CN109486831A - A kind of rouge radish anthocyanin biosynthetic controlling gene RsAN1 and its application - Google Patents
A kind of rouge radish anthocyanin biosynthetic controlling gene RsAN1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of rouge radish anthocyanin biosynthetic controlling genesRsAN1And its application,RsAN1Nucleotide sequence as shown in SEQ ID NO.1, coding amino acid sequence as shown in SEQ ID NO.2.RsAN1Expression quantity and rouge radish Anthocyanin Content positive correlation, by rouge radishRsAN1Clone, sequence analysis, expression analysis and the plant transfection of gene, obtain transient overexpressionRsAN1Rouge radish, can synthesize and accumulate a large amount of anthocyanin in cotyledon and blade, and wild type rouge radish blade and cotyledon can not synthesize anthocyanin.It is found by the functional analysis of gene, after being individually overexpressed RsAN1 gene in rouge radish, can significantly improve the accumulation of anthocyanin.Prove rouge radish geneRsAN1It is the positive regulatory factor of anthocyanin, the further improvement for rouge radish and other plants provides outstanding genetic resources.New thinking is provided for molecular breeding from now on, provides theoretical foundation and material to study the breed of variety etc. of rouge radish.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to a kind of rouge radish anthocyanin biosynthetic controlling
Gene RsAN1 and its application.
Background technique
Rouge radish is also referred to as turnip with red inside, is one local varieties of characteristic of Chongqing City Fuling District, due to its fleshy root from
Epidermis to internal aglow, anthocyanin rich in and it is well-known.Its meat is crisp, is suitable for the various radish products of processing, is used as bubble
Dish steep long it is not rotten, it is scarlet tender crisp.The counties such as Fuling, WuLong, the Nanchuan in Chongqing are the major production areas of rouge radish, have for more than 100 years
Plantation history.Rouge radish is high with respect to other turnip with red inside kind Anthocyanin Contents, in Chongqing region, often from rouge radish
Anthocyanin is extracted as natural colorant.But other provinces, city introduce a fine variety, due to soil, climate reasons, effect is bad.Due to
It is not clear to influence the mechanism that it accumulates anthocyanidin, significantly limits the production and popularization of rouge radish.Early-stage study shows
For the biosynthetic controlling of Anthocyanin mainly in transcriptional level, MYB and bHLH transcription factor plays anthocyanidin biosynthesis
Very important regulating and controlling effect affects the accumulating level of anthocyanidin.Rouge radish anthocyanidin it is a large amount of accumulation whether also by
The regulation of these two types of transcription factors, it is not clear at present.
It is both domestic and external studies have shown that anthocyanin synthesis mainly passes through structural gene in regulation anthocyanin anabolic process
Expression, so that the synthesis of the enzyme in anabolic process be made to be affected.It is putative at present to there are three classes to adjust anthocyanin
The transcription factor of synthesis, is R2R3-MYB, bHLH (basic Hlix-Loop-Hlix) and WD40 respectively, they interact altogether
With the biosynthesis for adjusting green glycosides of blossoming.
The myb transcription factor of plant is a very big family, they regulate and control the secondary metabolism process of plant.MYB
Class transcription factor is gained the name with there is one section of conservative DNA combination region structural domain in its structure.With DBD (DNA Binding
Domain) the most conservative, 1-3 not exclusively repetitive sequence R (Repeat) are generally comprised, each repeated fragment R is protected by 51-52
Amino acid residue and the intervening sequence composition kept.It include typical structural domain [D/E] Lx in the region R32[R/K]
x3Lx6Lx3R, referred to as bHLH Motif, it is can be with the region of bHLH one kind protein-interacting；C-terminal has a motif 6
Structural domain [R/K] Px [P/A/R] xx [F/Y].At least 154 MYB form huge transcription factor family in arabidopsis, this
A family is made of 25 subtribes.These transcription factors control cellular morphology in regulation secondary metabolism, mediate pathogen resistance, answer
Answering hormone signal etc. has highly important effect, participates in the PAP1 and PAP2 of arabidopsis anthocyanin biosynthesis transcriptional control
Belong to the 6th subtribe.Recently researches show that participate in the relevant MYB of anthocyanin synthesis transcriptional control to principally fall into R2R3-MYB class.
Although forefathers also have been reported that the relevant controlling gene MYB of anthocyanin biosynthesis, ordinary circumstance in carrot
Under be the expression analysis gene in tobacco arabidopsis isotype plant function, and when single expression myb gene anthocyanin product
Tired amount is little.So far there is not the relevant report of anthocyanin biosynthetic controlling gene in rouge radish also.
Summary of the invention
In view of the above shortcomings of the prior art, it is raw that the object of the present invention is to provide a kind of rouge radish anthocyanins
Object synthesis regulation gene RsAN1 (Anthocyanin 1) and its application, provide the pass that Anthocyanin Content regulates and controls in rouge radish
Key gene provides new genetic resources for the genetic modification that rouge radish accumulates a large amount of anthocyanins.
To achieve the above object, the present invention adopts the following technical scheme: a kind of rouge radish anthocyanin biosynthetic controlling
Gene RsAN1, nucleotide sequence is as shown in SEQ ID NO.1；The amino acid sequence that it is encoded is as shown in SEQ ID NO.2.
A kind of recombinant expression carrier, which is characterized in that the nucleotide sequence including the RsAN1.
A kind of plant cell or vegetable transformant comprising the recombinant expression carrier.
Application of the gene RsAN1 in regulation herbaceous plant anthocyanin synthesis.
A kind of breeding method of high-content anthocyanin rouge radish, comprising the following steps:
1) it takes rouge radish fleshy root to extract total serum IgE, then the first chain cDNA is synthesized by reverse transcription using total serum IgE as template；
2) cDNA obtained using step 1) is to draw with the column of nucleotides sequence shown in SEQID NO 3 and SEQID NO 4 as template
Object is expanded, and pcr amplification product is connected by the nucleotide sequence of obtained pcr amplification product as shown in SEQ ID NO.1
Recombinant expression carrier is constructed in expression vector；
3) recombinant expression carrier of building is converted to Agrobacterium GV3101, then with the Agrobacterium GV3101 to kermes
Radish carries out after infecting conversion, cultivates 4~5 days to get high-content anthocyanin rouge radish is arrived.
Further, the expression vector is pEAQ-HT.
Compared with prior art, the invention has the following beneficial effects:
1, in first identified of the present invention rouge radish anthocyanin biosynthesis crucial myb transcription factor RsAN1,
The expression quantity and rouge radish Anthocyanin Content positive correlation of RsAN1.Prove that rouge radish gene RsAN1 is anthocyanin
Positive regulatory factor, the further improvement for rouge radish and other plants provide outstanding genetic resources.
2, the present invention is transfected by clone, sequence analysis and the plant to rouge radish RsAN1 gene, obtains instantaneous super table
Up to the rouge radish of RsAN1, a large amount of anthocyanins can be synthesized and accumulated after 4~5 days in its blade and cotyledon, and the rouge of wild type
Rouge turnip leaves and cotyledon can not synthesize anthocyanin.Illustrate after being individually overexpressed RsAN1 gene in rouge radish, it can be significant
Raising anthocyanin accumulation.Theoretical foundation and material are provided to study the breed of variety etc. of rouge radish.And the present invention
Middle RsAN1 gene is generally applicable to herbaceous plant, provides a kind of effective technological means to regulate and control herbal anthocyanin,
It is with a wide range of applications.
Detailed description of the invention
Fig. 1 is that PCR amplification obtains the electrophoretogram of RsAN1 segment；
M is DNA Marker；A is RsAN1 gene open reading frame segment；
Fig. 2 is the structural analysis figure for the amino acid sequence that rouge radish RsAN1 is derived；
Fig. 3 is the evolutionary analysis figure of the amino acid sequence of rouge radish RsAN1 and the MYB albumen of other plants；
Fig. 4 be RsAN1 wild type rouge radish plant different parts expression；
Fig. 5 is the content of anthocyanin on rouge radish blade and cotyledon；
A and C is the cotyledon and blade of instantaneous conversion RsAN1, and B and D are the cotyledon and blade of negative control group.
Specific embodiment
Invention is further described in detail with attached drawing combined with specific embodiments below.Experiment side as described in the examples
Method is without special instruction, i.e., routinely experimental methods of molecular biology operates.
Rouge radish employed in following embodiments ' red heart 1 ', it is public by the green former development in agricultural science and technology in Chongqing City Fuling
Department provides；Bacillus coli DH 5 alpha and Agrobacterium GV3101 are bought from Shanghai Wei Di Bioisystech Co., Ltd, expression vector pEAQ-
HT is laboratory preservation.
1 × tbe buffer liquid, 6 × loading buffer, GoldviewTMNucleic acid dye, RNase inhibitor, dNTP
Mix, AMV reverse transcriptase, MgCl2, archaeal dna polymerase and agarose be purchased from Shanghai bioengineering Co., Ltd.Other raw materials used
Unless otherwise specified, as common commercially available.
The clone of 1 rouge radish gene RsAN1 of embodiment and expression vector establishment
(1) extraction and detection of rouge radish total serum IgE
The extraction of rouge radish total serum IgE is using Beijing Ai Delai Biotechnology Co., Ltd plant RNA extraction kit (tool
Body method is referring to specification).With remaining DNA in DNase I (TaKaRa) processing total serum IgE, specific method is referring to specification.With
The concentration mensuration and purity of RNA are measured in nucleic acid-protein instrument.
It draws 2 μ L RNA to be detected on 1.5% agarose gel electrophoresis, the OD of the RNA of extraction260/OD280Ratio
Between 1.80~2.00, integrality is preferable, can be used for reverse transcription.
The synthesis of (2) first chain cDNA
Using the total serum IgE for extracting rouge radish as template, MLV-Reverse Transcriptase kit is utilized
(Invitrogen company) synthesizes the first chain of cDNA, and kit specification progress is pressed in concrete operations.
The sequence preparating mixture (6 μ L) according to following template ribonucleic acid/primer: 2 μ L of template ribonucleic acid is prepared in centrifuge tube,
3 μ L, RNase-free ddH of Oligo (dT) Primer2O 1μL.In PCR instrument, 70 DEG C of heat preservation 10min are set rapidly afterwards
In rapid cooling 2min or more on ice.It is centrifuged the several seconds, obtains template ribonucleic acid/primer denaturing soln.
It prepares inverse transcription reaction liquid (10 μ L): 6 μ L, 5 × M-MLV Buffer of template ribonucleic acid/primer denaturing soln, 2 μ L,
0.5 μ L, RNase Inhibitor of dNTP Mixture (10mM), 0.25 μ L, RTase M-MLV 0.5 μ L, Nase-free
dH2O 0.75μL.In PCR instrument, 42 DEG C of heat preservation 1h.In PCR instrument, 70 DEG C of heat preservation 15min are subsequently placed in cooled on ice, obtain
CDNA solution.
(3) clone of gene RsAN1
The homologous comparison of Blast is carried out using the myb gene sequence of other species.Design corresponding primer RsAN1-F and
RsAN1-R, specific as follows:
RsAN1-F:5 '-ATGGAGGGTTCGTCCAAAGG-3 '
RsAN1-R:5 '-CTAATCAAGTTCAACAGTCTCTCC-3 '
Using obtained cDNA as template, using PrimeScriptTMRT-PCR Kit (TaKaRa company) carries out PCR amplification
Reaction.
PCR amplification system (50 μ L) are as follows: 5 0.5 μ L RsAN1-F of μ 10 × PCR of L buffer, 2 μ L dNTP Mixture
(forward primer), 0.5 μ L RsAN1-R (reverse primer), 3 μ L cDNA templates, 39 μ L Nuclease-free Water.
PCR program: 98 DEG C of 5min；98 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 1min 40s, 35 circulations；72℃7min.
PCR after reaction, takes a small amount of PCR product (2 μ L or so) to carry out agarose gel electrophoresis detection, as a result such as Fig. 1
It is shown.
It will be seen from figure 1 that the clip size that PCR amplification obtains is in 800bp or so.Use DNA gel QIAquick Gel Extraction Kit
(Agarose Gel DNA Purification Kit, TaKaRa company) recycling target fragment is recycled, recovery method base
Step in this reference kit specification carries out.
According to recovery product clip size and its effective concentration, the product of appropriate recovery purifying is taken to connect with cloning vector,
Carrier selects pMD19-T carrier (TaKaRa), and the molar ratio of target DNA and cloning vector is controlled in 3:1 or so.
Linked system: 1 μ L pMD19-T vector, 4 μ L DNA after purification, 5 μ L Ligation solution I.It is mixed
Constant temperature connects overnight at 16 DEG C after closing uniformly.
Competent cell DH5 α is taken out to be placed in ice from -80 DEG C and is melted, 2 μ L connection products is taken to be added to containing 50 μ L
In the centrifuge tube of competent cell, after ice bath 30min, 42 DEG C of water-bath heat shock 90s, ice bath 3min or more, is added 37 DEG C not immediately
Antibiotic 800 μ L of LB liquid medium, the 200rpm shaken cultivation 1h at 37 DEG C.Then it is centrifuged 2min at 4000rpm,
900 μ L supernatants are abandoned, remaining liq is suspended with pipette tips and precipitates and mixes, takes appropriate conversion fluid to be plated on and has coated X-gal/IPTG
LB solid plate culture medium (contain 100 μ g mL-1Amp) on, be inverted overnight incubation in 37 DEG C of insulating boxs after sealing plate, lead to
X-gal/IPTG indigo plant hickie screening white colony is crossed, and is chosen after being detected by bacterium solution PCR and is correctly cloned into Shenzhen Hua Da base
Because Science and Technology Ltd. carries out sequence verification.
(4) sequence is analyzed
It is right using ExPASy on-line analysis tool PI/Mw (http://www.expasy.ch/tools/dna.html)
The protein of LcMYB1 gene coding carries out amino acid analysis.As a result as shown in Fig. 2, the RsAN1 and MYB from other plants turns
The record factor is compared, and all with a relatively conservative R2R3 structural domain, one and bHLH transcription factor are contained on R3 structural domain
The site of interaction.Generally speaking, the N-terminal portion of the albumen and the homology of other albumen are higher, but C-terminal variation compared with
Greatly.
Using the amino acid alignment of homologous gene between 5.0 software of MEGA progress different plant species and construct phyletic evolution
Tree, as a result as shown in Figures 2 and 3, RsAN1 is closer with the relationship of arabidopsis AtPAP1 and AtPAP2.
(5) recombinant expression carrier is constructed
Expression vector pEAQ-HT is handled with Age I and Xho I double digestion, it is solidifying according to Takara agarose after digestion
Plastic recovery kit recycles expression vector pEAQ-HT large fragment.Again by the RsAN1 of correct sequence Age I and Xho I double digestion
Then processing is connected using Gibson package technique, connection product is finally converted escherichia coli DH5a, receive mycin from containing card
On the screening LB culture plate of (100mg/L) picking positive colony and carry out PCR detection and sequence verification to get to recombinant expression carry
Body pEAQ-RsAN1.
Wherein, single endonuclease digestion system are as follows: 5 μ L of pEAQ-HT or 19T-RsAN1 plasmid；Age I 1μL；Xho I 1μL；
Buffer 2μL；Sterile ddH2O complements to 20 μ L.37 DEG C of reaction 3h.
Gene RsAN1 amplimer:
pEAQRsAN1-F:5'-TATTCTGCCCAAATTCGCGAATGGAGGGTTCGTCCAAAG-3'
pEAQRsAN1-R:5'-TGAAACCAGAGTTAAAGGCCTCAAGTTCCAGTCTCTCCATC-3
Coupled reaction system: 3 μ L of RsAN1 segment；Linearized vector (pEAQ-HT recycles large fragment) 1-2 μ L；
Solution I 5μL；Sterile ddH2O complements to 10 μ L.2h is reacted in 16 DEG C of connections.
(6) RsAN1 expression quantity detects
Expression quantity detection of the RsAN1 in wild type ' red heart 1 ' rouge radish reacts (ABI using Real-Time PCR
7500Real-Time PCR System) it carries out.Reaction system according to Real-Time PCR Master Mix (TOYOBO,
Japan) specification, reaction volume are 20 μ L；Reaction condition is as follows: 50 DEG C, 60s；95 DEG C, 60s, 95 DEG C, 15s, 56 DEG C, 20s,
72 DEG C, 35s, 40 circulations；55-95 DEG C of melting curve analysis is carried out later.Each sample sets 3 repetitions.Lead to after reaction
Cross the specificity of melting curve identification product.Gene relative expression quantity analysis uses 2-△△CTMethod carries out data processing (Livak
And Schmittgen 2001), calculate the expression of RsAN1.All of above experiment is all provided with 3 repetitions, and ddH is used in experiment2O
For negative control；As a result as shown in Figure 4.
The primer of quantitative fluorescent PCR:
QRsAN1-F:5'-CGTTGTTCCTCTATGCCTTC-3'
QRsAN1-R:5'-TAGCAAACTCTCCCACCACA-3'
From fig. 4, it can be seen that RsAN1 has position expression (skin, the meat, petiole) expression of accumulation, and anthocyanin in anthocyanin
Accumulation it is more, gene expression amount is higher, it is seen then that the expression quantity of RsAN1 and the accumulation positive correlation of anthocyanin；And
Without the expression of RsAN1 in the blade of wild type rouge radish, also synthesis and accumulation without anthocyanin.
(7) genetic transformation of recombinant expression carrier
The recombinant expression carrier pEAQ-RsAN1 of building is transferred in Agrobacterium EHA105 by conventional freeze-thaw method, then is passed through
PEAQ-RsAN1 is transferred in wild type rouge radish by mediated by agriculture bacillus method, the specific steps are as follows:
By the Agrobacterium containing pEAQ-RsAN1 expression vector, it is inoculated in and receives the liquid LB of mycin (100mg/L) containing card
In culture medium, 28 DEG C, 300rpm is cultivated to OD600Between about 2, cultured bacterium solution is then centrifuged 5min in 4000rpm,
By the thallus of collection MAA (10mM MES (2- [N-morpholino] ethanesulfonic acid) pH 5.6,10mM
MgCl2, 100 μM of Acetosyringone) and suspension thalline OD600To 1.0 or so；It will be contained with the syringe without syringe needle
The Agrobacterium injection rouge radish cotyledon and vacuum side of blade of pEAQ-RsAN1 expression vector, while in rouge radish cotyledon and blade
It is upper to inject the Agrobacterium for containing empty carrier pEAQ-HT as negative control.It observes and takes pictures after 4~5d of injection, as a result such as Fig. 5 institute
Show.
From fig. 5, it can be seen that the transient overexpression RsAN1 in the cotyledon and blade of rouge radish, can make blade and son
A large amount of anthocyanins are accumulated in leaf, and without the synthesis of anthocyanin in the blade and cotyledon of negative control plant.Illustrate in kermes
Transient overexpression RsAN1 can promote the synthesis and accumulation of anthocyanin in radish.
Acquire above-mentioned sample analysis Anthocyanin Content, Anthocyanin Content measuring method are as follows: the measurement of Anthocyanin content is adopted
With pH differential method, the test method of reference Wrolstad etc. (1982), specific steps are as follows: take radish tissue 0.1g sample in 3mL
Leaching liquor (methanol；Water: concentrated hydrochloric acid 85:12:3) in, room temperature is protected from light sufficiently extraction (about 5~6 hours).Prepare 1 He of Buffer
Buffer 2, Buffer 1:0.2mol/L KCl-0.2mol/L HCl (25:67), pH=1；Buffer 2:1mol/L
NaAc-0.4mol/L HCl (100:150), pH=5.Take 0.5mL leaching liquor be separately added into test tube 2mL Buffer1 and
Buffer 2 measures their light absorption values at 530nm with ultraviolet specrophotometer.
Rule of thumb formula calculated result, formula are as follows: anthocyanin content (mg/g)=Δ OD530×5×3×445.2/
29600 × 0.1, wherein 5 be extension rate, 3 be extracting liquid volume, and 445.2 be the average molecular of Cy-3-G
Quality；29600 be mole specific absorptivity (mol of Cy-3-G-1.ml-1), 0.1 indicates example weight.
Be computed, in the turnip leaves and cotyledon of instantaneous conversion RsAN1 Anthocyanin Content be respectively 0.45mg/g and
0.28mg/g, and can't detect anthocyanin in the blade of negative control and cotyledon.
To sum up, by the way that when single expression RsAN1 gene, anthocyanin in rouge radish can be obviously promoted in rouge radish
Synthesis and accumulation, and then improve rouge radish in anthocyanin content, improve the quality of rouge radish.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
SEQUENCE LISTING
<110>Yangtze Normal University；
<120>a kind of rouge radish anthocyanin biosynthetic controlling gene RsAN1 and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 756
<212> DNA
<213>rouge radish (Raphanus sativus L.)
<400> 1
atggagggtt cgtccaaagg gttgagaaaa ggtgcatgga ctgctgaaga agatagtctc 60
ttgaggcaat gcattgataa gtatggagaa gggaaatggc accaagttcc tttaagagct 120
gagctcaatc ggtgcaggaa gagttgtaga ctaagatggt tgaactattt gaagccaagt 180
atcaagagag ggaaacttaa ctctgatgaa gttgatcttc ttcttcgcct tcataaactt 240
ttgggaaaca ggtggtcttt aattgctggt agattacccg gtcggactgc caatgatgta 300
aaaaattact ggaacaccca tttgagtaag aaacatgaac caggttgtaa gacccagatg 360
aaaaaagaga agagaaacat tccttgctct tctactacac tagcccaaaa aatcgacgtt 420
ttcaaacctc gacctcgatc cttcaccgtt aacaacggct gcagccatat cattggcatg 480
ccaaaacctg acgttgttcc tctatgcctt cgattcaaca acaccaaaaa tgtttgtgaa 540
aatattgcta catgtaacaa agatgacgat aaatctgagc ttgtttgtaa tttaatggat 600
ggtcagaata tgtggtggga gagtttgcta gatgagagcc aagatccagc tgctctctat 660
ccagaagcta cagcaacaaa aaaggccgta acctccgagt ttgacgttga tcacctttgg 720
agcctgttgg atggagagac tgttgaactt gattag 756
<210> 2
<211> 251
<212> PRT
<213>rouge radish (Raphanus sativus L.)
<400> 2
MEGSSKGLRK GAWTAEEDSL LRQCIDKYGE GKWHQVPLRA ELNRCRKSCR LRWLNYLKPS 60
IKRGKLNSDE VDLLLRLHKL LGNRWSLIAG RLPGRTANDV KNYWNTHLSK KHEPGCKTQM 120
KKEKRNIPCS STTLAQKIDV FKPRPRSFTV NNGCSHIIGM PKPDVVPLCL RFNNTKNVCE 180
NIATCNKDDD KSELVCNLMD GQNMWWESLL DESQDPAALY PEATATKKAV TSEFDVDHLW 240
SLLDGETVEL D 251
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
tattctgccc aaattcgcga atggagggtt cgtccaaag 39
<210> 4
<211> 41
<212> DNA
<213>artificial sequence
<400> 4
tgaaaccaga gttaaaggcc tcaagttcca gtctctccat c 41
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
cgttgttcct ctatgccttc 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
tagcaaactc tcccaccaca 20
Claims (7)
1. a kind of rouge radish anthocyanin biosynthetic controlling geneRsAN1, which is characterized in that its nucleotide sequence such as SEQ ID
Shown in NO.1.
2. rouge radish anthocyanin biosynthetic controlling gene according to claim 1RsAN1, the amino acid sequence of coding
As shown in SEQ ID NO.2.
3. a kind of recombinant expression carrier, which is characterized in that including described in claim 1RsAN1Nucleotide sequence.
4. a kind of plant cell or vegetable transformant comprising recombinant expression carrier described in claim 3.
5. application of the gene as described in claim 1 in regulation herbaceous plant anthocyanin synthesis.
6. a kind of breeding method of high-content anthocyanin rouge radish, which comprises the following steps:
1) it takes rouge radish fleshy root to extract total serum IgE, then the first chain cDNA is synthesized by reverse transcription using total serum IgE as template；
2) cDNA obtained using step 1) is template, with the column of nucleotides sequence shown in SEQID NO 3 and SEQID NO 4 be primer into
Row amplification, the nucleotide sequence of obtained pcr amplification product are connected into expression as shown in SEQ ID NO.1, and by pcr amplification product
Recombinant expression carrier is constructed in carrier；
3) recombinant expression carrier of building is converted to Agrobacterium GV3101, then with the Agrobacterium GV3101 to rouge radish
It carries out after infecting conversion, cultivates 4 ~ 5 days to get high-content anthocyanin rouge radish is arrived.
7. the breeding method of high-content anthocyanin rouge radish according to claim 6, which is characterized in that the expression vector
For pEAQ-HT.
Priority Applications (1)
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Cited By (3)
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| CN111733163A (en) * | 2019-08-07 | 2020-10-02 | 合肥工业大学 | IbMYB44 gene for regulating anthocyanin synthesis and recombinant expression vector and application thereof |
| CN111733165A (en) * | 2019-12-29 | 2020-10-02 | 合肥工业大学 | PyWRKY26 gene for promoting anthocyanin synthesis and recombinant expression vector and application thereof |
| CN111733164A (en) * | 2019-08-07 | 2020-10-02 | 合肥工业大学 | IbNAC56 gene for promoting anthocyanin synthesis and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733163A (en) * | 2019-08-07 | 2020-10-02 | 合肥工业大学 | IbMYB44 gene for regulating anthocyanin synthesis and recombinant expression vector and application thereof |
| CN111733164A (en) * | 2019-08-07 | 2020-10-02 | 合肥工业大学 | IbNAC56 gene for promoting anthocyanin synthesis and application thereof |
| CN111733164B (en) * | 2019-08-07 | 2022-05-03 | 合肥工业大学 | IbNAC56 gene for promoting anthocyanin synthesis and application thereof |
| CN111733163B (en) * | 2019-08-07 | 2022-05-03 | 合肥工业大学 | IbMYB44 gene for regulating anthocyanin synthesis and recombinant expression vector and application thereof |
| CN111733165A (en) * | 2019-12-29 | 2020-10-02 | 合肥工业大学 | PyWRKY26 gene for promoting anthocyanin synthesis and recombinant expression vector and application thereof |
| CN111733165B (en) * | 2019-12-29 | 2022-05-03 | 合肥工业大学 | PyWRKY26 gene for promoting anthocyanin synthesis and recombinant expression vector and application thereof |
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