CN114836431B

CN114836431B - Application of peony PsMYB1 gene in changing plant flower spot color and flower color

Info

Publication number: CN114836431B
Application number: CN202210364616.1A
Authority: CN
Inventors: 陶俊; 栾雨婷; 钱祎; 赵大球; 汤寓涵; 孙静
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2023-06-20
Anticipated expiration: 2042-04-08
Also published as: CN114836431A

Abstract

The invention discloses peonyPsMYB1The application of the gene in changing the color and luster of plant flower spots and flower color. The invention is realized by constructingPsMYB1The gene silencing vector is transformed into peony petals for expression, and after the petals grow for 7 days, observation is carried outPsMYB1The transgenic peony flower spot phenotype shows that the flower spot color is obviously lightened, the total anthocyanin content in the flower spot is obviously reduced compared with that of a wild type, the expression level of anthocyanin biosynthesis related genes in the flower spot is obviously reduced, and a new peony germplasm with light flower spots is created. The invention is also realized by the methodPsMYB1The gene over-expression vector is transformed into tobacco for expression, and after the plant flowers, the plant is observedPsMYB1The transgenic tobacco petals are provided with a phenotype, the color of the petals is obviously reddened, the total anthocyanin content in the petals is obviously increased compared with that of the wild tobacco petals, the expression level of anthocyanin biosynthesis related genes in the petals is obviously increased, and a new tobacco germplasm with bright color of the petals is created.

Description

Application of peony PsMYB1 gene in changing plant flower spot color and flower color

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to application of a peony PsMYB1 gene in changing the color and luster of plant flower spots.

Background

Petal stains are prevalent in ornamental plants and they are distributed in fixed morphology and location over flower organs, such as the petals or calyx bases of pansy (Viola x wittrockiana), oncidium (Oncidium hybridum) and peonies (Paeonia rockii) with dark spots (howling, wang Jian, li Qin, sheng, sun Haiyan, zhang Xuanbing. Plant plaque forming molecular mechanism research progress. Horticultural journal, 2014,41: 1485-1494). In angiosperms, plaque is also an important visual signal of the Plant to pollinating insects, promoting specific interactions between Plant and pollinators, playing a very important role in Plant reproduction and spread (Davies KM, albert NW, schwann ke. From landing lights to mimicry: the molecular regulation of flower coloration and mechanisms for pigmentation pattern Plant Biology,2012,39:619-638;Glover BJ,Walker RH,Moyroud E,Brockington SF.How to spot a flower.New Phytologist,2013,197:687-689).

Peony (Paeonia suffruticosa Andr.) is a perennial woody shrub of Paeoniaceae, is also a traditional ornamental and medicinal plant in China, enjoys the reputation of "king in flowers", and is known for its good moral meaning and profound cultural background. In recent years, peony is an emerging high-grade cut flower in the international market, and flower color largely determines ornamental value and commercial value. The existing cultivated peony has nine major color systems including red, powder, purple, white, yellow, black, green, blue and multiple colors. In addition to the rich flower types described above, peony with red or purple spots at the base is identified as a special strain, the number of which accounts for approximately 25% of the total peony varieties (Cheng FY. Advances in the breeding of tree peonies and a cultivar system for the cultivar group. International Journal of Plant Breeding,2007,1:89-104;Zhang JJ,Wang LS,Shu QY,Liu ZA,Li CH,Zhang J,Wei XL,Tian D.Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei tree peoney. Scientific Horticulture, 2007, 114:104-111), which is also less common in woody plants, however the mechanism of formation of this specific color pattern for peony is not yet known.

The MYB transcription factor family is one of the large transcription factor families widely existing in plants, is closely related to Plant growth and development, stress response (Baldoni E, genga A, cominelli E.plant MYB transcription factors: their role in drought response mechanisms.International Journal of Molecular Sciences,2015, 16:15811-15851), hormone synthesis, signal transduction and the like, and plays an important role in regulating Plant flavonoid biosynthesis (Liu JY, osbourn A, ma PD.MYB transcription factors as regulators of phenylpropanoid metabolism in plants.molecular Plant,2015,8:689-708;Ma DW,Constabel CP.MYB repressors as regulators of phenylpropanoid metabolism in plants.Trends in Plant Science,2019,24:275-289). MYB transcription factors can be classified into four types of 1R (R1/2, R3-MYB), 2R (R2R 3-MYB), 3R (R1R 2R 3-MYB) and 4R (4R 1/R2-like) according to the number of repeat domains, wherein the R2R3-MYB transcription factors are proved to be the main regulatory factors involved in regulating anthocyanin synthesis and plaque formation, and they generally promote the synthesis and accumulation of ornamental plant anthocyanin and the formation of plaque by enhancing the expression of key structural genes in flavonoid biosynthetic pathways.

At present, research on the formation of peony flower spots is mainly focused on the physiological basic level, and reports on the research on the molecular mechanism of the formation of peony flower spot characters are only one example. The transcription regulation related to peony flower color formation, which is participated by R2R3-MYB transcription factors, takes red-series and multi-series peony varieties as main research objects, for example, in a peony variety 'two arbor', a peony PsMYB57 gene and a PsMYB58 gene are cloned and overexpressed in tobacco, and the two genes can promote the accumulation of anthocyanin in peony petals (Zhang YZ, xu SZ, cheng YW, wang J, wang XX, liu RX, han JM.functional identification of PsMYB57 involved in anthocyanin regulation of tree peonoy.BMC Genetics,2020,21:124;Zhang YZ,Xu SZ,Ma HP,Duan XJ,Gao SX,Zhou XJ,Cheng YW.The R2R3-MYB gene PsMYB58 positively regulates anthocyanin biosynthesis in tree peony flowers.plant Physiology Biochemistry,2021, 164:279-288); in the peony complex variety 'Shima Nishiki', a peony PsMYB114L gene and a PsMYB12L gene are cloned and overexpressed in Arabidopsis thaliana and apple callus, and the two genes are found to significantly improve anthocyanin content in Arabidopsis thaliana plants and apple callus, and are inferred to serve as forward regulatory factors to promote anthocyanin accumulation in peony petals (Zhang XP, xu ZD, yu XY, zhao LY, zhao MY, han X, qi S.identification of two-animal R2R3-MYB transcription factors, psMYB114Land PsMYB12L, related to anthocyanin biosynthesis in Paeonia unification Journal of Molecular Sciences,2019, 20:1055). The result provides a theoretical basis for the molecular mechanism research of the formation of the peony flower spot character, however, the research on the MYB1 gene in the aspect of the regulation and control of the formation of the peony flower spot is not reported.

Therefore, the deep research on the peony PsMYB1 gene not only can enrich the bioinformatics resources of the species and expand the research field of peony molecular biology, but also can provide excellent gene resources and lay a theoretical foundation for obtaining the paeonia rockii by adopting a genetic engineering means in the future.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a novel peony PsMYB1 gene.

The invention also solves the technical problem of providing the protein encoded by the peony PsMYB1 gene.

The invention also solves the technical problem of providing an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the peony PsMYB1 gene.

The invention also solves the technical problem of providing the peony PsMYB1 gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain, and the application of the recombinant vector in the aspect of changing the plant flower spot color or changing the plant flower color.

The technical problem to be solved by the present invention is to provide a method for obtaining plants with light spots or a method for obtaining plants with vivid petals.

The technical problem to be finally solved by the present invention is to provide a method for identifying whether a plant obtained by the method has a plant with light-colored flower spots or a method for identifying whether a plant obtained by the method has a plant with vivid petals.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a peony PsMYB1 gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. Wherein SEQ ID NO.1 of the sequence Listing consists of 1274 bases. The gene PsMYB1 related to the formation of the flower spots can code PsMYB1 protein, and the protein has an amino acid sequence shown as SEQ ID NO.2, wherein the SEQ ID NO.2 in the sequence table consists of 327 amino acids.

The invention also discloses a protein encoded by the peony PsMYB1 gene, and the amino acid sequence of the protein is shown as SEQ ID NO.2.

The invention also comprises RACE amplification primers for cloning the full-length sequence of the peony PsMYB1 gene cDNA, wherein the sequences of the amplification primers are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

The invention also comprises an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain, which contains the peony PsMYB1 gene.

Wherein the recombinant vector comprises a gene silencing vector or an over-expression vector.

Wherein the gene silencing vector includes, but is not limited to, expression vector TRV2.

Wherein the over-expression vector includes, but is not limited to, the intermediate plant expression vector pCAMBIA2300.

The invention also comprises the peony PsMYB1 gene, the expression cassette, a recombinant vector, a recombinant cell or a recombinant strain, and the application of the recombinant vector in the aspect of changing the plant flower spot color or changing the plant flower color.

The invention also discloses a construction method of the recombinant vector, which comprises the following steps: amplifying the peony PsMYB1 gene specific fragment, and connecting with a gene silencing vector or an intermediate plant expression vector.

The invention also comprises a method for obtaining the plant with light-colored flower spots, and the peony PsMYB1 gene silencing vector is expressed in plant petals.

The invention also includes a method for identifying whether a plant has light-colored spots obtained by the method, comprising the steps of:

1) Identifying whether said plant comprises said gene silencing vector; or alternatively, the first and second heat exchangers may be,

2) Identifying whether the plant expresses the protein encoded by the peony PsMYB1 gene in low abundance.

The invention also includes a method of obtaining or identifying a plant having vivid petals according to the method, comprising the steps of:

1) Allowing or identifying whether the plant comprises said peony PsMYB1 gene; or (b)

2) So as to enable or identify whether the plant expresses the protein encoded by the peony PsMYB1 gene.

Among them, the plants of the present invention include, but are not limited to, peony or tobacco.

Among them, the present invention provides a method for obtaining plants with light spots or a method for obtaining plants with vivid petals, which comprises the steps of transgene, crossing, backcrossing or asexual reproduction.

According to the invention, through constructing the silencing vector of the peony PsMYB1 gene, transferring the TRV2-PsMYB1 silencing vector into peony petals by adopting an agrobacterium-mediated method, observing the flower plaque phenotype after the petals grow for 7 days, and finding that the color of the transgenic peony flower plaque is obviously light, and the wild peony flower plaque is dark red.

The invention also constructs an overexpression vector of the peony PsMYB1 gene, adopts an agrobacterium-mediated leaf disc method to transfer the pCAMBIA2300-PsMYB1 overexpression vector into tobacco, and after plants bloom, observes the phenotype of tobacco petals transformed with the PsMYB1 gene, and discovers that the petal color is obviously reddish, and wild tobacco petals are light pink, which indicates that the peony PsMYB1 gene has the function of changing the color and the flower color of plant flower spots.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: according to the invention, the constructed PsMYB1 gene silencing vector is transformed into peony petals for expression, the transgenic peony flower spots are found to be obviously lighter in color, the total anthocyanin content in the flower spots is obviously reduced compared with that of wild type, the expression level of anthocyanin biosynthesis related genes in the flower spots is obviously reduced, and a novel peony germplasm with light color flower spots is created. The constructed PsMYB1 gene overexpression vector is transformed into tobacco for expression, and the transgenic tobacco petals are found to obviously reddening, the total anthocyanin content in the petals is obviously increased compared with that of the wild type, the expression level of anthocyanin biosynthesis related genes in the petals is obviously increased, and new tobacco germplasm with bright petal colors is created.

Drawings

FIG. 1 shows the phenotype changes of the flower spots of the peony variety 'Haihuang' in different development periods;

FIG. 2 RACE results of the full length of the peony PsMYB1 gene cDNA are detected; wherein, M1 is DL 2000marker; m2, DL 5000 mark; 1:3' -RACE amplification products; 2:5' -RACE amplification products;

FIG. 3 homology alignment of the peony PsMYB1 protein with MYB proteins associated with anthocyanin biosynthesis in other species;

FIG. 4 phylogenetic tree analysis of the peony PsMYB1 protein with R2R3-MYB proteins associated with anthocyanin biosynthesis in other species;

FIG. 5 PCR identification of wild type and PsMYB1 gene-transferred peony petals;

FIG. 6 qRT-PCR identification of wild type and PsMYB1 Gene transferred peony petals: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 7 wild type and PsMYB1 gene transferred peony plaque phenotype;

fig. 8 comparison of color parameters a of wild type and PsMYB1 gene transferred peony plaques: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 9 comparison of total anthocyanin content in wild type and PsMYB1 gene-transferred peony plaques: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 10 expression levels of anthocyanin biosynthesis-related genes in wild-type and PsMYB 1-transgenic Vlady plaques: wherein CHS is chalcone synthase gene; DFR is a flavanonol-4-reductase gene; ANS is the anthocyanin synthase gene; different lowercase letters indicate significant differences (p < 0.05);

FIG. 11 PCR identification of wild type and PsMYB1 gene transferred tobacco plants;

FIG. 12 qRT-PCR identification of wild type and PsMYB1 Gene transferred tobacco plants: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 13 wild type and PsMYB1 gene transferred tobacco petal phenotype;

figure 14 comparison of values of wild type and PsMYB1 gene transferred tobacco petal colour parameters a: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 15 comparison of total anthocyanin content in wild type and PsMYB1 gene transferred tobacco petals: wherein different lowercase letters represent significant differences (p < 0.05);

FIG. 16 expression levels of anthocyanin biosynthesis-related genes in wild-type and PsMYB 1-transgenic tobacco petals: wherein CHS is chalcone synthase gene; CHI is chalcone isomerase gene; F3H is flavanone 3-hydroxylase gene; DFR is a flavanonol-4-reductase gene; ANS is the anthocyanin synthase gene; 3GT is a flavonoid-3-O-glucosyltransferase gene; different lowercase letters indicate significant differences (p < 0.05).

Detailed Description

The following detailed description of the present invention is given by way of specific examples, which are given for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

The experimental procedures, which are not specifically described in the following examples, were carried out according to conventional procedures, and materials, reagents, etc. used in the following examples, unless otherwise specified, were commercially available.

EXAMPLE 1 cloning of the full-Length cDNA sequence of the peony PsMYB1 Gene

1. Obtaining the cDNA sequence of the 3' end of the PsMYB1 Gene: the peony variety Haihuang (purchased from Shandong lotus) is selected as a material, and is firstly divided into 4 periods according to the change of petal shape in the plant development process, namely a color exposing period, a petal loosening period, a primary opening period and a full opening period (figure 1), and a peony petal mixed sample in the 4 periods is used as a material, and total RNA is extracted by adopting a MiniBEST Plant RNA Extraction Kit (TaKaRa) kit. The first strand of the cDNA was produced by reverse transcription of 3'full RACE Core Set Ver.2.0 (TaKaRa), the reverse transcription system being: 1. Mu.L of RNA, 1. Mu.L of 3' -RACE adapter, 1. Mu.L of dNTP mix (10 mM each), 2. Mu.L of 5 XM-MLV Buffer, 0.25. Mu. L RNase Inhibitor, 0.25. Mu. L Reverse Transcriptase M-MLV (RNase H) ^- )、4.5μL RNase Free ddH ₂ O; reverse transcription procedure: reacting at 42 ℃ for 60min, and extending at 70 ℃ for 15min. On this basis, 3' -RACE amplification was performed by two rounds of PCR. The first round PCR amplification system is as follows: 2. Mu.L of cDNA, 8. Mu.L of 1X cDNA Dilution Buffer II, 2. Mu.L of 3' -RACE Outer Primer, 2. Mu. L Gene specific Outer Primer (10. Mu.M) (5'-TTCACAAGCACCAATCTC-3' (SEQ ID NO. 3)), 5. Mu.L of 10 XLA PCR Buffer II (Mg) ⁺ Plus)、0.5μL LA DNA polymerase(5U/μL)、30.5μL RNase Free ddH ₂ O. The reaction conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 52℃for 30s, extension at 72℃for 60s, and cycling for 20 times; extending at 72℃for 10min. The second round PCR amplification system is as follows: 1 μL of the first round PCR amplification product, 8 μL of dNTP mix (2.5 mM each), 2 μL of 3' -RACE Inner Primer, 2 μ L Gene Specific Inner Primer (5'-TATCCGCTGAGGAAACTG-3' (SEQ ID NO. 4)), 5 μL of 10×LA PCR Buffer II (Mg) ⁺ Plus)、0.5μL LA DNA polymerase(5U/μL)、31.5μL RNase Free ddH ₂ O. The reaction conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 60s, and cycling for 30 times; extending at 72℃for 10min. The products were subjected to 1% agarose gel electrophoresis and the results are shown in FIG. 2.

2. Obtaining the cDNA sequence of the 5' end of the PsMYB1 gene: by smart ^TM RACE cDNA Amplification Kit User Manual (Clontech) first strand cDNA was produced by reverse transcription, which was divided into three steps, system one: 1. Mu.L RNA, 1. Mu.L 5' -RACE CDS Primer A, 9. Mu.L Deionizied H ₂ O. The reaction procedure is: reacting at 72 ℃ for 3min and then reversing at 42 DEG CShould be 2min. And a second system: 11. Mu.L of the First reaction solution, 4. Mu.L of 5 XFirst-Strand Buffer, 0.5. Mu.L of dithionite (100 mM), 1. Mu.L of dNTP mix (20 mM each), 0.5. Mu. L RNase Inhibitor, 2. Mu. L SMAR Tscribe Reverse Transcriptase, 1. Mu. L SMARTER IIA Oligonude. The reaction procedure is: after reaction at 42℃for 90min, reaction at 70℃for 10min. And (3) a system III: 20. Mu.L of reaction solution of system II and 50. Mu.L of Tricine-EDTA Buffer. The reaction procedure is: standing at 25deg.C for 15min to dilute cDNA. On the basis, the 5' -RACE is subjected to PCR amplification, and the reaction system is as follows: 2.5. Mu.L of 5'cDNA, 25. Mu.L of 2 XSeqAmp Buffer, 1. Mu. L SeqAmp DNA Polymerase, 5. Mu.L of 10 XUPM, 1. Mu.L 5'Gene Specific Primer (5'-AAGTTAGCAGTTTCCTCAGCGGATACGG-3' (SEQ ID NO. 5)), 15.5. Mu.L of RNase Free ddH ₂ O. The reaction conditions are as follows: reacting at 94 ℃ for 30s and 72 ℃ for 3min, and circulating for 5 times; reacting at 94 ℃ for 30s, 70 ℃ for 30s,72 ℃ for 3min, and circulating for 5 times; the reaction is carried out for 30s at 94 ℃, the annealing is carried out for 30s at 68 ℃ and the extension is carried out for 3min at 72 ℃, and the cycle is 25 times. The products were subjected to 1% agarose gel electrophoresis and the results are shown in FIG. 2. And splicing the RACE results to obtain the peony PsMYB1 gene, wherein the full-length nucleotide sequence of the gene is SEQ ID NO.1, and the coded amino acid sequence of the gene is SEQ ID NO.2.

Example 2 homology alignment of amino acid sequence deduced from peony PsMYB1 Gene with MYB protein related to anthocyanin biosynthesis and phylogenetic tree analysis

The amino acid sequence SEQ ID NO.2 of the peony 'marine yellow' PsMYB1 gene and the amino acid sequences of MYB genes related to anthocyanin biosynthesis pathways in other plants are respectively saved as TXT files, and then loaded into DNAMAN5.2.2 software for homology comparison, as shown in figure 3, the peony PsMYB1 protein and other anthocyanin biosynthesis related MYB proteins all contain R2 and R3 conserved domains at the N end. The amino acid sequence SEQ ID NO.2 of the peony 'marine yellow' PsMYB1 gene and the amino acid sequence of the R2R3-MYB gene related to anthocyanin biosynthesis pathway in other plants are respectively stored as TXT files and loaded into MEGA 7.0 software, a phylogenetic tree is constructed by utilizing an adjacent method (neighbor joining method), bootstrap is set to 1000, and the visualized result of the phylogenetic tree shows that the peony PsMYB1 and the R2R3-MYB transcription activator in other plants are gathered into one branch, and the branch is presumed to be used as the R2R3-MYB transcription activator to positively regulate anthocyanin biosynthesis (figure 4).

EXAMPLE 3 expression of peony PsMYB1 Gene silencing vector in peony petals

1. Construction of peony PsMYB1 gene silencing vector: primers containing the cleavage sites Xba I and BamH I were designed for amplification of PsMYB1 specific sequences (upstream primer: 5'-AAGGTTACCGAATTCTCTAGAACGGATACCAGGCCGGAG-3' (SEQ ID NO. 6), downstream primer: 5'-CGTGAGCTCGGTACCGGATCCATGGGTAGACCACCTTGCTGTG-3' (SEQ ID NO. 7)). PCR amplification system: 12.5. Mu.L 2X Phanta Flash Master Mix (Vazyme), 1. Mu.L Forward Primer, 1. Mu.L Reverse Primer, 2. Mu.L of the PsMYB1 gene-containing cDNA template of example 1, 8.5. Mu.L ddH ₂ O. The reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 60℃for 5s, extension at 72℃for 5s for 35 cycles; extending at 72℃for 1min. After the completion of the reaction, the PCR reaction mixture was analyzed by agarose gel electrophoresis, and a PsMYB 1-specific fragment containing the cleavage site was recovered using TSP601-DNA gel recovery kit (Tsingke). Taking a binary expression vector TRV2 plasmid (stored in a laboratory), and carrying out double digestion by using Xba I and BamH I (NEB), wherein the reaction system is as follows: 2.0. Mu.L of 10 XCutSmart Buffer, 7. Mu.L of TRV2 plasmid, 0.4. Mu.L of Xba I, 0.4. Mu.L of BamH I, 10.2. Mu.L of ddH ₂ O; the reaction was carried out at 37℃for 0.5h. The double digested products were analyzed by agarose gel electrophoresis, and the large fragment of purified plasmid TRV2 was recovered using TSP601-DNA gel recovery kit (Tsingke). By using

plus One step PCR Cloning Kit (Novoprotein) kit adopts a homologous recombination method to connect two recovered products, and the reaction system is as follows: 4.0. Mu.L of 5 Xreaction buffer, 1.0. Mu.L +.>

plus recombinase, 11 μl of large TRV2 fragment, 4 μl of PsMYB 1-specific fragment; after being connected in a metal bath at 50 ℃ for 15min, the mixture is cooled on ice, and 5 mu L of connection product is taken to be converted into 100 mu L of Trelief ^TM 5 alpha competent cells (Tsingke) were then cultured overnight on LB plates (containing Kan 50 mg/L) at 37℃and picked upPositive monoclonal amplification culture, extracting plasmid TRV2-PsMYB1, and performing double digestion and sequencing verification until the TRV2-PsMYB1 silencing vector is successfully constructed.

2. Peony petals are transformed by the peony PsMYB1 gene silencing vector: mu.L of TRV2-PsMYB1 silencing vector plasmid, TRV2 empty vector plasmid and TRV1 vector plasmid (stored in laboratory) were transformed into GV3101 (pSoup-p 19) competent cells (TOLOBIO), and then cultured on YEB plates (containing Rif 50mg/L and Kan 50 mg/L) at 28℃for 2d, positive monoclonal was selected in YEB liquid medium (containing Rif 50mg/L and Kan 50 mg/L), and cultured at 28℃at 200rpm overnight. Adding 2mL of the shaking bacteria solution into 50mL of liquid YEB containing the same antibiotics, and culturing under the same condition until OD ₆₀₀ =1.5. Pouring the shaken bacterial solution into a 50mL centrifuge tube, centrifuging at room temperature at 5000rpm for 10min, and discarding the supernatant for later use. Preparing resuspension (containing 10mM MES,10mM anhydrous magnesium chloride and 0.2mM acetosyringone) in sterilized triangular flask, adding resuspension to centrifuge tube to dissolve thallus, homogenizing with gun, and adjusting OD ₆₀₀ The bacterial solution was placed in the dark at room temperature for 1-3h to rejuvenate the cells. Mixing TRV2-PsMYB1 silencing vector bacterial liquid and TRV2 empty vector bacterial liquid with TRV1 vector bacterial liquid in equal volume for standby. Selecting peony petals in the color exposure period, taking petal discs (with the diameter of about 1.5 cm) with flower spots at the base parts, immersing in the bacterial liquid, vacuumizing for 8-10min to infect the petals, filtering the bacterial liquid after the infection is completed, taking out the petals, washing 2 times by using sterilized deionized water, sucking excessive water on the surfaces of the petals by using sterile filter paper, inoculating to a 1/2MS culture medium [1/2MS+6.66% agar+100 mg/LCb ]]Culturing in dark at 4deg.C for 24 hr, transferring into incubator, culturing in light (16 hr at 20deg.C and 8 hr at 16deg.C), and culturing for 7 days to obtain the petal of Paeonia suffruticosa with PsMYB1 gene.

Example 4 identification of peony petals transformed with peony PsMYB1 Gene

1. And (3) PCR identification: the peony petal DNA was extracted using a NuClean Plant Genomic DNA Kit (CWBIO) kit. On the basis, the peony beta-Tubulin (EF 608942) gene is taken as an internal reference (Forward Primer:5'-AGGTAAGATGAGCACCAAAG-3' (SEQ ID NO. 8) and Reverse Primer:5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 9)), and a TRV2 vector specific Primer is designed simultaneously(Forward Primer:5'-TTGTTACTCAAGGAAGCACGAT-3' (SEQ ID NO. 10), reverse Primer:5'-TCCCCTATGGTAAGACAATGAG-3' (SEQ ID NO. 11)) were subjected to PCR amplification. The reaction system: 12.5. Mu.L 2X Rapid Taq Master Mix (Vazyme), 1. Mu.L Forward Primer, 1. Mu.L Reverse Primer, 2. Mu.L DNA template, 8.5. Mu.L ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 52℃for 15s, extension at 72℃for 5s for a total of 35 cycles; extending at 72℃for 5min. After the reaction, the PCR reaction solution is subjected to gel electrophoresis detection. As can be seen from FIG. 5, a single and bright beta-Tubulin band was detected in the wild-type peony petals, the empty vector and the peony petals transformed with the PsMYB1 gene, whereas a single, bright and clear band with correct positions was detected only in the peony petals transformed with the PsMYB1 gene, and not in the wild-type peony and the empty vector, in the case of amplifying the PsMYB1 band.

2. qRT-PCR identification: total RNA was extracted using a MiniBEST Plant RNA Extraction Kit (TaKaRa) kit and reverse transcribed into cDNA using a HiScript III RT SuperMix for qPCR (+gDNA wind) (Vazyme) kit, with the following reaction system: 1.0. Mu.L RNA (1000 ng/. Mu.L), 4.0. Mu.L 4 XgDNA wind Mix, 11.0. Mu.L RNase Free dH ₂ O; the reaction conditions are as follows: the reaction was carried out at 42℃for 2min. After the reaction, 4.0. Mu.L of 5X HiScript III qRT SuperMix was added to the reaction mixture of the first step; the reaction conditions are as follows: the reaction was carried out at 37℃for 15min and at 85℃for 5s. For cDNA obtained by reverse transcription

SYBR qPCR SuperMix Plus (Novoprotein) kit was subjected to qRT-PCR detection. On the basis, the specific primers (Forward Primer:5'-GCTGTGATAAAATCGGAG-3' (SEQ ID NO. 12) and Reverse Primer:5'-TATTAGTTGGAACGGCTC-3' (SEQ ID NO. 13)) of the PsMYB1 gene are designed simultaneously by taking the peony beta-Tubulin (EF 608942) gene as an internal reference (Forward Primer:5'-AGGTAAGATGAGCACCAAAG-3' (SEQ ID NO. 8) and Reverse Primer:5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 9)). The reaction system: 2. Mu.L cDNA, 12.5. Mu.L +.>

SYBR qPCR SuperMix Plus、1μL Forward Primer、1μL Reverse Primer、8.5μL ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, extension at 72℃for 30s for 40 cycles; extending at 72℃for 10min. After the reaction is finished, 2 is adopted ^-△△Ct The method performs analysis of the relative expression level of genes. qRT-PCR identification showed that PsMYB1 had significantly lower expression levels in transgenic peony petals (FIG. 6).

Example 5 measurement of peony plaque color-dependent index of transgenic peony PsMYB1 Gene

After peony petals grow for 7 days, the phenotype change of the transgenic peony flower spots is observed, the wild type peony flower spots are found to be dark red, the PsMYB1 gene-transferred peony flower spots are found to be light red (figure 7), the index of the color correlation of the peony flower spots is further measured, the value of a value of the PsMYB1 gene-transferred peony flower spots is found to be obviously reduced compared with that of the wild type peony flower petals and empty carriers (figure 8), and the accumulation of the total anthocyanin content in the PsMYB1 gene-transferred peony flower spots is far lower than that of the wild type peony flower petals and empty carriers (figure 9), so that the PsMYB1 gene silencing function is shown to change the color of plant flower spots.

Example 6 detection of anthocyanin biosynthesis-related Gene expression level in peony plaque into which peony PsMYB1 Gene is transferred

Total RNA was extracted from peony petals using MiniBEST Plant RNA Extraction Kit (TaKaRa) kit, and was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wind) (Vazyme) kit, the reaction system was: 1.0. Mu.L RNA (1000 ng/. Mu.L), 4.0. Mu.L 4 XgDNA wind Mix, 11.0. Mu.L RNase Free dH ₂ O; the reaction conditions are as follows: the reaction was carried out at 42℃for 2min. After the reaction, 4.0. Mu.L of 5X HiScript III qRT SuperMix was added to the reaction mixture of the first step; the reaction conditions are as follows: the reaction was carried out at 37℃for 15min and at 85℃for 5s. The cDNA obtained by reverse transcription is adopted

SYBR qPCR SuperMix Plus (Novoprotein) was subjected to qRT-PCR detection using peony beta-Tubulin (EF 608942) as an internal reference gene (beta-Tubulin-F: 5' -AGGTAAGATG)AGCACCAAAG-3' (SEQ ID NO. 8), beta-Tubulin-R: 5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 9)), and detecting the expression levels of genes CHS, DFR and ANS related to anthocyanin biosynthesis, wherein the reaction system is as follows: 2. Mu.L cDNA, 12.5. Mu.L +.>

SYBR qPCR SuperMix Plus、1μL Forward Primer、1μL Reverse Primer、8.5μL ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, extension at 72℃for 30s for 40 cycles; extending at 7deg.C for 10min. Their specific primers are respectively: CHS-F:5'-GCCAAGGATTTAGCAGAG-3' (SEQ ID NO. 14), CHS-R:5'-TCAGCACCGACAATAACC-3' (SEQ ID NO. 15); DFR-F:5'-CGTTTTCACATCATCTGC-3' (SEQ ID NO. 16), DFR-R:5'-CATCTTGGACACGAAATACA-3' (SEQ ID NO. 17); ANS-F:5'-CAAACCAGCATCACCAAC-3' (SEQ ID NO. 18), ANS-R:5'-CTTTCCAAAAGCTCATCAGA-3' (SEQ ID NO. 19). Using equation 2 ^-△△CT The relative expression level of the gene was calculated by the method. As can be seen from fig. 10, the expression levels of CHS, DFR and ANS in the PsMYB1 gene-transferred peony plaques were significantly lower compared to wild-type peony.

EXAMPLE 7 expression of peony PsMYB1 Gene overexpression vector in tobacco

1. Construction of peony PsMYB1 gene overexpression vector: primers containing the cleavage site SaI for amplifying the PsMYB1 sequence were designed (upstream primer PsMYB1-F:5'-CGGGGATCCTCTAGAGTCGACATGGGTAGACCACCTTGCTGTG-3' (SEQ ID NO. 20), downstream primer PsMYB1-R:5'-CACCATGGTACTAGTGTCGACGAACAAACCAGCAGTTCCATCTAG-3' (SEQ ID NO. 21)). PCR amplification system: 12.5. Mu.L 2X Phanta Flash Master Mix (Vazyme), 1. Mu.L Forward Primer, 1. Mu.L Reverse Primer, 2. Mu.L of the PsMYB1 gene-containing cDNA template of example 1, 8.5. Mu.L ddH ₂ O. The reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 60℃for 5s, extension at 72℃for 10s for 35 cycles; extending at 72℃for 1min. After the completion of the reaction, the PCR reaction mixture was analyzed by agarose gel electrophoresis, and a large PsMYB1 fragment containing the cleavage site was recovered using TSP601-DNA gel recovery kit (Tsingke). The binary expression vector pCAMBIA2300 plasmid (stored in laboratory) was taken and subjected to single-use with SaI (NEB)Enzyme digestion, the reaction system is: 2.0. Mu.L of 10 XCutSmart Buffer, 7. Mu.L of pCAMBIA2300 plasmid, 0.4. Mu.L of SaI, 10.6. Mu.L of ddH ₂ O; the reaction was carried out at 37℃for 0.5h. The single digested product was analyzed by agarose gel electrophoresis, and a large fragment of the purified plasmid pCAMBIA2300 was recovered using TSP601-DNA gel recovery kit (Tsingke). By using

plus recombinase, 9 μl of pCAMBIA2300 large fragment, 6 μl of PsMYB1 large fragment; after being connected in a metal bath at 50 ℃ for 15min, the mixture is cooled on ice, and 5 mu L of connection product is taken to be converted into 100 mu L of Trelief ^TM 5 alpha competent cells (Tsingke) were then cultured overnight on LB plates (containing Kan 50 mg/L) at 37℃and positive monoclonal expansion was selected to extract plasmid pCAMBIA2300-PsMYB1, which was then subjected to single digestion and sequencing verification until the pCAMBIA2300-PsMYB1 overexpression vector was successfully constructed.

2. Transforming tobacco by using peony PsMYB1 gene overexpression vector: 5uL of pCAMBIA2300-PsMYB1 over-expression vector plasmid was used to transform GV3101 (pSoup-p 19) competent cells (TOLOBIO), followed by 2d incubation on YEB plates (containing Rif 50mg/L and Kan 50 mg/L) at 28℃and selection of positive monoclonal in YEB liquid medium (containing Rif 50mg/L and Kan 50 mg/L) at 28℃overnight at 200 rpm. Adding 2mL of the shaking bacteria solution into 50mL of liquid YEB containing the same antibiotics, and culturing under the same condition until OD ₆₀₀ =0.3-0.4. Pouring the shaken bacterial solution into a 50mL centrifuge tube, centrifuging at room temperature at 5000rpm for 10min, and discarding the supernatant for later use. Firstly, adding a proper amount of acetosyringone (20 mg/mL) into a sterilized small triangular flask, adding 5mL of MS0 (MS liquid basic culture medium, no agar and sucrose (Hopebio)) into a centrifuge tube, uniformly stirring the thallus with a gun, pouring the thallus into the small triangular flask with the proper amount of acetosyringone, and then adding MS0 to 50mL. Into another sterilized vial was added 50mL of MS0 (MS0 liquid minimal medium)Agar and sucrose free). Taking sterile seedling leaves of tobacco, cutting into small pieces (about 1cm multiplied by 1 cm), putting into a 50mL small triangular flask with MS0, cutting 100-150 leaves altogether, putting into a flask, pouring the leaves into a beaker covered with gauze, taking filtered leaves, adding 50mL of MS0+100 mu L acetosyringone (100 mu mol/mL) into the small three flasks, infecting for 8min, and continuously gently shaking during infection; after infection is completed, filtering out bacterial liquid, taking out leaf, sucking up the redundant bacterial liquid on the surface of the leaf by using sterile filter paper, inoculating the leaf to a co-culture medium [ MS+3.0 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+6.66% agar ]]Dark culture for 3d; after co-cultivation, transfer into resistant bud screening differentiation medium [ MS+3.0 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+6.66% agar+100 mg/L Cb+25mg/L G418 ]]Selecting culture for two weeks for secondary culture until bud formation; when the adventitious bud is grown to more than 2cm, the adventitious bud is cut off and transferred into rooting screening culture medium [1/2MS+0.3mg/L IBA+30g/L sucrose+6.66% agar+50 mg/LCb+8mg/L G418 ]]Rooting and screening. After 4-6 months of culture, the PsMYB1 gene-transferred tobacco can be obtained.

Example 8 identification of tobacco plants transformed with peony PsMYB1 Gene

1. And (3) PCR identification: tobacco leaf DNA was extracted using a NuClean Plant Genomic DNA Kit (CWBIO) kit. On the basis, PCR amplification was performed with the tobacco NtActin (AB 158612) gene as an internal reference (Forward Primer:5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO. 22), reverse Primer:5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO. 23)), while designing specific primers for the PsMYB1 gene (Forward Primer:5'-GCTGTGATAAAATCGGAG-3' (SEQ ID NO. 12), reverse Primer:5'-TATTAGTTGGAACGGCTC-3' (SEQ ID NO. 13)). The reaction system: 12.5. Mu.L 2X Rapid Taq Master Mix (Vazyme), 1. Mu.L Forward Primer, 1. Mu.L Reverse Primer, 2. Mu.L DNA template, 8.5. Mu.L ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 52℃for 15s, extension at 72℃for 5s for a total of 35 cycles; extending at 72℃for 5min. After the reaction, the PCR reaction solution is subjected to gel electrophoresis detection. As can be seen from FIG. 11, a single and bright NtActin band was detected in both wild-type tobacco and transgenic peony PsMYB1 gene tobacco, whereas in the case of amplifying PsMYB1 band, only in transgenic peony PsMYB1 gene tobaccoA single, bright, clear band was detected, not in wild type tobacco.

SYBR qPCR SuperMix Plus (Novoprotein) kit was subjected to qRT-PCR detection. Based on this, qRT-PCR was performed with the tobacco NtActin (AB 158612) gene as an internal reference (Forward Primer:5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO. 22), reverse Primer:5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO. 23)), while designing specific primers for the PsMYB1 gene (Forward Primer:5'-GCTGTGATAAAATCGGAG-3' (SEQ ID NO. 12), reverse Primer:5'-TATTAGTTGGAACGGCTC-3' (SEQ ID NO. 13)). The reaction system: 2 mu L cDNA, 12.5 mu L->

SYBR qPCR SuperMix Plus、1μL Forward Primer、1μL Reverse Primer、8.5μL ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, extension at 72℃for 30s for 40 cycles; extending at 72℃for 10min. After the reaction is finished, 2 is adopted ^-△△Ct The method performs analysis of the relative expression level of genes. qRT-PCR identification showed that PsMYB1 had significantly higher expression levels in transgenic tobacco (fig. 12).

Example 9 determination of tobacco petal color-dependent index of peony PsMYB1 Gene transferred

After tobacco flowers are opened, photographing and observing tobacco petals, finding that wild type tobacco petals are light pink, and the tobacco petals transformed with the PsMYB1 gene are bright red (figure 13), further determining the index of correlation of the colors of the tobacco petals, finding that the a value of the tobacco petals transformed with the PsMYB1 gene is obviously increased compared with that of the tobacco petals of the wild type (figure 14), and accumulating total anthocyanin in the tobacco petals transformed with the PsMYB1 gene is far higher than that of the tobacco petals of the wild type (figure 15), which shows that the over-expressed PsMYB1 gene has the function of changing the flower colors of plants.

EXAMPLE 10 detection of anthocyanin biosynthesis-related Gene expression level in tobacco petals transgenic for peony PsMYB1 Gene

Total RNA was extracted from tobacco petals using MiniBEST Plant RNA Extraction Kit (TaKaRa) kit, and was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wind) (Vazyme) kit, and the reaction system was: 1.0. Mu.L RNA (1000 ng/. Mu.L), 4.0. Mu.L 4 XgDNA wind Mix, 11.0. Mu.L RNase Free dH ₂ O; the reaction conditions are as follows: the reaction was carried out at 42℃for 2min. After the reaction, 4.0. Mu.L of 5X HiScript III qRT SuperMix was added to the reaction mixture of the first step; the reaction conditions are as follows: the reaction was carried out at 37℃for 15min and at 85℃for 5s. The cDNA obtained by reverse transcription is adopted

SYBR qPCR SuperMix Plus (Novoprotein) was tested by qRT-PCR using tobacco action (AB 158612) as an internal reference gene (action-F: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO. 22), actin-R:5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO. 23)), and detecting the expression levels of anthocyanin biosynthesis-related genes CHS (NM_ 001326166.1), CHI (NM_ 001325287.1), F3H (NM_ 001325083.2), DFR (NM_ 001325630.2), ANS (XM_ 016598316.1) and 3GT (NM_ 001326108.1), in the following reaction system: 2. Mu.L cDNA, 12.5. Mu.L +.>

SYBR qPCR SuperMix Plus、1μL Forward Primer、1μL Reverse Primer、8.5μL ddH ₂ O. The reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 55℃for 30s, extension at 72℃for 30s for 40 cycles; extending at 7deg.C for 10min. Their specific primers are respectively: CHS-F:5'-ACTGCGGTCACATTTCGT-3' (SEQ ID NO. 24), CHS-R:5'-ATCCGGGAGTAGGGTTTG-3' (SEQ ID N)O.25); CHI-F:5'-TTGAAGGGAAGTTTGTGAAG-3' (SEQ ID NO. 26), CHI-R:5'-AAGGGACCTGTGACGATA-3' (SEQ ID NO. 27); F3H-F:5'-CTACAGGGTGAAGTGGTC-3' (SEQ ID NO. 28), F3H-R:5'-CCCATTGCCTCTGATAGT-3' (SEQ ID NO. 29); DFR-F:5'-CGAGGACCCTGAGAATGA-3' (SEQ ID NO. 30), DFR-R:5'-CAGCCGATGAAGTGAAAA-3' (SEQ ID NO. 31); ANS-F:5'-GCCAAACAGATTAGGAAC-3' (SEQ ID NO. 32), ANS-R:5'-TCATTTGAAGCAGTAGGTC-3' (SEQ ID NO. 33); 3GT-F:5'-AAGTGGAACAATGAGTGC-3' (SEQ ID NO. 34), 3GT-R:5'-CTCCATTAGGTCCTTGAA-3' (SEQ ID NO. 35). Using equation 2 ^-△△CT The relative expression level of the gene was calculated by the method. As can be seen from fig. 16, the expression levels of CHS, CHI, F3H, DFR, ANS and 3GT in the PsMYB1 gene transferred tobacco petals were significantly higher compared to wild type tobacco.

In conclusion, the application of the peony PsMYB1 gene in the aspect of changing the color and luster of plant flower spots is provided, the constructed PsMYB1 gene silencing vector is transformed into peony petals for expression, the transgenic peony flower spots are found to be obviously lighter in color, the total anthocyanin content in the flower spots is obviously reduced compared with the wild type, the expression level of anthocyanin biosynthesis related genes in the flower spots is obviously reduced, and novel peony germplasm with light color flower spots is created. The constructed PsMYB1 gene overexpression vector is transformed into tobacco for expression, and the transgenic tobacco petals are obviously reddened, the total anthocyanin content in the petals is obviously increased compared with that of the wild type, the expression level of anthocyanin biosynthesis related genes in the petals is obviously increased, and new tobacco germplasm with bright petal colors is created.

Sequence listing

<110> university of Yangzhou

<120> application of peony PsMYB1 gene in changing plant flower spot color and flower color

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1274

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

tacgattgtg cgattgattt agcggccgcg aattcgccct tctaatacga ctcactatag 60

ggcaagcagt ggtatcaacg cagagtacat gggggtctca gcatttctta atagaaacaa 120

atagatctgt cgagtgagga agaagaagac ctgtgttgaa aaccatactt atctagggat 180

caacgttaag aaatgggtag accaccttgc tgtgataaaa tcggagtgaa gaaagggcca 240

tggactcccg aagaagatat cattctggtg tcttacattc aagaacatgg accagggaat 300

tggagagccg ttccaactaa tacaggattg cttagatgca gcaagagctg cagacttagg 360

tggactaact atctccggcc tggtatccgt agaggtaact tcactgatca agaggagaag 420

atgattatcc acctccaagc tcttttggga aatagatggg ctgccatagc ttcgtacctt 480

ccccaaagaa cagataatga tataaagaat tactggaata cccatctaaa aaagaagctc 540

aaaaagcttc aagcaggtgt tgatgatggg cacaaccaag atggcctagt ttcacaagca 600

ccaatctcaa agggacagtg ggagagaagg cttcaaacag atatccacat ggccaaacaa 660

gccctttgcg aggctttgtc cttggacaaa cctagcaata ctatttcacc cgagtcgaaa 720

atttgcccta attacaccgg accagctctc caatcatcta cctatgcctc cagtactgaa 780

aacatagcaa ggttgcttga aggttggatg agaaattcac ccaaatcagc ccaaaccaac 840

tcagatcaga attccttcat tcacaatccg gttccgaccg gttccagttc cagtgaaggg 900

gcactgagtg caacaactcc tgatgctttc gactcgcttt ttggcttcaa ttcttccacc 960

aattcggaag cctcgcaggc cgtatccgct gaggaaactg ctaacttcag tactcctgaa 1020

actagcctat tccaagatga aagcaaacca aatatggaga aaagtcgtgt cccgctcaca 1080

ttgttagaga aatggctctt cgatgatggt gctgctgctc aagggcagga tgatctaatt 1140

ggtatgccgc tagatggaac tgctggtttg ttctagaagg agttcaagtt tcaacctttg 1200

tgtttttctt atttgttttc tgttaggcct aaagctgatg agacttcttt ctagctagtt 1260

tgagttgaaa aaaa 1274

<210> 2

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Gly Arg Pro Pro Cys Cys Asp Lys Ile Gly Val Lys Lys Gly Pro

1 5 10 15

Trp Thr Pro Glu Glu Asp Ile Ile Leu Val Ser Tyr Ile Gln Glu His

20 25 30

Gly Pro Gly Asn Trp Arg Ala Val Pro Thr Asn Thr Gly Leu Leu Arg

35 40 45

Cys Ser Lys Ser Cys Arg Leu Arg Trp Thr Asn Tyr Leu Arg Pro Gly

50 55 60

Ile Arg Arg Gly Asn Phe Thr Asp Gln Glu Glu Lys Met Ile Ile His

65 70 75 80

Leu Gln Ala Leu Leu Gly Asn Arg Trp Ala Ala Ile Ala Ser Tyr Leu

85 90 95

Pro Gln Arg Thr Asp Asn Asp Ile Lys Asn Tyr Trp Asn Thr His Leu

100 105 110

Lys Lys Lys Leu Lys Lys Leu Gln Ala Gly Val Asp Asp Gly His Asn

115 120 125

Gln Asp Gly Leu Val Ser Gln Ala Pro Ile Ser Lys Gly Gln Trp Glu

130 135 140

Arg Arg Leu Gln Thr Asp Ile His Met Ala Lys Gln Ala Leu Cys Glu

145 150 155 160

Ala Leu Ser Leu Asp Lys Pro Ser Asn Thr Ile Ser Pro Glu Ser Lys

165 170 175

Ile Cys Pro Asn Tyr Thr Gly Pro Ala Leu Gln Ser Ser Thr Tyr Ala

180 185 190

Ser Ser Thr Glu Asn Ile Ala Arg Leu Leu Glu Gly Trp Met Arg Asn

195 200 205

Ser Pro Lys Ser Ala Gln Thr Asn Ser Asp Gln Asn Ser Phe Ile His

210 215 220

Asn Pro Val Pro Thr Gly Ser Ser Ser Ser Glu Gly Ala Leu Ser Ala

225 230 235 240

Thr Thr Pro Asp Ala Phe Asp Ser Leu Phe Gly Phe Asn Ser Ser Thr

245 250 255

Asn Ser Glu Ala Ser Gln Ala Val Ser Ala Glu Glu Thr Ala Asn Phe

260 265 270

Ser Thr Pro Glu Thr Ser Leu Phe Gln Asp Glu Ser Lys Pro Asn Met

275 280 285

Glu Lys Ser Arg Val Pro Leu Thr Leu Leu Glu Lys Trp Leu Phe Asp

290 295 300

Asp Gly Ala Ala Ala Gln Gly Gln Asp Asp Leu Ile Gly Met Pro Leu

305 310 315 320

Asp Gly Thr Ala Gly Leu Phe

325

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ttcacaagca ccaatctc 18

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tatccgctga ggaaactg 18

<210> 5

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aagttagcag tttcctcagc ggatacgg 28

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aaggttaccg aattctctag aacggatacc aggccggag 39

<210> 7

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cgtgagctcg gtaccggatc catgggtaga ccaccttgct gtg 43

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aggtaagatg agcaccaaag 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ggaggaatgt cacaaacact 20

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ttgttactca aggaagcacg at 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tcccctatgg taagacaatg ag 22

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gctgtgataa aatcggag 18

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tattagttgg aacggctc 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gccaaggatt tagcagag 18

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tcagcaccga caataacc 18

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cgttttcaca tcatctgc 18

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

catcttggac acgaaataca 20

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

caaaccagca tcaccaac 18

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ctttccaaaa gctcatcaga 20

<210> 20

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cggggatcct ctagagtcga catgggtaga ccaccttgct gtg 43

<210> 21

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

caccatggta ctagtgtcga cgaacaaacc agcagttcca tctag 45

<210> 22

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

tcctcatgca attcttcg 18

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

acctgcccat ctggtaac 18

<210> 24

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

actgcggtca catttcgt 18

<210> 25

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

atccgggagt agggtttg 18

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ttgaagggaa gtttgtgaag 20

<210> 27

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

aagggacctg tgacgata 18

<210> 28

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

ctacagggtg aagtggtc 18

<210> 29

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

cccattgcct ctgatagt 18

<210> 30

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

cgaggaccct gagaatga 18

<210> 31

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

cagccgatga agtgaaaa 18

<210> 32

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

gccaaacaga ttaggaac 18

<210> 33

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

tcatttgaag cagtaggtc 19

<210> 34

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

aagtggaaca atgagtgc 18

<210> 35

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

ctccattagg tccttgaa 18

Claims

1. Peony treePsMYB1The application of the gene in changing the color of peony flower spots or changing the flower color of peony is characterized in thatPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The application of the peony PsMYB1 protein in changing the color of peony plaques or changing the color of peony is characterized in that the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.

3. Peony containing coded peony PsMYB1 proteinPsMYB1The application of the gene expression cassette in changing the color of peony flower spots or changing the flower color of peony is characterized in thatPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.

4. Peony containing coded peony PsMYB1 proteinPsMYB1The application of the recombinant vector of the gene in changing the color of peony flower spots or changing the color of peony is characterized in thatPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.

5. The use of claim 4, wherein the recombinant vector comprises a gene silencing vector or an overexpression vector.

6. The use according to claim 4, wherein the construction method of the recombinant vector comprisesThe method comprises the following steps: augmenting peony according to claim 1PsMYB1The specific fragment of the gene is connected with a gene silencing vector or an over-expression vector.

7. Peony containing coded peony PsMYB1 proteinPsMYB1The application of the recombinant strain of the gene in changing the color of peony flower spots or changing the color of peony is characterized in thatPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.

8. A method for obtaining peony with light-colored flower spots, characterized in that peony is subjected toPsMYB1Gene silencing of the peonyPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

9. A method of identifying peony having a light colored plaque comprising the steps of:

1) Identifying that said peony comprises the gene silencing vector of claim 5; or alternatively, the first and second heat exchangers may be,

2) Identifying that the peony is expressed into peony PsMYB1 protein in low abundance, wherein the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.

10. A method for obtaining or identifying peony having vivid petals, comprising the steps of:

1) Allowing or identifying plants to overexpress peonyPsMYB1A gene; the peony is provided withPsMYB1The nucleotide sequence of the gene is shown as SEQ ID NO. 1; or (b)

2) Allowing or identifying plants to overexpress peonyPsMYB1A protein encoded by the gene; the amino acid sequence of the peony PsMYB1 protein is shown as SEQ ID NO.2.