CN116514942A

CN116514942A - Protein for regulating anthocyanin synthesis and fruit size in peony and encoding gene thereof

Info

Publication number: CN116514942A
Application number: CN202310686647.3A
Authority: CN
Inventors: 舒庆艳; 宋姝凡; 郝青; 李旸; 彭丽平; 刘政安; 姜小蕾
Original assignee: Institute of Botany of CAS; Qingdao Agricultural University
Current assignee: Institute of Botany of CAS; Qingdao Agricultural University
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2023-08-01
Anticipated expiration: 2043-06-09
Also published as: CN116514942B

Abstract

The invention discloses a protein for regulating anthocyanin synthesis and fruit size in peony and a coding gene thereof. The invention provides a protein, the amino acid sequence of which is shown as SEQ ID NO. 2. The nucleotide sequence of the coding gene of the protein is shown as SEQ ID NO. 1. The CDS of PsHY5 is obtained from a peony transcriptome database, and an open reading frame (open reading frame, ORF) of PsHY5 is cloned and successfully connected with a pSuper1300 over-expression vector. The tobacco transgenic strain is successfully obtained by transforming tobacco through agrobacterium tumefaciens-mediated leaf disc method to verify the biological functions of candidate genes, screening hygromycin and detecting by PCR. Compared with a control, the PsHY5 over-expressed tobacco can deepen red petals and green leaves and enlarge fruits, and the PsHY5 in the transient silencing peony petals is combined to obviously reduce the total anthocyanin content, so that the tobacco is presumed to be a transcription factor capable of positively regulating plant anthocyanin accumulation and affecting plant fruit growth.

Description

Protein for regulating anthocyanin synthesis and fruit size in peony and encoding gene thereof

Technical Field

The invention relates to the technical field of biology, in particular to a protein for regulating anthocyanin synthesis and fruit size in peony and a coding gene thereof.

Background

Peony is known as the king in flowers in China, which is one of ten traditional flowers in China and plays a very important role in garden application, flower production and the like. The flower colors are important ornamental characters of the peony, and the flower colors are gradually enriched through long natural selection and artificial breeding. In relation to the basic research of the color chemistry, the main substance of which petals are colored has been shown to be anthocyanin (Wang LS, hashimoto F, shiraishi A, aoki N, li JJ, sakata Y.chemical taxonomy of the Xibei tree peony from China by floral pigment. Journal of Plant Research,2004, 117:47-55.); the mechanisms of peony formation in pure flowers such as purple and yellow have both been reported and a large number of enzyme genes and regulatory genes (Zhou L, wang Y, ren L, shi Q, zheng B, miao K, guo X.Overexpress of Ps-CHI1, a homologue of the chalcone isomerase gene from tree peony (Paeonia suffruticosa), reduces the intensity of flower pigmentation in transgenic tobacco.plant Cell Tiss Organ Culture,2014, 116:285-295; zhang ZP, zhao LY, xu ZD, yu. Transcripta sequencing of Paeonia suffruticosa 'Shima Nishiki' to identify differentially expressed genes mediating double-color formation, plant Physiology and Biochemistry,2018, 123:114-124; qi Y, miao L, han L, zouHZ, miao K, wang Y.PsbHLH1, a novel transcription factor involved in regulating anthocyanin biosynthesis in tree peony (Paeonia suffruticosa), plant Physiology and Biochemistry,2020, 154:396-396). The applicant has developed many years of research around characteristic flower spots in peony petals, found key genes through various technical means, and determined that MYB-bHLH-WD40 is involved in the formation of peony flower spots through the formation of MBW complex regulated chalcone synthase genes (CHS) (Gu ZY, men SQ, zhu J, hao Q, tong NN, liu ZA, zhang HC, shu QY, wang LS. Chalcone synthase is ubiquitinated and degraded via interactions with a RING-H2 protein in petals of Paeonia 'HeXie'. Journal of Experimental Botany,2019a,70:4749-4762.; gu ZY, zhu J, hao Q, yuan YW, duan YW, men SQ, wang QY, hou QZ, liu ZA, shu QY, wang LS. A novel R2R3-MYB transcription factor contributes to petal blotch formation by regulating organ-specific expression of PsCHS in tree peony (Paeonia suffruticosa), plant and Cell Physiology,2019b,60:599-611. In addition, the applicant has obtained that after transformation of tobacco in response to light signal key regulatory factors, through overexpression, tobacco leaves, sepals, petals, pericarps and seeds can be colored, and thus multiple organ coloration can be regulated, and the obtained products are named as PsMYBM, which influences anthocyanin synthesis through interaction with light signal factors PsHY5 and transcription regulation PsCHS and PsF H (Geng DD, song SF, li Y, li TT, shu QY, hao Q.A novel transcription factor PsMYBM enhances the biosynthesis of anthocyanins in response to light in tree peoney. Industrial Crops and Products,2023, 200:116800.). Research on the color development and regulation mechanism of peony is focused on flavonoid synthesis regulation, but it is not clear how other regulation factors regulate anthocyanin synthesis, plant growth and development, fruiting and the like. Studies have shown that in apple and like species, the key protein HY5 in the optical signaling pathway is thought to be An upstream transcription factor of MYB10, binding directly to and activating the expression of the MYB10 promoter (An JP, liu X, li HH, you CX, wang XF, hao YJ.apple RING E3 library MdMIEL1 inhibits anthocyanin accumulation by ubiquitinating and degrading MdMYB1 protein.plant and Cell Physiology,2017, 58:1953-1962.). HY5 can interact with bHLH proteins such as PIF3 and the like to jointly regulate a downstream target gene of an anthocyanin synthesis pathway, so that anthocyanin synthesis is promoted (Shin J, park E, choi G.PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis, the Plant Journal,2007, 49:981-994.). The function of HY5 in peony, especially in anthocyanin synthesis regulation is unknown.

Disclosure of Invention

In view of the above, the invention provides a protein for regulating anthocyanin synthesis and fruit size by peony and a coding gene PsHY5 thereof.

The technical scheme of the invention is as follows:

a protein has an amino acid sequence shown in SEQ ID NO. 2.

The coding gene of the protein also belongs to the protection scope of the invention, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

The coding gene contains 471 nucleotides, which is shown as a sequence 1 in a sequence table; the coding sequence of the protein containing 156 amino acids is shown as a sequence 2 in a sequence table, the gene is named as PsHY5, and the coded protein is named as PsHY5.

The expression cassette, recombinant expression vector or recombinant bacteria containing the coding gene also belong to the protection scope of the invention.

The invention also provides a method for preparing the transgenic plant.

The method for preparing the transgenic plant provided by the invention comprises the following steps: introducing the coding gene into a starting plant to obtain a transgenic plant; transgenic plants have deeper petals and leaves, higher total anthocyanin content in the petals and/or increased fruit size and weight compared to the starting plant.

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 pSuper 1300.

The plant is tobacco.

The primer pair for amplifying the full length of the coding gene also belongs to the protection scope of the invention, wherein one primer sequence is shown as a sequence 3 in a sequence table, and the other primer sequence is shown as a sequence 4 in the sequence table.

The application of the protein in positive regulation of anthocyanin accumulation in plant petals and plant fruit growth also belongs to the protection scope of the invention.

The application of the coding gene in positively regulating anthocyanin accumulation in plant petals and plant fruit growth also belongs to the protection scope of the invention.

The plant is wild-type tobacco Nc89 (Nicotiana tabacum cv.nc89).

The CDS of PsHY5 is obtained from a peony transcriptome database, and an open reading frame (open reading frame, ORF) of PsHY5 is cloned and successfully connected with a pSuper1300 over-expression vector. The tobacco transgenic strain is successfully obtained by transforming tobacco through agrobacterium tumefaciens-mediated leaf disc method to verify the biological functions of candidate genes, screening hygromycin and detecting by PCR. Compared with a control, the PsHY5 over-expressed tobacco can deepen red petals and green leaves and enlarge fruits, and the PsHY5 in the transient silencing peony petals is combined to obviously reduce the total anthocyanin content, so that the tobacco is presumed to be a transcription factor capable of positively regulating plant anthocyanin accumulation and affecting plant fruit size. The invention lays a theoretical foundation for deeply understanding molecular mechanism of ornamental plant anthocyanin according to light synthesis, and provides reference for molecular breeding research of peony flower color improvement by adjusting illumination conditions.

Drawings

For purposes of illustration and not limitation, the invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 is 5 stages of development of the flowers of the peony variety 'twilight'.

FIG. 2 is a gel electrophoresis detection image of PCR products.

FIG. 3 is a prediction of the affinity/hydrophobicity, transmembrane structure, secondary and tertiary structure of PsHY 5; wherein a: prediction analysis of hydrophilicity/hydrophobicity; b: transmembrane predictive analysis; c: predictive analysis and analysis of signal peptides; d: predicting a secondary structure; e: three-level structure prediction analysis; f: conserved domain analysis.

FIG. 4 is a construction diagram of the recombinant vector pSuper1300-PsHY5.

FIG. 5 shows the result of agarose gel electrophoresis detection of the PCR product of the recombinant vector pSuper1300-PsHY5.

FIG. 6 is the result of converting pSuper1300-PsHY5 into tobacco; wherein a: a co-cultivation stage; b: callus stage; b: screening and culturing; d: inducing rooting; f: rooting; g: positive plants.

FIG. 7 shows the PCR detection results of PsHY5 transgenic tobacco.

FIG. 8 is a graph showing the phenotypic effects of each organ of the PsHY5 transgenic tobacco strain.

FIG. 9 shows the expression levels of PsHY5 in different lines of transgenic tobacco; asterisks indicate significant differences (P < 0.05).

FIG. 10 is a graph showing the results of wild type and PsHY5 transgenic tobacco flower color phenotype and pigment comparison analysis; asterisks indicate significant differences (P < 0.05).

FIG. 11 is a comparative analysis of wild-type and PsHY5 transgenic tobacco mature fruits; scale = 1cm; asterisks indicate significant differences (P < 0.05).

FIG. 12 is a PCR verification of pTRV2-PsHY5 recombinant vector.

FIG. 13 is a graph showing the phenotypic effects of each organ of the various lines of PsHY5 transgenic tobacco; asterisks indicate significant differences (P < 0.05).

Detailed Description

Plant material:

the peony variety 'twilight' (P. Suffruticosa 'Higurshi') is taken as an experimental material, purchased from Cao Zhou Bai Garden in the Mentha, shandong province, colored red, and planted in a Qingdao agricultural university garden and forest college experimental base. The developmental stages of the flowers were divided into 5 stages (fig. 1), designated S1 (tight flower buds, uncolored petals), S2 (colored petals, enlarged flower buds with open color), S3 (ready to bloom), S4 (bloom, gynoecial visible) and S5 (bloom), respectively.

Wild-type (WT) tobacco Nc89 (Nicotiana tabacum cv. Nc89) (non-patent literature describing wild-type (WT) tobacco Nc89 (Nicotiana tabacum cv. Nc89) is Physiol Mol Biol Plants,2021,27 (2): 237-249) seed stock is maintained in the laboratory. The WT tobacco seeds are evenly inoculated in a tissue culture bottle containing an MS culture medium, placed in an environment with the temperature of 28 ℃ and the relative humidity of 60 percent, the illumination intensity of 2500Lx and the photoperiod of 16h/8h for culture, transplanted to a artificial climate chamber of the garden and forest college of Qingdao university when 4-5 true leaves grow out of the plants, placed in the relative humidity of 80 percent and the light intensity of 250 mu mol m ^-2 s ^-1 Culturing at 25deg.C under light for 16 hr and at 18deg.C under dark for 8 hr.

Strains and vectors:

the Transl-T1 escherichia coli competent cells, fast-T1 escherichia coli chemocompetent cells (Fast-T1 competent cells) and pMD18-T vectors (pMD 18-T Vector) used in the invention are purchased from Nanjinouzan biotechnology Co., ltd; GV3101 Agrobacterium competent cells (GV 3101Chemically Competent Cell) were purchased from Shanghai Biotechnology Inc.

Enzyme and chemical reagent:

total RNA extraction kit (RNA prep Pure plant kit) for plants, genomic DNA extraction kit for plants, real-time fluorescent quantitative PCR kit (ChamQ Universal SYBR qPCR Master Mix) and real-time fluorescent quantitative reverse transcription kitRT SuperMix for qPCR (+gDNA wind)), 2× Taq Plus Master Mix II (Dye Plus), ultra GelRed, high fidelity enzyme ++>Max Master Mix (Dye Plus), homologous recombinase +.>Ultra One Step Cloning Kit, T4DNA ligase, DNA gel recovery kit, high purity plasmid DNA miniprep kit were all purchased from Nanjinopran Biotechnology Co., ltd; restriction enzymes, matchmaker Insert Check PCR Mix I, genome Walking Kit were purchased from Bao Ri doctor materials technology (Beijing) Co., ltd; high purity low electroosmotic agarose, DL 2000 DNAMaroer, purchased from catalpa, biotechnology Co., qingdao, optimaea; antibiotics Kan, amp, rif, cef, hyg and hormone NAA, 6-BA were purchased from Beijing Soy Bao technology Co.

Primer:

primer synthesis and sequencing are completed by Qingdao qing Ke catalpa, biological technology limited company.

Example 1, peony Cyanine Synthesis-controlling protein, its coding Gene PsHY5 and coding region clone Using the first PsHY5 Gene

A1802 bp full-length sequence was obtained from the transcriptome data Unigene (c 78548 _c0) and its open reading frame (open reading frame, ORF) 471bp was queried in the ORF Finder online software. The primer PsHY5-F/R (Table 1) was designed at both ends, and PCR amplification was performed using cDNA of the peony variety 'twilight' (P. Suffruticosa 'Higusshi') as a template.

PCR amplification of the target gene. PCR amplification System of target Gene (50. Mu.L): 0.5 mu LTaKaRa LA Taq (5U/. Mu.L), 5 mu.L 10 XLA Taq Buffer II (Mg ²⁺ Plus) (20 mM), 8. Mu.L dNTP mix (2.5 mM), 2. Mu.L cDNA, 2. Mu.L upstream primer (10. Mu.M), 2. Mu.L downstream primer (10. Mu.M) and 30.5. Mu.L sterilized water. The amplification procedure was: pre-denaturation at 95 ℃ for 5min;35 cycles (denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1 min); 72℃for 5min. The PCR products were subjected to agarose gel electrophoresis and the bands were observed by a gel imager, which showed that a single specific band was obtained around 471bp (FIG. 2).

And (5) recovering the product connection conversion. The PCR product was electrophoretically detected in 1% agarose gel, and the gel was cut by selecting a band with the correct length, and the target DNA was recovered by referring to the DNA gel recovery kit (Noruzan) instructions. The recovered target bands were cloned into the pMD18-T vector for sequencing. And comparing the sequencing result with transcriptome data in multiple sequences, wherein the result shows that the consistency of the overlapping region of the obtained gene sequence and the transcriptome sequence reaches 100%.

TABLE 1 primer sequences for cloning genes

The obtained gene sequence is 471bp, as shown in a sequence 1 in a sequence table, the gene is named as PsHY5, the gene codes protein containing 156 amino acids, as shown in a sequence 2 in the sequence table, and the protein is named as PsHY5.

2. Bioinformatics analysis of PsHY5 protein

Predicting the physicochemical property of the PsHY5 protein. The PsHY5 gene presumes the relative molecular weight 17423.51 of the coded protein, and the molecular formula is C ₇₂₃ H ₁₂₂₀ N ₂₃₈ O ₂₄₇ S ₇ . The total number of atoms is 2435, and the theoretical isoelectric point PI value is 9.30. The instability index was 64.92, from which the protein was presumed to be an unstable protein.

The protein transmembrane domain prediction results showed that PsHY5 does not have a protein transmembrane domain and does not belong to a membrane protein (fig. 3 a). The signal peptide prediction showed that PsHY5 was devoid of signal peptide and was a non-secreted protein (b in fig. 3). This suggests that the protein cannot be transported across the membrane and begins to function at the site of synthesis, possibly as a protein in the cytoplasm or organelle. NCBI conserved domain analysis showed that the conserved domain encoded by this sequence was bZIP superfamity (c in FIG. 3). The hydrophobicity analysis of the protein showed that PsHY5 had a maximum hydrophilicity/hydrophobicity of 1.522 and a minimum of-3.044, with more hydrophilic amino acid residues than hydrophobic amino acid residues throughout the peptide chain. At the same time, the protein has a very obvious alternating arrangement of hydrophilic and hydrophobic regions, psHY5 being a more hydrophilic protein as a whole (d in FIG. 3). The secondary structure prediction of PsHY5 showed that the amino acid composition of the product is 64.10% of alpha-helix (Hh), 2.56% of beta-sheet (Tt), 33.33% of random coil (Cc), and the product belongs to alpha-helix structure (e in FIG. 3). The PsHY5 protein was predicted for tertiary structure with a model similarity of 33.33% to the 6iak.1.a protein (f in fig. 3).

3. Verification of Gene function

1. Identification of PsHY5 Gene overexpression vector

The PsHY5 gene ORF region fragment was ligated with a Plant expression vector pSuper1300 (Plant Science,2022,317:111189, non-patent literature describing the Plant expression vector pSuper1300, saved by the present laboratory) to construct a recombinant vector pSuper1300-PsHY5 (FIG. 4). The specific method comprises the following steps:

plant RNA extraction and quantitative reverse transcription. The total RNA of petals at 1 mu g S2 period is used as a template and is referred toIII RT SuperMix for qPCR (+gDNA wind) and the synthesized cDNA was stored at-20℃for further use.

PCR amplification of the target gene. PCR amplification System of target Gene (50. Mu.L): 0.5 mu LTaKaRa LA Taq (5U/. Mu.L), 5 mu.L 10/Taq Buffer II (Mg ²⁺ Plus) (20 mM), 8. Mu.L dNTP mix (2.5 mM), 2. Mu.L cDNA, 2. Mu.L upstream primer (10. Mu.L), 2. Mu.L downstream primer (10. Mu.M) and 30.5. Mu.L sterilized water. The amplification procedure was: pre-denaturation at 95 ℃ for 5min;35 cycles (denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1 min); 72℃for 5min.

And (5) recovering the product connection conversion. The PCR product was electrophoretically detected in 1% agarose gel, and the gel was cut by selecting a band with the correct length, and the target DNA was recovered by referring to the DNA gel recovery kit (Noruzan) instructions. 4.0. Mu.L of the recovered product was gently blotted and mixed with 1.0. Mu.L of pEASY-T1 Cloning Vector and reacted at room temperature for 5 minutes. Uniformly mixing 5 mu L of the recombinant vector with 50 mu L of the transient-T1 escherichia coli competent cells in a slush state in a flick mode, and carrying out ice bath for 30min; heat shock at 42 ℃ for 45s; immediately place on ice for 2min. Add 500. Mu.L LB liquid medium and mix well, incubate at 200rpm,37℃for 1h. After centrifugation at 5000rpm for 6min, 100. Mu.L of the resuscitated solution was pipetted and spread evenly onto LB solid medium containing 50mg/L kanamycin (Kan), and the plates were placed in an incubator at 37℃for cultivation for 12-14h.

And (5) identifying and extracting the positive recombinant plasmid. The monoclonal colony is picked up to 500 mu L of LB liquid medium containing 50mg/LKan, and after incubation for 5h at the temperature of 37 ℃ at 200rpm, bacterial liquid PCR identification is carried out, and the system (15.0 mu L) is: 7.5. Mu.L 2X Taq PCR MasterMix, 0.6. Mu. L M13F (10. Mu.M), 0.6. Mu. L M13R (10. Mu.M), 5.7. Mu.L ddH ₂ O, 0.6. Mu.L of bacterial liquid. The amplification procedure was: pre-denaturation at 94 ℃ for 3min;30 cycles (94 ℃,30s;55 ℃,30s;72 ℃,1 min); 72℃for 10min. And (3) after electrophoresis detection, selecting bacterial liquid with correct band size for sequencing (in the family of the Optimaceae). The plasmid was extracted using a high purity plasmid DNA miniprep kit (Nuo Wei Zan) and stored at-20℃for further use.

Vector double cleavage and homologous recombination. The Super1300 vector was digested with restriction enzymes HindIII and KpnI, and the reaction system was as shown in Table 2:

TABLE 2 double restriction enzyme HindIII and KpnI Super1300 vector reaction System

And (3) performing enzyme digestion for 5 hours at a constant temperature of 37 ℃ to obtain the linearization vector of Super 1300.

The target fragment plasmid was subjected to target fragment PCR amplification again (same as above). After measuring the above concentrations, the formula was followed: optimal cloning vector usage= [0.02×cloning vector base pair number ] ng (0.03 pmol) optimal insert usage= [0.04×insert base pair number ] ng (0.06 pmol) the amount of DNA required for the recombination reaction was calculated. In order to ensure the accuracy of the sample addition, the linearized vector and the insert may be diluted appropriately before preparing the recombination reaction system, and the sample addition amount of each component is not less than 1. Mu.L. The following reaction systems (table 3) were formulated on ice:

TABLE 3 reaction System for fragment recombination reactions

Segment recombination reaction at 50 ℃ for 10min; cooling to 4 ℃ or immediately cooling on ice.

And (3) carrying out recombination reaction transformation. The ligation product of the recombination reaction is used as a template, fast-T1 chemically competent cells are used for high-efficiency transformation according to the specification of a C505-02/03 kit:

a. taking out Fast-T1 competent cells from-80 ℃, rapidly placing the competent cells on ice for melting, adding target DNA (plasmid or connection product), uniformly mixing the walls of the flick tube (avoiding sucking with a gun), and standing on the ice for 30min;

b, after heat shock for 30sec in a water bath at 42 ℃, rapidly placing the solution on ice for standing for 2min without shaking the centrifuge tube;

c. adding 900 mu L of LB liquid medium (without antibiotics) into the centrifuge tube, uniformly mixing, and then placing the mixture into a shaking table at 37 ℃ and 200rpm for resuscitation for 1h;

d.2,500g, centrifuging for 3min, discarding 900 mu L of supernatant, blowing and uniformly mixing the thalli with the rest culture medium, and uniformly coating on an LB solid culture medium plate containing Kan antibiotics.

e. The plate is placed in an incubator at 37 ℃ for 10min, and after the bacterial liquid is completely absorbed, the plate is inverted and cultured overnight.

And (5) identifying and extracting the positive recombinant plasmid. Amplifying and shaking culture the bacterial liquid sample of the recombinant product identified correctly overnight, adding 50% glycerol according to the ratio of 7:3, uniformly mixing, and preserving at-80 ℃ for later use; the recombinant product plasmid is preserved at-20 ℃ for standby.

Primer synthesis and sequencing were completed by Qingdao qing catalpa biological technology Co., ltd, and the primer sequences are shown in the following Table 4:

TABLE 4 primer sequence for constructing PsHY5 gene over-expression vector

The PsHY5 gene CDS region fragment was ligated with the plant expression vector pSuper1300 to construct a recombinant vector pSuper1300-PsHY5 (FIG. 4). PCR was performed using the recombinant plasmids of the recombinant vectors pSuper1300-PsHY5 as templates, pSuper1300-PsHY5-F and pSuper1300-PsHY5-R as primers, and the result of electrophoresis detection of the PCR products showed that there was a single correct band (FIG. 5). The comparison and analysis of the positive plasmid sequencing result shows that the sequence is consistent with the PsHY5 sequence, and the overexpression recombinant vector pSuper1300-PsHY5 is successfully constructed.

2. Agrobacterium tumefaciens mediated leaf disc method for transforming tobacco

And transforming agrobacterium to obtain positive clone and verify. With reference to GV3101Chemically Competent Cell, competent cells of Agrobacterium GV3101 were transformed by freeze thawing to obtain positive monoclonal Agrobacterium. After the transformation was completed, 900. Mu.LLB liquid medium was added thereto and incubated at 200rpm and 28℃for 2-3 hours. Is spread on LB solid medium containing 100mg/L Kan and 50mg/L rifampicin (Rif) and placed in a 28 ℃ incubator for inversion culture for 2-3d. The monoclonal colonies were picked up and incubated in LB liquid medium containing 100mg/LKan and 50mg/L Rif at 28℃for 8 hours at 200rpm, and positive recombinants were detected by PCR. Shake-culturing the bacterial liquid sample for overnight, adding 50% glycerol, mixing, and storing at-80deg.C.

Culturing aseptic seedlings of tobacco. Taking a proper amount of wild type seeds (wild type (WT) tobacco Nc89 (Nicotiana tabacum cv. Nc 89) non-patent documents: physiol Mol Biol Plants,2021,27 (2): 237-249, seed laboratory preservation.) in a 2mL centrifuge tube, adding 1mL 75% ethanol, uniformly mixing up and down, soaking for 2min, and discarding ethanol solution; adding 1mL of 2% sodium hypochlorite, mixing up and down, soaking for 5min, and discarding sodium hypochlorite solution; rinsing the seeds with sterilized water for 3-5 times, placing on sterile filter paper, and naturally drying the water. Tobacco seeds are evenly inoculated in a tissue culture bottle poured with an MS culture medium, placed at 28 ℃ and with relative humidity of 60%, and cultured in an environment with illumination intensity of 2500Lx and photoperiod of 16h/8h, and used for infection when 4-5 true leaves grow out from plants.

The agrobacterium tumefaciens-mediated leaf disc method for transforming tobacco comprises the following specific steps:

(1) Activating and preserving positive agrobacterium tumefaciens bacteria liquid, adding the activated bacteria liquid into an LB liquid culture medium containing 100mg/L Kan and 50mg/L Rif according to the proportion of 1:100, and carrying out shaking culture at 28 ℃ and 180rpm to obtain OD600 = 0.6;

(2) Centrifuging at 5000rpm at room temperature for 10min, collecting bacterial precipitate, and re-suspending bacterial with MS0 liquid culture medium (MS culture medium+sucrose 30g/L, pH of 5.8) to obtain impregnated leaf;

(3) Taking well-grown leaf (leaf 2-4), cutting off leaf tip, leaf margin and main vein, and cutting the rest into 1cm pieces ² Left and right small blocks;

(4) Placing the cut tobacco leaf small pieces into MS0 suspension, dip-dyeing for 10-15min, and lightly shaking the leaf back face downwards to facilitate the bacterial liquid to completely contact with the leaf edge wound;

(5) Taking out the infected leaves, placing the infected leaves on sterile filter paper, and sucking up residual surface bacterial liquid;

(6) The leaves are inoculated onto a solid co-culture medium MS0 (MS culture medium+sucrose 20 g/L+agar 7.5g/L, pH 5.8) and placed at 28 ℃ for light-shielding co-culture for 3d;

(7) After the co-cultivation is finished, cleaning agrobacterium remained on the surface of the leaf blade by using sterile water containing 500mg/L cephalosporin (Cef), sucking water by using sterile filter paper, transferring the leaf blade to a differentiation induction and screening culture medium MS1 (MS culture medium +6-BA (1.0 mg/L) +NAA (0.2 mg/L) +sucrose 20 g/L+agar 7.5g/L+Kan (100 mg/L) +Cef (200 mg/L), standing and cultivating at 28 ℃ with relative humidity of 50-60%, illumination intensity of 2500Lx and photoperiod of 16h/8h, and replacing the MS1 culture medium every ten days;

(8) After the regeneration buds are differentiated, the regeneration buds are separated from the calli when the differentiated seedlings grow to 2-3cm high, and the calli are transferred into an MS2 rooting culture medium (1/2 MS culture medium, sucrose 20g/L, agar 7.5g/L, kan (100 mg/L) +Cef (200 mg/L), pH is 5.8) for inducing rooting, and positive transgenic tobacco can root after 1-2 weeks.

(9) After the root system is developed, taking out the tobacco tissue culture seedling, and cleaning agar carried by the root by using sterile water. Planting it in turfy soil: vermiculite = 3:1 (V/V) and placing the culture medium in the same environment for film-covered culture for about 1 week, and then removing the film for normal culture until the seeds are mature. Collecting seeds, storing in a refrigerator at 4 ℃, and vernalizing at low temperature for later use.

3. Screening of PsHY5 over-expressed transgenic tobacco

Tobacco leaves are subjected to co-culture, screening culture, bud induction, rooting and transplanting culture to obtain Kan-resistant regenerated seedlings (figure 6, wherein a is a co-culture stage, b is a callus stage, b is a screening culture stage, d is an induced rooting stage, f is a rooting stage, and g is a positive plant). The DNA of the obtained transgenic tobacco is subjected to PCR detection, wild tobacco is taken as a negative control, pSuper1300-PsHY5 recombinant plasmid is taken as a positive control, the PsHY5 gene-transferred tobacco can be amplified to obtain a specific band (figure 7) with the same size as expected, and the control group does not amplify any fragment, so that the exogenous PsHY5 gene is primarily shown to be integrated into the tobacco genome.

Fluorescent quantitative PCR is performed on wild-type and transgenic plant tobacco petals, and the relative expression amounts of the target gene and related structural genes in anthocyanin biosynthesis pathways are verified. Three transgenic tobacco lines with high expression of the gene of interest were selected for phenotypic validation. The nutrition and reproductive growth conditions of wild and transgenic tobacco plants with seedling age of 12 weeks are compared, and data such as leaf color, flower color, sepal color, plant height, fruit length, width, weight, whole plant fruit number and the like are measured, and the organ phenotype is photographed and recorded.

The color phenotype and the petal anthocyanin content were determined as follows:

l of all samples was determined using a portable color difference meter (Hunter Associates Laboratory Inc., USA) ^* (lightness), a ^* (redness), b ^* (yellowness) value and according to formula C ^* ＝(a ^*2 +b ^*2 ) ^1/2 (McGuire, 1992) into C (chroma) values. Five random measurements were made for each set of samples, and the mean and standard deviation were taken as analytical data.

Extracting total flavone and total anthocyanin with organic solvent, and measuring with ultraviolet spectrophotometer UH 5300 (HITACHI, tokyo, japan). Extraction of total anthocyanin: samples of 0.2g were weighed, ground to a powder in liquid nitrogen, and cryoleached with 10mL of 1% hydrochloric acid-methanol solution (1 mol/L hydrochloric acid: 1mol/L methanol=1:99, v/v) until the petals became completely white. Determination of total anthocyanin content (TA): 1% hydrochloric acid-methanol solution was used as a reference solution inThe optical densities of the extracts were measured on a spectrophotometer at 530, 620, 650 nm. Calculating anthocyanin content of the extracting solution according to a formula: aλ= (a 530-a 620) -0.1 (a 650-a 620), ta= (aλ/ζ) × (v/m) ×1000000 (Ma and Cheng, 1984). Extraction of total flavonoids: the 1mol/l methanol solution is used to replace 1% hydrochloric acid-methanol solution to extract total flavone. By AlCl ₃ The total flavone content (TF) was determined by chromogenic method, rutin was used as a standard curve, and the absorbance at 510nm was determined by UV spectrophotometry. The total flavone content of the extracting solution is calculated by the formula: tf= (c×v3×v1)/(v2×m×10).

Harvesting after the fruits are ripe, and recording the length, width and weight of the ripe fruits. Comparing nutrition and reproductive growth conditions of wild and transgenic tobacco plants with seedling age of 12 weeks, measuring leaf color, flower color, sepal color, plant height, fruit length, width, weight, whole plant fruit number and other data, measuring, photographing and recording organ phenotype, wherein the flower color is measured by a color difference meter, the method is the same as that of the peony petals, the plant height, the fruit length and the width are measured by a ruler or a vernier caliper, the fruit number is visual count, and the fruit weight is weighed by a balance.

4. PsHY5 over-expression transgenic tobacco phenotype verification

Three transgenic tobacco line plants with high gene expression levels of interest were selected for phenotypic validation and designated OE-1, OE-2 and OE-3, respectively.

Comparing the nutritional and reproductive growth status of wild type and PsHY5 transgenic tobacco plants, a significant difference was found between the PsHY5 transgenic plants and the wild type plants (fig. 8). RNA is extracted from the transgenic tobacco strain, real-time fluorescent quantitative PCR identification is carried out, and the relative expression quantity of genes is verified. The results show that the relative expression level of PsHY5 is higher than that in wild tobacco, and the expression levels in three over-expression transgenic lines are OE-3> OE-2> OE-1 (figure 9) in sequence, so that the tobacco plants are further verified to be positive transgenic plants, and normal transcription is carried out.

The over-expressed transgenic tobacco petals show more vivid red color compared with wild type, the leaves are more deeply green, and the total anthocyanin content of the petals is higher (figure 8; figure 10); both the size and weight of the mature fruit were significantly increased compared to the wild type (fig. 11); the transgenic plants (OE-1, OE-2 and OE-3) are in sharp contrast to the wild-type control, regardless of flower color, plant fruit size, weight. In view of the fact that the tobacco can deepen red petals and green leaves and increase fruits through PsHY5 overexpression, the tobacco is primarily presumed to be a transcription factor capable of positively regulating plant anthocyanin accumulation and affecting plant fruit size.

5. Instantaneous silencing of PsHY5 in peony petals using VIGS

The specific fragment of PsHY5 is selected to 251bp, primers pTRV-PsHY5-F/R are designed at two ends of the specific fragment (Table 5), PCR amplification is carried out by taking cDNA of 'twilight' as a template, an electrophoresis detection result shows that clear single specific bands appear at about 250bp (figure 12), the size of the specific bands is consistent with that of a target gene, pTRV2-PsHY5 positive bacterial liquid is obtained through recovery, connection and transformation, sequencing is carried out, and the result shows that a peony PsHY5 gene interference fragment (251 bp) is obtained through successful cloning, and can be used for constructing a VIGS recombinant vector.

TABLE 5 primer sequences for PsHY5 Gene silencing

The underlined is the base cleavage site, upstream and downstream of the BamHI and XhoI cleavage sites, respectively.

The specific fragment cloned above was ligated to plant virus-mediated RNAi interference vector pTRV2 ligation (non-patent literature describing RNAi interference vector pTRV 2: shu Qingyan, zhu, mentha's, hairyveromyces, wang Qianyu, liu Zhengan, zen Xiuli, wang Liangsheng. 2018. Set up of the VIGS technology system based on peony flavonoid glycosyltransferase gene, gardening journal, 45 (1):

168-176), constructing a recombinant vector pTRV2-PsHY5, referring to GV3101Chemically Competent Cell instruction book, and transforming competent cells of the agrobacterium GV3101 by a freeze thawing method to obtain positive monoclonal agrobacterium. The recombinant plasmid of the recombinant vector pTRV2-PsHY5 is used as a template, PCR verification is carried out, PCR primers are shown in Table 5, and comparison and analysis of positive plasmid sequencing results show that the positive plasmid sequencing results are consistent with the original sequence, so that the silencing recombinant vector pTRV2-PsHY5 is successfully constructed.

Three types of agrobacterium containing pTRV1, pTRV2 and pTRV2-PsHY5 vectors were cultured at 28℃and 200rpm with shaking for 8 hours. PCR detection of the target gene bands (primers are shown in Table 5), and positive bacterial solutions with consistent sequencing are diluted into fresh LB culture medium according to the proportion of 1:100, and cultured at 28 ℃ and 200rpm overnight. The bacterial liquid treatment step comprises the following steps: the bacteria were collected by centrifugation at 4000rpm for 10min. By means of an infection liquid (10mM MES+200mM AS+10mM MgCl) ₂ Ph=5.6) resuspended cells, the pipettor was blown down and mixed until sterile clumps were obtained, and the spectrophotometer detected the concentration of the resuspension od600=1.5. According to pTRV1: pTRV2-PsHY 5=1:1, pTRV1: the bacteria solutions were mixed in respective volumes of ptrv2=1:1, and left to stand in the dark for 3 to 5 hours.

Petal and wafer treatment steps. And selecting flowers in the S2 period. Selecting middle-layer petals, and beating a petal wafer for each petal to ensure the consistency of the positions of the wafers as much as possible. The petals and the discs are immersed in the invasion liquid, and the petals and the discs are sucked and invaded by a vacuum pump. The first time to-0.08 and the second time to-0.07, the air is slowly discharged for 5min after being maintained for 90 s. After the infection treatment is finished, the petals or the discs are rinsed with distilled water. Then put on a disposable petri dish containing filter paper soaked by water, and cover to prevent water loss. The mixture was left at 8℃for 3 days. After 3 days, the mixture was taken out and transferred to 23℃for 7-8 days. The petal samples were photographed sequentially on the day and the samples were stored in liquid nitrogen.

As can be seen by comparing petals infected by pTRV empty vector and pTRV-PsHY5 silencing recombinant vector, the petals are obviously lighter in color (a in FIG. 13); petal CLEL a was measured using a colorimeter spectrophotometer CR-400 (Konica Minolta, inc., japan) ^* b ^* Values. In five periods of the variety, 5 flowers are selected respectively, the middle parts of petals near the axial surfaces of the flowers are taken for measurement, the petals are horizontally placed on a white board, a light collecting port faces to the measurement positions of the petals, and no light transmission is ensured. Finally, the average value of 5 indexes is taken to reflect the flower color phenotype of the period. L (L) ^* The larger the number of (c) is, the greater the luminance is; a, a ^* The size of (3) is changed from small to large, which indicates that the redness is increased and the greenness is reduced; b ^* An increase in the value indicates a decrease in blue, and a corresponding yellowIs a reinforcement of (a). According to the formula c= (a x 2+b x 2) 0.5 is converted into C (chroma) values. Five random measurements were made for each set of samples, and the mean and standard deviation were taken as analytical data. a, a ^* And L ^* Significantly reduced compared to control, C ^* Significantly increased compared to the control (b in fig. 13); the expression level of PsHY5 in pTRV-PsHY5 petals is obviously reduced compared with pTRV control, and the silencing efficiency reaches 73.40% (c in FIG. 13); there was also a very significant decrease in total anthocyanin content of the petals compared to the control (d in fig. 13). Given that silencing PsHY5 reduces anthocyanin content, it was again confirmed to be a transcription factor capable of positively regulating peony anthocyanin accumulation.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1.A protein has an amino acid sequence shown in SEQ ID NO. 2.

2. The protein-encoding gene of claim 1, wherein: the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

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

4. A method of making a transgenic plant comprising the steps of: introducing the coding gene of claim 2 into a starting plant to obtain a transgenic plant; transgenic plants have deeper petals and leaves, higher total anthocyanin content in the petals and/or increased fruit size and weight compared to the starting plant.

5. The method according to claim 5, characterized in that

6. The method according to claim 5, wherein: the plant is tobacco.

7. Amplifying the primer pair of the full length of the coding gene of claim 2 or 3, wherein one primer sequence is shown as a sequence 3 in a sequence table, and the other primer sequence is shown as a sequence 4 in the sequence table.

8. Use of the protein of claim 1 for positively regulating anthocyanin accumulation in plant petals and plant fruit growth.

9. Use of the coding gene according to claim 2 or 3 for positively regulating anthocyanin accumulation in plant petals and plant fruit growth.

10. Use according to claim 8 or 9, characterized in that: the plant is tobacco.