CN109486838A - A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis - Google Patents
A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
Abstract
The invention discloses a kind of transcription factor genes of regulation plant flavonoids synthesis, which is the MtbZIP10 gene identified from leguminous plant M. truncatula, and gene order is as shown in SEQ ID NO.1.Also, the invention discloses the function of the gene and its applications.The advantage of the invention is that, the present invention clones the transcription factor gene that can just regulate and control flavonoids synthesis from leguminous plant M. truncatula, and system identification is carried out to its function, it has found that the gene can activate multiple genes of flavonoid biosynthesis pathway, improves the content of flavonoids.
Description
Technical field
The invention belongs to gene clone technology field, specially a kind of transcription factor gene of regulation plant flavonoids synthesis
And application thereof.
Background technique
Flavonoids is that the major class in the root for being distributed widely in plant, stem, leaf, flower, fruit and kind skin is important
Secondary metabolites product, including flavonoids, isoflavones, flavonols, anthocyanidin and procyanidine etc..Wherein anthocyanidin has protection
From biological infestation and the function of attracting insect pollination, anthocyanidin can also respond biotic and abiotic stress, remove oxygen plant
Free radical and the injury for protecting the plants from high density illumination.Meanwhile anthocyanidin has at anticancer, disease-resistant and anti-oxidant aspect
Certain healthcare function.Another type procyanidine in flavonoids not only have protect the plants from herbivore and
Effect in terms of invading pathogens mechanism, and appropriate procyanidine can also effectively prevent livestock herbage flatulence simultaneously in herbage
Improve ruminant livestock nitrogen nutrition.Meanwhile procyanidine is beneficial to human health, therefore the metabolic regulation mechanism of flavonoids is plant
The research hotspot in secondary metabolism field.
Although regulate and control the transcription factor genes of the classes such as transcription factor such as MYB, bHLH and WDR of flavonoids biosynthesis by
Report, but other types of transcription factor research is relatively fewer.BZIP transcription factor (basicregion/
Leucinezippermotif, bZIP) it is found in people, animal, plant, microorganism and insect, it is to be distributed most extensively, most
Conservative a kind of transcription factor.Plant bZIP transcription factor also has a tissue expression specificity, the bZIP transcriptions of many plants because
Son constitutive expression in root, in the little or no expression of the expression quantity of stem and blade.Plant bZIP transcription factor participates in a variety of
Biological process, including seed maturation, germination, flower development, plant senescence, photomorphogenesis etc..Although in individual other plants
The transcription factor of bZIP family can participate in the biosynthesis of regulation anthocyanidin, but in M. truncatula, there is presently no
Any correlative study in relation to bZIP transcription factor regulation flavonoids route of synthesis, limits to the model plant flavonoid path
Research and application, so clone and identification have regulation flavonoids synthesis new function and class can be improved in plant
The bZIP class transcription factor gene of flavones content has important theory and application value.
Summary of the invention
It is insufficient for bZIP class transcription factor gene in flavonoid biosynthesis pathway in existing research, be difficult to largely improve
The problem of Flavonoid Content, the present invention provides a kind of transcription factor genes and application thereof of regulation plant flavonoids synthesis, should
Gene is a new gene, does not find the bZIP of regulation flavonoids (anthocyanidin and procyanidine) synthesis also in M. truncatula
Therefore family's transcription factor studies the bZIP transcription factor for the biosynthesis for regulating and controlling flavonoids in M. truncatula very
Necessary problem.
To achieve the goals above, technical solution provided by the invention is a kind of bZIP transcription factor provided by the invention
Gene, the bZIP transcription factor gene are MtbZIP10, and gene order is as shown in SEQ ID NO.1.The present invention is had found
MtbZIP10 gene be to clone to come from M. truncatula, be a kind of completely new plant bZIP transcription factor gene, enrich
The type of flavonoids bZIP transcription factor, simultaneously because bZIP transcription factor can just regulate and control function flavonoids, especially cyanine
The function of element and procyanidine, it is possible to improve application of the plant in resistance and nutritional quality.
Further, the protein of the bZIP transcription factor gene coding, amino acid sequence such as SEQ ID NO.2
It is shown.
Further, the purposes of the bZIP transcription factor gene is positive the biosynthesis pathway of flavonoids, especially
The key gene of the flavonoid paths such as CHS, DFR, ANS and MATE1, it was demonstrated that MtbZIP10 can by regulate and control these genes into
And adjusting function is played, this is the transcription factor base of the bZIP type of the first regulation flavonoid path found in M. truncatula
Cause.
Further, when expression of the MtbZIP10 gene in M. truncatula is suppressed, the anthocyanidin of aerial part
It is reduced with the proanthocyanidin in seed.
Further, the MtbZIP10 gene is in hairy middle overexpression of M. truncatula, hairy cyanine
Cellulose content is dramatically increased relative to unconverted control, is illustrated that the overexpression gene has and is improved the used of anthocyanin accumulation.
The present invention by adopting the above technical scheme, has the advantages that the present invention clones class Huang from M. truncatula
Ketone isopentenyl transferase genes, and system identification has been carried out to its function, it was found that gene mutation can influence to spend
The content of anthocyanidin can be improved in the content of green element and proanthocyanidin, the overexpression of the gene.
Detailed description of the invention
The multiple alignment of Fig. 1 MtbZIP10 and other plant bZIP transcription factor amino acid sequences is analyzed.Consensus
It indicates the completely the same amino acid of sequence, conservative amino acid: the dark-grey background pp of black font is emphasized with black and grey
100% is identical；It is identical that black font gray background represents 50%；Black font white background represents similarity below 50%.
The phylogenetic analysis of Fig. 2 plant bZIP transcription factor.Chadogram is constructed with DNAMAN, marks star after MtbZIP10
Number.Confidence level based on 1000 duplicate bootstrap supported value (%) estimation nodes.
The distribution expression pattern of Fig. 3 MtbZIP10.MtbZIP10 gene 10 days after root, stem, leaf, flower, beanpod and pollination,
12 days, 16 days, 20 days, 24 days, the expression in 36 days seeds.Data in graph form is mean+/-standard error, each experiment
It is repeated including three biology.
The relative expression quantity of anthocyanidin synthesis regulation key gene in Fig. 4 R108 and NF2620.ANS: anthocyanidin synthesis
Enzyme；CHS: chalcone synthase；CHI: enzyme, namely chalcone isomerase；DFR 1/2: dihydro flavonoids alcohol reductase；UGT72L1: glycosyl turns
Move enzyme；TT8:TT8 transcription factor；WD40:WD40 repetitive proteins；MATE1:MATE1 transhipment.Data in graph form is average value ± mark
Quasi- error, each experiment include that three biology repeat.
Fig. 5 confirms the building of plant expression vector using the method for PCR.Wherein left side is that DNA molecular amount marks；Right side is
The PCR of Agrobacterium positive colony containing MtbZIP10 gene is detected.
Anthocyanidin content measures in Fig. 6 transgenic hairy root.A, hairy under mutant NF260 background and its negative right
Anthocyanidin content according in.B, hairy under wild type A17 background and its anthocyanidin content in negative control
The influence that MtbZIP10 is expressed in Fig. 7 abscisic acid and illumination.A, C: at the appointed time with 50 μM of ABA in interval
R108 seedling is handled, the expression of MtbZIP10 is examined by semiquantitive PCR (A) and real-time fluorescence quantitative PCR (C).Without
The group of ABA processing is divided into 1.B, D: by R108 seedling dark processing 3 days at normal temperature, then respectively with illumination in 1,3,6,9,12 hour
Seedling is handled, the expression of MtbZIP10 is examined by semiquantitive PCR (A) and real-time fluorescence quantitative PCR (C).Dark processing
Group be divided into 1.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, all other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
One, main experimental materials and method
1.1CTAB method extracts M. truncatula genomic DNA
1) about 100mg M. truncatula organization material is taken, is placed in together with steel ball in 2mL centrifuge tube；Liquid nitrogen is added constantly to shake
It shakes, until organization material is sufficiently polished into powder.
2) 500 μ LCTAB extracting solutions (65 DEG C of preheatings) is added, vortex oscillation mixes well.
3) 65 DEG C of water-bath 30min of mixed liquor, during which constantly rock.
4) liquid to be mixed is down to room temperature, and isometric chloroform is added, is mixed by inversion.
5) 12000rpm is centrifuged 10min at room temperature.
6) mixed liquor is layered after being centrifuged, and careful Aspirate supernatant is placed in 1.5mL centrifuge tube.
7) isopropanol of isometric (about 500 μ L) is added, is mixed by inversion, stands 30min under room temperature.
8) 4 DEG C of 12000rpm are centrifuged 10min.
9) upper solution is abandoned, washs DNA precipitating with 70% ethanol solution.
10) after ethanol evaporation, 30-50 μ L sterile water dissolving DNA precipitating is added.- 20 DEG C of preservations.
1.2TRNzol-A+Method extracts M. truncatula RNA
1) about 200mg M. truncatula organization material is taken to be placed in 1.5mL RNase-free centrifuge tube, liquid nitrogen frozen, and
It is ground to powdery rapidly with the glass rod of sterilizing freezing.
2) 1mL TRNzol-A is added+Extracting solution, vortex oscillation mix mixed liquor, are stored at room temperature 5min.
3) by mixed liquor, 12000rmp is centrifuged 10min under the conditions of 4 DEG C.
4) Aspirate supernatant is into 1.5mL RNase-free centrifuge tube.
5) 200 μ L RNase-free chloroforms are added, are stored at room temperature 5min after being mixed by inversion repeatedly.
6) by mixed liquor, 12000rmp is centrifuged 10min under the conditions of 4 DEG C.
7) isometric RNase-free isopropyl is added into the new centrifuge tube of 1.5mL RNase-free in Aspirate supernatant
Alcohol is placed at room temperature for 10min after being mixed by inversion.
8) by mixed liquor, 12000rmp is centrifuged 10min under the conditions of 4 DEG C.
9) supernatant liquid is discarded, the processed ethanol solution of 75%DEPC is added and washs RNA precipitate repeatedly.
10) 7500rmp is centrifuged 5min under the conditions of 4 DEG C, discards liquid completely.
11) to ethyl alcohol evaporating completely, 30-50 μ L DEPC water is added, dissolves RNA precipitate.- 80 DEG C of preservations.
1.3 fluorescent quantitation qRT-PCR
SYBR Premix ExTaqII (the TliR NaseH provided according to Takara biomedical technology (Beijing) company
Plus) specification carries out fluorescent quantitation qRT-PCR.
Reaction solution is prepared on ice according to following table, carries out fluorescent quantitation qRT-PCR experiment.
QRT-PCR reaction system
The clone of 1.4MtbZIP10 gene and vector construction
The Prime STAR GXL Premix high-fidelity DNA provided according to Takara biomedical technology (Beijing) company
Polymerase specification carries out PCR reaction.According to Tiangeng biochemical technology company provide pEASY-T1Cloning Kit specification into
Row connection configures reaction system, 25 DEG C of reaction 20min according to table.Add A respectively at genetic fragment both ends, after by pCXSN plant table
Up to carrier Xcm Ι digestion with restriction enzyme.Segment after respective digestion is attached and is converted.
1.5 conversions M. truncatula hairy
1) activated MtbZIP10-pCXSN/Arqual I agrobacterium rhizogenes is spread evenly across and is resisted containing Kan and Strep
On the LB solid medium of raw element, 28 DEG C of inversion cultures are for 24 hours.
2) tip of a root is cut when root long of M. truncatula to 2cm or so, wound is gently dipped on Aqual I mycoderm,
Then it is arranged on not antibiotic F culture medium, is cultivated under light.
3) it after new root is grown, changes on the B5 solid medium containing Hyg resistance, continues to cultivate under light.
4) when growth of hair root is vigorous, the DNA of transgenic hairy root is extracted, screening transgenic is hairy positive.
5) positive hairy phenotype is observed, and the metabolite content of high expression strain is detected.
1.6 measuring the content of anthocyanidin
1) M. truncatula aerial part (stem and leaf) fresh tissue sample under the conditions of same growing environment is taken.
2) liquid nitrogen is added into organization material, is freeze-dried for 24 hours after grinding.10mg is accurately weighed, and 5 times of volumes are added
Methanol (hydrochloric acid for being 0.1% containing volume ratio), ultrasonic 30min, 12h under the conditions of being placed in 4 DEG C.
3) ultrasound 30min, 10000rmp are centrifuged 5min, take supernatant in new centrifuge tube again.
4) 750 μ L water and 750 μ L chloroforms are added, 10000rmp is centrifuged 5min after oscillation mixes, take supernatant in it is new from
In heart pipe.
5) it is detected at 530nm with ultraviolet specrophotometer.Using mole light absorption of Cyanidin -3-O- glucose as
The content of standard items estimation anthocyanidin.
The content of the 1.7 total flavonoids of measurement
1) the M. truncatula fresh tissue sample (including plant and seed) under same growing environment is taken, liquid nitrogen grinding is added
It is extremely powdered.
2) 50mg powder is accurately weighed, 1000 μ L, 80% methanol, ultrasonic 30min is added.
3) 12000rpm is centrifuged 20min, takes supernatant into new centrifuge tube.
4) 200 μ L supernatants are taken, 800 μ L ddH are added2O and 60 μ L 5%NaNO2, 5min is stood after mixing.
5) 60 μ L 10%AlCl are added3, 10min is stood after mixing.
6) 400 μ L 1mol/L NaOH solutions are added, add ddH2O is mended to 2mL (about 480 μ L).
7) it is read using ultraviolet specrophotometer in OD 510nm.Water is as blank control.Estimated with Kaempferol standard curve
Calculate the content of total flavonoids.
Two, main result
The clone of 2.1MtbZIP10 and bioinformatic analysis
Present invention ORF complete to MtbZIP10 gene is cloned and sequencing analysis, and discovery MtbZIP10 full length gene is
6542bp is made of two identical open reading frame tandem sequence repeats, contains 15 exons and 16 intrones.
MtbZIP10 gene encodes a protein sequence containing 709 amino acid；The a length of 2127bp of its transcript.MtbZIP10 transcription
The amino acid sequence of the factor and other 6 have verified that the ammonia that relevant bZIP transcription factor is synthesized to anthocyanidin and procyanidine
Base acid sequence carries out Multiple sequence alignments, find bZIP transcription factor that this 7 belong to different subfamilies conserved domain all
There are two conservative amino acid sites for tool, it is likely that related (Fig. 1) with regulation flavonoids biosynthesis.By MtbZIP10 transcribe because
The amino of 27 from species such as arabidopsis, the soybean and rice bZIP transcription factor of the amino acid sequence and known function of son
Acid sequence carries out Phylogenetic analysis, the bZIP that discovery MtbZIP10 transcription factor is synthesized with regulation anthocyanidin and procyanidine
Transcription factor clusters (Fig. 2).Further MtbZIP10 transcription factor is demonstrated from the angle of phyletic evolution to be likely to participate in regulation
Flavonoids metabolic pathway.
The identification of 2.2 mutant
The present invention carries out Phenotypic Observation, discovery and open country to the mutant NF2620 that obtained MtZIP10 mutates
Raw type R108 is compared, and the seedling hypocotyl part of the mutant and base of the plant lack anthocyanin accumulation, and the adaxial and its surface of leaf and
Red garland and the spot missing that abaxial side is formed by anthocyanin accumulation.In addition, with R108 kind skin due to procyanidine product
Yellowish-brown that is tired and showing is different, and NF2620 mutant sectors seed kernel seed coat colour is white.The above results show mutation
Body NF2620 causes anthocyanidin due to the mutation of MtZIP10 gene and procyanidin content is greatly decreased.
The distribution expression pattern of 2.3MtbZIP10
The present invention has detected expression of the MtbZIP10 gene in M. truncatula, has acquired M. truncatula respectively
Root, stem, leaf, flower and Post flowering 10 days, 12 days, 16 days, 20 days, 24 days, the seeds of 36 days harvests, extract its RNA.RNA reversion
CDNA is obtained after record, carries out fluorescent quantitation qRT-PCR experiment by internal reference of actin；In respectively being organized with wild type M. truncatula A17
The expression of MtbZIP10 gene is 1, and detection MtbZIP10 gene is sent out in mutant NF2620 different tissues and seed difference
Educate the relative expression levels in period.
The result shows that can detect the expression of MtbZIP10 at each position of M. truncatula, but expression quantity is lower.
Wherein, the expression highest (Fig. 3) in stem and 12 days seeds.Anthocyanidin mainly accumulates in the stem and leaf of M. truncatula,
Procyanidine accumulation highest in seed kind skin, the i.e. expression of MtbZIP10 gene and anthocyanidin and procyanidine accumulate
Position is consistent.
The expression analysis of flavonoids synthesis regulation key gene in 2.4 mutant NF2620
The present invention is by qRT-PCR to participation flavonoids synthesis in wild type and mutant M. truncatula 50d seedling leaves
Several key structure genes of approach and the expression of transcription factor have carried out quantitative analysis；Including CHS, CHI, ANS,
DFR1, DFR2, UGT72L1, TT8, WD40 and MATE1.
Compared with wild type, CHS, DFR1, DFR2 and ANS gene do not express (Fig. 4 A, C-E) in mutant；UGT72L1,
The expression quantity of TT8 and MATE1 all significantly reduces (Fig. 4 F-G, I), this illustrate in mutant NF2620 with flavonoids biosynthesis phase
The significant decrease of the expression quantity of the key structure gene and controlling gene of pass causes anthocyanidin and procyanidine in mutant
Lack.
2.5MtbZIP10 converts M. truncatula hairy
In order to further prove the function of MtbZIP10, by the driving of tobacco mosaic virus (TMV) CaMV 35S promoter
MtbZIP10 gene is connected on plant expression pCXSN；Recombinant plasmid is transferred in agrobacterium rhizogenes Arqual I again, is used
The method of PCR confirms gene successful expression (Fig. 5) in Agrobacterium.The agrobacterium rhizogenes of MtbZIP10-pCXSN will be had
Arqual I is transformed into hairy of mutant NF2620 and wild type M. truncatula A17, is obtained and is turned MtbZIP10 gene
Hairy of NF2620 and A17 hairy of overexpression MtbZIP10 gene.Zero load is also transformed into mutant simultaneously
It is used as negative control in NF2620 and wild type A17 hairy, observe and detects the complementary hairy stock system of positive gene and the positive
The accumulation of anthocyanidin in the hairy stock system of gene overexpression.
Present invention discover that the A17 of hairy of the NF2620 for turning MtbZIP10 gene and overexpression MtbZIP10 gene is hairy
The color of root is deeper than negative control, and anthocyanin accumulation is more than negative control (growth of hair root animated period in transgenic hairy root
Preceding observation).Hairy of the positive is chosen, 10mg is weighed and extracts its anthocyanidin progress assay.The result shows that turning MtbZIP10 base
Anthocyanidin content in the NF2620 of cause hairy remains basically stable with negative control, the A17 hair of overexpression MtbZIP10 gene
Anthocyanidin content in shape root is significantly higher than negative control.Illustrate MtbZIP10 gene overexpression in wild type M. truncatula
It can promote anthocyanin accumulation, while also further illustrate that MtbZIP10 transcription factor has the function of regulation anthocyanidin biosynthesis
(Fig. 6).
2.6MtbZIP10 the expression under illumination and abscisic acid inductive condition
The present invention also has detected whether MtbZIP10 gene is induced by abscisic acid, respectively in 0h, 1h, 3h, 6h, 9h and 12h
The abscisic acid for applying 100mg/L to wild type M. truncatula foreign aid, is detected respectively using semiquantitive PCR and quantitative fluorescent PCR
MtbZIP10 expression conditions (Fig. 7).The results show that the expression of MtbZIP10 gene and foreign aid's application abscisic acid be not obvious
Correlation (Fig. 7).The present invention has also carried out lighting process to wild type M. truncatula simultaneously.Semiquantitive PCR and quantitative fluorescent PCR
The result shows that: as the expression quantity of the increase MtbZIP10 gene of light application time increases (Fig. 7), illustrate that MtbZIP10 participates in light and is situated between
The anthocyanin accumulation led.
Three, final conclusion
1. M. truncatula is model legume and good forage-alfalfa sibling species, there is important grind
Study carefully value.The present invention demonstrates bZIP transcription factor MtbZIP10 gene in M. truncatula for the first time and encodes turning for a bZIP type
Record the factor, the accumulating level of the flavonoids such as expression of the MtbZIP10 gene in M. truncatula and anthocyanidin and condensed tannin
Correlation, MtbZIP10 are the key transcription factors for regulating and controlling the biosynthesis of M. truncatula flavonoids.
2. being widely present in plant using anthocyanidin and condensed tannin as the flavonoids of representative, not only there is protection
Plant also has the life of anticancer, disease-resistant and anti-oxidant aspect from the function of biology and abiotic infringement and attraction insect pollination
Object characteristic.The expression of the multiple genes of M. truncatula MtbZIP10 gene regulation flavonoid path, overexpression can be improved
The content of flavone compound provides candidate gene and beneficial reference for the metabolic engineering of plant flavonoids, while to change
Good alfalfa quality has established molecular basis.
Operation technique in the present embodiment without specified otherwise is the prior art, therefore excessive explanation herein.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>a kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2127
<212> DNA
<213>MtbZIP10 gene (Medicago truncatula)
<400> 1
atggcagata acagtcaaaa gacagaagat tttgacactg atgataaaaa taatcaatgt 60
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aaggatcaat gcaagactaa gggtcaatct gatgagcaca agactcttcg tcggctgatg 180
cagaatcgcg aggctgcaag gaaaagtagg ttaaggaaaa aggcttatgt gcaacaattg 240
gagaacagtc gacttaggct tgctcaaata gaacatgagc ttcaacaagt acgtcaacag 300
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tctgggttgt ggaagacacc tgcagaaaga tgtttcatgt ggcttggtgg attccgttca 600
tccgaacttc tcaagataat tagaaaccac ctcgaggcgt taacggatca gcaattgatg 660
gcgatcttca atctgcagca ttcttgtcaa caggctgaag atgcattatc tcaaggaatg 720
gaaggtttgc aacaatctct ttcagagaca ctttcctcca cgtccactgg atctggaaat 780
gttgttgagt atatgggtca aatggctctt tcaatggcca agctttccac actggagact 840
ttcattcatc aggcagatat cttgaggcaa caaacactgc aacagatgcg tcgaattttg 900
actgcgcacc aagctgctcg tgctctcctt gtcataaatg atttcatttc acgaatcaga 960
gctcttaatt cattatggca tgtgtttggt gcaaagtcta acaatgtcac tgttgttgca 1020
agtaacttgc agtatggaac attcaacagt aacattggtt cagcttcttc ttcaggcatg 1080
gcagataaca gtcaaaagac agaagatttt gacactgatg ataaaaataa tcaaagtggg 1140
aatgtcctta tttttctgtg tttatcaacc gtgtcttggt gcaatggtgt tggaaatgag 1200
gctcttgtag ttgttgaatc caaggatcaa tgcaagacta agggtcaatc tgatgagcac 1260
aagactcttc gtcggctgat gcagaatcgc gaggctgcaa ggaaaagtag gttaaggaaa 1320
aaggcttatg tgcaacaatt ggagaacagt cgacttaggc ttgctcaaat agaacatgag 1380
cttcaacaag tacgtcaaca gggtacattt gttgcacctg gagttacagc ggatcatggt 1440
cattcaattg ttggaaacag taatgcaggt tcggtagcat ttgatatgga ctatgctcgt 1500
tgggtagacg agcatcaacg actaatcaat gacataagat cagctataaa ttctcaaatg 1560
ggtgataatg aactgcatct tcttgttgat ggtgtcatgg tacattatga tgaattatac 1620
aagttgaaga gcataggtgc aaaggctgat gtatttcaca tactttctgg gttgtggaag 1680
acacctgcag aaagatgttt catgtggctt ggtggattcc gttcatccga acttctcaag 1740
ataattagaa accacctcga ggcgttaacg gatcagcaat tgatggcgat cttcaatctg 1800
cagcattctt gtcaacaggc tgaagatgca ttatctcaag gaatggaagg tttgcaacaa 1860
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ggtcaaatgg ctctttcaat ggccaagctt tccacactgg agactttcat tcatcaggca 1980
gatatcttga ggcaacaaac actgcaacag atgcgtcgaa ttttgactgc gcaccaagct 2040
gctcgtgctc tccttgtcat aaatgatttc atttcacgaa tcagagctct taattcatta 2100
tggtcagcat ttcctaaaga atactaa 2127
<210> 2
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<212> PRT
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Met Ala Asp Asn Ser Gln Lys Thr Glu Asp Phe Asp Thr Asp Asp Lys
1 5 10 15
Asn Asn Gln Cys Leu Ser Thr Val Ser Trp Cys Asn Gly Val Gly Asn
20 25 30
Glu Ala Leu Val Val Val Glu Ser Lys Asp Gln Cys Lys Thr Lys Gly
35 40 45
Gln Ser Asp Glu His Lys Thr Leu Arg Arg Leu Met Gln Asn Arg Glu
50 55 60
Ala Ala Arg Lys Ser Arg Leu Arg Lys Lys Ala Tyr Val Gln Gln Leu
65 70 75 80
Glu Asn Ser Arg Leu Arg Leu Ala Gln Ile Glu His Glu Leu Gln Gln
85 90 95
Val Arg Gln Gln Val Gln Gln Leu Lys Lys Ile Phe His Leu Val Lys
100 105 110
Leu Val Cys Ser Val Ala Phe Asp Met Asp Tyr Ala Arg Trp Val Asp
115 120 125
Glu His Gln Arg Leu Ile Asn Asp Ile Arg Ser Ala Ile Asn Ser Gln
130 135 140
Met Gly Asp Asn Glu Leu His Leu Leu Val Asp Gly Val Met Val His
145 150 155 160
Tyr Asp Glu Leu Tyr Lys Leu Lys Ser Ile Gly Ala Lys Ala Asp Val
165 170 175
Phe His Ile Leu Ser Gly Leu Trp Lys Thr Pro Ala Glu Arg Cys Phe
180 185 190
Met Trp Leu Gly Gly Phe Arg Ser Ser Glu Leu Leu Lys Ile Ile Arg
195 200 205
Asn His Leu Glu Ala Leu Thr Asp Gln Gln Leu Met Ala Ile Phe Asn
210 215 220
Leu Gln His Ser Cys Gln Gln Ala Glu Asp Ala Leu Ser Gln Gly Met
225 230 235 240
Glu Gly Leu Gln Gln Ser Leu Ser Glu Thr Leu Ser Ser Thr Ser Thr
245 250 255
Gly Ser Gly Asn Val Val Glu Tyr Met Gly Gln Met Ala Leu Ser Met
260 265 270
Ala Lys Leu Ser Thr Leu Glu Thr Phe Ile His Gln Ala Asp Ile Leu
275 280 285
Arg Gln Gln Thr Leu Gln Gln Met Arg Arg Ile Leu Thr Ala His Gln
290 295 300
Ala Ala Arg Ala Leu Leu Val Ile Asn Asp Phe Ile Ser Arg Ile Arg
305 310 315 320
Ala Leu Asn Ser Leu Trp His Val Phe Gly Ala Lys Ser Asn Asn Val
325 330 335
Thr Val Val Ala Ser Asn Leu Gln Tyr Gly Thr Phe Asn Ser Asn Ile
340 345 350
Gly Ser Ala Ser Ser Ser Gly Met Ala Asp Asn Ser Gln Lys Thr Glu
355 360 365
Asp Phe Asp Thr Asp Asp Lys Asn Asn Gln Ser Gly Asn Val Leu Ile
370 375 380
Phe Leu Cys Leu Ser Thr Val Ser Trp Cys Asn Gly Val Gly Asn Glu
385 390 395 400
Ala Leu Val Val Val Glu Ser Lys Asp Gln Cys Lys Thr Lys Gly Gln
405 410 415
Ser Asp Glu His Lys Thr Leu Arg Arg Leu Met Gln Asn Arg Glu Ala
420 425 430
Ala Arg Lys Ser Arg Leu Arg Lys Lys Ala Tyr Val Gln Gln Leu Glu
435 440 445
Asn Ser Arg Leu Arg Leu Ala Gln Ile Glu His Glu Leu Gln Gln Val
450 455 460
Arg Gln Gln Gly Thr Phe Val Ala Pro Gly Val Thr Ala Asp His Gly
465 470 475 480
His Ser Ile Val Gly Asn Ser Asn Ala Gly Ser Val Ala Phe Asp Met
485 490 495
Asp Tyr Ala Arg Trp Val Asp Glu His Gln Arg Leu Ile Asn Asp Ile
500 505 510
Arg Ser Ala Ile Asn Ser Gln Met Gly Asp Asn Glu Leu His Leu Leu
515 520 525
Val Asp Gly Val Met Val His Tyr Asp Glu Leu Tyr Lys Leu Lys Ser
530 535 540
Ile Gly Ala Lys Ala Asp Val Phe His Ile Leu Ser Gly Leu Trp Lys
545 550 555 560
Thr Pro Ala Glu Arg Cys Phe Met Trp Leu Gly Gly Phe Arg Ser Ser
565 570 575
Glu Leu Leu Lys Ile Ile Arg Asn His Leu Glu Ala Leu Thr Asp Gln
580 585 590
Gln Leu Met Ala Ile Phe Asn Leu Gln His Ser Cys Gln Gln Ala Glu
595 600 605
Asp Ala Leu Ser Gln Gly Met Glu Gly Leu Gln Gln Ser Leu Ser Glu
610 615 620
Thr Leu Ser Ser Thr Ser Thr Gly Ser Gly Asn Val Val Glu Tyr Met
625 630 635 640
Gly Gln Met Ala Leu Ser Met Ala Lys Leu Ser Thr Leu Glu Thr Phe
645 650 655
Ile His Gln Ala Asp Ile Leu Arg Gln Gln Thr Leu Gln Gln Met Arg
660 665 670
Arg Ile Leu Thr Ala His Gln Ala Ala Arg Ala Leu Leu Val Ile Asn
675 680 685
Asp Phe Ile Ser Arg Ile Arg Ala Leu Asn Ser Leu Trp Ser Ala Phe
690 695 700
Pro Lys Glu Tyr
705
Claims (4)
1. a kind of transcription factor gene of regulation plant flavonoids synthesis, which is characterized in that the transcription factor gene is labeled as
MtbZIP10, the gene order is as shown in SEQ ID NO.1.
2. the protein that the transcription factor gene of claim 1 encodes, which is characterized in that its amino acid sequence such as SEQ ID
Shown in NO.2.
3. a kind of purposes of gene described in claim 1, which is characterized in that its purposes is to inhibit to be somebody's turn to do in M. truncatula mutant
When the expression of gene, anthocyanidin in M. truncatula aerial part flavonoids is caused to significantly reduce, seed Procyanidins obviously subtract
It is few.
4. a kind of purposes of gene described in claim 1, which is characterized in that its purposes is the method mediated using agrobacterium rhizogenes
By MtbZIP10 gene in hairy middle overexpression of M. truncatula, the content of its anthocyanidin can be improved, show
MtbZIP10 transcription factor regulates and controls the biosynthesis of anthocyanidin in hairy.
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Cited By (1)
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| CN114891807A (en) * | 2022-05-30 | 2022-08-12 | 中国农业科学院北京畜牧兽医研究所 | Application of CYP450 gene in regulation and control of synthesis of soybean isoflavone |
| CN114891807B (en) * | 2022-05-30 | 2024-01-26 | 中国农业科学院北京畜牧兽医研究所 | Application of CYP450 gene in regulating and controlling soybean isoflavone synthesis |
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