CN114836437B

CN114836437B - Application of peony PsMYB4 gene in changing color and size of plant petal color spots

Info

Publication number: CN114836437B
Application number: CN202210559684.3A
Authority: CN
Inventors: 栾雨婷; 赵大球; 陶俊; 许聪; 孙静
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-06-20
Anticipated expiration: 2042-05-20
Also published as: CN114836437A

Abstract

The invention discloses peonyPsMYB4The application of the gene in changing the color and size of plant petal stain. The peony is provided withPsMYB4The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention also discloses silent peonyPsMYB4The application of the vector, the expression cassette, the recombinant bacteria or the cells of the gene in changing the color and the size of plant petal color spots. The invention also discloses a method for obtaining the peony with the larger petal stain and a method for identifying the peony with the larger petal stain obtained by the method. The invention is realized by constructingPsMYB4The gene silencing vector is transformed into peony petals for expression, and after the petals grow for 7 days, observation is carried outPsMYB4The transgenic peony petals have the color-changing phenotype, the color-changing is obviously larger than that of wild type, the color-changing color is obviously deepened, the total anthocyanin content in the color-changing is obviously increased, the expression level of anthocyanin biosynthesis related genes in the color-changing is obviously increased, and a new peony germplasm with larger petals is created.

Description

Application of peony PsMYB4 gene in changing color and size of plant petal color spots

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to application of a peony PsMYB4 gene in changing color and size of plant petal color spots.

Background

The peony (Paeonia suffruticosa Andr.) is a perennial woody plant of Paeoniaceae Paeonia, which is an important ornamental plant and medicinal plant native to China, is one of the traditional 'ten flowers' of China, and has extremely high ornamental value and economic value. The deep color spots exist on the base parts of the petals of part of peony varieties, and besides the ornamental value, the characteristic becomes an important basis for the identification and classification of the variety groups of the peony varieties. Color-wise classification, the stains can be classified into deep purple, red and brown 3 classes (Li Jia. Chinese peony variety, emotions, 2005, 26-39); classified in size, the color spots can be classified into super-large, medium and small 4 types according to the ratio of the color spots on petals (Zhao Na, yuan Tao. Preliminary study of the morphology of the primary color spots of the northwest peony variety, chinese agronomic bulletin 2013, 29 (16): 192-197).

Previous studies have shown that anthocyanin is a decisive pigment group for the color and size of peony petals, and that the anthocyanin in the color spots comprises six of pelargonidin-3-O-glucoside (Pg 3G), pelargonidin-3, 5-O-diglucoside (pelargonidin-3, 5-di-O-glucoside, pg3G 5G), cyanidin-3-O-glucoside (cyanidin-3-O-glucoside), cy 3G), cyanidin-3, 5-O-diglucoside (cyanidin-3, 5-di-O-glucoside, cy3G 5G), paeoniflorin-3-O-glucoside (peonidin-3-O-glucoside, pn 3G), and paeoniflorin-3, 5-O-diglucoside (peonidin-3, 5-di-O-glucoside, pn3G 5G) (Wang LS, hashimoto F, shiraishi A, aoki N, li JJ, shimizu K.Phenetics in tree peony species from China by flower pigment cluster analysis. Journal of Plant Research,2001, 114:213-221;Wang LS,Hashimoto F,Shiraishi A,Aoki N,Sakata Y.Chemical taxonomy ofthe Xibei tree peony from China by floral pigmentation.Journal of Plant Research,2004, 117:47-55;Zhang JJ,Wang LS,Shu QY,Liu ZA,Li CH,Zhang J,Wei XL,Tian D.Comparison of anthocyanins in non-blotches and blotches ofthe petals ofXibei tree pepey. Scintia Horticulate, 2007, 114:104-111). It is thought that the cells in the stained area possess much higher anthocyanin synthesis capacity than the cells in the non-stained area, and this difference is related to the interaction of endogenous and exogenous environmental factors, structural genes and regulatory genes. Among them, the R2R3-MYB family transcription factors are the most widely studied class for regulating gene-mediated stain formation (Quattrocchio F, wing JF, van der Woude K, mol JN, koes R.analysis of bHLH and MYB domain proteins: species specific regulatory differences are caused by divergent evo lution of target anthocyanin genes.plant Journal,1998, 13:475-488). Currently, the functions of the oncidium OgMYB1 (Chiou CY, yeh KW. Differential expression of MYB gene (OgMYBl) determines color patterning in floral tissue ofOncidium Gower Ramsey. Plant Molecular Biology,2008, 66:379-388), the lilium oriental LhMYB12 (Yamagishi M. Oriental hybrid lily Sorbonne homologue of LhMYB12regulates anthocyanin biosyntheses in flower tepals and tepal spots. Molecular diagnostics, 2010, 28:381-389), the gentian GtMYB3 (Nakatsuka T, saito M, yamada E, nishihara M.production of picotee-type flowers in Japanese gentian by CRES-T.plant Biotechnology,2011, 28:173-180) and the Chinese character ClMYB1 (Jiang P, rausher M.Two genetic changes in cis-regulatory elements caused evolution ofpetal spot position in, plant, 2018, 4:14-22) in the forward regulation and control formation were studied successively. Only the PsMYB12 transcription factor is reported in peony to positively regulate the formation of purple color spots on the base of petals, but researches on how R2R3-MYB family transcription factors playing a negative regulation role influence the color and the size of the color spots of peony petals have not been reported.

Recently, we isolated 1R 2R3-MYB family transcription factor from peony full-length transcriptome library, constructed a evolutionary tree from the amino acid sequence of the gene and the MYB transcription factor family of Arabidopsis thaliana, found that the gene has the closest relationship with the Arabidopsis thaliana AtMYB4 transcription inhibitor (FIG. 1), and therefore we named PsMYB4. At present, the application of the peony PsMYB4 gene in changing the color and size of plant petal color spots has not been reported, so that the deep research of the PsMYB4 gene is developed, the bioinformatics resource of the species can be enriched, the research field of peony molecular biology is expanded, and excellent gene resources can be provided for obtaining peony with changeable color and size spots by adopting a genetic engineering means in the future, and a theoretical basis is laid.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing application of peony PsMYB4 gene in changing color and size of plant petal color spots.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a peony PsMYB4 gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention also discloses a protein encoded by the peony PsMYB4 gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also comprises an amplification primer for cloning the full-length sequence of the peony PsMYB4 gene cDNA, wherein the sequence of the amplification primer is shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also comprises an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain, which contains the peony PsMYB4 gene.

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

The invention also discloses the peony PsMYB4 gene, the expression cassette, a recombinant vector, a recombinant cell or recombinant strain and application of the recombinant vector in changing the color and size of plant petal color spots.

Wherein the plant includes, but is not limited to, peony.

The invention also discloses a construction method of the recombinant vector, which comprises the following steps: amplifying the peony PsMYB4 gene fragment and connecting the fragment with a binary expression vector TRV2 plasmid.

The invention also includes a method for obtaining a plant with larger petal stain, wherein the PsMYB4 gene or the gene silencing vector is expressed in plant petals.

The invention also includes a method for identifying plants with larger petal stain obtained by the method, comprising the steps of:

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

2) Determining whether the plant expresses the protein encoded by the peony PsMYB4 gene of claim 2 in low abundance.

According to the invention, a silencing vector of the peony PsMYB4 gene is constructed, the TRV2-PsMYB4 silencing vector is transferred into peony petals by adopting an agrobacterium-mediated method, after the petals grow for 7 days, the flower petal color spot phenotype is observed, the color of the transgenic peony petals is obviously enlarged compared with that of the wild type, the color of the color spot is obviously reddish compared with that of the wild type, the total anthocyanin content in the color spot is obviously increased, and the expression level of anthocyanin biosynthesis related genes in the color spot is obviously increased, so that the peony PsMYB4 gene has the function of changing the color and size of the flower petal color spots of plants.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: according to the invention, the constructed PsMYB4 gene silencing vector is transformed into peony petals for expression, so that the color of the color spots of the transgenic peony petals is obviously increased compared with that of the wild type, the color of the color spots is obviously reddened compared with that of the wild type, the total anthocyanin content in the color spots is obviously increased, the expression level of anthocyanin biosynthesis related genes in the color spots is obviously increased, and a novel peony germplasm with larger petal color spots is created.

Drawings

FIG. 1A tree of evolution constructed from the Paeonia suffruticosa PsMYB4 amino acid sequence and the MYB transcription factor family of Arabidopsis thaliana.

FIG. 2 detection of full-length amplification result of peony PsMYB4 gene cDNA; wherein M: DL2000 marker;1: full-length amplification product.

FIG. 3 homology alignment of the peony PsMYB4 protein with MYB proteins associated with anthocyanin biosynthesis in other species.

FIG. 4 PCR identification of wild type and PsMYB4 gene transferred peony petals.

FIG. 5 qRT-PCR identification of wild type and PsMYB4 gene-transferred peony petals: wherein different lowercase letters indicate significant differences (p < 0.05).

FIG. 6 wild type and PsMYB4 gene transferred peony petal stain phenotype.

FIG. 7 comparison of the sizes of the peony petals spots of wild type and PsMYB4 gene transferred: wherein different lowercase letters indicate significant differences (p < 0.05).

Fig. 8 comparison of color parameters a of wild type and PsMYB4 gene transformed peony petals: wherein different lowercase letters indicate significant differences (p < 0.05).

FIG. 9 comparison of total anthocyanin content in wild-type and PsMYB4 gene-transferred peony petals stain: wherein different lowercase letters indicate significant differences (p < 0.05).

FIG. 10 expression levels of anthocyanin biosynthesis-related genes in wild-type and PsMYB 4-transgenic peony petal stain: wherein F3H is flavanone-3-hydroxyl transferase gene; FLS is a flavonol synthase gene; DFR is a flavanonol-4-reductase gene; ANS is the anthocyanin synthase gene; 3GT is anthocyanin 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 PsMYB4 Gene

Obtaining the full-length sequence of the PsMYB4 gene cDNA: the peony variety 'Haihuang' petal sample is selected as a material, and a MiniBEST P1ant RNA Extraction Kit (TaKaRa) kit is adopted to extract total RNA. Using PrimeScript ^TM II 1st Strand cDNA Synthesis Kit (TaKaRa) reverse transcription produces the first strand of cDNA, the first round of reverse transcription system is: 1. Mu.L RNA, 1. Mu. L Oligo dT Primer (50. Mu.M), 1. Mu.L dNTP mix (10 mM each), 7. Mu.L RNase Free ddH ₂ O; the reaction conditions are as follows: the reaction was carried out at 65℃for 5min and rapidly cooled on ice. The second round reverse transcription system is: 10. Mu.L of the first round reaction solution, 4. Mu.L of 5X PrimeScript II Buffer, 0.5. Mu. L RNase Inhibitor (40U/. Mu.L), 1μL PrimeScript II RTase(200U/μL)、4.5μL RNase Free ddH ₂ O; the reaction conditions are as follows: the reaction was carried out at 42℃for 60min and at 95℃for 5min, and cooled on ice. The PCR amplification system is as follows: 12.5. Mu.L of 2X Phanta Flash Master Mix (Vazyme), 1. Mu.L of Forward Primer (5'-ACTTAGTTCTTCTACTCGTG-3' (SEQ ID NO. 3)), 1. Mu.L of Reverse Primer (5'-CTCCATTATGTTCACTGC-3' (SEQ ID NO. 4)), 2. Mu.L of second round PCR amplification product, 8.5. Mu.L of 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. The products were subjected to 1% agarose gel electrophoresis and the results are shown in FIG. 2.

EXAMPLE 2 homology alignment of amino acid sequence deduced from peony PsMYB4 Gene with MYB protein related to anthocyanin biosynthesis

The amino acid sequences deduced from MYB genes related to anthocyanin biosynthesis pathways in peony and 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 PsMYB4 protein and other anthocyanin biosynthesis related MYB proteins both contain R2 and R3 conserved domains at the N end, the R3 conserved domain also contains potential binding sites for interaction with bHLH family transcription factors, and at the C end of the PsMYB4 protein, 4 conserved inhibitory motifs, namely C1 motif, C2 motif, C3 motif and C4 motif, exist, and the inhibitory motifs endow the PsMYB4 protein with the capacity of negatively regulating anthocyanin biosynthesis (figure 3).

EXAMPLE 3 expression of peony PsMYB4 Gene silencing vector in peony petals

Construction of peony PsMYB4 gene silencing vector: primers containing the cleavage sites Xba I and BamH I were designed for amplification of PsMYB4 specific sequences (upstream primer: 5'-AAGGTTACCGAATTCTCTAGAGCCTAAGAAATCATAACCCGTGTT-3' (SEQ ID NO. 5), downstream primer: 5'-CGTGAGCTCGGTACCGGATCCTTAGTCCCCCTTGTCAACACCA-3' (SEQ ID NO. 6)). 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 PsMYB4 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,a total of 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 4-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 BamHI (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 4-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 positive monoclonal expansion was selected to extract plasmid TRV2-PsMYB4, followed by double digestion and sequencing verification until the TRV2-PsMYB4 silencing vector was successfully constructed.

Peony petals are transformed by the peony PsMYB4 gene silencing vector: mu.L of each of the TRV2-PsMYB4 silencing vector plasmid, the TRV2 empty vector plasmid and the TRV1 vector plasmid (stored in a laboratory) was transformed into competent cells (TOLOBIO) of Agrobacterium strain GV3101 (pSoup-p 19) (TOLOBIO Co.), followed by culturing at 28℃for 2d on a YEB plate (containing Rif50 mg/L and Kan 50 mg/L), and positive monoclonal was selected in YEB liquid medium (containing Rif50 mg/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 =1.5. The shaken bacteria were poured into 50mL centrifuge tubes, centrifuged at 5000rpm for 10min at room temperature and the supernatant was discarded for use. First atPreparing resuspension (containing 10mM MES, 10mM anhydrous magnesium chloride and 0.2mM acetosyringone) in sterilized triangular flask, adding into centrifuge tube, dissolving thallus in the resuspension, 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-PsMYB4 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 color 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 the petals for 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/L Cb ]]Culturing in dark at 4deg.C for 24 hr, transferring into incubator, culturing in light (light at 20deg.C for 16 hr and dark at 16deg.C for 8 hr), and culturing for 7 days to obtain the petal of Paeonia suffruticosa with PsMYB4 gene.

Example 4 identification of peony petals transformed with peony PsMYB4 Gene

And (3) PCR identification: the peony petal DNA was extracted using a NuClean Plant Genomic DNA Kit (CWBIO) kit. On the basis, PCR amplification was performed with the peony beta-Tubulin (EF 608942) gene as an internal reference (Forward Primer:5'-AGGTAAGATGAGCACCAAAG-3' (SEQ ID NO. 7), reverse Primer:5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 8)), while designing TRV2 vector-specific primers (Forward Primer:5'-TTGTTACTCAAGGAAGCACGAT-3' (SEQ ID NO. 9), reverse Primer:5'-TCCCCTATGGTAAGACAATGAG-3' (SEQ ID NO. 10)). 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. 4, a single bright beta-Tubulin band was detected in all of the wild-type peony petals, empty vector and transgenic peony with PsMYB4 gene, whereas a single bright clear band with correct position was detected in only the transgenic peony with PsMYB4 gene in the case of amplifying PsMYB4 band, and in wild-type peonyAnd empty carriers were not detected.

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 XgDNAwiter 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'-GAAAGCCCATACAAACAAAG-3' (SEQ ID NO. 11) and Reverse Primer:5'-TTAGGAAGTGACCGCCAG-3' (SEQ ID NO. 12)) of the PsMYB4 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. 7) and Reverse Primer:5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 8)). The reaction system: 2. Mu.L cDNA, 12.5. Mu.L +.>

SYBR qPCR SuperMix Plus、1 μL 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 ^-ΔΔCt The method performs analysis of the relative expression level of genes. qRT-PCR identification showed that PsMYB4 had significantly lower expression levels in transgenic peony petals (FIG. 5).

Example 5 determination of peony petal stain size and color correlation index of peony transformed with the PsMYB4 Gene

After peony petals grow for 7 days, the change of the color spot phenotype of the transgenic peony petals is observed, the color of the color spot of the transgenic peony petals is larger and darker compared with that of wild type peony petals (figure 6), the color spot size and the color correlation index of the peony petals are further measured, the color spot size of the transgenic peony petals is increased by 30.43 percent compared with that of the wild type peony petals (figure 7), the color spot correlation index a is increased by 14.06 percent compared with that of the wild type peony petals (figure 8), and the accumulation of the total anthocyanin content in the color spot of the transgenic peony petals of the PsMYB4 gene is far higher than that of the wild type peony petals (figure 9), so that the silencing PsMYB4 gene has the function of changing the color and the size of the plant petal color spot.

Example 6 detection of anthocyanin biosynthesis-related Gene expression level in peony petal stain transformed with peony PsMYB4 Gene

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 XgDNAwiter 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, and the expression levels of genes CHS, DFR and ANS involved in anthocyanin biosynthesis were detected using peony beta-Tubulin (EF 608942) as an internal reference gene (beta-Tubulin-F: 5'-AGGTAAGATGAGCACCAAAG-3' (SEQ ID NO. 7) and beta-Tubulin-R: 5'-GGAGGAATGTCACAAACACT-3' (SEQ ID NO. 8)), 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: F3H-F:5' -TCCTGAATCTAAGTCTGTGG-3' (SEQ ID NO. 13), F3H-R:5'-CCTCATTAGCTTCCCTGTA-3' (SEQ ID NO. 14); FLS-F:5'-GCTGAGTTCATCCGACCA-3' (SEQ ID NO. 15), FLS-R:5'-CCCATTCTTTGCTTGCAC-3' (SEQ ID NO. 16); DFR-F:5'-CGTTTTCACATCATCTGC-3' (SEQ ID NO. 17), DFR-R:5'-CATCTTGGACACGAAATACA-3' (SEQ ID NO. 18); ANS-F:5'-CAAACCAGCATCACCAAC-3' (SEQ ID NO. 19), ANS-R:5'-CTTTCCAAAAGCTCATCAGA-3' (SEQ ID NO. 20) 3GT-F:5'-TTCATCACTACCGAAGCC-3' (SEQ ID NO. 21), 3GT-R:5'-GAAAGACGAGCAACGACG-3' (SEQ ID NO. 22). Using equation 2 ^-ΔΔCT 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 PsMYB4 gene-transferred peony petal stain were significantly lower compared to wild-type peony.

In conclusion, the application of the peony PsMYB4 gene in the aspect of changing the color and the size of plant petal color spots is provided, and the constructed PsMYB4 gene silencing vector is transformed into the peony petals to be expressed, so that the transgenic peony petals are found to be obviously larger in color compared with wild type color, the total anthocyanin content in the color spots is obviously increased, the expression level of anthocyanin biosynthesis related genes in the color spots is obviously increased, and novel peony germplasm with larger petal color spots is created.

Sequence listing

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<120> application of peony PsMYB4 gene in changing color and size of plant petal stain

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<213> Artificial sequence (Artificial Sequence)

<400> 3

acttagttct tctactcgtg 20

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctccattatg ttcactgc 18

<210> 5

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aaggttaccg aattctctag agcctaagaa atcataaccc gtgtt 45

<210> 6

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgtgagctcg gtaccggatc cttagtcccc cttgtcaaca cca 43

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aggtaagatg agcaccaaag 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ggaggaatgt cacaaacact 20

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ttgttactca aggaagcacg at 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcccctatgg taagacaatg ag 22

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gaaagcccat acaaacaaag 20

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ttaggaagtg accgccag 18

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tcctgaatct aagtctgtgg 20

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

cctcattagc ttccctgta 19

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gctgagttca tccgacca 18

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cccattcttt gcttgcac 18

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

cgttttcaca tcatctgc 18

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

catcttggac acgaaataca 20

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

caaaccagca tcaccaac 18

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ctttccaaaa gctcatcaga 20

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ttcatcacta ccgaagcc 18

<210> 22

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gaaagacgag caacgacg 18

Claims

1. Peony treePsMYB4The gene is characterized in that the peonyPsMYB4The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The peony PsMYB4 protein is characterized in that the amino acid sequence of the peony PsMYB4 protein is shown as SEQ ID NO. 2.

3. Cloning of the peony of claim 1PsMYB4The amplification primer of the cDNA full-length sequence of the gene is characterized in that the sequence of the amplification primer is shown as SEQ ID NO.3 and SEQ ID NO. 4.

4. An expression cassette, recombinant vector or recombinant strain comprising the peony of claim 1PsMYB4And (3) a gene.

5. The recombinant vector of claim 4, wherein the recombinant vector comprises a gene silencing vector or an over-expression vector.

6. The peony of claim 1PsMYB4The application of the gene, the peony PsMYB4 protein of claim 2, the expression cassette, the recombinant vector or the recombinant strain of claim 4 in changing the color and the size of plant petal color spots is characterized in that the plant is peony.

7. The method for constructing a recombinant vector according to claim 5, comprising the steps of: expansion ofAdding the peony of claim 1PsMYB4The gene fragment is connected with a binary expression vector TRV2 plasmid.

8. A method for obtaining peony with larger petal stain, characterized by making claim 1PsMYB4And (3) gene silencing.

9. A method of identifying peony having relatively large petal stain 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) Determining that the peony low abundance expresses the peony PsMYB4 protein of claim 2.