CN108795950A - A kind of strawberry anthocyanin related gene FvMYB17 and its application - Google Patents
A kind of strawberry anthocyanin related gene FvMYB17 and its application Download PDFInfo
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- CN108795950A CN108795950A CN201810632980.5A CN201810632980A CN108795950A CN 108795950 A CN108795950 A CN 108795950A CN 201810632980 A CN201810632980 A CN 201810632980A CN 108795950 A CN108795950 A CN 108795950A
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- fvmyb17
- gene
- anthocyanin
- strawberry
- leu
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Abstract
The invention belongs to the genetic engineering fields in molecular biology, and in particular to a kind of strawberry anthocyanin related gene FvMYB17 and its application, the nucleotide sequence such as SEQ ID NO of FvMYB17：Shown in 1；Amino acid sequence such as sequence table SEQ ID NO:Shown in 2；Research shows that the gene has the function of promoting the synthesis of arabidopsis anthocyanin.The present invention is to provide theoretical foundation and technological means to the regulation and control of fruit colour using technique for gene engineering, has prodigious application value.
Description
Technical field
The invention belongs to molecular biology, gene engineering technology fields, and in particular to a kind of strawberry anthocyanin related gene
FvMYB17 and its application.
Background technology
Strawberry is herbaceos perennial, rose family Fragaria, is one of maximum berry of consumption figure in the world.Fragaria
It is short in growth cycle, be easy breeding, plant is smaller, facilitates a kind of fruit (Tan Changhua etc., 2003) of management.Due to strawberry fruit
Special fragrance and high nutritive value, therefore liked by consumers in general.There is a large amount of cyanine in strawberry fruit
Glycosides, so as to cause the variation of fruit color.Myb transcription factor has extremely important effect in the secondary metabolism of plant simultaneously.
Myb transcription factor is gained the name because it contains one section of conservative Myb structural domain, its N-terminal is highly conserved, and is contained
There are the concatenated 1-3 R structural domains not exclusively repeated, i.e. R1, R2 and R3.It can be classified as following 4 according to contained R quantity difference
Class：1R-MYB (MYB-related), R2R3-MYB, 3R-MYB, 4R-MYB (Niu Yiling etc., 2016).Each R can form 3 α-
Spiral, wherein the 2nd, 3 alpha-helix forms helix turn helix (Wang Xiqing etc., 2003), third spiral can identify dye
The major groove of colour solid.The 3 conservative trp residues opened containing 17-19 acids apart in each MYB structural domain, they
With hydrophobic effect and to maintaining helix turn helix to have important role.
In model plant arabidopsis the 4th subtribe of R2R3-MYB families include AtMYB3, AtMYB4, AtMYB6 and
AtMYB7 genes.Jin (2000) has found that AtMYB4 Arabidopsis Mutants enhance the tolerance of UV-B.It is homologous with said gene
Another Gene A tMYB7 may also be new as one in arabidopsis UV protection factors (Fornal é et al.,
2014).By forefathers' research as can be seen that mainly in negative regulation flavonoids and ultraviolet in the 4th subgroup of arabidopsis MYB families
Line protection plays an important role.But it have been found that strawberry FvMYB17 can promote the synthesis of anthocyanin, this and model plant are quasi- southern
The function of homologous gene is entirely different in mustard.Functional study is carried out to the gene, illustrates its anthocyanin Regulation Mechanism, it can be advantageous
In the synthesis for understanding strawberry anthocyanin in terms of molecule.
Invention content
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of anthocyanin synthetic gene-strawberry genes
FvMYB17 and its application.The technical scheme is that the isolated FvMYB17 bases from diploid forest strawberry ' Ruegen '
Cause builds the plant over-express vector of the gene, by agrobacterium-mediated transformation, by the genetic transformation to arabidopsis, to realize
The functional analysis of FvMYB17 genes.
The present invention provides a kind of strawberry anthocyanin related gene FvMYB17, the cDNA sequence such as SEQ NO.1 of the gene
It is shown；The protein of strawberry anthocyanin synthesis related gene FvMYB17 codings, the amino acid sequence such as SEQ of the gene code
ID NO：Shown in 2；
The present invention provides a kind of primer for expanding strawberry anthocyanin related gene FvMYB17, including：
FvMYB17-F:5 '-AAAGGTACCATGAGGAAACCCTGCTGCGA-3 '
FvMYB17-R:5 '-AAAGAATTCTCTGAAGAGAGGAAGCGTGG-3 '
Wherein, preceding 3 bases at 5 ends ' of primer are protection base, and back to back 6 bases are restriction enzyme sites, 9 before 5 ends '
A base is to be not belonging to the gene order of FvMYB17 needed for structure over-express vector.FvMYB17-R primers melt due to structure GFP
It closes expression vector to need, eliminates terminator codon.
The present invention provides a kind of plant over-express vector of strawberry anthocyanin related gene FvMYB17, the load
Body is pRI101-GFP-CaMV35S-FvMYB17.
The present invention provides a kind of strawberry anthocyanin related gene FvMYB17 in the application for improving plant Anthocyanin Content.
A kind of expression vector pRI101-GFP-CaMV35S-FvMYB17 of present invention offer is improving plant Anthocyanin Content
Application.
The present invention also provides a kind of plant expression vector pRI101- containing strawberry anthocyanin gene FvMYB17
It is transferred to by agriculture bacillus mediated method in arabidopsis by CaMV35S-FvMYB17-GFP.
In fact, any type can use the expression vector that foreign gene imports in plant, the present invention preferentially selects
The pRI101-CaMV35S-GFP for being.
The beneficial effects of the invention are as follows：Using existing plant gene engineering technology, the present invention has cloned strawberry anthocyanin phase
Correlation gene FvMYB17, and the gene is transferred to by arabidopsis by Agrobacterium-medialed transformation method, it is proved by comparative analysis,
Anthocyanin Content in rotaring gene plant blade significantly improves.
Description of the drawings
Fig. 1：The amplification of FvMYB17 gene cDNA sequences.Wherein M is DL2000
Fig. 2：Kpn1 and EcoR1 is to pRI101-CaMV35S-FvMYB17-GFP recombinant vector double digestion verification results.Its
Middle M is DL2000.
Fig. 3：For the upgrowth situation of normal growing conditions Transfer-gen plant and wild type.Wherein WT is unconverted quasi- south
Mustard, OE-1/OE-2 are two independent transgenic lines.
Specific implementation mode：
The clone of embodiment 1, strawberry anthocyanin synthetic gene FvMYB17
(1) it is examination material with diploid forest strawberry ' Ruegen '.
(2) RNA is extracted：Using modified CTAB method, extract the total serum IgE in material is later with RNA with reverse transcription reagent box
Template reverse transcription obtains the first chains of cDNA.
(3) clone of gene：Using the first chains of fruit cDNA of reverse transcription as template, using primers F vMYB17-F and
FvMYB17-R carries out PCR amplification, recycles PCR product, obtains the target fragment of 765bp.
FvMYB17-F:5 '-AAAGGTACCATGAGGAAACCCTGCTGCGA-3 '
FvMYB17-R:5 '-AAAGAATTCTCTGAAGAGAGGAAGCGTGG-3 '
Wherein, preceding 3 bases at 5 ends ' of primer are protection base, and back to back 6 bases are restriction enzyme sites,
9 bases are to be not belonging to the gene order of FvMYB17 needed for structure over-express vector before 5 ends '.
The structure of example 2, plant expression vector
(1) plant expression vector pRI101-CaMV35S-GFP is utilized, Kpn1 and EcoR1 (being purchased from TaKaRa companies) are selected
The PMD18-T to pRI101-CaMV35S-GFP and containing target gene (being purchased from TaKaRa companies) carries out double digestion respectively；It returns
Body large fragment and target gene small fragment are recorded, with conversion E. coli competent DH5a (purchases after the connection overnight of T4DNA ligases
From Beijing Quanshijin Biotechnology Co., Ltd), it can carry out double digestion verification after identifying recon.
(2) it chooses double digestion and verifies correct recombinant plasmid, the sequencing of Song Jinwei intelligence bio tech ltd.It converts later
Agrobacterium GV3101 competent cells, the obtained Agrobacterium containing recombinant plasmid are used for arabidopsis thaliana transformation.
Example 3, transgenosis functional verification-transformation of Arabidopsis thaliana screening and phenotypic analysis
3.1 transformation of Arabidopsis thaliana
When arabidopsis (Colombia's wild type) inflorescence is formed, inflorescence top is cut to induce the generation of side inflorescence,
Material is sprinkled profoundly water before conversion.Select the Agrobacterium GV3101 positive colonies containing target gene FvMYB17.Be inoculated in containing
In the YEP fluid nutrient mediums of Kan (kanamycins) 50mg/L and Rif (rifampin) 25mg/L, 28 DEG C, 200rpm shaken cultivations
For 24 hours, the cultured bacterium solutions of 1ml are taken, 50ml liquid YEP fluid nutrient mediums are added, are positioned over 28 DEG C, 200rpm shaken cultivations make
OD 600=0.8 or so.
Bacterium solution is transferred in sterile 50ml centrifuge tubes, 5000rpm, 5min are collected by centrifugation strain, then with equal volume
Agrobacterium is resuspended in MS re-suspension liquids (1/2MS+5%Sucrose, pH=5.7), and surfactant Silwet is added.After one month
T is harvested0Positive plant is filtered out on the culture medium that the card containing 30mg/L receives mycin for seed, to harvest T1For seed,
For later observations phenotype.
3.2 arabidopsis positive strain phenotypic evaluations
By transgenic arabidopsis T2(transgenosis of pRI101-CaMV35S-FvMYB17-GFP expression vectors is carried for plant
Arabidopsis) and wildtype Arabidopsis thaliana under equal conditions cultivated under the conditions of (16h illumination/8h dark).WT is acquired after 7 weeks
With the blade of transfer-gen plant, the measurement of anthocyanin total amount is carried out, measurement result is as shown in table 1.
Table 1：
| Phenotype | Anthocyanin Content (nmol/g FW) |
| Wildtype Arabidopsis thaliana | 0.26±0.05 |
| Transgenic line OE-1 | 0.87±0.16 |
| Transgenic line OE-2 | 0.81±0.08 |
It cultivates under the same conditions, culture carries out anthocyanin measurement, as seen from the above table, transgenic arabidopsis after 7 weeks
Anthocyanin Content be respectively 0.87 ± 0.16nmol/g FW and 0.81 0.08nmol/g FW.And the flower of wildtype Arabidopsis thaliana
Green glycosides content is only 0.26 ± 0.05nmol/g FW.Therefore, the results showed that, strawberry FvMYB17 can promote transgenic arabidopsis
Anthocyanin accumulates in blade.
Sequence table
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<120>A kind of strawberry anthocyanin related gene FvMYB17 and its application
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225 230 235 240
Lys Thr Asp Ile Phe Ala Ser Ser Thr Thr Leu Pro Leu Phe Arg Glx
245 250 255
Claims (3)
1. strawberry anthocyanin related gene FvMYB17 is used to improve the purposes of Anthocyanin Content in plant, it is characterised in that：It is described
The nucleotide sequence of FvMYB17 such as SEQ ID NO：Shown in 1.
2. purposes according to claim 1, it is characterised in that：The amino acid sequence such as SEQ ID of the FvMYB17 codings
NO：Shown in 2.
3. according to the purposes described in claim 1-2, it is characterised in that：The plant is arabidopsis.
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| CN108707623A (en) * | 2018-06-19 | 2018-10-26 | 沈阳农业大学 | A kind of strawberry apical meristem related gene FvMYB17 and its application |
| 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 |
| CN114525284A (en) * | 2022-01-21 | 2022-05-24 | 长江师范学院 | Red-peel longan anthocyanin biosynthesis regulation gene DlMYB1-HP and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN114525284A (en) * | 2022-01-21 | 2022-05-24 | 长江师范学院 | Red-peel longan anthocyanin biosynthesis regulation gene DlMYB1-HP 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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