CN106589087A - Dendrobium bHLH transcription factor and encoding protein and application thereof - Google Patents
Dendrobium bHLH transcription factor and encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses a dendrobium bHLH transcription factor and an encoding protein and application thereof. The dendrobium bHLH transcription factor is protein shown in a or b, wherein a is protein formed by an amino acid sequence shown in a sequence 1 in a sequence table, and b is protein formed by the amino acid sequence shown in the sequence 1 in the sequence table through substitution and/or deletion and/or addition of one or more amino acid residues, relevant to anthocyanin synthesis and derived from a. The transcription factor DhbHLH1 is obtained from dendrobium and belongs to a bHLH transcription factor family for positively regulating anthocyanin synthesis in monocotyledons. By means of reverse transcription RT-PCR, a complete encoding frame of the gene is obtained, an excessive expression vector is established later, dendrobium pedals are homologically and instantly converted, and it is found that the transcription factor has the capacity of promoting anthocyanin accumulation.
Description
Technical field
The present invention relates to biological technical field, more particularly to Herba Dendrobii bHLH transcription factor and its encoding proteins and application.
Background technology
BHLH albumen is the transcription factor extended familys being widely present in animals and plants, with the spiral shell comprising about 60 aminoacid
The bHLH domains of rotation-ring-spiral.MYB interaction area of the bHLH transcription factor comprising a N-terminal, it is mutual with myb transcription factor albumen
Make, and the small molecule of C-terminal combines ACT-like domains.Arabidopsiss contain 162 bHLH transcription factor, according to amino acid structure
Similarity is divided into different subgroups.The transcription factor of plant bHLH IIIf subgroups is proved to participate in regulation and control flavonoid biosynthesiss
Approach.BHLH generally forms complex with R2R3-MYB and WD40 transcription factor and participates in the biological conjunction of flavonoid in transcriptional level control
Into enzyme gene expression, bHLH class transcription factor GL3, EGL3 such as in arabidopsiss, TT8, MYC1；The Lc of Semen Maydiss；Antirrhinum majus L.
DELILA；AN1 and JAF13 of petunia etc..And the expression control of such bHLH genes can often change flavonoid metabolism way
Footpath.Overexpression bHLH genes can make the accumulation of anthocyanidin in the plant such as arabidopsiss, Semen Maydiss, petunia, Herba Medicaginiss.
Herba Dendrobii orchid tint is gorgeous, and flower pattern is various, long fresh-keeping period, is a kind of high flowers of ornamental value, in cut-flower, basin
The aspects such as flower, corsage, bouquet, pantry flower are used widely.The CF of petal, sepal and lip is that Herba Dendrobii is most main
The fancy points wanted.Herba Dendrobii orchid tint mainly has kermesinus, aubergine, pink, yellow, green and white etc., and has rich
Rich coloring mode, such as pure color, there are decorative pattern, lip color special.Anthocyanidin is the primary pigments in Herba Dendrobii flower, control
The formation of the color such as purplish red, pink, pink.In recent years breeder focuses on changing Herba Dendrobii orchid tint and flower coloring mode.But
Conventional hybridization breeding requires comprehensively to collect very much breeding resources, and expected breeding objective difficult to realize.With biotechnology
Develop rapidly, improveing pattern and the new pattern Herba Dendrobii orchid species of directive breeding using technique for gene engineering will be possibly realized.And turn
The record factor can open up thinking as the effective means of the accumulation of regulation and control anthocyanidin battalion amount for Herba Dendrobii pattern molecular breeding.So
And, not yet report with regard to the biosynthetic transcriptional control research of anthocyanidin in Herba Dendrobii.So far, cyanine is regulated and controled in Herba Dendrobii
The bHLH gene orders and function of element synthesis has not been reported.
The content of the invention
In order to make up the deficiency in above field, the invention provides Herba Dendrobii bHLH transcription factor and its encoding proteins with should
With.
Herba Dendrobii bHLH transcription factor provided by the present invention, entitled DhbHLH1, from Herba Dendrobii orchid species
Dendrobium Blue sapphine No.3。
It is an object of the present invention to provide a kind of albumen, is following protein a) or b)：
A) protein that the aminoacid sequence shown in sequence in sequence table 1 is constituted；
B) by the aminoacid sequence shown in sequence in sequence table 1 through the replacement of one or several amino acid residues and/or
Disappearance and/or addition and by a) derived from the protein related to anthocyanidin synthesis.
The encoding gene of the albumen falls within protection scope of the present invention.
The encoding gene of the albumen is following 1) or 2) or 3) or 4) shown：
1) its nucleotide sequence is DNA molecular shown in sequence 2 in sequence table；
2) its nucleotide sequence is DNA molecular shown in sequence 3 in sequence table；
3) under strict conditions with 1) or 2) DNA molecular of the DNA molecule hybridize for limiting；
1) or 2) or 3) 4) there is the DNA molecular of more than 90% homology with the DNA molecular for limiting.
It is DhbHLH1 by the unnamed gene, the albumen for being encoded is named as DhbHLH1.The DhbHLH1 genes of acquisition are complete
Long cDNA is 2109bp, as shown in sequence 2 in sequence table；Wherein, in sequence table sequence 2 from 5 ' ends the 21st to the 2015th
Position is open reading frame, and open reading frame part is 1995bp, as shown in sequence 3 in sequence table；Sequence 1 in its polynucleotide
Shown albumen, sequence 1 is made up of 665 aminoacid sequences in sequence table, and this aminoacid sequence has conservative MYB interactions
Area, bHLH domains and ACT-like domains, other plant anthocyanidin synthesis correlation bHLH has higher conservative.
Expression cassette containing the encoding gene, recombinant expression carrier, transgenic cell line or recombinant bacterium fall within this
Bright protection domain.
The primer pair for expanding the encoding gene total length or its arbitrary fragment falls within protection scope of the present invention, described to draw
Thing centering, in a primer sequence such as sequence table shown in sequence 4, in another primer sequence such as sequence table shown in sequence 5.
Application of the described albumen, the encoding gene in regulation and control anthocyanidin synthesis falls within the protection model of the present invention
Enclose.
Transcription factor DhbHLH1 provided by the present invention is obtained from Herba Dendrobii, is belonged in monocotyledon and is just adjusted
The bHLH transcription factor families of control anthocyanidin synthesis.By reverse transcription RT-PCR, the complete encoder block of the gene is obtained, subsequently
Overexpression carrier and homologous instantaneous conversion Herba Dendrobii petal are built, it is found that the transcription factor has the energy for promoting anthocyanin accumulation
Power.
Description of the drawings
Fig. 1 is the bHLH amino acid alignment figures of DhbHLH1 and regulation and control anthocyanidin synthesis in the plant reported；
PebHLH1, iris Phalaenopsis equestris；LhbHLH2, oriental hybrid lily Lilium hybrid division
I；AtTT8, arabidopsiss Arabidopsis thaliana；PhAN1, petunia Petunia × hybrida, NtAn1a, Nicotiana tabacum L.
Nicotiana tabacum；ZmLc, Semen Maydiss Zea mays.
Fig. 2 is expression of the DhbHLH1 in different colours Herba Dendrobii petal；BS No.3,‘Blue sapphine
No.3’；RB,‘Red bull’；PS,‘Pink stripe’；BWD,‘Burana white dove’.
Fig. 3 is particle gun instantaneous conversion result.A, converts recombiant plasmid pCXSN-DhbHLH1；B, converts empty plasmid
pCXSN；C, conversion empty plasmid pCAMBIA (1303)；Scale is 200 μm.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, and as a result makes even
Average.
The clone of embodiment 1, Herba Dendrobii DhbHLH1 genes
Dongfang Shi Canaan Orchis planting peasant (is purchased from Herba Dendrobii orchid species Dendrobium Blue sapphine No.3
Specialty Co-operative Organization, catalog number is D20) flower be material, using Tiangeng polysaccharide polyphenol plant total RNA extraction reagent box (purchase
From TIANGEN Biotech (Beijing) Co., Ltd., catalog number is DP441) method extraction total serum IgE, using Thermo
Scientific reverse transcription reagent box (purchased from the silent winged generation that science and technology of match, catalog number is K1622) synthesis cDNA, as mould
Plate.Design full-length cDNA amplification primer：F:5'-AACCAACGAAACCAATCACCA-3'；R:5'-
AACAAAGGCCAGCATCTACAGA-3'。
Expanded with above-mentioned template, the response procedures be 94 DEG C of 3min, 94 DEG C of 30S, 56 DEG C of 30S, 72 DEG C of 2min 30S,
30 circulations, 72 DEG C.Archaeal dna polymerase used is Takara ExTaq enzymes.Amplification system：The μ L of Extaq enzymes 0.4,10 ×
The μ L of Extaqbuffer 5, dNTP mix 4 μ L, forward primer and each 1 μ L of reverse primer, template cDNA1 μ L, are supplied with sterilized water
The μ L of total system 50.Amplified production carries out 1% sepharose electrophoresis, reclaims purpose band and is cloned into pMD 18-T carriers (purchased from treasured
Biological engineering (Dalian) company limited, catalog number be 6011) on be sequenced.
As a result Fig. 1 is seen.The cDNA of the DhbHLH1 full length genes of acquisition is 2109bp, as shown in sequence 2 in sequence table；Its
In, sequence 2 is open reading frame from 5 ' ends the 21st to the 2015th in sequence table, and open reading frame part is 1995bp,
As shown in sequence 3 in sequence table；Albumen in its polynucleotide shown in sequence 1, sequence 1 is by 665 aminoacid in sequence table
Sequence is constituted, and this aminoacid sequence has conservative MYB interactions area, bHLH domains and ACT-like domains, other plant
Anthocyanidin synthesis correlation bHLH has higher conservative.Jing sequence alignments, the DhbHLH1 and iris PebHLH1 for delivering
70.2%, 43.5% is respectively with the amino acid identity of arabidopsiss AtTT8.It is DhbHLH1 by the unnamed gene, is encoded
Albumen be named as DhbHLH1.
The expression of embodiment 2, DhbHLH1 genes in the Herba Dendrobii petal of different colours compares
Choose 3 stages during Herba Dendrobii flower growth：First stage is little flower bud phase (～0.5 × 1.0-1.5cm:
It is wide × high), second stage be big flower bud phase (1.5-2.0cm), phase III just to open the phase (flower is just opened).It is many using Tiangeng
Sugared polyphenol plant total RNA extraction reagent box method extract respectively Herba Dendrobii orchid species ' Blue sapphine No.3 ' (bluish violet),
' Redbull ' (aubergine) (being purchased from Dongfang Shi Canaan Orchis planting farmer specialized cooperative society, catalog number is D4), ' Pink
Stripe ' (pink) (being purchased from Dongfang Shi Canaan Orchis planting farmer specialized cooperative society, catalog number is D10), ' Burana
3 developments of whitedove ' (white) (being purchased from Dongfang Shi Canaan Orchis planting farmer specialized cooperative society, catalog number is D16)
Total serum IgE in the petal in stage, is that cDNA (uses Thermo Scientific reverse transcription reagent box, synthesis by total serum IgE reverse transcription
cDNA).It is ((big purchased from precious biological engineering using Takara SYBR Premix Ex TaqTM II (Tli RNaseH Plus)
Even) company limited, catalog number is RR420A) fluorescence quantitative kit analyzes DhbHLH1 genes in different colours Herba Dendrobii
Expression in petal.Primer used by real-time fluorescence quantitative PCR detection DhbHLH1 genes is as follows：
DhbHLH1-qF：5′-AATGTGTTGGGGTCAAAAGTATC-3′；
DhbHLH1-qR：5′-TGCAGACGTGCTAATTCCTGT-3′.
The amplified reaction program of real-time fluorescence quantitative PCR：95℃3min；95 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 30s, 40 are followed
Ring.
The relative expression levels of DhbHLH1 genes are normalized with 18S genes as internal reference, use 2–△CTMethod represents gene
Relative quantification.
As a result Fig. 2 is seen.In bluish violet, aubergine and pink petal, DhbHLH1 genes expression before flowering
Increase, when opening at the beginning of phase III flower, expression drastically declines.And DhbHLH1 genes are in bluish violet and aubergine petal
Expression is more much higher than its expression in pink and white petal.
Embodiment 3, the structure of Herba Dendrobii DhbHLH1 expression vectors and functional analyses
With the pMD 18-T-DhbHLH1 plasmids that have been sequenced as template, (have purchased from precious biological engineering (Dalian) with Extaq enzymes
Limit company, catalog number is RR001A) amplification (amplification primers F used:5'-AACCAACGAAACCAATCACCA-3'；R:
5'-AACAAAGGCCAGCATCTACAGA-3', primer is with embodiment 1) go out the DhbHLH1 comprising complete ORF and termination codon
Fragment, by TA clones pCXSN (T- carriers) (Chen S, Songkumarn P, Liu J, Wang G.A are connected to
versatile zero background T-Vector system for gene cloning and functional
genomics.Plant Physiology,2009,150:1111-1121) on, and be sequenced confirmation connection it is errorless, extraction builds
Overexpression plasmid pCXSN-DhbHLH1 it is standby.
Using GJ-1000 high pneumatic gene guns by pCXSN-DhHL and control plasmid pCXSN be unloaded and pCAMBIA1303
(Hajdukiewicz P,Svab Z,Maliga P.The small,versatile pPZP family of
Agrobacterium binary vectors for plant transformation.Plant Mol.Biol.,1994,25
(6):989-994) ' Burana charming ' (are purchased from Dongfang Shi Canaan Orchis planting agriculture to unloaded bombardment importing Herba Dendrobii orchid species
People Specialty Co-operative Organization, catalog number is D5) white petal in.Biolistic bombardment parameter is：Pressure 7MPa, target distance
55cm, vacuum is -0.085MPa.Petal is transferred in MS culture medium after bombardment, is sealed.At 25 DEG C, 16h light/8h is dark
Photoperiod under cultivate 2 days, observed with Stereo microscope.
As a result Fig. 3 is seen.Transient expression DhbHLH1 genes cause white Petal to generate the pigment spot of purple.Turn
Change the unloaded petals of pCXSN and do not produce any pigment spot.Petal after conversion pCAMBIA1303, Jing GUS dyeing is produced
The speckle of blueness is given birth to, has shown transient expression success.
Sequence table
<110>Tropical Crop Variety Resource Institute of Chinese Academy of Tropical Agricultural Sciences
<120>Herba Dendrobii bHLH transcription factor and its encoding proteins and application
<130> P160687/RKY
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 665
<212> PRT
<213>Herba Dendrobii orchid species Dendrobium Blue sapphine No. 3
<400> 1
Met Glu Glu Leu Pro Leu Asn Arg Thr Lys Leu Gln Gln Met Leu Gln
1 5 10 15
Thr Ile Val Gln Asn Ile Gly Trp Thr Tyr Ser Leu Phe Trp Lys Leu
20 25 30
Cys Pro Gln Gln Gly Leu Leu Val Trp Gly Asp Gly His Tyr Asn Gly
35 40 45
Ser Ile Lys Thr Arg Lys Thr Met Gln Pro Thr Ala Val Ser Ser Glu
50 55 60
Val Ser Pro Thr Glu Arg Ser Glu Gln Leu Lys Glu Leu Tyr Gln Phe
65 70 75 80
Leu Ala Ala Asp Ala Glu Ser Ser Glu Arg Arg Arg Pro Gly Leu Ala
85 90 95
Leu Ser Pro Glu Asp Leu Thr Glu Cys Glu Trp Phe Tyr Leu Met Cys
100 105 110
Phe Ser Leu Ser Phe Ala Pro Gly Cys Gly Leu Pro Gly Lys Ala Phe
115 120 125
Glu Thr Gln Asn Ser Ile Trp Leu Thr Lys Ala Asn Asp Ala Lys Ser
130 135 140
Lys Val Phe Ser Arg Thr Ile Leu Ala Lys Ser Ala Gly Ile Gln Thr
145 150 155 160
Val Val Cys Ile Pro Leu Thr Asn Gly Val Leu Glu Leu Gly Thr Glu
165 170 175
Glu Leu Val Lys Glu Asp Thr Arg Leu Thr Gln Gln Ala Arg Asn Ile
180 185 190
Phe Ala Asp Ile His Val Asn Ser Pro Ile Ser Ala Cys Ser Glu His
195 200 205
Ser Thr Ser Ser Pro Ala Leu Gln Arg Glu Thr Leu Pro Met Thr His
210 215 220
Gly Met Asp Gly Leu Cys Gln Pro Cys Glu Glu Val Asp Asn Glu Lys
225 230 235 240
Glu Asp Ser Asn Asn Gly Glu Ser Pro Glu Arg Glu Ile Met Lys Ala
245 250 255
Tyr Ser Glu Ser Glu Leu Glu Ile Gly Arg Asn Ile Ser Val Gln Thr
260 265 270
Asn Phe Pro Arg Met Ile Ala Thr Asn Leu Ser Met Thr Lys Ser Glu
275 280 285
Ser Ile Lys Phe Gly Phe Ala Asp Asn Gly Ile Asn Asp Ser Glu Met
290 295 300
Ser Gln Gln Lys Asn Pro Glu Val Ala His Lys Cys Ser Ser Val His
305 310 315 320
Ser Phe Glu Gln Asp Glu Gln Glu Ser Asp Leu Ala Trp Tyr Leu Leu
325 330 335
His Asp Gly Ser Cys Asn Asp Leu Gln His Ser Ser Glu Ser Met Gln
340 345 350
Val Asp Glu Leu Pro Pro Gln Asp Val His Tyr Ser Glu Thr Asp Ala
355 360 365
Thr Ile Leu His Asp Asn Thr Thr Asp Phe Ser Val Gly Ser Leu Ile
370 375 380
Ser Gln Leu Ser Leu Ser Asp Thr Ser Ser Phe Ser Lys Trp Phe Cys
385 390 395 400
Glu Asn Gln Ile Phe Ser Lys Thr Gly Ala Ser Gln Trp Lys Leu Lys
405 410 415
Tyr Ile Leu Phe Ile Leu Pro Thr Ile His Asn Lys Lys Tyr Val Asn
420 425 430
Ser Val His Leu Lys Gly Lys Glu Asn Gly Asp Trp Lys Val Ile Leu
435 440 445
Gln Asp Glu Thr Asn Gly Asn Arg Val Leu Ala Glu Arg Arg Arg Arg
450 455 460
Glu Lys Leu Asn Lys Arg Phe Met Ser Leu Arg Ala Leu Val Pro Cys
465 470 475 480
Val Thr Lys Met Asp Lys Ala Ser Ile Leu Ala Asp Thr Ile Glu Tyr
485 490 495
Val Arg His Leu Thr Arg Arg Ile His Asp Leu Glu Leu Gln Asn Asn
500 505 510
Lys Ile Ser Asp Val Leu Gly Ser Lys Val Ser Asp Pro His Asn Gln
515 520 525
Ile Lys Thr Leu Ala Glu Arg Leu Glu Thr Asn Cys Ser Thr Ile Ser
530 535 540
Ala Ser Ile Thr Lys Ser Lys Gln Glu Leu Ala Arg Leu Gln Gly Thr
545 550 555 560
Lys Lys Arg Arg Thr Leu Gln Cys Thr Asn Tyr Ile Glu Glu Ala Thr
565 570 575
Lys Ala Val Asp Asp Thr Ser Ile Ser Ile Gln Val Ser Ile Ile Glu
580 585 590
Ser Asn Ala Val Leu Gly Leu Gln Cys Pro Tyr Lys Asn Gly Leu Leu
595 600 605
Val Arg Ile Met Lys Lys Leu Asp Glu Leu Thr Leu Glu Ile Thr Ser
610 615 620
Leu Asn Ser Thr Ile Val Asn Gly Asn Phe Ile Ala Glu Phe Lys Ala
625 630 635 640
Lys Ile Lys Gly Pro Arg Gly Arg Arg Ala Asn Val Glu Glu Ile Lys
645 650 655
Trp Ala Ile His Gln Leu Leu Ser His
660 665
<210> 2
<211> 2109
<212> DNA
<213>Herba Dendrobii orchid species Dendrobium Blue sapphine No. 3
<400> 2
aaccaacgaa accaatcacc atggaagagt tgccattgaa cagaaccaag cttcaacaaa 60
tgctccagac tattgttcag aacatcgggt ggacttacag cctcttctgg aagctctgcc 120
cgcagcaagg gttgctggtg tggggggatg gccactacaa cggttccata aagacgagga 180
agacaatgca gccgacggcg gtgagctcgg aggtgtcgcc gactgagagg agtgaacagt 240
tgaaggaact gtatcagttc ctcgcagccg atggagagag tagcgagcgc cgccgtccag 300
gccttgctct gtcgcctgag gatttaactg agtgcgaatg gttctatctc atgtgctttt 360
ctttgtcctt tgctcctggt tgtgggcttc caggtaaggc atttgaaacg caaaatagca 420
tttggcttac aaaagcaaat gatgctaaga gcaaagtctt ctctagaact attcttgcca 480
agagcgcagg cattcagaca gtagtgtgca taccgctcac taatggcgtc cttgagcttg 540
gaacagaaga actggtcaaa gaagacacaa gacttacaca gcaagcaagg aatatatttg 600
ctgatattca cgtgaactca cctatatcag cctgctcgga gcactctacg tcgagcccgg 660
cattgcaatg ggaaaccttg cccatgactc atggcatgga tggactctgc caaccatgtg 720
aagaagtaaa taatgaaaaa gaagatagca ataatggaga gagtcctgaa cgtgaaatta 780
tgaaggcata ttcagaatcg gagcttgaga ttggaagaaa tatttcagtg cagacaaatt 840
ttcctagaat gattgctacc aatttgtcaa tgactaaatc tgaaagtatt aagtttggtt 900
ttgctgacaa tggcattaac gactcggaga tgtctcagca aaagaatccg gaagtagctc 960
ataaatgcag cagtgtccat tcttttgaac aagatgaaca agagtcagat ttagcttggt 1020
accttttgca tgatggttct tgtaatgacc ttcaacattc atcagagagc atgcaggttg 1080
atgagttacc tccgcaagat gttcactact cagaaacaga tgctaccatc ctgcatgata 1140
atactaccga tttctcagtg gggagcctta tcagtcaact cagcctctca gatacatcat 1200
cattctccaa gtggttttgt gaaaatcaaa tcttttcaaa gaccggagcc tcccagtgga 1260
aactgaaata tatcctattc atccttccca ctatacacaa taaaaaatat gtaaattctg 1320
ttcatttgaa aggtaaagag aatggagact ggaaggtaat acttcaggat gaaacaaatg 1380
gaaatcgtgt gcttgcagaa cgaagaagaa gagagaagtt gaacaaaagg ttcatgagtc 1440
ttcgtgcatt ggttccatgt gtgacaaaga tggataaggc ttcaatctta gccgatacaa 1500
tcgaatatgt cagacaccta acacgacgta tacacgatct tgaattacaa aacaacaaaa 1560
tatcaaatgt gttggggtca aaagtatcag atccgcacaa tcaaattaag acactggccg 1620
agagattgga gacaaactgt agcacaattt cagccggtat cactaaaagt aaacaggaat 1680
tagcacgtct gcaaggcaca aagaagcgca gaacattaca atgcacaaat aacattgagg 1740
aagcaacaaa agcagtggat gataccagca taagcattca agtttccata attgagtcca 1800
atgctgtatt aggactgcag tgcccttaca agaatggact acttgtcagg attatgaaga 1860
aacttgatga actcactttg gagatcactt ctctcaactc tacaattgtc aatggcaatt 1920
tcattgcaga atttaaggca aagatcaaag gaccttgtgg aaggagagcc aacgtcgaag 1980
aaataaaatg ggcaattcat caactcttat cccattaata tttctaagaa atgtatgcga 2040
gacttggtag aagaggatcc ttagaatctt cttagtgctt acttccttct gtagatgctg 2100
gcctttgtt 2109
<210> 3
<211> 1995
<212> DNA
<213>Herba Dendrobii orchid species Dendrobium Blue sapphine No. 3
<400> 3
atggaagagt tgccattgaa cagaaccaag cttcaacaaa tgctccagac tattgttcag 60
aacatcgggt ggacttacag cctcttctgg aagctctgcc cgcagcaagg gttgttggtg 120
tggggggatg gccactacaa cggttccata aagacgagga agacaatgca gccgacggcg 180
gtgagctcgg aggtgtcgcc gactgagagg agtgaacagt tgaaggaact gtatcagttc 240
ctcgcagccg atgcagagag tagcgagcgc cgccgtccag gccttgctct gtcgcctgag 300
gatttaactg agtgcgaatg gttctatctc atgtgctttt ctttgtcctt tgctcctggt 360
tgtgggcttc caggtaaggc atttgaaacg caaaatagca tttggcttac aaaagcaaat 420
gatgctaaga gcaaagtctt ctctagaact attcttgcca agagcgcagg cattcagaca 480
gtagtgtgca taccgctcac taatggcgtc cttgagcttg gaacagaaga actggtcaaa 540
gaagacacaa gacttacaca gcaagcaagg aatatatttg ctgatattca cgtgaactca 600
cctatatcag cctgctcgga gcactctacg tcgagcccgg cattgcaacg ggaaaccttg 660
cccatgactc atggcatgga tggactctgc caaccatgtg aagaagtaga taatgaaaaa 720
gaagatagca ataatggaga gagtcctgaa cgtgaaatta tgaaggcata ttctgaatcg 780
gagcttgaga ttggaagaaa tatttcagtg cagacaaatt ttcctagaat gattgctacc 840
aatttgtcaa tgactaagtc tgaaagtatt aagtttggtt ttgctgacaa tggcattaac 900
gactcggaga tgtctcagca aaagaatccg gaagtagctc ataaatgcag cagtgtccat 960
tcttttgaac aagatgaaca agagtcagat ttagcttggt accttttgca tgatggttct 1020
tgtaatgacc ttcaacattc atcagagagc atgcaggttg atgagttacc tccgcaagat 1080
gttcactact cagaaacaga tgctaccatc ctgcatgata atactaccga tttctcagtg 1140
gggagcctta tcagtcaact cagcctctca gatacatcat cattctccaa gtggttttgt 1200
gaaaatcaaa tcttttcaaa gaccggagcc tcccagtgga agctgaaata catcctattc 1260
atccttccca ctatacacaa taaaaaatat gtaaattctg ttcatttgaa aggtaaagag 1320
aatggagact ggaaggtaat acttcaggat gaaacaaatg gaaatcgtgt gcttgcagaa 1380
cgaagaagaa gagagaagtt gaacaaaagg ttcatgagtc ttcgtgcatt ggttccatgt 1440
gtgacaaaga tggataaggc ttcaatctta gccgatacaa tcgaatatgt cagacaccta 1500
acacgacgta tacacgatct tgaattacaa aacaacaaaa tatcagatgt gttggggtca 1560
aaagtatcag atccgcacaa tcaaattaag acactggccg agagattgga gacaaactgt 1620
agcacaattt cagccagtat cactaaaagt aaacaggaat tagcacgtct gcaaggcaca 1680
aaaaagcgca gaacattaca atgcacaaat tacattgagg aagcaacaaa agcagtggat 1740
gataccagca taagcattca agtttccata attgagtcca atgctgtatt aggactgcag 1800
tgcccttaca agaatggact acttgtcagg attatgaaga aacttgatga actcactttg 1860
gagatcactt ctctcaactc tacaattgtc aatggcaatt tcattgcaga atttaaggca 1920
aagatcaaag gaccccgtgg aaggagagcc aacgttgaag aaataaaatg ggcaattcat 1980
caactcttat cccat 1995
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>CDNA amplimer F
<400> 4
aaccaacgaa accaatcacc a 21
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>CDNA amplimer R
<400> 5
aacaaaggcc agcatctaca ga 22
Claims (6)
1. a kind of albumen, is following protein a) or b)：
A) protein that the aminoacid sequence shown in sequence in sequence table 1 is constituted；
B) replacement by the aminoacid sequence shown in sequence in sequence table 1 through one or several amino acid residues and/or disappearance
And/or addition and by a) derived from the protein related to anthocyanidin synthesis.
2. the encoding gene of albumen described in claim 1.
3. encoding gene according to claim 2, it is characterised in that：The encoding gene for it is following 1) or 2) or 3) or 4)
It is shown：
1) its nucleotide sequence is DNA molecular shown in sequence 2 in sequence table；
2) its nucleotide sequence is DNA molecular shown in sequence 3 in sequence table；
3) under strict conditions with 1) or 2) DNA molecular of the DNA molecule hybridize for limiting；
1) or 2) or 3) 4) there is the DNA molecular of more than 90% homology with the DNA molecular for limiting.
4. the expression cassette, recombinant expression carrier, transgenic cell line or recombinant bacterium containing encoding gene described in Claims 2 or 3.
5. the primer pair of encoding gene total length or its arbitrary fragment described in Claims 2 or 3, in the primer pair, one are expanded
In primer sequence such as sequence table shown in sequence 4, in another primer sequence such as sequence table shown in sequence 5.
6. application of the albumen, encoding gene described in Claims 2 or 3 described in claim 1 in regulation and control anthocyanidin synthesis.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153978A (en) * | 2020-01-20 | 2020-05-15 | 中国农业科学院生物技术研究所 | RH2 protein and coding gene and application thereof |
| CN111733166A (en) * | 2020-06-18 | 2020-10-02 | 中国农业科学院郑州果树研究所 | Vitis davidii anthocyanin synthetic gene VdbHLH037 and application thereof |
| CN112010957A (en) * | 2020-09-23 | 2020-12-01 | 金天国际医疗科技有限公司 | bHLH transcription factor for promoting anthocyanin synthesis in lotus and application thereof |
| CN114014917A (en) * | 2021-10-28 | 2022-02-08 | 上海交通大学 | FvbHLH36 protein and coding gene and application thereof |
| CN117343156A (en) * | 2023-12-04 | 2024-01-05 | 中国农业科学院作物科学研究所 | Tartary buckwheat-derived bHLH transcription factor, coding gene and application thereof |
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| CN102372769A (en) * | 2011-11-03 | 2012-03-14 | 中国科学院研究生院 | Artemisia apiacea bHLH transcription factor as well as encoding gene and application thereof |
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| CHONGHUI LI等: "Anthocyanin biosynthesis regulation of DhbHLH1 and DhbHLH1 in Dendrobium hybrids petals", 《PLANT PHYSIOLOGY AND BIOCHEMISTRY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153978A (en) * | 2020-01-20 | 2020-05-15 | 中国农业科学院生物技术研究所 | RH2 protein and coding gene and application thereof |
| CN111153978B (en) * | 2020-01-20 | 2021-02-26 | 中国农业科学院生物技术研究所 | RH2 protein and coding gene and application thereof |
| CN111733166A (en) * | 2020-06-18 | 2020-10-02 | 中国农业科学院郑州果树研究所 | Vitis davidii anthocyanin synthetic gene VdbHLH037 and application thereof |
| CN112010957A (en) * | 2020-09-23 | 2020-12-01 | 金天国际医疗科技有限公司 | bHLH transcription factor for promoting anthocyanin synthesis in lotus and application thereof |
| CN114014917A (en) * | 2021-10-28 | 2022-02-08 | 上海交通大学 | FvbHLH36 protein and coding gene and application thereof |
| CN114014917B (en) * | 2021-10-28 | 2023-09-08 | 上海交通大学 | FvbHLH36 protein, and encoding gene and application thereof |
| CN117343156A (en) * | 2023-12-04 | 2024-01-05 | 中国农业科学院作物科学研究所 | Tartary buckwheat-derived bHLH transcription factor, coding gene and application thereof |
| CN117343156B (en) * | 2023-12-04 | 2024-03-01 | 中国农业科学院作物科学研究所 | Tartary buckwheat-derived bHLH transcription factor, coding gene and application thereof |
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Application publication date: 20170426 |