CN108795950B - Strawberry anthocyanin related gene FvMYB17 and application thereof - Google Patents
Strawberry anthocyanin related gene FvMYB17 and application thereof Download PDFInfo
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- CN108795950B CN108795950B CN201810632980.5A CN201810632980A CN108795950B CN 108795950 B CN108795950 B CN 108795950B CN 201810632980 A CN201810632980 A CN 201810632980A CN 108795950 B CN108795950 B CN 108795950B
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Abstract
The invention belongs to the field of genetic engineering in molecular biology, and particularly relates to a strawberry anthocyanin related gene FvMYB17 and application thereof, wherein the nucleotide sequence of FvMYB17 is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence is shown as a sequence table SEQ ID NO. 2; research shows that the gene has the function of promoting synthesis of arabidopsis anthocyanin. The invention provides theoretical basis and technical means for regulating and controlling fruit color by utilizing genetic engineering technology, and has great application value.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a strawberry anthocyanin related gene FvMYB17 and application thereof.
Background
Strawberry is a perennial herb, the genus strawberry of the family rosaceae, and is one of the largest-consuming berries in the world. Strawberry belongs to a fruit (Tantanhua, 2003) with short growth cycle, easy propagation, small plant and convenient management. The strawberry is popular with consumers due to the special fragrance and the extremely high nutritional value of the strawberry. A large amount of anthocyanin is present in strawberry fruit, resulting in a change in fruit color. Meanwhile, MYB transcription factors play an extremely important role in secondary metabolism of plants.
MYB transcription factors are named for their containing a conserved Myb domain, which is highly conserved at the N-terminus and contains 1-3R domains in tandem, i.e., R1, R2, and R3, which are not completely repeated. The R can be divided into the following 4 types according to the different contained R numbers: 1R-MYB (MYB-related), R2R3-MYB, 3R-MYB, 4R-MYB (Niuyelin et al, 2016). Each R may form 3 alpha-helices, of which the 2 nd and 3 rd alpha-helices form helix-turn-helices (king shiqing et al, 2003), the third helix may identify the major groove of the chromosome. Each MYB domain contains 3 conserved tryptophan residues spaced 17-19 amino acids apart, which have hydrophobic effects and are important for maintaining helix-turn-helix.
The fourth subfamily of the R2R3-MYB family in model plants Arabidopsis includes the AtMYB3, AtMYB4, AtMYB6 and AtMYB7 genes. Jin (2000) found that the AtMYB4 Arabidopsis mutant has increased tolerance to UV-B. Another gene AtMYB7 homologous to the above gene may also be a novel UV protection factor in Arabidopsis (Fornal et al, 2014). It can be seen from previous studies that the fourth subgroup of the Arabidopsis MYB family plays an important role mainly in the negative regulation of flavonoids and UV protection. However, we found that strawberry FvMYB17 can promote anthocyanin synthesis, which is completely different from the function of homologous genes in model plant arabidopsis thaliana. The function of the gene is researched, the anthocyanin regulation mechanism is clarified, and the synthesis of the strawberry anthocyanin can be understood in the aspect of molecules.
Disclosure of Invention
The invention aims to provide an anthocyanin synthetic gene-strawberry gene FvMYB17 and application thereof, aiming at the defects of the prior art. The technical scheme of the invention is that FvMYB17 gene is separated from diploid forest strawberry 'Ruegen', a plant over-expression vector of the gene is constructed, and the gene is transformed into Arabidopsis thaliana by an agrobacterium-mediated method, so that the functional analysis of the FvMYB17 gene is realized.
The invention provides a strawberry anthocyanin related gene FvMYB17, wherein the cDNA sequence of the gene is shown in SEQ NO. 1; protein coded by a strawberry anthocyanin synthesis related gene FvMYB17, wherein the amino acid sequence coded by the gene is shown in SEQ ID NO:2 is shown in the specification;
the invention provides a primer for amplifying a strawberry anthocyanin related gene FvMYB17, which comprises the following components:
FvMYB17-F:5＇－AAAGGTACCATGAGGAAACCCTGCTGCGA－3＇
FvMYB17-R:5＇－AAAGAATTCTCTGAAGAGAGGAAGCGTGG－3＇
wherein, the first 3 bases at the 5 'end of the primer are protection bases, the next 6 bases are enzyme cutting sites, and the first 9 bases at the 5' end are gene sequences which are required for constructing an over-expression vector and do not belong to FvMYB 17. FvMYB17-R primers the stop codon was removed as needed for the construction of GFP fusion expression vectors.
The invention provides a plant overexpression vector of a strawberry anthocyanin-related gene FvMYB17, which is a plant overexpression vector
The vector is pRI101-GFP-CaMV35S-FvMYB 17.
The invention provides application of a strawberry anthocyanin related gene FvMYB17 in improving the content of plant anthocyanin.
The invention provides application of an expression vector pRI101-GFP-CaMV35S-FvMYB17 in improving the content of plant anthocyanin.
The invention also provides a plant expression vector pRI101-CaMV35S-FvMYB17-GFP containing the strawberry anthocyanin gene FvMYB17, which is transferred into arabidopsis thaliana by an agrobacterium-mediated method.
Practically, any expression vector for introducing a foreign gene into a plant can be used, and pRI101-CaMV35S-GFP is preferably used in the present invention.
The invention has the beneficial effects that: by utilizing the existing plant genetic engineering technology, the strawberry anthocyanin related gene FvMYB17 is cloned, the gene is transferred into arabidopsis thaliana by an agrobacterium-mediated transformation method, and comparative analysis proves that the anthocyanin content in the leaves of the transgenic plant is obviously improved.
Drawings
FIG. 1: amplification result of FvMYB17 gene cDNA sequence. Wherein M is DL2000
FIG. 2: and (3) carrying out double enzyme digestion verification on the pRI101-CaMV35S-FvMYB17-GFP recombinant vector by using Kpn1 and EcoR 1. Wherein M is DL 2000.
FIG. 3: the growth conditions of transgenic plants and wild type under normal growth conditions. Wherein WT is untransformed Arabidopsis thaliana and OE-1/OE-2 are two independent transgenic lines.
The specific implementation mode is as follows:
example 1 cloning of strawberry anthocyanin Synthesis Gene FvMYB17
(1) Diploid forest strawberry 'Ruegen' is used as a test material.
(2) RNA extraction: extracting total RNA in a material by adopting an improved CTAB method, and then carrying out reverse transcription by using a reverse transcription kit and taking the RNA as a template to obtain a first strand of cDNA.
(3) Cloning of the genes: and (3) carrying out PCR amplification by using a reverse transcription fruit cDNA first chain as a template and using primers FvMYB17-F and FvMYB17-R, and recovering a PCR product to obtain a 765bp target fragment.
FvMYB17-F:5＇－AAAGGTACCATGAGGAAACCCTGCTGCGA－3＇
FvMYB17-R:5＇－AAAGAATTCTCTGAAGAGAGGAAGCGTGG－3＇
Wherein, the first 3 bases at the 5' end of the primer are protection bases, the next 6 bases are enzyme cutting sites,
the first 9 bases at the 5' end are gene sequences required for constructing an over-expression vector and do not belong to FvMYB 17.
Example 2 construction of plant expression vectors
(1) The method comprises the following steps of utilizing a plant expression vector pRI101-CaMV35S-GFP, selecting 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 a target gene respectively; and recovering the large vector fragment and the small target gene fragment, connecting the large vector fragment and the small target gene fragment with T4DNA ligase overnight, transforming escherichia coli competence DH5a (purchased from Beijing all-type gold biotechnology, Inc.), and identifying the recon to perform double enzyme digestion verification.
(2) Selecting a recombinant plasmid with correct double enzyme digestion verification, and sending the recombinant plasmid to Jinzhi Biotechnology Limited for sequencing. Then agrobacterium GV3101 competent cells are transformed, and the obtained agrobacterium containing recombinant plasmid is used for transforming Arabidopsis thaliana.
Example 3 verification of transgene function-Arabidopsis transformation screening and phenotypic analysis
3.1 Arabidopsis transformation
When an arabidopsis thaliana (colombia wild-type) inflorescence was formed, the tip of the inflorescence was subtracted to induce the formation of lateral inflorescences, and the material was watered thoroughly before transformation. An Agrobacterium GV3101 positive clone containing the gene of interest FvMYB17 was selected. Inoculating to YEP liquid medium containing Kan (kanamycin) 50mg/L and Rif (rifampicin) 25mg/L, shaking-culturing at 28 deg.C and 200rpm for 24h, collecting 1ml of cultured bacterial liquid, adding 50ml of liquid YEP liquid medium, and shaking-culturing at 28 deg.C and 200rpm to make OD 600 about 0.8.
Transferring the bacterial liquid into a sterile 50ml centrifuge tube, centrifuging at 5000rpm for 5min to collect strains, then re-suspending the agrobacterium with an equal volume of MS re-suspension (1/2MS + 5% Sucrose, pH 5.7), and adding a surfactant Silwet. One month later, harvest T0The generation seeds are screened out positive plants on a culture medium containing 30mg/L of kanamycin, so that T is harvested1Generation seeds for later observation of phenotype.
3.2 phenotypic identification of Arabidopsis Positive lines
Transgenic Arabidopsis thaliana T2The generation plant (transgenic Arabidopsis thaliana with pRI101-CaMV35S-FvMYB17-GFP expression vector) and the wild type Arabidopsis thaliana were cultured under the same conditions (16h light/8 h dark). Leaves of WT and transgenic plants were harvested 7 weeks later, and the total anthocyanin amount was measured, and the measurement results are shown in Table 1.
Table 1:
| phenotype | Anthocyanin content (nmol/g FW) |
| Wild type Arabidopsis thaliana | 0.26±0.05 |
| Transgenic line OE-1 | 0.87±0.16 |
| Transgenic line OE-2 | 0.81±0.08 |
When the transgenic Arabidopsis thaliana was cultured under the same conditions and then cultured for 7 weeks, the anthocyanin content was measured, and it was found from the above table that the anthocyanin content in the transgenic Arabidopsis thaliana was 0.87. + -. 0.16nmol/g FW and 0.810.08 nmol/g FW, respectively. While the anthocyanin content of the wild type Arabidopsis thaliana is only 0.26 + -0.05 nmol/g FW. Therefore, the results show that strawberry FvMYB17 can promote anthocyanin accumulation in transgenic Arabidopsis leaves.
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His Leu His Leu Arg Ile Ser Asp Pro His Glu Met Ser Val Gly Thr
225 230 235 240
Lys Thr Asp Ile Phe Ala Ser Ser Thr Thr Leu Pro Leu Phe Arg Glx
245 250 255
Claims (1)
1. Use of a strawberry anthocyanin-related gene FvMYB17 for increasing anthocyanin content in a plant, characterized in that: the nucleotide sequence of the FvMYB17 is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence encoded by FvMYB17 is shown in SEQ ID NO:2 is shown in the specification; the plant is arabidopsis thaliana.
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| CN108707623B (en) * | 2018-06-19 | 2021-04-13 | 沈阳农业大学 | Strawberry apical meristem related gene FvMYB17 and application thereof |
| CN113563441A (en) * | 2021-08-18 | 2021-10-29 | 沈阳农业大学 | Strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof |
| CN114525284B (en) * | 2022-01-21 | 2023-09-19 | 长江师范学院 | Red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof |
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