Strawberry anthocyanin-related gene FvMYB17 and application thereof
An anthocyanin and gene technology, applied in the field of genetic engineering and molecular biology, to achieve the effect of increasing anthocyanin content
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Embodiment 1
[0021] Embodiment 1, the cloning of strawberry anthocyanin synthesis gene FvMYB17
[0022] (1) The diploid forest strawberry 'Ruegen' was used as the test material.
[0023] (2) RNA extraction: the modified CTAB method was used to extract the total RNA in the material, and then the first strand of cDNA was obtained by reverse transcription with RNA as a template using a reverse transcription kit.
[0024] (3) Cloning of the gene: using the first strand of reverse-transcribed fruit cDNA as a template, PCR amplification was performed using primers FvMYB17-F and FvMYB17-R, and the PCR product was recovered to obtain a 765bp target fragment.
[0025] FvMYB17-F:5'-AAAGGTACCATGAGGAAACCCTGCTGCGA-3'
[0026] FvMYB17-R:5'-AAAGAATTCTCTGAAGAGAGGAAGCGTGG-3'
[0027] Among them, the first 3 bases at the 5' end of the primer are protective bases, and the next 6 bases are enzyme cutting sites,
[0028] The first 9 bases at the 5' end are required for constructing an overexpression vector ...
example 2
[0029] Example 2, the construction of plant expression vector
[0030] (1) Using the plant expression vector pRI101-CaMV35S-GFP, select Kpn1 and EcoR1 (purchased from TaKaRa Company) to carry out double enzyme digestion on pRI101-CaMV35S-GFP and PMD18-T (purchased from TaKaRa Company) containing the target gene respectively; The large fragment of the vector and the small fragment of the target gene were ligated overnight with T4 DNA ligase and then transformed into Escherichia coli competent DH5a (purchased from Beijing Quanshijin Biotechnology Co., Ltd.).
[0031] (2) Select the correct recombinant plasmid verified by double enzyme digestion and send it to Jinweizhi Biotechnology Co., Ltd. for sequencing. Afterwards, Agrobacterium GV3101 competent cells were transformed, and the obtained Agrobacterium containing the recombinant plasmid was used to transform Arabidopsis.
example 3
[0032] Example 3. Verification of transgene function—transformation screening and phenotypic analysis of Arabidopsis thaliana
[0033] 3.1 Arabidopsis transformation
[0034] When Arabidopsis (Colombia wild-type) inflorescences were formed, the inflorescence tips were trimmed to induce lateral inflorescences, and the material was watered thoroughly before transformation. Agrobacterium GV3101 positive clones containing the target gene FvMYB17 were selected. Inoculate in YEP liquid medium containing Kan (kanamycin) 50mg / L and Rif (rifampicin) 25mg / L, culture at 28°C, 200rpm shaking for 24h, take 1ml cultured bacteria liquid, add 50ml liquid YEP The liquid culture medium was placed at 28° C. and cultured with shaking at 200 rpm, so that OD 600 = about 0.8.
[0035] Transfer the bacteria solution into a sterile 50ml centrifuge tube, centrifuge at 5000rpm for 5min to collect the bacteria, then resuspend Agrobacterium with the same volume of MS resuspension (1 / 2MS+5%Sucrose, pH=5....
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