CN109486838B - Transcription factor gene for regulating plant flavonoid synthesis and application thereof - Google Patents
Transcription factor gene for regulating plant flavonoid synthesis and application thereof Download PDFInfo
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- 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 transcription factor gene for regulating and controlling plant flavonoid synthesis, which is MtbZIP10 gene identified from medicago truncatula belonging to leguminous plants, and the gene sequence is shown as SEQ ID NO. 1. Furthermore, the invention discloses the function and application of the gene. The invention has the advantages that transcription factor genes which can positively regulate and control the synthesis of flavonoid compounds are cloned from the medicago truncatula L.of leguminous plants, the functions of the transcription factor genes are systematically identified, and the genes are found to activate a plurality of genes of a flavonoid biosynthesis pathway and improve the content of the flavonoid compounds.
Description
Technical Field
The invention belongs to the technical field of gene cloning, and particularly relates to a transcription factor gene for regulating and controlling plant flavonoid synthesis and application thereof.
Background
Flavonoid compounds are a large group of important secondary metabolites widely distributed in roots, stems, leaves, flowers, fruits and seed coats of plants, including flavonoids, isoflavones, flavonols, anthocyanidins, procyanidins, and the like. The anthocyanin has the functions of protecting plants from biological attack and attracting insects to pollinate, and can respond to biotic and abiotic stress, eliminate oxygen free radicals and protect plants from damage caused by high-density illumination. Meanwhile, the anthocyanin has certain health care functions in the aspects of cancer resistance, disease resistance and oxidation resistance. The other procyanidin in the flavonoid compound has the function of protecting plants from the invasion mechanism of herbivores and pathogenic bacteria, and the appropriate amount of procyanidin can effectively prevent livestock pasture from flatulence in the pasture and improve nitrogen nutrition of ruminant livestock. Meanwhile, procyanidine is beneficial to human health, so the metabolism regulation mechanism of flavonoid is a research hotspot in the field of plant secondary metabolism.
Although transcription factor genes of transcription factors regulating flavonoid biosynthesis, such as MYB, bHLH, and WDR, have been reported, other types of transcription factors have been relatively poorly studied. bZIP transcription factors (bZIPs) are found in humans, animals, plants, microorganisms and insects, and are the most widely distributed and conserved transcription factors. Plant bZIP transcription factors are also tissue expression specific, and many plant bZIP transcription factors are constitutively expressed in roots with little or no expression in stems and leaves. Plant bZIP transcription factors are involved in a variety of biological processes including seed maturation, germination, flower development, plant senescence, photomorphogenesis, and the like. Although bZIP family transcription factors in other individual plants can participate in regulation of anthocyanin biosynthesis, no related research on bZIP transcription factor regulation flavonoid synthesis pathway exists in medicago truncatula, and research and application of the bZIP transcription factor regulation flavonoid synthesis pathway are limited, so that cloning and identifying bZIP transcription factor genes which have a novel function of regulating flavonoid synthesis and can improve flavonoid content in plant plants have important theoretical and application values.
Disclosure of Invention
Aiming at the problems that bZIP transcription factor genes in flavonoid biosynthesis approaches are insufficient and flavonoid content is difficult to greatly improve in the existing research, the invention provides a transcription factor gene for regulating plant flavonoid synthesis and application thereof.
In order to achieve the purpose, the technical scheme provided by the invention is that the bZIP transcription factor gene is MtbZIP10, and the gene sequence of the bZIP transcription factor gene is shown in SEQ ID No. 1. The MtbZIP10 gene discovered by the invention is cloned from medicago truncatula, is a brand new plant bZIP transcription factor gene, enriches the variety of flavonoid bZIP transcription factors, and can improve the application of plants in resistance and nutritional quality because the bZIP transcription factors can positively regulate and control the functions of flavonoid, especially anthocyanin and procyanidine.
Furthermore, the amino acid sequence of the protein coded by the bZIP transcription factor gene is shown as SEQ ID NO. 2.
Furthermore, the bZIP transcription factor gene is used for positively regulating flavonoid biosynthesis pathways, particularly key genes of flavonoid pathways such as CHS, DFR, ANS, MATE1 and the like, and the MtbZIP10 is proved to play a regulating function by regulating the genes, and is the bZIP type transcription factor gene which is found in medicago truncatula and is used for regulating the flavonoid pathways.
Furthermore, when the expression of the MtbZIP10 gene in medicago truncatula is inhibited, the anthocyanin in the aerial part and the proanthocyanidin in the seeds are reduced.
Furthermore, when the MtbZIP10 gene is over-expressed in the hairy root of medicago truncatula, the anthocyanin content of the hairy root is obviously increased relative to an untransformed control, which indicates that the over-expression of the gene is used for improving the accumulation of anthocyanin.
By adopting the technical scheme, the invention has the following beneficial effects that the flavonoid isopentenyl transferase gene is cloned from the medicago truncatula, the function of the flavonoid isopentenyl transferase gene is systematically identified, the mutation of the gene is found to influence the contents of anthocyanin and proanthocyanidin, and the overexpression of the gene can improve the content of anthocyanin.
Drawings
FIG. 1 multiple alignment analysis of MtbZIP10 with other plant bZIP transcription factor amino acid sequences. Consensus indicates amino acids with perfect sequence identity, with conserved amino acids highlighted in black and grey: black font dark grey background represents 100% identity; black font off-white background represents 50% identity; the black font white background represents that the similarity is below 50%.
FIG. 2 phylogenetic tree analysis of plant bZIP transcription factors. DNAMAN was used to construct an evolutionary tree, which was marked with an asterisk after MtbZIP 10. The reliability of the node is estimated based on the bootstrap support (%) of 1000 repetitions.
FIG. 3 tissue expression profile of MtbZIP 10. Expression of the MtbZIP10 gene in seeds 10, 12, 16, 20, 24, 36 days after root, stem, leaf, flower, pod and pollination. Data are shown as mean ± standard error, with three biological replicates included in each experiment.
FIG. 4 shows that anthocyanin synthesis in R108 and NF2620 regulates relative expression of key genes. An ANS: an anthocyanin synthase; CHS: a chalcone synthase; CHI: chalcone isomerase; DFR 1/2: dihydroflavonol reductase; UGT72L 1: a glycosyltransferase; TT 8: TT8 transcription factor; WD 40: WD40 repeat protein; MATE 1: MATE1 transporter. Data are shown as mean ± standard error, with three biological replicates included in each experiment.
FIG. 5 confirmation of the construction of the plant expression vector using PCR. Wherein the left side is a DNA molecular weight marker; the right side is the PCR assay of agrobacterium positive clones containing MtbZIP10 gene.
FIG. 6 anthocyanin content assay in transgenic hairy roots. A, anthocyanin content in hairy roots against the background of mutant NF260 and its negative control. B, anthocyanin content in hairy roots against the background of wild type a17 and its negative control.
FIG. 7 Effect of abscisic acid and light on MtbZIP10 expression. A, C: r108 seedlings were treated with 50 μ M ABA at the indicated time intervals and the expression level of MtbZIP10 was examined by semi-quantitative pcr (a) and real-time fluorescent quantitative pcr (c). The fraction without ABA treatment was 1. B, D: r108 seedlings are treated in the dark at normal temperature for 3 days, then the seedlings are respectively treated by light for 1, 3, 6, 9 and 12 hours, and the expression level of MtbZIP10 is tested by semiquantitative PCR (A) and real-time fluorescent quantitative PCR (C). The dark-treated fraction was 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
First, main experimental materials and methods
1.1CTAB method for extracting medicago truncatula genome DNA
1) Taking about 100mg of medicago truncatula tissue material, and putting the medicago truncatula tissue material and a steel ball into a 2mL centrifuge tube; liquid nitrogen was added and the shaking was continued until the tissue material was sufficiently ground into powder.
2) Adding 500 μ of LCTAB extract (preheated at 65 deg.C), vortexing, and mixing.
3) The mixture was subjected to a 65 ℃ water bath for 30min while shaking.
4) And cooling the mixed solution to room temperature, adding chloroform with the same volume, and reversing and uniformly mixing.
5) Centrifuge at 12000rpm for 10min at room temperature.
6) After centrifugation, the mixture was separated into layers, and the supernatant was carefully pipetted into a 1.5mL centrifuge tube.
7) An equal volume (about 500. mu.L) of isopropanol was added, mixed by inversion, and allowed to stand at room temperature for 30 min.
8) Centrifuge at 12000rpm for 10min at 4 ℃.
9) The upper solution was discarded, and the DNA precipitate was washed with a 70% ethanol solution.
10) After the ethanol is evaporated, 30-50. mu.L of sterile water is added to dissolve the DNA precipitate. Storing at-20 deg.C.
1.2TRNzol-A+Method for extracting medicago truncatula RNA
1) Approximately 200mg of medicago truncatula tissue material was placed in a 1.5mL RNase-free centrifuge tube, frozen in liquid nitrogen, and rapidly ground to powder using a sterilized frozen glass rod.
2) 1mL TRNzol-A was added+Vortex the extract, mix the mixture evenly, and stand for 5min at room temperature.
3) The mixture was centrifuged at 12000rmp for 10min at 4 ℃.
4) The supernatant was pipetted into a 1.5mL RNase-free centrifuge tube.
5) Adding 200 μ L RNase-free chloroform, repeatedly reversing, mixing, and standing at room temperature for 5 min.
6) The mixture was centrifuged at 12000rmp for 10min at 4 ℃.
7) And (3) sucking the supernatant into a 1.5mL RNase-free new centrifuge tube, adding the same volume of RNase-free isopropanol, reversing, uniformly mixing, and standing at room temperature for 10 min.
8) The mixture was centrifuged at 12000rmp for 10min at 4 ℃.
9) The supernatant liquid was discarded, and the RNA precipitate was washed repeatedly with 75% DEPC-treated ethanol solution.
10) Centrifuging at 7500rmp for 5min at 4 deg.C, and completely discarding the liquid.
11) After the ethanol was completely evaporated, 30-50. mu.L of DEPC water was added to dissolve the RNA precipitate. Storing at-80 deg.C.
1.3 fluorescent quantitation qRT-PCR
Fluorescent quantitative qRT-PCR was performed according to the SYBR Premix ExTaqII (TliR NaseH Plus) instructions supplied by Takara biomedical technologies (Beijing).
Reaction solutions were prepared on ice according to the following table and subjected to fluorescent quantitative qRT-PCR experiments.
qRT-PCR reaction system
1.4 cloning and vector construction of MtbZIP10 Gene
The PCR reaction was performed according to the Prime STAR GXL Premix high fidelity DNA polymerase instructions provided by Takara biomedical technologies (Beijing) Inc. The reaction system was prepared according to the protocol of pEASY-T1Cloning Kit (Takara Biochemical science) provided by Tiangen Biochemical Co., Ltd and reacted at 25 ℃ for 20 min. A is added at two ends of the gene fragment respectively, and then the pCXSN plant expression vector is cut by Xcm I restriction enzyme. And connecting and transforming the fragments subjected to respective enzyme digestion.
1.5 transformation of hairy root of Medicago truncatula
1) The activated MtbZIP10-pCXSN/Arqual I agrobacterium rhizogenes is evenly spread on an LB solid culture medium containing Kan and Strep antibiotics, and is inversely cultured for 24h at 28 ℃.
2) Cutting off the root tip when the root of the medicago truncatula grows to about 2cm, slightly dipping the wound on Aqual I mycoderm, then arranging on an F culture medium without antibiotics, and culturing under light.
3) After new roots grow out, the culture medium is changed to a B5 solid medium containing Hyg resistance, and the culture is continued under the light.
4) When the hairy root grows vigorously, DNA of the transgenic hairy root is extracted, and transgenic positive hairy roots are screened.
5) And observing positive hairy root phenotype, and detecting the metabolite content of the high-expression strain.
1.6 determination of the anthocyanin content
1) Fresh tissue samples of aerial parts (stems and leaves) of Medicago truncatula under the same growth environment were taken.
2) Liquid nitrogen was added to the tissue material, ground and freeze-dried for 24 h. Accurately weighing 10mg, adding 5 times volume of methanol (containing 0.1% hydrochloric acid), ultrasonic treating for 30min, and standing at 4 deg.C for 12 hr.
3) Performing ultrasonic treatment for 30min again, centrifuging for 5min at 10000rmp, and collecting supernatant in a new centrifuge tube.
4) Adding 750 μ L water and 750 μ L chloroform, shaking, mixing, centrifuging at 10000rmp for 5min, and collecting supernatant in a new centrifuge tube.
5) Detection was performed with an ultraviolet spectrophotometer at 530 nm. The content of anthocyanin is estimated by taking the molar light absorption of cyanidin-3-O-glucose as a standard substance.
1.7 determination of Total Flavonoids content
1) Taking a fresh tissue sample (including plants and seeds) of the medicago truncatula under the same growth environment, adding liquid nitrogen, and grinding into powder.
2) 50mg of the powder is accurately weighed, 1000 mul of 80% methanol is added, and the mixture is subjected to ultrasonic treatment for 30 min.
3) Centrifuging at 12000rpm for 20min, and taking the supernatant into a new centrifuge tube.
4) Collecting 200 μ L supernatant, adding 800 μ L ddH2O and 60. mu.L of 5% NaNO2And standing for 5min after mixing uniformly.
5) 60 μ L of 10% AlCl was added3Mixing, and standing for 10 min.
6) 400 μ L of 1mol/L NaOH solution was added, followed by ddH2O was made up to 2mL (about 480. mu.L).
7) Read at OD 510nm using a UV spectrophotometer. Water was used as a blank control. The total flavonoid content was estimated using kaempferol standard curve.
Second, the main results
2.1 cloning and bioinformatic analysis of MtbZIP10
The invention clones and analyzes the complete ORF of the MtbZIP10 gene, and finds that the full length of the MtbZIP10 gene is 6542bp, consists of two identical open reading frames which are connected in series and repeated, and contains 15 exons and 16 introns. The MtbZIP10 gene encodes a protein sequence containing 709 amino acids; the transcript length is 2127 bp. The amino acid sequence of MtbZIP10 transcription factor and the amino acid sequences of another 6 bZIP transcription factors that have been verified to be related to anthocyanin and procyanidine synthesis were subjected to multiple sequence alignment, and it was found that the 7 bZIP transcription factors belonging to different subfamilies all have two conserved amino acid sites in the conserved domain, and are likely to be involved in regulating flavonoid biosynthesis (fig. 1). Phylogenetic tree analysis is carried out on the amino acid sequence of the MtbZIP10 transcription factor and the amino acid sequences of 27 bZIP transcription factors from arabidopsis, soybean, rice and other species with known functions, and the MtbZIP10 transcription factor is clustered with the bZIP transcription factor for regulating and controlling the synthesis of anthocyanin and procyanidine (figure 2). It is further demonstrated from a phylogenetic perspective that MtbZIP10 transcription factor is likely involved in regulating flavonoid metabolic pathways.
2.2 identification of mutants
The phenotype observation of the mutant NF2620 with the mutation of the obtained MtZIP10 shows that compared with the wild type R108, the seedling hypocotyl part and the plant stem part of the mutant lack anthocyanin accumulation, and the paraxial surface and the abaxial surface of the leaf lack red flower rings and spots formed by anthocyanin accumulation. In addition, the NF2620 mutant partial seed coat appeared white in color, unlike the yellow-brown color exhibited by the R108 seed coat due to procyanidin accumulation. The results show that the mutant NF2620 has greatly reduced anthocyanin and procyanidine contents due to mutation of the MtZIP10 gene.
2.3 tissue expression profiling of MtbZIP10
The invention detects the expression of MtbZIP10 gene in alfalfa, collects the roots, stems, leaves, flowers and seeds harvested 10 days, 12 days, 16 days, 20 days, 24 days and 36 days after flowering, respectively, and extracts RNA thereof. Carrying out reverse transcription on RNA to obtain cDNA, and carrying out a fluorescence quantitative qRT-PCR experiment by taking actin as an internal reference; the expression level of MtbZIP10 gene in each tissue of wild medicago truncatula A17 is 1, and the relative expression level of MtbZIP10 gene in different tissues and different development periods of the mutant NF2620 is detected.
The results show that the expression of MtbZIP10 can be detected in all parts of medicago truncatula, but the expression level is low. Among them, the expression level was highest in the stem and in the 12-day seed (FIG. 3). The anthocyanin is mainly accumulated in stems and leaves of medicago truncatula, and the accumulation amount of the procyanidine in seed coats is the highest, namely the expression condition of the MtbZIP10 gene is consistent with the accumulation position of the anthocyanin and the procyanidine.
2.4 expression analysis of flavonoid Synthesis regulatory Key Gene in mutant NF2620
Quantitative analysis is carried out on expression levels of several key structural genes and transcription factors participating in a flavonoid synthesis way in leaves of 50d seedlings of wild type and mutant medicago truncatula L.var.truncatula L.and transcription factors by qRT-PCR; including CHS, CHI, ANS, DFR1, DFR2, UGT72L1, TT8, WD40 and MATE 1.
CHS, DFR1, DFR2, and ANS genes were not expressed in the mutant compared to the wild type (FIG. 4A, C-E); the expression levels of UGT72L1, TT8 and MATE1 were all significantly reduced (FIG. 4F-G, I), which indicates that significant reduction in the expression levels of key structural and regulatory genes associated with flavonoid biosynthesis in mutant NF2620 results in a lack of anthocyanidins and procyanidins in the mutant.
2.5MtbZIP10 transformation of hairy root of Medicago truncatula
To further demonstrate the function of MtbZIP10, the MtbZIP10 gene driven by the tobacco mosaic virus CaMV 35S promoter was ligated to plant expression pCXSN; the recombinant plasmid was then transferred into Agrobacterium rhizogenes Arqual I, and successful expression of the gene in Agrobacterium was confirmed using PCR (FIG. 5). The Agrobacterium rhizogenes Arqual I with MtbZIP10-pCXSN is transformed into hairy roots of mutant NF2620 and wild-type Medicago truncatula A17 to obtain NF2620 hairy roots of MtbZIP10 gene transfer and A17 hairy roots of over-expressing MtbZIP10 gene. And simultaneously, the unloaded gene is also transformed into mutant NF2620 and wild type A17 hairy roots to be used as a negative control, and the accumulation conditions of anthocyanin in a positive gene complementation hairy root strain and a positive gene over-expression hairy root strain are observed and detected.
The invention finds that the colors of NF2620 hairy roots transformed with MtbZIP10 gene and A17 hairy roots over-expressing MtbZIP10 gene are darker than that of negative control, and the anthocyanin accumulation in the transgenic hairy roots is more than that of the negative control (observed before the hairy roots grow vigorously). And (4) selecting positive hairy roots, weighing 10mg of the positive hairy roots, and extracting anthocyanin of the positive hairy roots for content determination. The result shows that the anthocyanin content in NF2620 hairy roots of the MtbZIP10 transferred gene is basically equal to that of a negative control, and the anthocyanin content in A17 hairy roots of the MtbZIP10 overexpressed gene is obviously higher than that of the negative control. The overexpression of the MtbZIP10 gene in wild medicago truncatula can promote the accumulation of anthocyanin, and the MtbZIP10 transcription factor has the function of regulating anthocyanin biosynthesis (figure 6).
2.6 expression of MtbZIP10 under light and abscisic acid-induced conditions
The invention also detects whether the MtbZIP10 gene is induced by abscisic acid, 100mg/L of abscisic acid is applied to wild type medicago truncatula for 0h, 1h, 3h, 6h, 9h and 12h respectively, and the MtbZIP10 gene expression condition is detected by utilizing semi-quantitative PCR and fluorescent quantitative PCR (figure 7). The results show that the expression of the MtbZIP10 gene did not significantly correlate with the exopathic application of abscisic acid (figure 7). The invention also carries out illumination treatment on wild medicago truncatula. The results of semi-quantitative PCR and fluorescent quantitative PCR show that: the expression level of MtbZIP10 gene increased with the increase of illumination time (FIG. 7), indicating that MtbZIP10 is involved in light-mediated anthocyanin accumulation.
Third, the final conclusion
1. The medicago truncatula is a leguminous model plant and also a kindred species of the good pasture, namely medicago sativa, and has important research value. The invention firstly proves that the bZIP transcription factor MtbZIP10 gene codes a bZIP type transcription factor in medicago truncatula, the expression level of the MtbZIP10 gene in the medicago truncatula is related to the accumulation level of flavonoids such as anthocyanin and condensed tannin, and the MtbZIP10 is a key transcription factor for regulating and controlling the biosynthesis of the flavonoids in the medicago truncatula.
2. Flavonoid compounds represented by anthocyanidin and condensed tannin widely exist in plants, and have the functions of protecting the plants from biological and non-biological invasion and attracting insects to pollinate, and also have the biological characteristics of resisting cancer, resisting diseases and resisting oxidation. The alfalfa MtbZIP10 gene of the tribulus terrestris regulates the expression of a plurality of genes in the flavonoid pathway, the overexpression of the alfalfa MtbZIP10 gene can improve the content of flavonoid compounds, candidate genes and beneficial references are provided for the metabolic engineering of plant flavonoids, and a molecular foundation is laid for improving the alfalfa quality.
The operation methods not specifically described in the present embodiment are conventional techniques, and thus are not explained in detail herein.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> transcription factor gene for regulating and controlling plant flavonoid synthesis and application thereof
<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
ttatcaaccg tgtcttggtg caatggtgtt ggaaatgagg ctcttgtagt tgttgaatcc 120
aaggatcaat gcaagactaa gggtcaatct gatgagcaca agactcttcg tcggctgatg 180
cagaatcgcg aggctgcaag gaaaagtagg ttaaggaaaa aggcttatgt gcaacaattg 240
gagaacagtc gacttaggct tgctcaaata gaacatgagc ttcaacaagt acgtcaacag 300
gtccaacaac ttaaaaagat ttttcatttg gtaaaattgg tttgttcggt agcatttgat 360
atggactatg ctcgttgggt agacgagcat caacgactaa tcaatgacat aagatcagct 420
ataaattctc aaatgggtga taatgaactg catcttcttg ttgatggtgt catggtacat 480
tatgatgaat tatacaagtt gaagagcata ggtgcaaagg ctgatgtatt tcacatactt 540
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
tctctttcag agacactttc ctccacgtcc actggatctg gaaatgttgt tgagtatatg 1920
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
<211> 708
<212> PRT
<213> MtbZIP10 protein (Medicago truncatula)
<400> 2
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 (2)
1. The application of a transcription factor gene MtbZIP10 for regulating plant flavonoid synthesis is characterized in that the nucleotide sequence of the gene MtbZIP10 is shown as SEQ ID No.1, and the application is that the inhibition of the expression of MtbZIP10 in medicago truncatula leads to the reduction of anthocyanin in the overground part flavonoid of the medicago truncatula and the reduction of proanthocyanidin in seeds.
2. The application of a transcription factor gene MtbZIP10 for regulating plant flavonoid synthesis is characterized in that the nucleotide sequence of the gene MtbZIP10 is shown as SEQ ID No.1, and the application is to excessively express the MtbZIP10 gene in hairy roots of medicago truncatula so as to improve the content of anthocyanin.
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