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

Active Publication Date: 2018-11-13
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But we found that strawberry FvMYB17 can promote the synthesis of anthocyanins, which is c

Method used

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  • Strawberry anthocyanin-related gene FvMYB17 and application thereof
  • Strawberry anthocyanin-related gene FvMYB17 and application thereof
  • Strawberry anthocyanin-related gene FvMYB17 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention belongs to the field of genetic engineering in molecular biology, and specifically relates to a strawberry anthocyanin-related gene FvMYB17 and an application thereof. A nucleotide sequence of the FvMYB17 is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2; the gene is indicated to have the function of promoting arabidopsis thaliana anthocyanin synthesis through research. A theoretical basis and technical means are provided for regulation of fruit color by using a genetic engineering technology, and great application value is achieved.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and specifically relates to a strawberry anthocyanin-related gene FvMYB17 and an application thereof. Background technique [0002] Strawberry is a perennial herb, Rosaceae, and is one of the largest berries consumed in the world. Strawberry is a kind of fruit with short growth cycle, easy reproduction, small plant and convenient management (Tan Changhua et al., 2003). Due to the special aroma and high nutritional value of strawberry fruit, it is loved by consumers. There are a lot of anthocyanins in strawberry fruit, which cause the change of fruit color. At the same time, MYB transcription factor plays an extremely important role in the secondary metabolism of plants. [0003] MYB transcription factor is named because it contains a conserved Myb domain, its N-terminus is highly conserved, and contains incompletely repeated tandem 1-3 R domains, namely R1, R...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/825
Inventor 张俊祥石伟佳张志宏王保田雷莹莹于双
Owner SHENYANG AGRI UNIV
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