A RsMYB3 transcription factor related to anthocyanin synthesis in rose petal spots and its application

Abstract

The present invention relates to the technical field of plant genetic engineering, and in particular to a RsMYB3 transcription factor related to anthocyanin synthesis in rose petal spots and its application. The present invention cloned the MYB transcription inhibitor RsMYB3 involved in the synthesis of anthocyanin in rose petal spots, and the transcription inhibitor can negatively regulate anthocyanin synthesis; overexpression of the transcription inhibitor RsMYB3 inhibits anthocyanin synthesis in petals, and the expression of anthocyanin synthesis-related genes is reduced. The transcription inhibitor RsMYB3 provided by the present invention provides a new gene and method for regulating anthocyanin synthesis, petal color and petal spot in plants.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to an RsMYB3 transcription factor related to anthocyanin synthesis in rose petal spots and an application thereof. Background Art

[0002] Flower color is an important visual signal for the interaction between plants and pollinators, and has important functions in ecology and evolution (Davies KM, Albert NW, Schwinn K E. From landing lights to mimicry: themolecular regulation of flower colouration and mechanisms for pigmentation patterning[J]. Functional Plant Biology, 2012, 39(8): 619-638.). Changes in flower color may cause changes in pollinators, leading to reproductive isolation and speciation (Heuschen B, Gumbert A, LunauK. A generalised mimicry system involving angiosperm flower colour, pollenand bumblebees innate colour preferences[J]. Plant Systematics and Evolution,2005, 252(3-4): 121-137;Yuan YW, Sagawa JM, Frost L, et al. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers ( Mimulus )[J]. NewPhytologist, 2014, 204(4): 1013-1027; Liang M, Chen W, LaFountain AM, et al. Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers[J]. Science, 2023, 379(6632): 576-582).

[0003] Petal color spots (also called petal spots, abbreviated as flower spots), such as stripes, spots, patches, etc., are a unique color pattern on petals, which is widely present in angiosperms and has important significance for the interaction between plants and pollinators (Martins TR, Berg JJ, Blinka S, et al. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formationin Clarkia gracilis (Onagraceae)[J]. New Phytologist, 2013, 197(3): 958-969.). In addition, petal color spots also enrich the variation of flower color, improving the ornamental value and commercial value of plants. Therefore, studying the molecular mechanism of petal color spot formation is of great significance for understanding the evolution and germplasm innovation of ornamental plants.

[0004] Modern roses are world-famous ornamental plants, and flower color is one of their most important ornamental traits. Some varieties have dark red or brown spots at the base of the petals, which improves the ornamental and commercial value of modern roses. Rose varieties with petal spots break the color morphology of common modern rose varieties, with advantages such as large number of flowers, long flowering period, rich color combinations, and repeatable blooming. In addition, the color of the flowers changes from dark to light as the flowers bloom, making them popular in the market. However, the mechanism of the formation of rose petal spots is still unclear.

[0005] Studies have shown that the formation of petal spots is due to the accumulation of pigment components in specific areas on the petals or calyx. The temporal and spatial expression differences of structural genes affect the accumulation of pigment substances. These structural genes are controlled by a variety of transcription factors. Among them, R2R3-MYB transcription factors play an important role in regulating the formation of petal spots. Mimulus )、Petunia( Petunia hybrida ),lily( Lilium spp.), Cattleya (Rhyncholaeliocattleya Beauty Girl 'KOVA'), Clakia gracilis , Phalaenopsis ( Phalaenopsis 'Panda') and tree peonies ( Paeonia rockii) and other plants, and identified the R2R3-MYB transcription factor that regulates petal coloration (Yuan YW, Sagawa JM, Frost L, et al. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers ( Mimulus )[J]. New Phytologist, 2014, 204(4): 1013-1027; Gu Z, Zhu J, Hao Q, etal. A novel R2R3-MYB transcription factor contributes to petal blotchformation by regulating organ-specific expression of PcS in tree peony( Paeonia suffruticosa )[J]. Plant and Cell Physiology, 2019, 60(3): 599-611; LiB, Zheng B, Wang J, et al. New insight into the molecular mechanism of colordifferentiation among floral segments in orchids[J]. Communications Biology, 2020, 3(1): 1-13; Hsu CC, Chen YY, Tsai WC, et al. Three R2R3-MYBtranscription factors regulate distinct floral pigmentation patterningin Phalaenopsis spp.[J]. Plant Physiology, 2015, 168(1): 175-191; Yamagishi M, Uchiyama H, Handa T. Floral pigmentation pattern in Oriental hybrid lily( Liliumspp.) cultivar 'Dizzy' is caused by transcriptional regulation ofanthocyanin biosynthesis genes[J]. Journal of Plant Physiology, 2018, 228:85-91;Luan Y, Tang Y, Wang X, et al. Tree peony R2R3-MYB transcription factorPsMYB30 promotes petal blotch formation by activating the transcription ofthe anthocyanin synthase gene[J]. Plant and Cell Physiology, 2022, 63(8):1101-1116;Luan Y, Chen Z, Tang Y, et al. Tree peony PsMYB44 negativelyregulates petal blotch distribution by inhibiting dihydroflavonol-4-reductasegene expression[J]. Annals of Botany, 2023, 131(2): 323-334;Martins T R,Jiang P, Rausher M D. How petals change their spots: cis-regulatory re-wiringin Clarkia(Onagraceae)[J]. New Phytologist, 2017, 216(2): 510-518;Albert N W,Lewis D H, Zhang H, et al. Members of an R2R3-MYB transcription factor familyin Petunia are developmentally and environmentally regulated to controlcomplex floral and vegetative pigmentation patterning[J]. The Plant Journal,2011, 65(5): 771-784;Lin R C, Rausher M D. R2R3-MYB genes control petalpigmentation patterning in Clarkia gracilis ssp. sonomensis (Onagraceae)[J]. NewPhytologist, 2021, 229(2): 1147-1162;Ding B, Patterson E L, Holalu S V, etal. Two MYB proteins in a self-organizing activator-inhibitor system producespotted pigmentation patterns[J]. Current Biology, 2020, 30(5): 802-814;Yamagishi M. Involvement of a LhMYB18 transcription factor in largeanthocyanin spot formation on the flower tepals of the Asiatic hybrid lily( Liliumspp.) cultivar "Grand Cru"[J]. Molecular Breeding, 2018, 38(5).) These transcription factors can affect the formation and distribution of anthocyanins by regulating the expression of structural genes, ultimately forming different flower color patterns. However, there are no reports on the role of R2R3-MYB transcription factors in regulating the formation of rose petal color spots. Therefore, the study of the molecular regulatory mechanism of rose petal color spot formation will provide a basis for the targeted creation of new rose varieties with color spots, and also provide a reference for the study of the regulatory mechanism of petal color spot formation in other ornamental plants. Summary of the invention

[0006] The invention provides a RsMYB3 transcription factor related to anthocyanin synthesis in rose petal color spots and application thereof.

[0007] The present invention is based on modern rose varieties Rosa The transcriptome data of 'Sunset Babylon Eyes' were used to clone the CDS sequence of the MYB transcription repressor RsMYB3 involved in anthocyanin synthesis in petal spots based on the annotation results and the difference in the expression of MYB transcription factors in petal spots and non-spots. The open reading frame of the RsMYB3 transcription factor is 549 bp long, encoding a total of 182 amino acids, with R2R3 and bHLH conserved domains. Multiple sequence alignment and phylogenetic analysis found that the RsMYB3 protein of the modern rose variety 'Sunset Babylon Eyes' is most closely related to the strawberry FaMYB1 protein and belongs to the FaMYB1-like transcription repressor. qRT-PCR analysis showed that RsMYB3 The expression level of the gene in the petal spots of the modern rose variety 'SunsetBabylon Eyes' is higher than that in the non-spots, and the expression level increases first and then decreases with the different opening periods of the flowers. RsMYB3 The gene was transferred into tobacco for overexpression, and it was found that the flower color of the transgenic strain became significantly lighter, the anthocyanin content was significantly reduced, and the relative expression levels of structural genes related to anthocyanin synthesis were also significantly reduced, indicating that RsMYB3 has a negative regulatory effect on the synthesis of anthocyanins in the petal color spots, and can be used to regulate the synthesis of anthocyanins in the petals and affect the formation of petal color spots.

[0008] Specifically, the present invention provides the technical solution described below.

[0009] In a first aspect, the present invention provides a RsMYB3 protein having an amino acid sequence as shown in SEQ ID NO.1.

[0010] Preferably, the amino acid sequence of the RsMYB3 protein is as shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the RsMYB3 protein described above.

[0012] According to the amino acid sequence of the RsMYB3 protein provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above RsMYB3 protein. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the RsMYB3 protein is not unique, and all nucleic acid molecules capable of encoding the production of the RsMYB3 protein are within the scope of protection of the present invention.

[0013] In some specific embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO. 2. Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2.

[0014] In a third aspect, the present invention provides a recombinant nucleic acid molecule, which comprises the nucleic acid molecule described above.

[0015] Preferably, the recombinant nucleic acid molecule is an expression cassette comprising the nucleic acid molecule described above.

[0016] Preferably, the expression cassette can be obtained by connecting a transcription and / or translation regulatory element such as a promoter upstream of the nucleic acid molecule, and / or connecting a transcription and / or translation regulatory element such as a terminator downstream of the nucleic acid molecule.

[0017] In a fourth aspect, the present invention provides a vector, wherein the vector comprises the nucleic acid molecule described above or comprises the recombinant nucleic acid molecule.

[0018] The vectors mentioned above include but are not limited to plasmid vectors, phage vectors, viral vectors, etc.

[0019] In a fifth aspect, the present invention provides a host cell, the host cell, the nucleic acid molecule or the recombinant nucleic acid molecule or the vector.

[0020] The host cells mentioned above include microbial cells or non-reproductive plant cells. The microbial cells may be bacteria or fungi, wherein bacteria include Escherichia coli, Agrobacterium, etc., and fungi include yeast, etc. The non-reproductive plant cells are plant cells that cannot reproduce into complete plant individuals.

[0021] In a sixth aspect, the present invention provides the use of the above-mentioned RsMYB3 protein or the nucleic acid molecule or the recombinant nucleic acid molecule or the vector or the host cell in promoting the formation of color spots on plant petals.

[0022] In the above application, the formation of color spots on the petals of the plant is promoted by increasing the expression level and / or activity of the RsMYB3 protein in the plant.

[0023] In a seventh aspect, the present invention provides use of the above-mentioned RsMYB3 protein or the nucleic acid molecule or the recombinant nucleic acid molecule or the vector or the host cell in cultivating plant varieties with petal color spots.

[0024] In the above application, the plant variety with petal spots is cultivated by increasing the expression amount and / or activity of the RsMYB3 protein in the plant. Preferably, the plant variety with petal spots is cultivated by increasing the expression amount and / or activity of the RsMYB3 protein in the petals of the plant.

[0025] In an eighth aspect, the present invention provides use of the above-mentioned RsMYB3 protein or the nucleic acid molecule or the recombinant nucleic acid molecule or the vector or the host cell in regulating the synthesis of anthocyanins in plant petals.

[0026] In the above application, the synthesis of anthocyanins in plant petals is inhibited by increasing the expression level and / or activity of RsMYB3 protein in the plant; or, the content of anthocyanins in plant petals is reduced by increasing the expression level and / or activity of RsMYB3 protein in the plant.

[0027] In a ninth aspect, the present invention provides the use of the above-mentioned RsMYB3 protein or the nucleic acid molecule or the recombinant nucleic acid molecule or the vector or the host cell in regulating the expression of genes related to anthocyanin synthesis in plant petals.

[0028] Preferably, the anthocyanin synthesis-related gene is selected from oeLh , F3H , Nf , NtS One or more of .

[0029] In a tenth aspect, the present invention provides the use of the above-mentioned RsMYB3 protein or the nucleic acid molecule or the recombinant nucleic acid molecule or the vector or the host cell in regulating the color of plant petals.

[0030] In the above application, the color of the plant petals becomes lighter by increasing the expression level and / or activity of the RsMYB3 protein in the plant.

[0031] In the above application, the plant is a plant of the genus Rosa or Nicotiana.

[0032] Preferably, the plant of the genus Rosa is Chinese rose, rose or rose; and the plant of the genus Nicotiana is tobacco.

[0033] In an eleventh aspect, the present invention provides a method for constructing a transgenic plant, the method comprising: introducing the above-mentioned nucleic acid molecule or the vector into a plant to obtain a transgenic plant.

[0034] Preferably, the petal color of the transgenic plant becomes lighter due to the introduction of the nucleic acid molecule or the vector described above, and / or the anthocyanin synthesis in the petals of the transgenic plant is reduced due to the introduction of the nucleic acid molecule or the vector described above, and / or the petal color spots of the transgenic plant are formed due to the introduction of the nucleic acid molecule or the vector described above.

[0035] Preferably, the plant is a plant of the genus Rosa or Nicotiana.

[0036] Preferably, the plant of the genus Rosa is Chinese rose, rose or rose; and the plant of the genus Nicotiana is tobacco.

[0037] The beneficial effects of the present invention include at least: the present invention cloned the MYB transcription inhibitor RsMYB3 involved in the synthesis of anthocyanins in petal color spots in roses, and the transcription inhibitor can negatively regulate anthocyanin synthesis; overexpression of the transcription inhibitor RsMYB3 inhibits anthocyanin synthesis in petals, reduces the expression of anthocyanin synthesis-related genes, and changes the color of petals. The transcription inhibitor RsMYB3 provided by the present invention provides a new gene and method for regulating anthocyanin synthesis, petal color, and petal color spots in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 It is a modern rose variety in the embodiment of the present invention. Rosa Flowers and petals of 'Sunset Babylon Eyes' at different developmental stages; the two petals at different stages are the front and back of the petals.

[0040] Figure 2 This is the modern rose variety 'Sunset Babylon Eyes' in the embodiment of the present invention. RsMYB3 Gene CDS sequence amplification result detection; where M is DL2000 marker; 1 is sequence amplification product.

[0041] Figure 3The figure shows the sequence alignment of the RsMYB3 protein in the present invention and the MYB proteins related to anthocyanin synthesis in other plants.

[0042] Figure 4 This is a phylogenetic analysis of RsMYB3 in the modern rose variety 'Sunset Babylon Eyes' in the examples of the present invention.

[0043] Figure 5 In the embodiment of the present invention RsMYB3 The expression levels of genes in the petal spots and non-spots of the modern rose variety 'Sunset Babylon Eyes'; ** indicates significant differences at the 0.01 level.

[0044] Figure 6 This is the detection of anthocyanin content in petal spots and non-spots of rose flowers at different opening stages in the embodiments of the present invention.

[0045] Figure 7 The wild-type tobacco (CK) and some transgenic RsMYB3 Flower color phenotype of genetic tobacco, the scale bar is 1 cm.

[0046] Figure 8 In the embodiment of the present invention RsMYB3 Flower color phenotype and anthocyanin content of transgenic tobacco; CK represents wild-type plants; #11, #36, #14, and #34 are transgenic positive plants tested; * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively; the scale bar is 1 cm.

[0047] Fig. 9 In the embodiment of the present invention RsMYB3 Analysis of relative expression levels of structural genes related to anthocyanin synthesis in transgenic tobacco. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example RsMYB3 Gene cloning, overexpression and functional analysis

[0050] 1. Experimental Materials and Methods

[0051] 1.1 Experimental Materials

[0052] Plant material: The flowers of the modern rose variety 'Sunset Babylon Eyes' were divided into four different developmental stages ( Figure 1 ), respectively: during the bud stage, no spots appear at the base of the petals (S1); during the bud stage, spots begin to appear at the base, and the non-spotted part is yellow-green (S2); during the semi-open stage, the color of the base spots deepens, and the non-spotted part turns yellow (S3); during the blooming stage, the base spots become larger and the flowers are fully open (S4). The petals of the flowers at different developmental stages were taken. For each sample, 15 flowers were collected in the S1, S3 and S4 periods, and 30 flowers were collected in the S2 period. The spotted and non-spotted parts of the petals were sampled separately. The spotted parts of the petals were S2d, S3d, and S4d, and the non-spotted parts were S2u, S3u, and S4u. Each sample was biologically replicated three times. The samples were quick-frozen in liquid nitrogen and stored at -80 o C. Refrigerator.

[0053] 'Honghua Dajinyuan' tobacco ( Nicotiana tabacum 'Honghuadajinyuan') was provided by the Tobacco Research Institute, Chinese Academy of Agricultural Sciences.

[0054] Main reagents: OMEGA plant total RNA extraction kit, Escherichia coli DH5α, Agrobacterium tumefaciens strain GV3101 were purchased from Beijing Biodi Biotechnology Co., Ltd., cDNA first-strand synthesis kit, PrimeSTAR ® Max DNA Polymerase, restriction endonuclease, ligase and DL2000 DNA Marker were purchased from Takara, and agarose gel recovery kit was purchased from BIOMIGA. Primer synthesis and sequencing were completed by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0055] 1.2 Experimental methods

[0056] 1.2.1 RsMYB3 Cloning and sequencing of gene CDS sequences

[0057] Total RNA was extracted from the petals of the modern rose variety 'Sunset Babylon Eyes' using the OMEGA total RNA extraction kit and reverse transcribed into cDNA. RsMYB3The cloning primers were designed using Primer3Web (https: / / primer3.ut.ee / ) online primer design software. The primer sequences are shown in Table 1. PCR amplification was performed using cDNA of rose petals of 'SunsetBabylon Eyes' as a template. The reaction system was: PrimeSTAR Max Premix (2×) 25 μL, upstream primer 1 μL, downstream primer 1 μL, cDNA 1 μL, ddH2O 22 μL. The PCR reaction program was: 98 o C 10s; 62 o C 5 s, 35 cycles, 72 o C 5 s. After amplification, the amplified product was subjected to 1% agarose gel electrophoresis to recover the target fragment. The specific operation steps refer to the instructions of the BIOMIGA kit. The recovered target fragment was connected to the TOPO vector and transformed into the competent E. coli DH5α. Then the positive clone was picked for shaking. The correct bacterial solution identified by PCR was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0058] 1.2.2 RsMYB3 Gene sequence alignment and phylogenetic tree construction

[0059] DNAMAN 8.0 software was used for gene sequence alignment analysis. After sequence alignment using MUSCLE in MEGA X software, the Neighbor-Joining method (bootstrap value was 1000) was used to construct the MYB phylogenetic tree. iTOL (https: / / itol.embl.de / ) was used to further beautify the phylogenetic tree.

[0060] 1.2.3 RsMYB3 Gene expression analysis

[0061] Total RNA was extracted from petal spotted and non-spotted samples of the modern rose variety 'Sunset Baylon Eyes' at different opening stages, and the total RNA was converted to cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Japan). The LightCycler 480II fluorescence quantitative PCR instrument (Roche, Switzerland) was used according to the NovoStart ®The real-time fluorescence quantitative reaction was performed according to the instructions of SYBR High-Sensitivity qPCR SuperMix (Novoprotein, China). A 20 μL reaction system was used, as follows: 10 μL SYBR qPCR Master Mix, 1 μL cDNA template (100 ng / μL), 0.4 μL upstream / downstream primers (10 μM), and ddH2O was used for completion. The reaction procedure was as follows: 95 o C 1min; 95 o C 20 s, 60 o C 45 s, 40 cycles. Primers were designed using the online software PrimerQuest Tool (https: / / sg.idtdna.com / PrimerQuest / Home / Index). The primers used are shown in Table 1. PP2A As the internal reference gene, the relative expression of different genes was used as 2 -ΔΔCt Method calculation. Each sample was repeated three times biologically and three times technically. The expression level in the S1 period was used as the control, that is, the expression level in the S1 period was 1.

[0062] 1.2.4 RsMYB3 Construction of gene recombinant plasmid and transformation of Agrobacterium

[0063] (1) Construction of recombinant plasmid

[0064] The target gene was cleaved by restriction endonucleases XbaⅠ and SacⅠ RsMYB3 The cloning vector and pSuper1300 The vector plasmid was double-digested and incubated at 37°C for 3-4 h. After adding DNA Loading buffer, 1% agarose gel electrophoresis was performed. After confirming that the fragment was correct, gel excision and recovery were performed. T4 DNA ligase was used to connect the target fragment and the linear vector to complete the recombinant plasmid. pSuper1300:RsMYB3 's construction.

[0065] (2) Transformation of Agrobacterium with recombinant plasmid

[0066] Take out the competent Agrobacterium GV3101 stored in the -80°C refrigerator, place it on ice to thaw, add 10 μL of recombinant plasmid to the competent Agrobacterium (100 μL), mix gently, ice bath for 5 min, quick freeze in liquid nitrogen for 5 min, heat shock in a 37°C water bath for 5 min, ice bath for 5 min, add 700 μL LB liquid culture medium, shake and culture at 28°C 200 rpm for 2-3 h, take an appropriate amount of bacterial liquid and spread it on the solid culture medium of LB + 50 mg / L kanamycin + 20 mg / L rifampicin, and place it in a 28°C incubator for 2-3 d.

[0067] (3) Identification of Agrobacterium transformants

[0068] Single colonies were picked and cultured in LB+50 mg / L kanamycin+20 mg / L rifampicin liquid culture medium for 12 h (28°C, 200 rpm), and the bacterial solution was identified by PCR, and the correctly identified bacterial solution was sequenced.

[0069] 1.2.5 Genetic transformation of tobacco

[0070] Will pSuper1300:RsMYB3 The overexpression vector was transferred into tobacco leaves by leaf disc transformation method. The specific method is as follows:

[0071] (1) Cut the leaves of tobacco 'Honghua Dajinyuan' sterile seedlings into leaf discs of about 1 cm × 1 cm, avoiding the main veins. Spread the leaf discs flat on the pre-culture medium and pre-culture for 2 days. Pre-culture medium: MS + 25 g / L sucrose + 7 g / L agar, pH = 5.6-5.8.

[0072] (2) Place the pre-cultured leaf disc into the prepared pSuper1300:RsMYB3 Plasmid Agrobacterium infection solution (OD 600 = 0.6-0.8), shake gently and infect for 10-15 min.

[0073] (3) Use filter paper to absorb excess bacterial liquid on the leaf surface and spread it flat on the co-culture medium so that the leaf cut contacts the medium. Co-culture for 2 days in the dark. Co-culture medium: MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 25 g / L sucrose + 7 g / L agar, pH = 5.6-5.8.

[0074] (4) After the co-cultivation, the leaves were transferred and spread onto the resistant differentiation medium and placed under 16 h light (25 o C) and 8 h darkness (25 o C) Culture under the following conditions: Resistance differentiation medium: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L agar, add 200 mg / L timentin, 30 mg / L hygromycin, pH = 5.8.

[0075] (5) After the resistant buds grow out, transfer them to a rooting medium with resistance for cultivation. Resistance rooting medium: MS + 0.3 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar, with 200 mg / L timentin and 30 mg / L hygromycin, pH = 5.8.

[0076] (6) After the resistant seedlings have taken root, move them out of the pots and place them in a greenhouse for cultivation.

[0077] (7) Using T0 generation transgenic tobacco as material, total DNA was extracted from leaves and resistant seedlings were identified by PCR. After flowering, RNA was extracted from petals and reverse transcribed into cDNA. Gene expression was verified by fluorescence quantitative PCR.

[0078] 1.2.6 Determination of tobacco anthocyanins and fluorescence quantitative PCR detection

[0079] Determination of the total content of anthocyanins in tobacco petals: Take about 0.1 g of tobacco petals at full flowering stage, grind them into powder with liquid nitrogen, add 1 mL of methanol (containing 1% hydrochloric acid), vortex and place them at 4 o C in the dark for 24 h, and vortexed and mixed once every 6 h. o C, centrifuge at 10000 g for 5 min, transfer the supernatant to a new centrifuge tube, and filter the extract through a 0.2 μm filter membrane. Use a UV-visible spectrophotometer to measure the OD value of the extract at 530 nm and 657 nm, and perform three biological replicates for each sample. The anthocyanin content is calculated as follows: Q = (A 530 -0.25×A 657 ) / m, where Q is the anthocyanin content, A 530 , A 657 are the OD values ​​at 530 nm and 657 nm respectively, and m is the sample mass.

[0080] Using the cDNA reverse transcribed from tobacco petals as a template, fluorescence quantitative PCR was used to detect the CHS, CHI, F3H, F3'H, DFR, ANS, UFGT Changes in gene expression, with empty vector transgenic tobacco as control and internal reference gene as NtActin The primer sequences are shown in Table 1. The different samples were repeated 3 times, using 2 -ΔΔCt Methods: Analyze the relative expression of genes. The detection and analysis methods are the same as 1.2.3.

[0081] Table 1 Primers used for gene cloning and gene expression quantification

[0082]

[0083] 2. Experimental results

[0084] 2.1 RsMYB3 Gene cloning and sequence analysis

[0085] Using the cDNA of the petals of the modern rose variety 'Sunset Babylon Eyes' as a template, RsMYB3 The CDS sequence of the gene was cloned and the amplification results were as follows Figure 2As shown. After sequencing comparison, it was found that RsMYB3 The CDS sequence is 549 bp in length and encodes 182 amino acids.

[0086] RsMYB3 The CDS sequence of the gene (SEQ ID NO.2) is as follows:

[0087] .

[0088] The deduced amino acid sequence (SEQ ID NO.1) is as follows:

[0089] MRKPCCEKTEMTKGAWSIQEDQKLIDYIQKHGEGCWNSLPKAAGLRRCGKSCRLRWINYLRPDLKRGSFGEDEEDLIIRLHTLLGNRWSLIAGRLPGRTDNEVKNYWNSHLKKKLLNIGTTTLRPNKPREINHAPYNKLVKYFNDMDDEVVDEVSADSASTGISSSLVPKLNLDLSLSLALE.

[0090] Protein sequence comparison of RsMYB3 and MYB transcription factors in other species revealed that the N-terminus of RsMYB3 contained an R2R3 domain and a bHLH binding domain ( Figure 3 ), which belongs to the R2R3-MYB transcription factor ( Figure 3 ). Phylogenetic analysis revealed that ( Figure 4 ), RsMYB3 and other MYB transcription factors that negatively regulate anthocyanin biosynthesis in plants are clustered together with strawberry ( Fragaria × ananassa ) The sequence similarity of FaMYB1 is the highest and the relationship is the closest. Their functions may be similar and they belong to the FaMYB1-like transcriptional repressor.

[0091] 2.2 RsMYB3 Gene expression analysis

[0092] RsMYB3 The results of the expression analysis of the gene in the petal spots and non-spots at different stages of flower development of the modern rose variety 'Sunset Babylon Eyes' showed that as the petal spots appeared, RsMYB3 The expression levels of genes in the petal spots were significantly higher than those in the non-spots at the same time, and the overall expression levels of genes showed a trend of first increasing and then decreasing, with the highest expression levels in the S3 period ( Figure 5 ).

[0093] In order to study the accumulation of anthocyanins in petal spots and non-spots of rose varieties with color spots at different developmental stages, the anthocyanin components of rose flowers of 'Sunset Babylon Eyes' at four stages of blooming were determined and analyzed using high performance liquid chromatography (HPLC). Figure 6 ), anthocyanins were only detected in the petal spots during S2-S4, and no anthocyanins accumulated in the non-spots during S1 and each period. Three main anthocyanin components were detected in the spots, namely cyanidin 3,5- O -Diglucoside, pelargonidin 3,5- O -Diglucoside and cyanidin 3- O -Glucoside. In the pigmented area, as the pigmented spots began to appear and the flowers opened, the contents of the three anthocyanins gradually increased, reaching the highest value in the S3 period, and gradually decreased after the flowers fully opened.

[0094] RsMYB3 The expression of the gene in the petal spots and non-spots is related to the accumulation of anthocyanins. There is a feedback regulation mechanism in the accumulation and synthesis of anthocyanins in the spots, which can inhibit the further increase of anthocyanin content. In many plants, R2R3-MYB that inhibits anthocyanin synthesis is often co-expressed with the activating R2R3-MYB to avoid excessive accumulation of anthocyanins.

[0095] The above results show that RsMYB3 The expression levels of the gene in the spotted and non-spotted parts of the petals of the 'Sunset Babylon Eyes' rose at different opening stages showed a similar trend and were positively correlated with the anthocyanin contents.

[0096] 2.3 RsMYB3 GMO Verification

[0097] 2.3.1 Obtaining genetically modified tobacco

[0098] The constructed overexpression vector was stably transformed into tobacco 'Honghua Dajinyuan' by Agrobacterium-mediated leaf disc transformation, and 36 flowering antibiotic-positive seedlings were obtained ( Figure 7 ). PCR identification was performed on the positive seedlings after antibiotic screening. No target band was amplified in the control plants, but the target band of about 550 bp was amplified in all 36 PCR-positive plants, indicating that RsMYB3 The gene was successfully transferred into 36 positive plants.

[0099] 2.3.2 Detection of flower color phenotype, anthocyanin content and expression of anthocyanin structural genes in transgenic tobacco

[0100] Compared with the control plants, overexpression RsMYB3 The flower color of tobacco plants became significantly lighter, white or light pink, and the anthocyanin content in the petals was also significantly reduced ( Figure 8 ). The relative expression levels of structural genes related to anthocyanin synthesis pathway in tobacco were detected and analyzed. The results showed that compared with the control plants, NtF3H, NtF3'H, NtDFR, NtANS The relative expression levels of the genes in each transgenic tobacco plant were significantly reduced ( Fig. 9 ).

[0101] The above results show that RsMYB3 It is a key regulatory gene that inhibits the synthesis and accumulation of anthocyanins in rose petal color spots.

[0102] The present invention cloned candidate genes related to anthocyanin synthesis from the petals of the rose variety 'Sunset Babylon Eyes' with petal spots RsMYB3 Gene, belongs to R2R3-MYB transcription factor. RsMYB3 has a high homology with strawberry FaMYB1 and is a FaMYB1-like inhibitory transcription factor. Overexpression in tobacco RsMYB3 , the petal color became lighter, the anthocyanin content decreased, and the expression of genes related to anthocyanin synthesis decreased, indicating RsMYB3 It is a key regulatory gene that inhibits the synthesis of anthocyanins in rose petal color spots.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of RsMYB3 protein or a nucleic acid molecule encoding RsMYB3 protein or a vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule or the vector in promoting the formation of rose petal spots or cultivating rose varieties with petal spots; in, The amino acid sequence of the RsMYB3 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

3. Application of RsMYB3 protein or a nucleic acid molecule encoding RsMYB3 protein or a vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule or the vector in regulating anthocyanin synthesis in rose or tobacco petals; in, The amino acid sequence of the RsMYB3 protein is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

5. Use of RsMYB3 protein or a nucleic acid molecule encoding RsMYB3 protein or a vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule or the vector in regulating the expression of genes related to anthocyanin synthesis in tobacco petals; in, The amino acid sequence of the RsMYB3 protein is shown in SEQ ID NO.1; The anthocyanin synthesis-related genes are one or more genes selected from NtF3H, NtF3'H, NtDFR, and NtANS.

6. The use according to claim 5, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

7. Use of RsMYB3 protein or a nucleic acid molecule encoding RsMYB3 protein or a vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule or the vector in regulating the color of rose or tobacco petals.

8. A method for constructing a transgenic rose or tobacco, characterized in that: The method comprises: introducing a nucleic acid molecule encoding RsMYB3 protein or a vector containing the nucleic acid molecule into rose or tobacco to obtain transgenic rose or tobacco; Wherein, the amino acid sequence of the RsMYB3 protein is shown as SEQ ID NO.

1.

9. The method according to claim 8, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

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