CN113005139B - Application of transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin - Google Patents

Abstract

The invention discloses application of a transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin, wherein an amino acid sequence of the transcription factor PsMYB1 is shown as SEQ ID No. 1. The PsMYB1 can activate expression of PsDFR and PsANS promoters independently, and can form a complex with PsbHLH1 and PsWD40-1 to improve promoter activity of PsDFR and PsANS, so that synthesis of anthocyanin in peony petals is promoted.

Description

Application of transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a MYB transcription factor related to synthesis of peony petal anthocyanin and application thereof.

Background

Peony (Paeonia suffruticosa Andrews) is a deciduous shrub of Paeoniaceae (Paeoniaceae) and Paeonia (Paeonia) peony group (Sect. Moutan DC.), is an important traditional ornamental plant and medicinal plant in China, and has extremely high ornamental value and economic value. Flower color is an important quality character of ornamental plants. The peony flower color can be roughly divided into 9 large color systems such as red, pink, purple, white, yellow, black, green, blue, compound color and the like according to the traditional color system. However, due to the close hybridization in the population of the long-term collected products, the problem of the same color of the peony varieties in China is getting worse. The molecular mechanism of peony flower formation is clarified, and efficient molecular biology is utilized to develop peony flower breeding.

Researches show that the flavonoid is a decisive pigment group formed by peony flower color, and the differences among different color systems are mainly concentrated on the components and the content of anthocyanin. The anthocyanin synthesis pathway is a branch of the flavonoid synthesis pathway derived from phenylpropanoid metabolism, and involves the interaction of multiple structural genes, regulatory genes and environmental factors. Among them, the R2R3 MYB protein, which contains both DNA binding regions (MYB motifs) of R2 and R3, is one of the largest families in plant transcription factors and is also the most widely involved regulator in anthocyanin biosynthesis. At present, the known model plant Arabidopsis MYB family has 124 genes, can be divided into 22 subclasses according to conserved elements, and MYB proteins for regulating and controlling flavonoid synthesis belong to 1-7 of Stracke classification, wherein PAP1, PAP2, MYB113 and MYB114 genes can directly regulate and control the transcription level of anthocyanin synthase genes, so that the synthesis of anthocyanin in cells is promoted. In addition, a large number of studies show that the regulatory action of MYB transcription factors has obvious tissue specificity; in addition to positive regulation, MYB members exist which have negative regulation effects on anthocyanin synthesis; it can not only be used for controlling gene expression by forming MBW complex together with bHLH and WDR transcription factors, but also can be used for independently controlling.

At present, the research on the formation of MYB transcription factors related to peony flower color is still in the initial stage, most of the research focuses on the aspects of candidate gene screening, transgene function verification and the like, and the regulation and control effects on structural genes in anthocyanin synthesis pathways and the interaction relationship among the transcription factors are not clear.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide an application of a MYB transcription factor related to the regulation and control of peony petal anthocyanin synthesis.

The technical scheme of the invention is as follows: the application of a transcription factor PsMYB1 or a gene coding the transcription factor PsMYB1 in regulating and controlling peony petal anthocyanin synthesis is disclosed, and the amino acid sequence of the transcription factor PsMYB1 is shown in SEQ ID No. 1.

Further, the application is the application of the transcription factor PsMYB1 or a gene coding the transcription factor PsMYB1 in activating a peony PsDFR gene or/and PsANS gene promoter.

Further, the nucleotide sequence of the gene coding the transcription factor PsMYB1 is shown as SEQ ID No. 2.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, original peony varieties 'Yubanbai' (white flower), 'Zhao powder' (pink flower) and 'Heihuakui' (deep purple red flower) are selected as test materials, 2 MYB transcription factors PsMYB1 and PsMYB2 which are likely to participate in synthesis and regulation of peony anthocyanin are screened based on transcriptome data and bioinformatics analysis, the cDNA full lengths of PsMYB1 and PsMYB2 are separated by adopting a method combining RACE-PCR and RT-PCR, gene expression pattern analysis is carried out, and PsMYB1 is found to play a key role in regulation and control of anthocyanin synthesis. Through a yeast single hybrid technology, a dual-luciferase test and an in vivo imaging test, the PsMYB1 can be proved to be capable of binding to promoter regions of PsDFR and PsANS to activate the expression of the PsDFR and PsANS, and can also be involved in forming an MBW complex, enhancing the promoter activity of PsDFR and PsANS and promoting the synthesis of anthocyanin. The invention provides thinking and basis for disclosing a transcription regulation and control mechanism for peony flower color formation, and provides an important target gene for ornamental plant flower color molecular breeding. By utilizing the function of promoting expression of downstream structural genes in anthocyanin synthesis pathway of PsMYB1, the new flower variety with variant flower color can be cultivated by using the PsMYB1 as an exogenous gene through a transgenic technology.

Drawings

FIG. 1 is a tree evolution analysis of the peony MYB gene family;

FIG. 2 shows the expression patterns of the transcription factors PsMYB1, PsMYB2 and PsMYB3 in the coloring process of the peony flowers of different color lines;

FIG. 3 shows the analysis of the multiplex sequence alignment of the transcription factors PsMYB1, PsMYB2 and PsMYB 3;

FIG. 4 is a diagram of the yeast two-hybrid (Y2H) system for analyzing the interaction between transcription factors;

FIG. 5 shows bimolecular fluorescence complementation (BiFC) validation of the interaction of PsMYB1, PsbHLH1 and PsWD 40-1;

FIG. 6 shows the selection of recombinant yeast on selective medium SD/Leu/AbAr;

FIG. 7 is a dual luciferase assay to analyze the regulatory effect of transcription factors on promoters;

FIG. 8 shows the regulation of promoter by transcription factor analyzed by luciferase complementation imaging.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

1. Materials and methods

1.1 test materials

The study takes peony white flower variety 'Yuban Bai' (P.suffruticosa 'Yuban'), pink flower variety 'Zhao powder' (P.suffruticosa 'Zhuao Fen') and deep-violet flower variety 'Black flower Kui' (P.suffruticosa 'Heihua Kui') as test materials, and is collected from Yuquan mountain peony planting bases of China forestry science research institute. According to the division of the growth and development annual cycle of the peony and the observation of the bud development and the flower coloring process of the peony, the flower blooming process is divided into 4 stages: level 1, hard bud Stage (Stage 1: uncolored compact bud); grade 2, strike through (Stage 2: lightly colored, flower about to bloom); grade 3, initial blooming period (Stage 3: flower initial blooming); stage 4, full bloom Stage (Stage 4: full bloom of flowers, with anthers exposed). Collecting three varieties of petals in different development periods, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C. The used seeds of Nicotiana benthamiana are stored in the laboratory, the seeds are sown in a substrate and cultured in a light incubator with the photoperiod of 14h/10h day/night, and 4-6 true leaves are grown on the seeds for the next experiment.

1.2 vectors, reagents and strains

Carrier: cloning vectors pMD19-T, yeast double hybrid vectors pGADT-7 and pGBKT-7 were purchased from TaKaRa company; the bimolecular fluorescent complementary vectors pUC-SPYNE and pUC-SPYCE were purchased from the vast Ling plasmid platform; the yeast single hybrid vector pAbAi, the dual-luciferase vector pGreenII0800-Luc and pGreenII62-SK are stored in the laboratory.

The kit comprises: RNA extraction kit (RN33-PLANTpure general plant total RNA rapid extraction kit) purchased from Beijing Ederly Biotech limited; reverse transcription kit (

One-Step gDNA Removal and cDNAsynthes SuperMix) from Kyoto Total gold, Beijing; RACE Kit (SMARTer RACE 5 '/3' Kit) and fluorescent quantitative Kit (TB Green^TMPremix Ex Taq^TM) The Plasmid Extraction Kit (MiniBEST Plasmid Purification Kit Ver.4.0) and the Gel recovery Kit (MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0) were purchased from TaKaRa company; the genomic DNA extraction kit is purchased from Beijing Tiangen Biochemical technology Co.

The strain is as follows: coli strain DH5 a and Agrobacterium strain GV3101, yeast strain Y2H and Y1H were purchased from Shanghai Diego Biotechnology Ltd.

Reagents and drugs: YPDA medium, two-lacking SD/-Trp-Leu medium, four-lacking SD/-Ade/-His/-Leu/-Trp medium, SD/-Leu medium, and SD/-Ura medium were purchased from Kuibop technologies, Beijing. The preparation steps are as follows:

1) YPDA medium (1L):

adding 20g/L peptone and 10g/L yeast extract into deionized water, diluting to 935mL, adjusting pH to 5.8, sterilizing at 121 deg.C for 15min, cooling to 55 deg.C, and adding 50mL 40% glucose and 15mL 0.2% adenine.

2) Deficient yeast medium (1L):

8g of the SD + defective amino acid mixture was added to 900mL of deionized water and dissolved with stirring. The pH was adjusted to 5.8 and autoclaved at 121 ℃ for 15 minutes. The sterilized liquid medium was stored in a refrigerator at 4 ℃ in the dark, and 100mL of a filter-sterilized 20% glucose solution, or other carbon source, was added before use.

3) LB medium (1L)

The following components were dissolved in 900mL of water: 10g tryptone, 5g yeast extract, 5g NaCl; adjusting pH to 7.0 with 1mL of 1mol/L NaOH, adding deionized water to constant volume of 1L, and steam sterilizing at high pressure for 20 min; if a solid culture medium is prepared, agar needs to be added in an amount of 12 g/L.

4) Other reagents:

①1×PEG/LiAc:8mL 50％PEG3350，1mL 10×TE，1mL 1mol/L LiAc。

x-alpha-gal: dissolve 60mg of X- α -gal in 3mL of DMF (N, N-dimethylformamide) to a final concentration of 20 mg/mL.

③ golden basidin A (AbA): the resulting solution was dissolved in methanol to give a final concentration of 1 mg/mL.

Ampicillin (100 mg/mL): dissolving 1g of ampicillin sodium salt in enough water, and finally metering to 10 mL. Subpackaging into small parts and storing at-20 ℃. It is often added to the growth medium at a final concentration of 25. mu.g/mL to 50. mu.g/mL.

Kanamycin (10 mg/mL): dissolve 100mg kanamycin in sufficient water and finally make up to 10 mL. Subpackaging into small parts and storing at-20 ℃. It is often added to the growth medium at a final concentration of 10. mu.g/mL to 50. mu.g/mL.

1.3 screening and analysis of members of the peony MYB Gene family

1.3.1 screening of members of the peony MYB Gene family

The MYB domain PF00249 sequence downloaded by using a Pfam (http:// Pfam. sanger. ac. uk /) website is used as a query sequence (query), and tBLASTN search is carried out on a previous acquired 'Bluekui' petal transcript group database through local BLAST. The obtained Unigene sequence was subjected to domain analysis and verification in Pfam and CDD (https:// www.ncbi.nlm.nih.gov/Structure/CDD/wrpsb. cgi) online databases, and sequences with domain integrity less than 30% were removed.

1.3.2 phylogenetic analysis

Arabidopsis MYB gene family sequences were downloaded from the TAIR website (https:// www.arabidopsis.org/index. The protein sequences of Arabidopsis thaliana and putative peony MYB family members were subjected to multiple sequence alignment using the software MUSCLE (http:// www.ebi.ac.uk/Tools/msa/MUSCLE /) with default parameters, and phylogenetic trees were constructed using the neighbor-joining method of MEGA7.0 (NJ).

1.4 Total RNA extraction

RNA extraction was performed according to the instruction of RN33-PLANTpure general plant Total RNA Rapid extraction kit (Beijing Eldely Biotech Co., Ltd.). The specific method comprises the following steps:

1) 500 μ L of lysate RLT was transferred to a 1.5mL centrifuge tube, 50 μ L of PLAntaid was added and mixed for use.

2) Grinding proper amount of peony petals in liquid nitrogen into fine powder, transferring 50mg of fine powder into the centrifuge tube containing RLT and PLAntaid, immediately and violently oscillating for 20 seconds by a vortex instrument, and fully cracking.

3) The lysate was centrifuged at 13,000rpm for 5-10 minutes to precipitate the uncleavable debris and the polysaccharide polyphenol bound PLAntaid.

4) Transfer the lysate supernatant to a new centrifuge tube. Half the volume of the supernatant was added absolute ethanol (0.5 vol), at which time precipitation may occur, but without affecting the extraction process, and was immediately whipped into a uniform mass without centrifugation.

5) Immediately, the mixture (less than 720. mu.L each time, more than two additions can be made) is added to an adsorption column RA (which is placed in a collection tube) and centrifuged at 13,000rpm for 2 minutes, and the waste liquid is discarded.

6) Add 700. mu.L of deproteinized solution, leave at room temperature for 1 minute, centrifuge at 13,000rpm for 30 seconds, and discard the waste.

7) 500. mu.L of the rinsing solution RW was added, and centrifuged at 13,000rpm for 30 seconds, and the waste liquid was discarded. Add 500. mu.L of the rinse RW and repeat.

8) The adsorption column RA was returned to the empty collection tube and centrifuged at 13,000rpm for 2 minutes to remove the rinse as much as possible so as not to inhibit downstream reactions due to residual ethanol in the rinse.

9) The adsorption column RA was taken out and put into an RNase free centrifuge tube, and 30 to 50. mu.L of RNase free water (previously heated in a water bath at 70 to 90 ℃ C. to increase the yield) was added to the middle part of the adsorption membrane according to the expected RNA yield, and the membrane was left at room temperature for 1 minute and centrifuged at 12,000rpm for 1 minute.

10) If the expected RNA yield is > 30. mu.g, step 9 is repeated with 30-50. mu.L RNase free water and the two washes are combined.

1.5 reverse transcription

First strand cDNA Synthesis

One-Step gDNA Removal and cDNA Synthesis SuperMix (Beijing Quanyu gold). The method comprises the following specific steps:

a reaction mixture was prepared according to the genomic DNA removal system shown in Table 1, and gently mixed. Incubating in 42 deg.C constant temperature water bath for 30min, and incubating for 15min if it is used for fluorescent quantitative reaction. Heating at 85 deg.C for 5s to deactivate

RT and gDNA remover.

TABLE 1 gDNA removal and reverse transcription reaction System

1.6 fluorescent quantitative PCR

Specific primers were designed based on the screened Unigene sequence using NCBI's Primer-Blast online software. Reference fluorescent quantitation kit TB Green^TMPremix Ex Taq^TM(TaKaRa) in the Specification, PsPP2A was used as an internal reference gene, and 2-. DELTA.CT was performed by Roche Light

And (3) analyzing the relative expression quantity of the candidate gene on a 480 real-time fluorescence quantitative PCR instrument. 3 biological replicates and 3 technical replicates were set up for each quantification experiment. The primer sequences are shown in Table 2.

TABLE 2 primers for fluorescent quantitative PCR

1.7 Gene cloning and sequence analysis

The first strand of cDNA reverse-transcribed and synthesized by total RNA extracted from petals of 'Bluekui' peony at full bloom stage is taken as a template, and 5 'RACE and 3' RACE specific primers of PsMYB1 and PsMYB2 are respectively designed according to Unigene sequences. Amplification of the 5 'and 3' end sequences was performed according to the SMARTER RACE 5 '/3' kit (TaKaRa) instructions. PCR amplification products were recovered by 1.2% agarose gel electrophoresis. And connecting a strip with the same size as the expected fragment to a pMD19-T vector, transforming to an escherichia coli competent cell, and performing sequencing analysis after blue-white screening and bacterial liquid PCR identification.

Predicting the gene open reading frame and the conserved structural domain of the coding protein by using a CD-Search and ORF tool on NCBI; predicting basic physicochemical properties of target gene coding protein on line by utilizing a Prot-Param tool in an ExPasy server; gene sequence homology comparison is completed by DNAMAN software; and (4) constructing a phylogenetic tree by using MEGA7.0 software. The sequences of the relevant primers are shown in Table 3.

TABLE 3 primers used for RACE-PCR

1.8 Yeast two-hybrid

1.8.1 Bait vector and Prey vector construction

According to Matchmaker^TMGold Yeast Two-hybrid (Clontech) instructions, PsMYB1 and PsMYB2, and open reading frames of bHLH and WD40 transcription factors which are screened and possibly participate in the peony petal anthocyanin biosynthesis regulation are respectively carried on a pGBKT7 vector containing a GAL4 binding DNA domain (BD) and a pGADT7 vector containing a GAL4 Activation Domain (AD). The primers used are shown in Table 4.

TABLE 4 primers for Yeast diheterous

1.8.2 Co-transformation of Bait and Prey plasmids

Plasmids carrying PsMYB, PsbHLH and PsWD40 respectively are combined in pairs by a PEG/LiAc method, transformed into a Y2HGold yeast strain, sequentially coated on a SD medium lacking leucine and tryptophan (SD/-Leu/-Trp), and then screened on the SD medium lacking adenine, histidine, leucine and tryptophan (SD/-Ade/-His/-Leu/-Trp). Finally, the positive interaction obtained is analyzed by a method for verifying the activity of the beta-galactosidase by using X-alpha-Gal. The conversion steps are as follows:

1) taking 100 mu L of ice-thawed Y2HGold competent cells, sequentially adding precooled target plasmid, positive control (pGBKT7-53+ pGADT7-T) and negative control (pGBKT7+ pGBKT7-lam)2-5 mu g, Carrier DNA (95-100 ℃, 5min, quick ice bath, repeated once) 10 mu L, PEG/LiAc 500 mu L, sucking and beating for several times, mixing uniformly, and water-bathing at 30 ℃ for 30min (turning 6-8 times for mixing uniformly when 15 min).

2) Placing the tube in 42 deg.C water bath for 15min (turning 6-8 times when 7.5min, and mixing well).

3) Centrifuging at 5000rpm for 40s, discarding the supernatant, ddH₂O400. mu.L of the suspension was resuspended, centrifuged for 30s and the supernatant discarded.

4)ddH₂O50. mu.L of the suspension was resuspended, plated, and cultured at 29 ℃ for 48-96 h.

1.9 bimolecular fluorescence complementation

ORF sequences of PsMYB1, PsbHLH1 and PsWD40-1 with the stop codon removed were constructed between the BamHI and XhoI cleavage sites of pUC-SPYNE (pUC-SPYCE) by means of seamless cloning. The primers used are shown in Table 5.

TABLE 5 primers for bimolecular fluorescence complementation test

1.10 Agrobacterium mediated transient transformation of Bunshi tobacco leaf and fluorescence observation

pSPYNE/PsbHLH1, pSPYCE/PsMYB1, pSPYNE/PsWD40-1, pSPYCE/PsWD40-1, empty vector plasmid pSPYNE, empty vector plasmid pSPYCE, positive control plasmid pSPYNE/bZIP63, positive control plasmid pSPYCE/bZIP63 and nuclear marker plasmid GHD7-CFP were transformed into Agrobacterium GV3101 by heat shock method, respectively. After 2-3 days, positive clones are obtained through colony PCR identification, and single colonies are selected and inoculated in LB liquid culture medium (Kan 100mg/mL, Rif 100mg/mL) and cultured in a shaking table at 28 ℃ for overnight shaking at 200 rpm. 100 mu L of fresh bacterial liquid is inoculated into 50mL LB liquid culture medium for expanding culture until the strain grows to logarithmic phase, and then the strain is centrifuged for 5min at the normal temperature of 5000 Xg. The supernatant was removed, excess LB medium was minimized, and prepared infection buffer (10mM MES,10mM MgCl)₂200mM AS, pH5.6) and the bacterial cell concentration was adjusted to 0.75 OD 600. And uniformly mixing the bacterial liquid 1:1 in equal amount according to different combinations. Standing in the dark for 2-3h, injecting the back of the ben's tobacco leaves in the 4-6 leaf stage by using a 1mL injector without a needle, keeping moisture of the injected tobacco, culturing overnight in the dark, culturing in an illumination incubator for 2-3 days, and observing. After 3d, the leaves are cut into small squares of about 5mm, the squares are placed on a glass slide, and the YFP fluorescence expression is observed by a laser confocal microscope (Zeiss LSM 510Meta, Jena, Germany). The YFP fluorescence excitation wavelength is 514nm, and the receiving wave band is 500-560 nm. The excitation wavelength of the chloroplast autofluorescence is 633nm, and the receiving wavelength range is 650-720 nm. All transient expression experiments were repeated three times.

1.11 Yeast Monohybrid

1.11.1 vector construction

Connecting promoter sequences of 8 key structural genes (PsCHS, PsCHI, PsF3H, PsF 3' H, PsFLS, PsFNS, PsDFR and PsANS) biosynthesized by the peocyanin to a pAbAi vector to construct a yeast single hybrid decoy vector (Bait); CDS sequences of three transcription factors of peony MYB, bHLH and WD40 are connected to a pGADT-7 vector to construct a yeast single-hybrid Prey vector (Pre y). And searching by BioEdit software, and selecting BstBI/BbsI to perform single enzyme digestion on the pAbAi recombinant plasmid. The linearized product was recovered by electrophoresis on a 1% agarose gel.

1.11.2Bait strain transformation and strain identification

And transferring the linearized fragments of pAbAi-CHS, pAbAi-CHI, pAbAi-F3H, pAbAi-F3' H and the like into Y1H competent cells, coating the competent cells on an SD/-Ura solid culture medium, and carrying out inverted culture in an incubator at 30 ℃ for 3-5 d. The conversion comprises the following specific operation steps:

1) taking 100 mu l of ice-thawed Y1HGold competent cells, sequentially adding 1-5 mu g (the volume is not higher than 15 mu l) of precooled linear pBait-Abai target plasmid, 10 mu l of Carrier DNA (95-100 ℃, 5min, quick ice bath and repeated once) and 500 mu l of PEG/LiAc, sucking and beating for several times, and uniformly mixing, and carrying out 30-degree water bath for 30min (turning 6-8 times and uniformly mixing when 15 min).

2) Placing the tube in 42 deg.C water bath for 15min (turning 6-8 times when 7.5min, and mixing well).

3) Centrifuging at 5000rpm for 40s, discarding the supernatant, ddH₂O400. mu.l of the suspension was resuspended, centrifuged for 30s and the supernatant discarded.

4)ddH₂O50. mu.l of the suspension was resuspended, coated with SD/-Ura plates and incubated at 29 ℃ for 72 h.

5) 5-10 single clones were picked and confirmed to integrate pBait-Abai into the Y1HGold genome by colony PCR validation using Matchmaker Insert Check PCR Mix 1. Meanwhile, untransformed Y1HGold monoclonal was used as a negative control. Colony PCR analysis was expected to result in a positive bait strain of 1.35Kb + insert size, while the negative control had no band.

6) The PCR positive strain is streaked on an SD/-Ura plate, cultured at 29 ℃ for 72h, and preserved at 4 ℃, and the strain is the Y1HGold [ Bait/AbAi ] strain.

1.11.3 screening of background concentration of recombinant Yeast strain Y1H (pAbAi-Bait) AbA

To eliminate false positives during the experiment, the optimum concentration of AbA needs to be selected as follows:

1) selecting a positive monoclonal with the length of 2-3mm from the strains with correct sequencing, dissolving the positive monoclonal in 0.9% NaCl (w/v), and adjusting the OD600 to about 0.002 by using an ultraviolet spectrophotometer;

2) preparing SD/-Ura/AbA nutrient-deficient culture medium with the concentration of 0ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL, 350ng/mL, 400ng/mL, 450ng/mL, 500ng/mL, 550ng/mL, 600ng/mL, 650ng/mL, 700ng/mL, 750ng/mL, 800ng/mL, 850ng/mL, 900ng/mL and 950ng/mL in advance, sucking 100 microliter of bacterial liquid, sequentially coating the bacterial liquid on culture media with different concentrations, simultaneously using p53-Abai as a positive control, and placing the culture media in an inverted incubator at 30 ℃ for 2-3 d;

3) relevant studies have shown that suitable AbA concentrations for inhibiting yeast growth are typically 100-200ng/mL, and if not effective, increase the concentration to 500-1000 ng/mL. If the gene still can grow normally on the nutrient-deficient culture medium with AbA concentration of 1000, the gene is not suitable for detection by using an AbA system.

1.11.4 Gene and protein interaction detection

1) Streaking strains Y1H (pAbAi-CHS, pAbAi-CHI, pAbAi-F3H, pAbAi-F3' H, pAbAi-FLS, pAbAi-FNS, pAbAi-DFR, pAbAi-ANS and positive control p53-AbAi) on SD/Ura plates for resuscitation, growing single colonies after 2-3d, and picking single colonies of about 3mm to prepare competent cells;

2) transferring recombinant plasmids pGADT7-MYB1, pGADT7-bHLH1 and pGADT7-WD40-1 into yeast competent cells Y1H (pAbAi-CHS, pAbAi-CHI, pAbAi-F3H, pAbAi-F3' H and the like), transferring positive control plasmids pGADT7-53 into corresponding Y1H (p53-AbAi) competence, coating the transformed competence on two culture media of SD/-Leu (SD medium lacking leucine) and SD/-Leu/AbA (screened AbA concentration), and culturing in a 30 ℃ constant temperature culture box for 3-5 d;

3) in addition, negative controls were set, i.e., empty pGADT7 plasmids were sequentially transferred into yeast competent Y1H (pAbAi-CHS, pAbAi-CHI, pAbAi-F3H, pAbAi-F3' H, etc.), spread on two media, SD/-Leu (SD medium lacking leucine) and SD/-Leu/AbA (screened ABA concentration), and cultured in 30 ℃ incubator for 3-5 d;

4) the growth of all combinations on SD/-Leu and SD/-Leu/AbA medium was observed, and if normal yeast cells were able to grow on both media, it was shown that there was an interaction between the protein of interest and the promoter.

1.12 Dual luciferase assay

1.12.1 expression vector construction

The sequences of PsMYB1, PsbHLH1 and PsWD40-1 were respectively constructed between two enzyme cutting sites (BamHI and KpnI) of pGreenII62-SK by means of seamless cloning, and plant over-expression vectors 35S: PsMYB1, 35S: PsbHLH1 and 35S: PsWD40-1 driven by 35S promoters were obtained. Promoter sequences of 8 key structural genes PsCHS, PsCHI, PsF3H, PsF 3' H, PsFLS, PsFNS, PsDFR and PsANS in peony anthocyanin biosynthesis are respectively constructed between two enzyme cutting sites (SalI and BamHI) of pGreenII0800-LUC, and expression vectors are obtained.

1.12.2 transient transformation of tobacco and luciferase Activity assay

1) And (4) preparing infection bacterial liquid.

The constructed vector, the empty vector pGreen II62-SK and the empty vector pGreen II0800-LUC are transferred into agrobacterium GV3101 by a heat shock method. After 2-3 days, positive clones are obtained through colony PCR identification, and single colonies are selected and inoculated in LB liquid culture medium (Kan 100mg/mL, Rif 100mg/mL) and cultured in a shaking table at 28 ℃ for overnight shaking at 200 rpm. 100 mu L of fresh bacterial liquid is inoculated into 50ml of LB liquid culture medium for expanding culture until the strain grows to the logarithmic phase, and then the strain is centrifuged for 5min at the normal temperature of 5000 Xg. The supernatant was removed, excess LB medium was minimized, and prepared infection buffer (10mM MES,10mM MgCl)₂200mM AS, pH5.6) and the bacterial cell suspension was adjusted to an OD600 of 0.75. The different types of combinations containing promoters and transcription factors were mixed in equal amounts at 1: 1. Standing in the dark for 2-3h, injecting the back of the Bentoni tobacco leaves at the 4-6 leaf stage by using a 1mL injector without a needle, keeping the moisture of the injected tobacco, culturing overnight in the dark, placing in an illumination incubator for culturing for 2-3 days, and sampling to determine the LUC and REN activities of the leaves.

2) Determination of luciferase Activity.

First, a leaf blade having a diameter of 3mm (48 to 72 hours after the tobacco is instantaneously transferred) was taken out by a punch, and 380. mu.L of 1 XPLB buffer was added to a small precooled mortar to sufficiently grind the leaf blade. Pipette 2.5. mu.L of the slurry into a new 100. mu.L of 1 XPLB buffer, mix well for dilution, and place on ice for use.

Secondly, the detection instrument is a Module Luminometer (Promega, USA), the instrument is opened, the protocol is selected, the Run Promega protocol is selected, the DLR-O-INL is selected, and the operation is carried out in a dark room.

③ Add 5. mu.L of sample solution to the new centrifuge tube, aspirate 45. mu.L of LARII gently and pump 2-3 times (without violent aspiration), centrifuge slightly with a small centrifuge rotor, place in the instrument, and click to measure, at which time the lower strip is read for about 10 s.

After reading the strip, the tube was removed without touching the screen, and 50 μ L of Stop x Glo substrate (diluted 50 times) was added and rapidly aspirated 10-15 times, and the tube was slightly centrifuged with a small centrifuge rotor. Place the instrument and click the assay, at which point the strip is read a second time.

Fifthly, after reading the strips, displaying the measurement results RL1, RL2 and Ratio on a screen, and after the measurement of one sample is finished, continuing the steps 6 and 7, and measuring at least 3 leaves by the same strain. The measurement completion log data.

1.12.3 fluorescent observation

The in vivo Imaging System Newton 7.0Bio Plant Imaging System (Vilber Lourmat, France) was turned on in advance, with pre-cooling for 30 minutes; taking off infected tobacco leaves, and uniformly spraying prepared luciferase spray liquid on the back; placing in the dark at room temperature for 5-10 min; and (4) putting the tobacco into an imaging system, carrying out fluorescence imaging and taking a picture.

2. Results

2.1 screening and expression Pattern analysis of peony MYB transcription factors

123 non-redundant peony MYB gene family members are screened out through local tBLASTN search and conservative domain identification analysis. The classification into 4 types according to the number of incomplete repeat regions (R) includes 60R 2R3 MYB, 57 MYB-related, 4R 1R2R3-MYB and 2 4R-MYB. According to the division of an arabidopsis R2R3-MYB protein family, 60R 2R3-MYB proteins of peony are combined with a phylogenetic tree, and a bootstrap value is set to be 1000. As shown in FIG. 1, members selected from PsMYB1, PsMYB2, PsMYB3, AtMYB113, AtMYB114 and the like, which play an important role in anthocyanin synthesis, were clustered into the 6 th subfamily, and it was presumed that they had similar functions in peony.

Carrying out fluorescence quantitative analysis on expression patterns of PsMYB1, PsMYB2 and PsMYB3 transcription factors in the formation processes of different flowers of peony, and as shown in figure 2, the expression of PsMYB1 is closely related to anthocyanin synthesis, almost no expression is carried out in 'Yubanbai' petals without anthocyanin, a large amount of expression is carried out in 'Zhao powder' and 'Heihuakui' petals with anthocyanin accumulation, the expression amount in 'Heihuakui' is far higher than that in 'Zhao powder', and the expression amount rapidly rises from a hard bud stage along with the coloring process of the petals, and rapidly falls after the initial bloom stage reaches a maximum value; in contrast, the expression level of PsMYB2 was highest in 'yubaobai', and very low in 'zhao fen' and 'hei kui'; and the expression level of PsMYB3 in 3 varieties of petals is very low, and no obvious change rule exists.

2.2 cloning and sequence analysis of PsMYB1 and PsMYB2

cDNA reverse-transcribed from total RNA of petals of a deep purple red flower variety 'Black Huakui' at the full bloom stage is taken as a template, and a method combining RACE-PCR and RT-PCR is adopted to clone the full length of the cDNA of PsMYB1 and PsMYB 2. The results show (FIG. 3) that the full lengths of the ORFs of PsMYB1 and PsMYB2 are 648bp and 561bp, respectively, and 215 amino acids and 186 amino acids are coded respectively. The sequence similarity of the compounds and the known petunia PhAN2, gerbera GhMYB10, apple MdMYB, grape VlMYBA1 and waxberry MrMYB1 for regulating anthocyanin biosynthesis is between 40% and 50%. Meanwhile, the N-terminal amino acid sequences of PsMYB1 and PsMYB2 both contain conserved R2 and R3 regions, and the R3 domain contains a [ DE ] Lx2[ RK ] x3Lx6Lx3R motif which can interact with bHLH transcription factors, so that the two proteins can possibly interact with certain flower color-related bHLH transcription factors; the C-terminal amino acid sequence contains a conserved domain ([ K/R ] P [ Q/R ] P [ Q/R ]) related to anthocyanin biosynthesis, and further shows that PsMYB1 and PsMYB2 can be involved in regulating and controlling peony petal anthocyanin synthesis.

2.3 analysis of interaction between PsMYB1 and PsMYB2 and bHLH and WD40 transcription factors

The yeast two-hybrid results show (FIG. 4) that only the AD-PsbHLH1+ BD-PsMYB1 and AD-PsbHLH1+ BD-PsWD40-1 transformed yeast cells grew normally and appeared blue on the four-deficient medium containing X- α -Gal and AbA (SD-Ade-His-Leu-Trp). To further validate the results, a bimolecular fluorescence complementation test (BiFC) was performed in Nicotiana benthamiana. As shown in FIG. 5, YFP fluorescence signal in nucleus was observed after injecting only Agrobacterium containing the combination PsbHLH1-nYFP + PsMYB1-cYFP and the combination PsbHLH1-nYFP + PsWD40-1-cYFP into tobacco leaves, and YFP fluorescence signal was stronger compared to the positive control (bZIP63-nYFP + bZIP 63-cYFP). The above results demonstrate that PsMYB1 can form an MBW complex with PsbHLH1 and PsWD40-1, and the interaction modes are that PsMYB1 and PsWD40-1 interact with PsbHLH1 respectively, but PsMYB1 cannot interact with PsWD 40-1.

2.4 transcription factor activation assay

The yeast single-hybrid results (FIG. 6) show that, in addition to the positive control, the combination of Y1H [ pGADT7-PsMYB1+ pAbAi-DFR ], Y1H [ pGADT7-PsMYB1+ pAbAi-ANS ], Y1H [ pGADT7-PsbHLH1+ pAbAi-DFR ] and Y1H [ pGADT7-PsbHLH1+ pAbAi-ANS ] can grow a single colony on the plate, indicating that the PsMYB1 protein can recognize the promoters of the downstream structural genes PsDFR and PsANS in the anthocyanin synthesis pathway to generate protein-DNA interaction as a transcription factor, thereby activating the expression of the downstream reporter gene AbAr.

The results of the dual luciferase assay (FIG. 7) show that 35S: PsMYB1 alone directly activates the activity of PsDFR and PsANS promoter fragments compared to the empty vector pGreen II 62-SK. The activity of the PsDFR promoter in the 35S-PsMYB 1 and 35S-PsbHLH 1 co-injected leaves is respectively improved by 2.8 times and 3.2 times, and the activity of the PsANS promoter is respectively improved by 4.6 times and 6.3 times; in the leaves injected with 35S PsMYB1, 35S PsbHLH1 and 35S PsWD40-1, the activity of both PsDFR and PsANS promoters reaches the maximum value, the activity of the PsDFR promoter is respectively improved by 4 times and 4.8 times, and the activity of the PsANS promoter is respectively improved by 4.9 times and 6.7 times.

In vivo imaging experiments also confirmed this conclusion, as shown in FIG. 8A, when PsMYB1+ PsDFR/PsANS, PsbHLH1+ PsDFR/PsANS, PsMYB1+ PsbHLH1+ PsDFR/PsANS and PsMYB1+ PsbHLH1+ PsWD40-1+ PsDFR/PsANS were injected respectively, a clear red signal was observed, demonstrating that transcription factors and promoters bind well, and that when MYB1+ PsbHLH1+ PsWD40-1+ PsDFR/PsANS combination, the red signal was significantly enhanced.

The results show that the PsMYB1 can activate the expression of PsDFR and PsANS promoters independently, and can also form a complex with PsbHLH1 and PsWD40-1 to improve the promoter activity of PsDFR and PsANS, so that the synthesis of anthocyanin in peony petals is promoted.

Sequence listing

<110> forestry research institute of China forestry science research institute

Application of transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 215

<212> PRT

<213> peony (Paeonia suffruticosa)

<400> 1

Met Glu Gly Arg Ile Leu Gly Leu Arg Lys Gly Ala Trp Thr His Glu

1 5 10 15

Glu Asp Cys Leu Leu Arg Asn Cys Val Glu Lys Tyr Gly Gln Glu Gln

20 25 30

Trp His Leu Val Pro Leu Arg Ala Gly Leu Asn Arg Cys Arg Lys Ser

35 40 45

Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn Ile His Arg Gly

50 55 60

Lys Phe Ala Leu Asp Glu Val Asp Leu Ile Ile Arg Leu His Lys Leu

65 70 75 80

Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Ile Pro Gly Arg Thr

85 90 95

Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr His Met Arg Lys Lys Met

100 105 110

Val Ser Cys Arg Glu Val Ile Asn Asp Lys Ile Gln Gln Arg Trp Lys

115 120 125

Ser Asn Ile Ile Lys Pro Gln Pro Arg Thr Phe Thr Lys Ser Leu Ser

130 135 140

Trp Leu Arg Gly Lys Ser Thr Leu Val Phe Asp Lys Leu Gln Val Lys

145 150 155 160

Asp Ser Thr Arg Thr Glu Pro Pro Ala Ile Leu His Leu Pro Pro Gln

165 170 175

Thr Asp Asp Ile Gln Leu Leu Asp Phe Asp Asn Glu Gly Asn Lys Leu

180 185 190

Ser Thr Asn Gly Pro Met Val Cys Phe Glu Ser Leu Trp Asp Glu Glu

195 200 205

Val Ala Pro Asn Thr Asn Gly

210 215

<210> 2

<211> 648

<212> DNA

<213> peony (Paeonia suffruticosa)

<400> 2

atggaagggc gcatcttggg actgagaaag ggtgcatgga cccatgaaga agattgcctt 60

cttagaaact gcgttgaaaa atacggacaa gagcagtggc atctagtgcc tctccgagca 120

ggattgaaca ggtgcaggaa gagctgcaga ttgaggtggt tgaactatct caggccaaat 180

atccatagag gaaaatttgc attagatgaa gtagatctta tcattaggct tcataagctc 240

ttgggtaaca gatggtcatt gattgctggt agaattccag gaagaactgc aaatgatgtg 300

aagaactact ggaataccca catgcgcaaa aagatggttt cttgccgaga agtgatcaat 360

gataaaatcc agcagagatg gaaatccaac attataaagc ctcaacctcg gacgttcacc 420

aaaagtctat catggttgag gggaaaatct accttagtat ttgataagtt acaagtaaaa 480

gactctactc gtacagagcc accagccata ctgcatcttc cacctcaaac agatgatatt 540

cagttgttgg actttgataa tgaaggtaat aaattatcca ccaatgggcc aatggtatgc 600

tttgaaagcc tctgggatga ggaagttgca ccaaacacga acggctga 648

Claims (3)

1. The application of a transcription factor PsMYB1 or a gene coding the transcription factor PsMYB1 in regulating and controlling peony petal anthocyanin synthesis is disclosed, and the amino acid sequence of the transcription factor PsMYB1 is shown in SEQ ID No. 1.

2. The use of claim 1, wherein the use is of the transcription factor PsMYB1 or a gene encoding the transcription factor PsMYB1 for activating the peony PsDFR gene or/and PsANS gene promoter.

3. The use according to claim 1 or 2, wherein the nucleotide sequence of the gene encoding the transcription factor PsMYB1 is shown in SEQ ID No. 2.

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