASEpro promoter fused with GUS and GFP. Among them, TOPO enzyme and LR enzyme were purchased from Invitrogen Company, and pKGWFS7 and pK7FWG0 vectors were purchased from Ghent University.
[0025] F1: 5'-CACCGTGAATAGGATGGCGAGAAGAG-3' (SEQ ID NO: 2)
[0026] R1: 5'-AAGAATTTTTCTTGAAGGCTCTTTG-3' (SEQ ID NO: 3)
Embodiment 2
[0027] Example 2, the acquisition of transgenic plants
[0028] (1) Cultivate Arabidopsis thaliana that can be infected by Agrobacterium:
[0029] The plump col-0 Arabidopsis thaliana seeds were selected, sterilized with 15% sodium hypochlorite solution or 75% absolute ethanol for 15 minutes, replaced with water 4 times, and evenly placed on sterilized MS solid medium. Disproportionate in a refrigerator at 4°C for 2-3 days, continue to cultivate for 10 days in a long-day incubator at 22°C with 16 hours of light / 8 hours of darkness to form seedlings, transfer the seedlings to the soil and cultivate until bolting.
[0030] (2) Agrobacterium transformation:
[0031] The pKGWFS7 and pK7FWG0 vectors with the EASEpro promoter fragment were transformed into Agrobacterium GV3101 competent cells by electroshock method, coated with solid LB medium with 50 μg / L rifampicin and spectinomycin, and cultured at 28°C for 2 days in the dark , screen for positive clones. The obtained positive...
Embodiment 3
[0036] Example 3. Activity analysis of EASpro promoter in anther tissue
[0037] The flowers and seedlings at different reproductive stages of positive transgenic Arabidopsis were taken and treated with pre-cooled 90% acetone for 20 minutes; the acetone was aspirated and discarded, and 50 mM phosphate buffer (pH 7.2) was added to rinse twice; the phosphate buffer was aspirated and discarded, Add an appropriate amount of GUS staining solution (50 mM phosphate buffer at pH 7.2, potassium ferricyanide 2 mM, potassium ferrocyanide 2 mM, X-gluc 1 mM) to completely immerse the plant material in the staining solution, and vacuum for 1 hr; Put it into a 37°C incubator for dyeing. After the plant material has developed color, aspirate the GUS staining solution, add 1 mL of 70% ethanol, and mix gently to decolorize. Observe and photograph under a stereo microscope.
[0038] The results showed that the GUS staining was darker in the flowers closer to the middle of the inflorescence, whi...
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