Application of Chinese rose MYB transcription factor RcMYB1a in regulation and control of plant flower color

Abstract

The invention belongs to the field of genetic engineering technology breeding, and discloses application of a Chinese rose MYB transcription factor RcMYB1a in regulating plant flower color. The nucleotide sequence of the RcMYB1a transcription factor gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the RcMYB1a transcription factor gene is as shown in SEQ ID NO. 2. The RcMYB1a protein belongs to the S6 subfamily of R2R3-MYBs and has a potential function of positively regulating and controlling anthocyanin synthesis, and the overexpression of RcMYB1a promotes the anthocyanin synthesis in tobaccos and'iceberg 'Chinese roses, so that the flower color is deepened. As an important gene resource, the rosa chinensis MYB transcription factor RcMYB1a provided by the invention not only can provide a basis for rosa chinensis color breeding, but also can be applied to breeding of other flower plants.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering breeding technology, and specifically relates to the application of rose MYB transcription factor RcMYB1a in regulating plant flower color. Background Art

[0002] Rose (Rosa chinensis) is a plant of the genus Rosa in the family Rosaceae. It is the flower with the highest influence and economic value in the world, and flower color is an important breeding goal.

[0003] Anthocyanin is the main coloring substance in rose petals. Studies have found that the biosynthesis of anthocyanin is mainly regulated by three types of transcription factors, namely MYB, bHLH and WD40 proteins. These three types of transcription factors directly regulate the expression of downstream genes alone or in combination with each other or by forming MBW complexes. Among them, R2R3-MYB is the most common member in the study of anthocyanin synthesis regulation, but there are relatively few studies on the positive regulator MYB of anthocyanin in roses.

[0004] Therefore, the development and utilization of MYB transcription factors related to rose flower color is an issue that needs to be urgently addressed by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an application of a rose MYB transcription factor RcMYB1a in regulating plant flower color.

[0006] One of the purposes of the present invention is to provide a rose MYB transcription factor RcMYB1a.

[0007] The second purpose of the present invention is to provide biological materials related to the RcMYB1a gene.

[0008] The third object of the present invention is to provide the application of the RcMYB1a transcription factor in regulating plant flower color.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] A rose MYB transcription factor RcMYB1a, characterized in that the nucleotide sequence of the transcription factor is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2.

[0011] The biological material related to the RcMYB1a transcription factor gene is characterized by comprising any one of the following (A1) to (A5):

[0012] (A1) Recombinant cloning vector containing RcMYB1a gene;

[0013] (A2) Recombinant plant expression vector containing RcMYB1a gene;

[0014] (A3) a recombinant plant expression vector obtained by connecting a tag to the N-terminus and / or the C-terminus of the gene described in (A2);

[0015] (A4) a bioengineered bacterium containing the recombinant vector described in (A1) or (A2);

[0016] (A5) A transgenic plant containing the recombinant plant expression vector described in (A2) or (A3).

[0017] The plant expression vector containing the RcMYB1a gene described in (A2) and / or (A3) refers to a DNA capable of expressing the above-mentioned RcMYB1a gene in a host cell, and the DNA may contain not only a promoter for initiating transcription of the RcMYB1a gene, but also a terminator for terminating transcription of the RcMYB1a gene.

[0018] The plant expression vector of the present invention is pCAMBIA1305.1-GFP.

[0019] The bioengineering bacteria described in the present invention is Agrobacterium tumefaciens GV3101.

[0020] The application of the above-mentioned RcMYB1a transcription factor in regulating plant flower color.

[0021] A method for regulating plant flower color, characterized in that a plant expression vector containing a RcMYB1a transcription factor gene is introduced into a recipient plant;

[0022] The regulation is to increase the anthocyanin content in the flowers of the recipient plant;

[0023] The receptor plant is rose or tobacco.

[0024] The recombinant plant overexpression vector containing the RcMYB1a gene is used to transform plant tissues or cells through Agrobacterium-mediated method, and the transformed plant tissues are cultured into plants.

[0025] The present invention has the following beneficial effects:

[0026] (1) The present invention cloned the RcMYB1a gene from rose petals, constructed the pCAMBIA1305.1-RcMYB1a-GFP plant overexpression vector, transformed the Agrobacterium tumefaciens competent cell GV3101, and genetically transformed tobacco with the recombinant plant expression vector containing the RcMYB1a gene using the Agrobacterium-mediated method. It was found that the overexpression of the RcMYB1a gene increased the anthocyanin content in the tobacco corolla.

[0027] (2) After transiently transforming the RcMYB1a gene into rose 'Iceberg', it was found that anthocyanins accumulated in the petals of 'Iceberg'.

[0028] (3) The RcMYB1a transcription factor gene provided by the present invention can be used as an excellent gene resource and widely used in the field of genetic breeding of roses or other flower plants, which is of great significance for flower color breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A phylogenetic tree of the protein encoded by the RcMYB1a gene provided by the present invention and the Arabidopsis thaliana R2R3-MYB protein;

[0030] Figure 2 The subcellular localization map of the protein encoded by the RcMYB1a gene provided by the present invention, wherein GFP is green fluorescence;

[0031] Autofluorescence is chloroplast autofluorescence; Bright is bright field; Merge is the fusion of green fluorescence, chloroplast autofluorescence and bright field.

[0032] Figure 3 This is a phenotypic diagram of the corolla coloring of RcMYB1a gene-transfected tobacco provided by the present invention, wherein WT is the wild type, EV is the pCAMBIA1305.1-GFP empty vector, and OE is the RcMYB1a gene-transfected tobacco.

[0033] Figure 4 This is a phenotype diagram of the petal coloring of the RcMYB1a gene-transfected 'Iceberg' provided by the present invention, wherein WT is the wild type, EV is the pCAMBIA1305.1-GFP empty vector, and OE is the RcMYB1a gene-transfected.

[0034] Figure 5 This is the expression pattern of anthocyanin synthesis-related genes in the petals of the 'Iceberg' transgenic plant with the RcMYB1a gene in the present invention, where WT is the wild type and OE is the transgenic. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the embodiments and drawings, but the embodiments of the present invention are not limited thereto. 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.

[0036] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0038] Example 1 Phylogenetic tree of RcMYB1a and Arabidopsis thaliana R2R3-MYB proteins

[0039] Download 126 R2R3-MYBs protein sequences from the Arabidopsis TAIR website and save them in fasta format;

[0040] MEGA7.0 was used to perform multiple sequence alignment of RcMYB1a and 126 Arabidopsis R2R3-MYBs proteins, and then an evolutionary tree was drawn;

[0041] The construction of phylogenetic trees was carried out using the Neighbor-Joining method and the Bootstrap test.

[0042] From the evolutionary tree results ( Figure 1 ), RcMYB1a protein is clustered with the Arabidopsis S6 subfamily, which includes AtMYB113, AtMYB114, AtMYB75, and AtMYB90. These four transcription factor proteins have been shown to positively regulate the synthesis of plant anthocyanins. Therefore, it can be speculated that RcMYB1a also has the function of promoting anthocyanin synthesis in regulating plant flower color.

[0043] Example 2: Cloning of the Rose RcMYB1a Gene

[0044] SnapGene was used to design specific primers for cloning the RcMYB1a gene. A 708 bp band was amplified from rose cDNA by RT-PCR. The PCR product was recovered and sequenced to obtain the RcMYB1a gene.

[0045] The primer sequences used for gene cloning are as follows:

[0046] RcMYB1a-F1: 5'-ATGGAGGGTTTCGGGCTGA-3'

[0047] RcMYB1a-R1: 5'-TCATGCGTAGAAGTTGTTGAC-3'

[0048] The ORF region of the RcMYB1a gene contains 708 bases and the sequence is as follows:

[0049] ATGGAGGGGTTTCGGCGTGAGAAAAGGTGCATGGACTAAAGAGGAAGATGAACT

[0050] TCTGAGACAGGTCATCGAAAAGCATGGAGAAGGAAAATGGCATCAGGTTCCTTTCA

[0051] AAGCAGGCTTAAACAGATGCAGGAAGAGCTGTAGACTGAGGTGGCTAAATTATTTG

[0052] AAGCCAAATATCAAGAGAGGAGAGTTTACAGTTGATGAAGTTGATATGATCATCAG

[0053] ACTTCATAAGCTTCTAGGAAACAGGTGGTCCTTAATTTCTGGAAGACTACCGGGAAG

[0054] AACAGCCAACGATGTAAAGAACTATTGGAATACTTATCAACGGAAAAAGAATCAAA

[0055] AGATGACTTCAGGCGCAAAAAAAATGAAAGATAAATCCCAAAAAAACACAATCGC

[0056] CCCTTTGGTTGTAAGACCTCGACCACGAACCTTCATCAAAAGTTTGAATTTTTTGGA

[0057] AAGAGATGCCAATTTAGAGCATATTCATTCAGAAGAGAATTCTTCCACTTCTTTACC

[0058] AACAGCACCACCACAAACTCTAGAATTAGAGAATGTAATTGATTGGTGGAAAGTTG

[0059] TATCTGAAGACAGTACAGGAAGCATTGATAGAACAACATGTTCTAGTCTTGGTTTAG

[0060] AGGACGACTTCTTCACAAACTTCTGGGTTGAAGATATGGTACAATTGTCAACTATAG

[0061] ATGGCCATGATCTAGTCAACAACTTCTACGCATGA

[0062] The RcMYB1a gene encodes 235 amino acids, and the sequence is as follows:

[0063] MEGFGVRKGAWTKEEDELLRQVIEKHGEGKWHQVPFKAGLNRCRKSCRLRWLNYLKPNIKRGEFTVDEVDMIIRLHKLLGNRWSLISGRLPGRTANDVKNYWNTYQRKKNQKMTSGA KKMKDKSQKNTIAPLVVRPRPRTFIKSLNFLERDANLEHIHSEENSSTSLPTAPPQTLELENVIDWWKVVSEDSTGSIDRTTCSSLGLEDDFFTNFWVEDMVQLSTIDGHDLVNNFYA

[0064] Example 2 Subcellular localization detection

[0065] The pCAMBIA1305.1-GFP vector was double-digested with restriction endonucleases XbaⅠ and BamHⅠ, and the RcMYB1a without the stop codon was ligated to the linearized vector. The pCAMBIA1305.1-RcMYB1a-GFP recombinant plant expression vector was constructed by homologous recombination, and the expression was driven by the CaMV35S promoter.

[0066] The vector was constructed using the homologous recombination kit ClonExpress II One Step Cloning Kit (Novozymes, China), and the operation steps were referred to the kit manual.

[0067] The homology arm primer sequences for the construction of pCAMBIA1305.1-RcMYB1a-GFP vector are as follows:

[0068] RcMYB1a-F2:5'-aagtccggagctagc tctaga ATGGAGGGTTTCGGCGTG-3'

[0069] RcMYB1a-R2:5'-gcccttgctcaccat ggatcc TGCGTAGAAGTTGTTGACTAGATCATG-3'

[0070] The underlined parts in the homology arm primers are the restriction sites of XbaⅠ and BamHI.

[0071] The constructed recombinant plant expression vector pCAMBIA1305.1-RcMYB1a-GFP was transformed into Agrobacterium tumefaciens competent cell GV3101 and cultured in 10 mL LB medium containing 50 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kana) at 28°C and 200 rpm for 16 h.

[0072] Pipette 50 μL of bacterial solution and inoculate into a new 10 mL LB liquid medium, which contains LB + Rif (50 mg / L) + Kana (50 mg / L) + 10 mM MES (PH = 5.6) + 40 μM acetosyringone (AS), and culture at 28 °C with shaking until OD 600 is 1.5.

[0073] The cells were collected by centrifugation at 3200 g for 10 min, the supernatant was discarded, and the cells were washed with 10 mM magnesium chloride (MgCl 2 ) Resuspend the cells and adjust the OD of each bacterial solution using a spectrophotometer 600 is 1.5.

[0074] The leaves of Nicotiana benthamiana were injected with a 1 ml syringe, and GFP fluorescence was observed under a laser confocal microscope after culturing for 2-3 days.

[0075] Subcellular localization detection results Figure 2 As shown, the green fluorescence of the control empty vector pCAMBIA1305.1-GFP was distributed throughout the epidermal cells, while the fluorescence was only detected in the nucleus of tobacco leaves transformed with the RcMYB1a gene, which indicates that RcMYB1a meets the general characteristics of transcription factors and its action site is in the nucleus.

[0076] Example 3 Genetic transformation of tobacco

[0077] Pre-culture: In an ultra-clean workbench, the pre-sterile cultured common tobacco (N. tabacum) leaves were cut into 1 cm × 1 cm size and placed on M1 medium (MS + 1 mg / L 6BA + 0.05 mg / L NAA) for 2 days.

[0078] Prepare infection solution: Meanwhile, culture 50 mL of Agrobacterium containing RcMYB1a gene in a shaking incubator at 28°C until the OD600 is about 0.7;

[0079] The cells were collected by centrifugation at 5000 rpm for 5 min and the cell pellet was suspended in an equal volume (50 mL) of 1 / 2 MS infiltration medium (pH 5.7) under sterile conditions.

[0080] Co-cultivation: Place the pre-cultured leaves in the resuspended Agrobacterium solution for infection for 5 minutes (during this period, the bottle can be gently shaken to accelerate the infection), put them back on the M1 medium, and culture them in the dark in a 28°C incubator for 2 days.

[0081] Screening culture: In an ultra-clean workbench, change the co-cultivated leaves to the screening medium M2 (1 / 2MS + 1mg / L6BA + 0.05mg / L NAA + 50mg / L G-418 + 200mg / L Timentin) and culture them in the culture room under normal light for 7 to 14 days. Callus formation and differentiation of resistant buds can be observed around the leaves.

[0082] Rooting culture: cut off the resistant buds in the clean bench and change to the rooting medium M3 (1 / 2MS + 0.05mg / LNAA + 50mg / L G-418 + 200mg / L Timentin). After the resistant buds take root, move them to the flower pot.

[0083] The DNA of transgenic tobacco seedlings was extracted and positive seedlings were identified.

[0084] By observing the performance of transgenic positive tobacco corolla, it was found that the color of the transgenic RcMYB1a tobacco corolla was darker compared with the control (wild type and empty vector tobacco).

[0085] Example 4 Instantaneous transformation of rose 'Iceberg'

[0086] The preparation method of infection solution is consistent with subcellular localization.

[0087] The infection solution was injected into the 'Iceberg' flower buds at the S2 stage (sepals slightly opened, petals visible) using a 1 mL syringe, and the phenotype of the flowers after opening was observed 2-3 days later.

[0088] The best time to inject is in the evening.

[0089] When the injected 'Iceberg' flowers opened, it was found that compared with the control (no injection and injection of empty vector), the petals of the 'Iceberg' flowers that transiently overexpressed RcMYB1a accumulated a large amount of anthocyanin, and the petals turned red ( Figure 4 ).

[0090] RNA was extracted from the petals of the RcMYB1a-transfected 'Iceberg' plant and reverse transcribed into cDNA. The expression of structural genes in the anthocyanin biosynthesis pathway was detected by fluorescence quantitative PCR (RT-qPCR). The results showed that overexpression of RcMYB1a significantly increased the expression of the early structural gene CHS and the late structural genes F3'H, DFR, ANS, and UFGT ( Figure 5 ).

[0091] Therefore, the present invention uses the rose MYB transcription factor RcMYB1a in regulating plant flower color. RcMYB1a belongs to the S6 subfamily of R2R3-MYBs and positively regulates the formation of plant flower color. The corolla color of common tobacco plants with stable overexpression of RcMYB1a deepens, and the petals of the 'iceberg' plant with transient overexpression of RcMYB1a also accumulate anthocyanins.

[0092] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

Claims

1. A method for regulating plant flower color, characterized in that: Introducing a plant expression vector containing the RcMYB1a transcription factor gene into a recipient plant; the nucleotide sequence of the RcMYB1a gene is shown in SEQ ID NO.1; The amino acid sequence of the protein encoded by the RcMYB1a gene is shown in SEQ ID NO.2; The regulation is to increase the anthocyanin content in the flowers of the recipient plant; The receptor plant is rose or tobacco.

2. The method according to claim 1, characterized in that: The recombinant plant overexpression vector containing the RcMYB1a gene is transformed into plant tissues or cells through Agrobacterium-mediated method, and the transformed plant tissues are cultured into plants.

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