CN107936104B

CN107936104B - Peony PsMYB12 transcription factor and coding gene and application thereof

Info

Publication number: CN107936104B
Application number: CN201711479630.1A
Authority: CN
Inventors: 舒庆艳; 朱瑾; 刘政安; 王亮生
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-03-24
Anticipated expiration: 2037-12-29
Also published as: CN107936104A

Abstract

The invention discloses a peony PsMYB12 transcription factor, and a coding gene and application thereof. The peony PsMYB12 transcription factor provided by the invention is a protein of a) or b) as follows: a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; b) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table, is related to peony stain formation and/or anthocyanin synthesis and is derived from a). Through VIGS silencing and heterologous over-transformation analysis, the results show that the gene can regulate and control the expression of key enzyme genes in the anthocyanin synthesis pathway. The method is supposed to play an important role in synthesis and accumulation of anthocyanin in peony spots and formation of the spots. The invention clones the specific expression gene in the speckles for the first time and verifies the function of the gene, lays a foundation for a molecular mechanism of the formation of the speckles, and provides a technical basis and an idea for deeply analyzing the synthesis and regulation of peony flavonoids.

Description

Peony PsMYB12 transcription factor and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a peony PsMYB12 transcription factor, and a coding gene and application thereof.

Background

Peony (Paeonia suffruticosa Andrews) is a deciduous shrub of Paeoniaceae and Paeonia (Paeonia) peony group (Sec. Moutana DC), becomes a traditional famous flower in China due to luxurious posture and rich colors of the flower, and plays an important role in the development history of flowers in China and even the world. The peony flower color can be roughly divided into 9 large color systems of red, pink, purple, white, yellow, black, green, blue and compound color according to the traditional color system. The color spot at the base of the peony petal is one of the important characteristics, and besides the great ornamental value, the color spot becomes an important basis for the classification and evolution research of peony breed groups. It is worth mentioning that the bases of the petals of Paeonia rockii (P.rockii) and part of Paeonia yunnanensis (P.delayvaii) as well as a considerable part of the cultivars (Central plains, Japan and European and American populations) carry significant color spots. The color spots of the northwest peony variety are more abundant, and different color spots such as black, black purple, brownish red, purple and the like are seen.

The basic research on the chemistry of the peony flower spot color is deeper. Researches show that the non-spot part anthocyanin of most varieties of petals of the peony in northwest consists of Pn3G5G, Cy3G5G and Cy3G, the Pn3G content is very low, and the peony almost does not contain Pg type pigment; the Cy-type dye is synthesized in a large amount at the base of the petal, and it is inferred that glycosylation, methylation, high Cy content and the formation thereof at the base of the petal are important causes for the characteristic that the northwest population has purple spots. Zhang et al (2007) (Zhang JJ, WanglS, Shu QY, Liu ZA, Li CH, Zhang J, Wei XL, Tian DK (2007) company of anti-cynanins non-blobs and blobs of the pets of Xibei tree peon. Sci Hortic114,104-111.) the composition of non-blobs and blobs of 35 northwest peony varieties was analyzed by high performance liquid chromatography, and 6 kinds of anthocyanin were detected in the blobs and the blobs, namely Pn3G5G, Pn3G, Cy3G5G, Cy3G, Pg3G5G and Pg 3G; but the content of the pigment is greatly different, and the content of the pigment in the spots is obviously higher than that of the non-spots; cy-type glucoside is used as main component in the spot, and 3G-type pigment is used as main component; the non-macula contains Pn type glycoside as main ingredient, and 3G5G type pigment as main ingredient. Cy-type pigments are synthesized in large amounts at the base of peony petals, resulting in the formation of a mottled color. In addition, preliminary study on the base color spot forms of the petals of the peony variety in the northwest China (2013) preliminary study on the base color spot forms of the petals of the peony variety in the northwest China (Zhaonao, Yuan.) Chinese agriculture report 29:192 + 197) provides a morphological basis for studying the mechanism of forming the peony color spots.

The molecular mechanism research of peony spot color formation is only reported a few, 1573 genes which are differentially expressed from non-spots are obtained by analyzing the transcriptome sequence of a peony purple-spot variety ' Jinrong ', 933 up-regulated expression genes and 640 down-regulated expression genes are obtained from purple spots, and the fluorescent quantitative analysis is carried out on the expression of enzyme genes in an anthocyanin synthesis pathway in the spots and the non-spots, and the result shows that the expression of PsCHS, PsF3' H, PsDFR and PsANS in the spots is remarkably higher than that of the non-spots, and the formation of the spot color is presumed to be caused by the common high expression of the four genes. In addition, through analysis of transcriptome of paeonia rockii, paeonia ostii' and hybrid species F1 generation petals, CHS, DFR, ANS and GST are presumed to have important roles in the formation of the speckled color of the paeonia rockii, and 2R3-MYB regulate the formation of the speckled color through the regulation of CHS, ANS and GST, but all lack experimental evidence. Functional identification and research on genes for spot color formation are not carried out, and particularly, no report is found on genes of a regulatory factor MYB family for regulating the spot color formation.

The families of the repeated MYB are divided into 4 types according to the number of repeated MYB structural domains, namely 1R-MYB, R2R3-MYB, 3R-MYB and 4R-MYB, wherein the R2R3-MYB transcription factor is one of the largest transcription factor families in plants, is proved to be widely involved in the secondary metabolism of the plants, the response of hormones and environmental factors and has important regulation effects on the growth, development and stress-resistant response of the plants; is also a main regulation factor which is discovered at present and is involved in regulating anthocyanin synthesis and plant stain formation.

The MYB transcription factor has tissue specificity on the regulation and control of anthocyanin synthesis related enzyme genes; one MYB regulatory factor can regulate enzyme genes in a plurality of anthocyanin synthesis paths, and the enzyme gene in a certain anthocyanin synthesis path can also be regulated by the plurality of MYB regulatory factors; MYB regulating factors are used for positively regulating and controlling most of anthocyanin synthesis, and occasionally have negative regulation and control; the MBW complex can be formed by the co-action of the bHLH and WDR transcription factors to regulate the expression of genes, and can also be independently regulated.

Disclosure of Invention

In order to make up for the deficiencies of the above fields, the invention provides a peony MYB transcription factor, named as PsMYB12, derived from the peony variety 'Qinghai lake silver wave' (Paeonia suffruticosa 'Qing hai hu yin bo').

The peony transcription factor PsMYB12 provided by the invention is a protein of a) or b) as follows:

a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

b) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table, is related to peony stain formation and/or anthocyanin synthesis and is derived from a).

The coding gene of the protein also belongs to the protection scope of the invention.

The coding gene is shown as the following 1) or 2) or 3) or 4):

1) the nucleotide sequence of the DNA molecule is a DNA molecule shown in a sequence 2 in a sequence table;

2) the nucleotide sequence is a DNA molecule shown in a sequence 3 in a sequence table;

3) DNA molecules which hybridize under stringent conditions with the DNA molecules defined in 1) or 2);

4) DNA molecules having a homology of 90% or more with the DNA molecules defined in 1) or 2) or 3).

The expression cassette, the recombinant expression vector, the transgenic cell line or the recombinant strain containing the coding gene also belong to the protection scope of the invention.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: introducing the coding gene into a starting plant to obtain a transgenic plant; compared with the original plant, the color of the petals of the transgenic plant is changed and/or the expression quantity of the genes related to the flavonoid synthetic pathway in the petals is changed.

The coding gene is introduced through a recombinant expression vector, and the recombinant expression vector is obtained by inserting the coding gene into a multiple cloning site of a starting vector pCXSN (T-vector);

the starting plant is control tobacco; the transgenic plant is transgenic tobacco.

Compared with the control tobacco, the color of the petals of the transgenic tobacco is darker; compared with the control tobacco, the expression level of flavonoid synthesis pathway related genes NtCHS and/or NtDFR and/or NtANS in petals of the transgenic tobacco is improved.

The VIGS silencing system for identifying the peony flavonoid synthetic pathway related genes also belongs to the protection scope of the invention.

A VIGS silencing system for identifying peony flavonoid synthetic pathway-related genes, comprising: inserting a target gene into the VIGS silencing vector to obtain a recombinant vector; the target gene is a nucleotide sequence shown as a sequence 2 in a sequence table and/or a nucleotide sequence shown as a sequence 3 in the sequence table.

The VIGS silencing vector is a tobacco rattle virus TRV2 vector; the peony flavonoid synthetic pathway related gene is PsCHS and/or PsCHI and/or PsF3H and/or PsF3' H and/or PsFLS and/or PsANS and/or PsWD40 and/or bHLH.

The application of the protein or the coding gene in regulating anthocyanin synthesis also belongs to the protection scope of the invention.

The invention obtains a partial sequence of a MYB family gene from a peony species Qinghai lake silver wave petal transcriptome, obtains a full-length name PsMYB12 by cloning according to RACE technology, and specifically expresses the gene in peony spots through fluorescent quantitative analysis, and the expression level is the highest in S2 stage. The eukaryotic expression vector is constructed to transform tobacco, so that the expression quantity of NtCHS, NtDFR and NtANS in the tobacco can be activated, the anthocyanin content in tobacco petals is improved, and the anthocyanidin in the transgenic tobacco petals is unevenly distributed. By using virus-mediated gene silencing PsMYB12, key enzyme genes in a flavonoid synthesis pathway can be reduced, including PsCHS, PsCHI, PsANS and the like. Therefore, the gene is proved to have an important role in the formation of peony spots.

The PsMYB12 specifically expressed in peony piebald belongs to R2R3-MYB family gene, the ORF length of the gene is 834bp, 278aa is speculated to be coded, the homology with the amino acid sequence of known species is not very high and can reach 56% at most, and the clustering analysis shows that the gene belongs to S5 subfamily and can possibly regulate and control the synthesis of procyanidine. Through VIGS silencing and heterologous over-transformation analysis, the results show that the gene can regulate and control the expression of key enzyme genes in the anthocyanin synthesis pathway. The method is supposed to play an important role in synthesis and accumulation of anthocyanin in peony spots and formation of the spots. The invention clones the specific expression gene in the speckles for the first time and verifies the function of the gene, lays a foundation for a molecular mechanism of the formation of the speckles, and provides a technical basis and an idea for deeply analyzing the synthesis and regulation of peony flavonoids.

Drawings

FIG. 1 shows the blooming process and spot coloration of the flowers of the peony variety Qinghai lake Yinbao.

FIG. 2 is an ORF amplification electrophoresis detection map, with DNA marker on the left and target gene ORF-834bp on the right.

FIG. 3 is an alignment of the PsMYB12 homologous amino acid sequences (underlined represents the R2R3 domain).

FIG. 4 is a cluster analysis of PsMYB12 with homologous sequences from other species in public databases.

FIG. 5 shows the expression of PsMYB12 in different stages of the development of the silver wave spot in Qinghai lake.

FIG. 6 is analysis of expression level of genes related to flavonoid synthesis in plaques before and after petal of VIGS silencing PsMYB 12; p < 0.05; p < 0.01.

FIG. 7 is an Agrobacterium-mediated transformation of tobacco with the PsMYB12 gene, wherein A: co-culturing process; b: inducing a differentiation process; c: differentiating the cluster buds for six weeks; d: inducing rooting process.

FIG. 8 is an identification of tobacco positive plants transformed with PsMYB 12. Lane 1-6: transgenic tobacco of interest, WT: wild type control.

FIG. 9 shows the result of expression analysis of PsMYB12 in leaves and flowers of transgenic positive lines.

Figure 10 is the transgenic and control tobacco flower phenotype. CK is the control, and No.3, No.5 and No.7 are the transgenic lines.

FIG. 11 shows the expression of key genes for flavonoid synthesis in transgenic tobacco and control petals.

Detailed Description

The peony variety ` Qinghai lake silver wave ` (Paeonia suffruticosa ` Qing Hai Hu Yin Bo `) used was from the resource garden of Beijing plantary, institute of plant, academy of sciences in China (non-patent documents describing the peony variety ` Qinghai lake silver wave ` (P. suffruticosa ` Qing Hai Hu Yin Bo `) are Zhang J, Wang L S, Shu QY, Liu Z A, Li C H, Zhang J, Wei X L, Tian D K.2007. company of anticancer in-waters and blotches of the peptides of Xibei treseon. Sci Hortic, 114: 104-111)). Petals of different developmental stages (S1-S4). Firstly, dividing the petals into 4 periods (S1-S4) (figure 1) according to the petal coloring process and the flower opening process, namely the S1 period, wherein the whole petals are yellow green, and spots do not appear; in stage S2, light red peach-shaped spots appear, and petals at non-spots are still yellow green; at the stage of S3, the color of the spots turns dark red, and the petals at the non-spot parts turn white; at stage S4, the color of the spots remained purple red, the shape of the spots was spindle-shaped, and the petals at the non-spots were still white.

The non-patent documents of the seeds of tobacco Nc89(Nicotiana tabacum cv. Nc89) used in this study (the seeds of this tobacco Nc89(Nicotiana tabacum cv. Nc89) are Du H, Wu J, Ji KX, Zeng QY, Bhuiyad MW, Su S, Shu QY, Ren HX, LiZA, Wang LS.2015.methylation media treated by an anticancer O-methyl fusion enzyme, is fermented in tobacco fusion in Paeonia J ExpB66 (21):6563-77), and are stored in the laboratory.

Example 1 cloning of the peony PsMYB12 Gene

First, Gene cloning

The method utilizes petals of 'Qinghai lake silver wave' S2 phase of the paeonia rockii variety as materials to clone genes.

(1) Plant total RNA extraction

The method for extracting the total RNA of the plant material by referring to the product specification of the RNAprep Pure plant kit of Tiangen company comprises the following steps:

1) taking 50-100mg of petals of S2 phase, quickly and fully grinding into powder in liquid nitrogen, immediately pouring into a 1.5mL EP tube, adding 500 μ L of SL (adding 5% mercaptoethanol before use), immediately whirling, violently shaking and uniformly mixing;

2) centrifuging at 12,000rpm for 2 min;

3) transferring the supernatant to a filter column CS in a collecting tube, centrifuging at 12,000rpm for 2min, carefully sucking the supernatant in the collecting tube to a new 1.5mL EP tube without RNase, and preventing the suction head from contacting with cell debris sediment in the collecting tube as much as possible;

4) slowly adding 0.4 times of the volume of the supernatant of absolute ethyl alcohol, mixing (at this time, precipitation may occur), transferring the obtained solution and the precipitation into an adsorption column CR3, centrifuging at 12,000rpm for 15s, pouring off the waste liquid in a collecting pipe, and placing an adsorption column CR3 into the collecting pipe;

5) adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12000rpm for 15s, pouring off waste liquid in the collection tube, and placing adsorption column CR3 into the collection tube;

6) preparing DNase I working solution: putting 10 mu L of DNase I stock solution into a new EP tube without RNase, adding 70 mu L of RDD solution, and gently mixing;

7) adding 80 μ L DNase I working solution into adsorption column CR3, standing at room temperature for 15 min;

8) adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12000rpm for 15s, pouring off waste liquid in the collection tube, and placing adsorption column CR3 into the collection tube;

9) adding 500 μ L of rinsing solution RW (ethanol before use) into adsorption column CR3, centrifuging at 12,000rpm for 15s, removing waste liquid in the collection tube, and placing adsorption column CR3 into the collection tube;

10) repeating the step 9;

11) centrifuging at 12,000rpm for 2min, placing adsorption column CR3 into a new RNase-free EP tube of 1.5mL, suspending 30-50 μ L sterilized DEPC-H in the middle of the adsorption membrane₂O, standing at room temperature for 2min, and centrifuging at 12,000rpm for 1min to obtain an RNA solution;

12) the quality of RNA was checked by agarose electrophoresis (7-15kb band for mRNA and hnRNA, 5kb band for 28SrRNA, 2kb band for 18S rRNA, and 0.1-0.3kb band for 5S rRNA. Bands do not clearly indicate degradation of RNA).

13) The final concentration of RNA was determined by a Nanodrop 2000(Thermo) spectrophotometer and stored at-70 ℃ until use.

(2) Reverse transcription

Reverse transcription was performed according to the Kit instructions (SMARTer RACE 5 '/3' Kit, TaKaRa). The reverse transcription system and procedure are shown in tables 1, 2 and 3.

TABLE 1 reverse transcription System I

TABLE 2 reverse transcription System II

1) The reverse transcription system II is reacted for 3min at 72 ℃ and then for 2min at 42 ℃. After cooling, a brief centrifugation concentrated the reaction at the bottom of the tube.

TABLE 3 reverse transcription System III

2) Mixing the system I, II with the system III, reacting at 42 deg.C for 90min, and reacting at 70 deg.C for 10 min. After the reaction, 90. mu.L of Tricine-EDTA Buffer was added and stored at-20 ℃ for further use.

3) After obtaining the 3 'and 5' end sequences of the gene, ORF is obtained by amplifying cDNA obtained by reverse transcription of RNA extracted from petal spots and non-spots by high fidelity Takara LA Taq polymerase. Primers for amplification of the ORFs were as follows: PsMYB12F (Forward primer (5 '-3')): ATGGGAAGGGCTCCTTGTTGTTCAAA, respectively; PsMYB 12R: ATAAGTGATATCTACTGCTGCTGCTGCTGC are provided. The PCR reaction systems and procedures are shown in tables 4-1, 4-2 and 5.

TABLE 4-1 PCR reaction System I

TABLE 4-2 PCR reaction System II

TABLE 5 PCR reaction procedure

Second, target DNA band recovery and ligation

(1) Recovery of the strip

The amplified target band was recovered using the easy pure Quick Gel Extraction Kit (all-type gold), and the recovery method was performed according to the Kit instructions. The specific method comprises the following steps:

1) the DNA band of interest in the agarose gel is excised, placed in a clean 1.5mL centrifuge tube, and the gel weight, e.g., 100mg gel, can be regarded as 100. mu.L, and so on.

2) Adding 3 times of GSB gel solution, placing in 55 deg.C water bath to completely dissolve the gel, and adding 1 time of isopropanol after melting;

3) cooling the melted gel to room temperature, placing into a centrifugal column, standing at room temperature for 1min, centrifuging at 10,000 Xg for 1min, and discarding the effluent;

4) adding 650 μ L WB solution, centrifuging at 10,000 Xg for 1min, and discarding the effluent;

5) centrifuging at 10,000 Xg for 2min to remove the residual WB solution;

6) placing the adsorption column in a new 1.5mL centrifuge tube, opening the cover, standing for 1min to volatilize the residual ethanol, adding 30 μ L EB solution (preheated at 65 ℃) to the center of the column, and standing for 1min at room temperature;

7) centrifuging at 10,000 Xg for 1min to elute DNA, detecting by electrophoresis and recovering the result as shown in FIG. 2, and storing in a refrigerator at-20 deg.C for use

(2) Ligation and transformation of target DNA bands

The ligation of the target band was transformed according to the manufacturer's instructions by the following method:

1) the recovered product (4.0. mu.L) and 1.0. mu.L of pEASY-T3 Cloning Vector (available from all-purpose gold Biotechnology Co., Ltd.) were added to the centrifuge tube, and the mixture was reacted at room temperature (20 ℃ C. -37 ℃ C.) for 5min, and after the reaction was completed, the centrifuge tube was placed on ice.

2) The ligation product was added to a solution containing 50. mu.L of Transl-T1 E.coli competent cells (purchased from all-grass Biotechnology Ltd.) added to the competent cells immediately after thawing, gently mixed and ice-cooled for 30 min.

3) Heat shock at 42 ℃ for 30s and immediately on ice for 2 min.

4) Add 300. mu.L of LB medium equilibrated to room temperature, incubate at 37 ℃ for 1h at 200 rpm.

5) 300. mu.L of the bacterial suspension was uniformly applied to the prepared resistant medium and cultured overnight at 37 ℃.

6) White monoclonal colonies were picked to 400. mu.L of LB medium supplemented with 0.1mg/mL kanamycin (Kan), incubated at 200rpm and 37 ℃ for 2 h.

The LB medium formula:

in 1,000mL of the system, 10g of peptone, 5g of yeast extract and 10g of sodium chloride were added, and the pH was 7.0. And if the culture medium is a solid culture medium, adding 15g of agar powder, and sterilizing at high temperature and high pressure.

Third, PCR method for identifying positive recombinants and sequencing

mu.L of the bacterial solution was used as a template for PCR reaction, and M13 forward and reverse primers (M13F:5'TGTAAAACGACGGCCAGT 3'; M13R:5'CAGGAAACAGCTATGACC3') were used to identify recombinants, and the reaction system and procedure are shown in tables 6 and 7, respectively.

Table 6 PCR reaction system for identifying recombinants is as follows:

TABLE 7 PCR reaction procedure for identifying recombinants

The product was detected by 1.0% agarose electrophoresis at 100V and stained with EB. The amplified target band was excised and recovered with the Easypure Quick Gel Extraction Kit (Beijing Omegano gold Biotechnology Co., Ltd.), and 4. mu.L of the recovered product and 1. mu.L of pEASY-T3 vector were ligated at 25 ℃ for 15 min. After the connection is finished, positive clones are transformed and screened, and are used for sequencing after being identified by PCR. The sequence accuracy was verified by sequencing data from 10 independent clones pEASY-T3, wherein the sequence data from more than 5 clones were identical and were considered to represent the correct sequence information.

As a result: the cDNA of the obtained gene full length is 1081bp, and is shown as a sequence 2 in a sequence table; wherein, the 1 st site to the 834 th site from the 5' end of the sequence 2 in the sequence table is an open reading frame, and the open reading frame part is 834bp which is shown as a sequence 3 in the sequence table; the protein shown in a sequence 1 in a coding sequence table, wherein the sequence 1 consists of 278 amino acid sequences. The gene was named PsMYB12, and the protein encoded by the gene was named PsMYB 12.

The amino acid sequence of the protein is subjected to BlastP analysis by online software (https:// blast. ncbi. nlm. nih. gov /), and the result shows that the similarity of the protein and the known species is below 56%, in particular, the similarity of the protein and the known species is 56% with wild strawberry (Fragaria viscosa) (NP-001295449), the similarity of the protein and the wild strawberry (Theobroma cacao) TT2 like MYB is 55%, the similarity of the protein and the wild strawberry (Vibrio) MYBPA1 (NP-001268160) is 52%, and the like. Multiple comparisons were made with homologous amino acid sequences of known species including Arabidopsis thaliana (Arabidopsis thaliana) AtMYB12(DQ224277), grape (Vitis vinifera) VvMYBF1(ACT88298), apple (Malus domestica) MdMYB22(AAZ20438), hybrid lily (Lilium hybrid) Ljmyb12(BAF74782), and African daisy (Gerbera hybrid) GhMYB1(CAD87007), all of which contain R2\ R3 domain (FIG. 3).

The cloned peony PsMYB12 amino acid sequence and MYB 5-7 subfamily sequence cloned from known species are analyzed and a phylogenetic tree is constructed (figure 4), and the results show that three family members are respectively polymerized into one, S7 subfamily is polymerized into one, synthesis of flavone and flavonol is regulated, the homologous relation between the same family members is shown, S5 subfamily members are dispersedly polymerized into three, PsMYB12 of peony and grape VvMBPA1(NM _001281231) are polymerized into one, the peony PsMYB12 and grape VvMBPA1(NM _001281231) belong to S5 subfamily, and synthesis of Proanthocyanidins (Proanthocyanidins) is presumed to be regulated.

Example 2 comparison of expression levels of the PsMYB12 Gene in different stages of peony petal development and in different tissues

The process from the colorless period to the full-bloom period of the paeonia rockii variety 'Qinghai lake silver wave' is divided into 4 stages (figure 1), wherein the first two stages are sampled 1 time every two weeks, and the second two stages are sampled once every one week. Petals from 3 different plants were taken each time and repeated as 3 independent experiments. All samples were subjected to total RNA extraction and reverse transcribed into cDNA as template for fluorescent quantitative PCR.

First, RNA extraction

The RNA extraction method was the same as in example 1.

Second, reverse transcription

The reverse transcription was performed according to the product specification of FastQuant RT Kit from Tiangen, as follows:

since the concentrations of RNA extracted from samples at various stages are inconsistent and greatly different, in order to keep the concentration of cDNA in the product consistent, the final inversion amount is preset to be 800ng, and a 20. mu.L reaction system is established.

The method comprises the following steps:

1) template and reagent thawing

Thawing template RNA on ice, 5 XgDNA Buffer, FQ-RT Primer Mix, 10 Xfast RTbuffer, RNase-Free ddH₂O was thawed at room temperature (15-25 ℃ C.), and placed on ice immediately after thawing. Each solution was vortexed and mixed well before use, and briefly centrifuged to collect the liquid remaining on the tube wall.

2) Preparation of genomic DNA removal System

The mixture was prepared on ice according to the genomic DNA removal system of Table 8 and thoroughly mixed. Briefly centrifuged and placed at 42 ℃. Incubate for 3 min. Then placed on ice.

TABLE 8 gDNA removal reaction System

3) Reverse transcription system

A mixed solution was prepared according to the reverse transcription reaction system of Table 9.

TABLE 9 reverse transcription reaction System

4) And adding the reverse transcription system mixed solution into the reaction system in the gDNA removing step, and fully and uniformly mixing. Incubate at 42 ℃ for 15 min.

5) After incubation at 95 ℃ for 3min, the cDNA is placed on ice and can be used for subsequent experiments or stored at low temperature.

Third, fluorescent quantitative PCR

Fluorescent quantitation was performed using the SuperReal PreMix Plus (SYBR Green) kit (available from Tiangen Biochemical technology, Inc. (Beijing). This was carried out by LT STEPONE PLUS (LIFE TECHNOLOGIES, USA) qPCR instrument. Relative expression analysis of mRNA transcripts three technical and biological replicates were performed, respectively, with the peony PsTublin gene (EF608942) as the reference gene. Primer sequences are shown in Table 10.

TABLE 10 peony PsMYB12 Gene cloning and List of primers used for fluorescent quantitation

Fourthly, obtaining a result:

a specially expressed MYB family gene is obtained from 'Qinghai lake silver wave' petal spots and named as PsMYB12, primers are designed, fluorescent quantitative analysis is carried out on the gene at different periods of petal spots and non-spot spots, the gene is specifically expressed in the spots, a peak value appears at the S2 period, and the relative expression amount reaches 1101 +/-43.4 (figure 5). It is speculated that this PsMYB12 may be an important regulatory gene regulating plaque color formation.

Example 3 silencing of VIGS PsMYB12

The Tobacco Rattle Virus (TRV) is a virus composed of two chains, TRV1 and TRV 2. The VIGS vector used in the invention is derived from a Tobacco brittle fracture virus (TRV), TRV1/TRV2 is a donation given by the teaching of the university of Chinese agriculture ornamental plant postharvest and adversity physiology laboratory horse male (non-patent documents recorded with the vector are Tian J, Pei H X, Zhang S, Chen J W, Chen W, Yang R Y, Meng Y L, You J, Gao J P, Ma N.2014.TRV-GFP: amplified to baco viral vector for infection and visualization of gene function J Top Bot 65: 311 cake 322), which carries a Kana screening marker and a 35S promoter, and pTRV2 carries multiple cloning sites such as BamH I and Xho I. The engineered viruses were used to perform relevant studies. TRV1 mainly plays a role in virus operation in plants, and TRV2 is an expression vector and contains a plurality of cloning sites.

Cloning PsMYB12 to obtain a 200bp fragment, adding BamH I and Xho I double enzyme cutting sites to respectively replace a multi-cloning site part on a TRV2 vector, constructing a TRV2-PsMYB12 plant expression vector, transforming escherichia coli DH5 α and agrobacterium GV3101 to obtain positive clones, and verifying a correct sequence through sequencing to prove that the construction is successful, cutting a flower (at S3 stage) from the plant for vacuum infection, sampling 4 days after infection, separating spots from non-spots, and using the spots for fluorescent quantitative analysis.

(1) Construction of VIGS expression vector

Designing primers according to a cDNA sequence of a target gene PsMYB12, cloning a target gene fragment, adding enzyme cutting sites BamH I and Xho I at two ends of the target gene, and showing the sequence of a primer Myb12F/R in a table 10.

The PCR amplification reaction system and procedure were the same as for cloning the complete ORF in the examples. The target band obtained by amplification is connected with pEASY-T after being cut and recovered₃Vector (purchased from holo-gold biotechnology limited), transformed into Transl-T1 Escherichia coli Competent cells (Trans1-T1 Phage resist chemical company Cell, purchased from holo-gold biotechnology limited), coated with LB Resistant plate containing Amp (0.1mg/mL), cultured overnight at 37 ℃, white single colony was picked up, PCR detection was performed after 2h of shaking, and the plasmid was verified to be correct in sequence, and after verification, double digestion with restriction enzymes BamH I and Xho I was performed and recovered. At the same time, carrying out double enzyme digestion of BamH I and Xho I on VIGS expression vector TRV2, recovering, and separating target fragment and lineThe sex vectors are connected by T4DNA ligase to construct a VIGS expression vector, an enzyme digestion connection system and reaction conditions are as shown in tables 12 and 13, a connection product is transformed into DH5 α escherichia coli cells (purchased from Kangjikang, century Biotechnology Co., Ltd.) and then the correct expression vector plasmid is verified by sequencing, GV3101 agrobacterium competent cells (purchased from Beijing Bomaide Gene technology Co., Ltd.) are transformed by a freeze-thaw method to obtain positive single-clone agrobacterium, and the bacterial liquid is added with 50% of glycerol and is uniformly mixed and stored at-80 ℃ for later use.

(2) The peony bud is infected by an air pumping method (refer to Tian J, Pei H X, Zhang S, Chen J W, Chen W, YangR Y, Meng Y L, You J, Gao J P, Ma N (2014) TRV-GFP: a modified tomato viral vector for efficacy and vitality analysis of gene function J Exp Bot 65,311 + 322.), and the specific method is as follows:

1) activating the preserved positive monoclonal bacteria liquid, adding the activated bacteria liquid into LB liquid culture medium containing Kan (0.1mg/mL) and Rif (0.05mg/mL) according to the proportion of 1:100, performing shaking culture at 28 ℃ and 180rpm until OD is reached₆₀₀＝1.0；

2) Centrifuging at 6,000rpm at room temperature for 5min, collecting thallus, and discarding supernatant;

3) preparation of the staining solution (10mM MgCl)₂20mM acetosyringone, 10mM MES, pH 5.6), and sucking the thallus by a gun until the thallus is evenly suspended in the infection liquid;

4) adjustment of OD of resuspended suspension with infecting solution₆₀₀1.0 OD of the bacterial suspension pTRV2-MYB12, pTRV1 and pTRV2₆₀₀The values are the same;

5) mixing pTRV2-MYB12 and pTRV1, and pTRV2 and pTRV1 bacterial solutions at a volume ratio of 1:1 respectively, and standing in the dark for 4 h;

6) cutting peony flower with 5cm of flower stalk, randomly distributing in different treatments, immersing in bacteria solution, vacuumizing to 0.7atm (atmospheric pressure), treating under negative pressure for 10min, and slowly deflating for 20 min. Three flowers were treated each, i.e. repeated 3 times;

7) rinsing the sucked petals with deionized water to remove redundant bacteria liquid;

8) immersing flower stalks in deionized water, culturing at 8 deg.C and humidity of about 60% in dark for 1 d; then transferring to 23 ℃, culturing for 3 days with the humidity of about 60%;

9) sampling after infecting for 4 days, separating the spots and non-spots of the petals, taking the spots and the non-spots as RNA extraction detection samples, and quickly freezing and storing the samples at minus 80 ℃ by liquid nitrogen. The expression level of the gene was then tested for changes relative to the blank. The primer PsMYB12 for fluorescent quantitative determination is referred to as PsMYB12-qF/R in Table 10, and the other primers are referred to as Table 11. The reaction system and procedure were the same as in example 2.

TABLE 11 List of primers used for quantitative assay of fluorescent PsMYB12 silencing by VIGS

The results show that: after transient silencing of PsMYB12 by using VIGS technology, the expression level is reduced by 54.0%, the expression level of PsCHS is reduced by 82.7%, and the expression levels of PsCHI, PsF3H, PsF3' H, PsFLS, PsANS and PsWD40 are reduced by 24.0%, 39.4%, 30.5%, 48.4%, 31.1% and 28.6% respectively; the reduction in PsDFR was minimal, 2.1%. The expression level of bHLH was not only not decreased but also increased by 8.8%. Statistical analysis shows that the expression quantity of PsCHS, PsFLS and PsANS in the plaques is down-regulated, and the difference is very significant; expression was downregulated by PsCHI and PsF3H, with significant differences (fig. 6).

Example 3 construction of eukaryotic overexpression vector and verification of PsMYB12 Gene function by transformation of tobacco

Construction of eukaryotic over-expression vector

The eukaryotic over-expression vector is PSN1301 (publicly available from plant research institute of Chinese academy of sciences, and non-patent documents describing the eukaryotic over-expression vector PSN1301 are Liuqing, Tangjian, Zeanjuan, yellow and bright, Chunjianhua, yellow sengjust, 2014, the cloning of Arabidopsis AtVQ29 gene and the construction of plant expression vector, the modern biomedicine progress 14(35): 6814) 6817), and carries tobacco mosaic virus promoter CaMV35S, hygromycin resistance marker gene and multiple cloning sites. Designing a primer according to a target gene sequence, cloning a target gene fragment ORF, adding Xba I and Kpn I enzyme cutting sites at the 5 '/3' end respectively, and carrying out double enzyme cutting on the empty vector and the DNA fragment respectively, wherein an enzyme cutting reaction system is shown in Table 12. The primers for amplifying the ORF of the target gene fragment are as follows: PsMYB12F (Forward primer (5 '-3')): ATGGGAAGGGCTCCTTGTTGTTCAAA, respectively; PsMYB12R (Reverprimer (5 '-3')): ATAAGTGATATCTACTGCTGCTGCTGCTGC are provided.

TABLE 12 double enzyme digestion System composition and dosage

The reaction conditions are 37 ℃ and 2-4 h.

Recovery of the double cleavage products the gene of interest was ligated into eukaryotic overexpression vectors under the influence of T4DNA ligase as described in example 1 above, the ligation system is shown in Table 13.

TABLE 13 ligation System composition and amounts

The reaction conditions were 25 ℃ for 15 min.

Secondly, transforming agrobacterium to obtain positive clone and sequencing verification

Sequencing to verify correct extracted plasmids, and transforming agrobacterium GV3101 competent cells by a freeze-thaw method to obtain positive agrobacterium monoclonal, wherein the method comprises the following steps:

A. melting GV3101 Agrobacterium tumefaciens competent cells stored at-80 deg.C in ice water bath or at room temperature;

B. adding 1 mu g of plasmid DNA to be transformed into the just thawed competent cell suspension under the aseptic condition, gently mixing uniformly, and standing for 10min in ice bath;

C. placing the centrifugal tube in liquid nitrogen for quick freezing for 5 min;

D. then placing the centrifuge tube in 37 deg.C water bath rapidly and keeping for 5 min;

E. placing the centrifuge tube back into ice and maintaining for 5 min;

F. adding 800 μ L of LB liquid culture medium without antibiotic under aseptic condition, and performing shake culture at 28 deg.C for 2-3 h;

g.5,000rpm centrifugation for 1min to collect the thallus, removing 600. mu.L of liquid, resuspending the thallus, evenly spreading in a solid culture medium containing 0.1mg/mL kanamycin (Kan) and 0.05mg/mL rifampicin (Rif), and culturing at 28 ℃ for 48 h;

H. single colonies were picked and cultured with shaking in LB liquid medium containing Kan (0.1mg/mL) and Rif (0.05mg/mL) at 28 ℃ for 3 hours for PCR detection. The PCR detection method was the same as in example 1. The primer used was MYB12F/R as shown in Table 10.

Third, transforming tobacco

(1) The tobacco seed disinfection steps are as follows:

A. a certain amount of seeds was taken and placed in a 5mL sterile EP tube.

B. Adding 3mL of 75% ethanol, mixing for 1min by turning upside down, and removing the supernatant.

C. Adding 3mL of absolute ethyl alcohol, turning upside down, mixing for 3min, and removing supernatant. This was repeated three times.

The steps are carried out in a sterile super clean bench. The tobacco seeds are directly inoculated in an MS culture medium and are placed in a tissue culture room for culture.

(2) Tobacco transformation by leaf disc method

A. Activating the preserved positive monoclonal bacteria liquid, adding the activated bacteria liquid into LB liquid culture medium containing Kan (0.1mg/mL) and Rif (0.05mg/mL) according to the proportion of 1:100, performing shaking culture at 28 ℃ and 180rpm until OD is reached₆₀₀＝0.5；

B. Centrifuging at room temperature of 6,000rpm for 4min, collecting thallus precipitate, re-suspending and cleaning with MS-1 liquid culture medium to remove residual antibiotics, re-suspending thallus in equivalent MS-1 liquid culture medium after centrifuging for ready-to-contaminate leaves;

C. collecting well-grown leaf of sterile tobacco seedling, cutting off leaf edge and main vein, and cutting the rest into 1cm²The left and right small blocks;

D. putting the cut tobacco leaf small blocks into MS-1 suspended bacterial liquid, dip-dyeing for 10min, and slightly shaking to facilitate the bacterial liquid to completely contact with leaf edge wounds;

E. placing the impregnated leaves on sterile filter paper, and sucking residual bacteria liquid;

F. inoculating the leaf of the dry-absorbed bacterial liquid to a co-culture medium MS-1 flat plate without antibiotics, and placing the flat plate in a 23 ℃ environment to be shaded for co-culture for 2 days;

G. after the co-culture is finished, washing residual agrobacterium on the surface of the leaf by using sterile water containing 200mg/mL of cefradycin (Cef) and 200mg/mL of carbenicillin (Carb), sucking water by using sterile filter paper, transferring the leaf to a differentiation culture medium MS-2, and performing illumination culture at 26 ℃;

H. after the cluster buds are differentiated, when the differentiated seedlings grow to be 2-3cm high, the cluster buds are cut off and transferred into 1/2MS rooting culture medium for inducing rooting.

MS-1 culture medium: MS basic ingredient +30g/L sucrose, pH 5.8

MS-2 differentiation medium: MS-1+ BA (1.0mg/L) + NAA (0.2mg/L) + Hyg (20mg/L) + Cef (200mg/L) + Carb (200mg/L), pH 5.8

Rooting culture medium: 1/2MS +20g/L sucrose, pH 5.8.

(3) PCR detection of transformed plants:

the DNA extraction of the transformed plant leaves adopts an improved CTAB method, and the specific steps are as follows:

adding 0.6% (v/v) mercaptoethanol into CTAB extracting solution, and preheating in a 65 ℃ water bath kettle;

B. weighing about 0.2g of leaf, grinding into powder with liquid nitrogen, adding about 0.01g of polyvinylpyrrolidone (PVP), and packaging into 2mL EP tubes containing about 100 mg;

C. adding 800 μ L preheated CTAB extractive solution into each EP tube, shaking vigorously, mixing, placing into 65 deg.C water bath for 60min, and shaking gently every 10 min;

D. taking out the EP tube, cooling to room temperature, adding 800 μ L chloroform/isoamyl alcohol, shaking thoroughly for 10min, centrifuging at 12,000rpm for 5 min;

E. taking the centrifuged supernatant into a new 2mL EP tube, adding equal volume of chloroform/isoamylol, fully shaking for 10min, and centrifuging at 12,000rpm for 5 min;

F. after centrifugation, the supernatant was removed to a 1.5mL EP tube, and 2/3 volumes of isopropanol (pre-cooled in a-20 ℃ freezer) were added, gently shaken well and then precipitated in a 4 ℃ freezer for 40min, and then centrifuged at 12,000rpm for 10 min;

G. centrifuging, pouring out supernatant, adding 1mL of 70% ethanol, washing precipitate, and centrifuging at 12,000rpm for 1 min;

H. repeating the step 7 for cleaning once;

I. the supernatant was decanted and the DNA was dried at room temperature, and after about 20-30min (not overdrying), 100. mu.L of double distilled water was added to dissolve the DNA.

The DNA concentration was measured with an ultraviolet spectrophotometer, the DNA quality was checked with 1.0% agarose gel electrophoresis, and recorded by taking pictures after EB staining. The DNA was stored at-20 ℃ until use.

The PCR system and procedure were the same as in example 1, the PCR product detection method was the same as in example 2.4, and the detection primer was 12GF/R, as shown in Table 14:

TABLE 14 fluorescent quantitative analysis primer list for flavonoid synthetic pathway related genes in transgenic tobacco plants

As a result: the eukaryotic vector PSN1301 with the PsMYB12 gene is transferred into wild tobacco by an agrobacterium-mediated method, resistant tobacco is obtained by screening hygromycin through the processes of co-culture, differentiation, root induction and the like (figure 7), and 6 strains of the PsMYB12 gene-transferred positive seedlings are obtained through PCR identification (figure 8).

And (3) transplanting the positive plants into seedlings, and carrying out fluorescence quantitative analysis on the expression quantity of PsMYB12 in leaves and flowers of the positive plants to obtain 3 strains with higher expression quantities, wherein the strains are respectively numbered as No.3, No.5 and No.7 (figure 9). The exogenous peony PsMYB12 is proved to be capable of being transcribed normally and highly expressed in tobacco, and the relative expression amount in the leaves of the transgenic line is 2403.9 +/-235.7 (No.3), 2235.8 +/-585.6 (No.5) and 13871.0 +/-949.5 (No.7) times of that of the control non-transgenic line; the expression level in petals was 6834.3. + -. 152.2 times that of the control (No.3), 3015.9. + -. 83.0(No.5), and 20074.5. + -. 776.4(No. 7).

Expression analysis of flavonoid synthetic pathway genes in transgenic positive plant petals:

the transgenic tobacco and the control flower were photographed and observed to see that the pigments in the petals of the transgenic line were not uniformly distributed, as in

line

7, 2 petals in one flower were darker, 3 petals were lighter, the junction between the petals was darker, and the anthocyanidin tended to extend downward along the tube and to be non-uniformly distributed in the tube (fig. 10).

Quantitative fluorescence analysis is carried out on expression of genes related to flavonoid synthetic pathways in petals of transgenic plants and control plants, and expression levels of NtCHS, NtDFR and NtANS in tobacco petals can be up-regulated after over-expression of PsMYB12 gene is found (figure 11).

Sequence listing

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Claims

1. A protein is a protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The encoding gene of claim 2, wherein: the coding gene is shown in the following 1) or 2):

2) the nucleotide sequence is a DNA molecule shown in a sequence 3 in a sequence table.

4. An expression cassette, recombinant expression vector or recombinant bacterium comprising the coding gene of claim 2 or 3.

5. A method of making a transgenic plant comprising the steps of: introducing the coding gene of claim 2 or 3 into a starting plant to obtain a transgenic plant; compared with the original plant, the color of the petals of the transgenic plant is changed and/or the expression quantity of the genes related to the flavonoid synthetic pathway in the petals is changed.

6. The method of claim 5, wherein:

the coding gene is introduced through a recombinant expression vector, and the recombinant expression vector is obtained by inserting the coding gene into a starting vector pCXSN;

7. The method of claim 6, wherein: compared with the control tobacco, the pigment in the petals of the transgenic tobacco is not uniformly distributed; compared with control tobacco, the gene related to flavonoid synthetic pathway in petals of the transgenic tobaccoNtCHSAnd/orNtDFRAnd/orNtANSThe expression level of (3) is increased.

8. The VIGS silencing system for identifying the peony flavonoid synthetic pathway related genes is characterized in that: comprises the following steps: inserting a target gene into the VIGS silencing vector to obtain a recombinant vector; the target gene is a nucleotide sequence shown as a sequence 2 in a sequence table and/or a nucleotide sequence shown as a sequence 3 in the sequence table.

9. The VIGS silencing system for identifying peony flavonoid synthetic pathway-associated genes as claimed in claim 8, wherein: the VIGS silencing vector is a tobacco rattle virus TRV2 vector; the peony flavonoid synthetic pathway related gene isPsCHSAnd/orPsCHIAnd/orPsF3HAnd/orPsF3'HAnd/orPsFLSAnd/orPsANSAnd/orPsWD40And/or bHLH.

10. Use of the protein of claim 1, the gene encoding the protein of claim 2 or 3 for modulating anthocyanin synthesis.