Gene HfMYB90 for regulating and controlling hemerocallis fulva anthocyanin synthesis and application of gene HfMYB90

Abstract

The invention belongs to the field of plant gene breeding, and particularly relates to an MYB90 gene for regulating and controlling hemerocallis fulva anthocyanin synthesis and application of the MYB90 gene. The nucleotide sequence of the hemerocallis fulva MYB90 gene is shown as SEQ ID NO.1, the amino acid sequence of the hemerocallis fulva MYB90 gene is shown as SEQ ID NO.2, it is found for the first time that the hemerocallis fulva MYB90 gene can regulate and control synthesis of anthocyanin in tobacco flowers, and colors of tobacco leaves, stems, flowers and other positions can be deepened through overexpression of the hemerocallis fulva MYB90 gene. The hemerocallis fulva MYB90 gene provided by the invention not only provides a theoretical reference for research of hemerocallis fulva flower color regulation, but also provides an important gene resource for future flower color breeding work.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and specifically relates to anthocyanin regulatory genes, and in particular to a gene MYB90 for regulating the synthesis of anthocyanins in daylilies and an application thereof. Background Art

[0002] Daylily (Hemerocallis fulva) is a perennial monocotyledonous plant with high ornamental value. It is a widely used garden plant. Daylily hybrid breeding has rich flower color variation. At present, the molecular mechanism of daylily flower color formation is still unclear. This study plays a vital role in the directional breeding of daylily flower color.

[0003] Flower color is one of the most obvious phenotypes of ornamental plants. Anthocyanins play an important role in the growth and development of plants and can affect the color of flowers. They are also involved in the development process of plants and affect biological processes such as cell differentiation and tissue formation.

[0004] The synthesis of anthocyanins is regulated by transcription factors (TFs) during plant development. Among them, MYB transcription factors are the most important regulatory factors in plant anthocyanin synthesis and have been widely verified in plants such as Arabidopsis thaliala and lily (Lilium brownii). Summary of the Invention

[0005] This invention discloses a key transcription factor gene, HfMYB90, that regulates anthocyanin synthesis in daylilies, and its applications, opening up new avenues for studying the mechanisms of flower color formation in ornamental garden plants. Through cloning, sequence analysis, and functional identification of HfMYB90, this invention further elucidates the genetic regulatory network for daylily flower color, providing core gene resources and technical references for targeted molecular breeding of daylily flower color, with significant theoretical significance and promising industrial applications.

[0006] One of the objectives of the present invention is to provide a Hemerocallis HfMYB90 gene.

[0007] The second purpose of the present invention is to obtain tobacco transformed plants containing the Hemerocallis HfMYB90 gene.

[0008] The third object of the present invention is to provide an application of Hemerocallis HfMYB90 protein in regulating the synthesis of plant anthocyanins.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A daylily HfMYB90, characterized in that the gene sequence is shown as SEQ ID NO.1.

[0011] The protein sequence encoded by the daylily HfMYB90 is shown in SEQ ID NO.2.

[0012] The biological material related to the Hemerocallis HfMYB90 gene is characterized by comprising any one of the following (A1) to (A3):

[0013] (A1) Plant expression vector containing the Hemerocallis HfMYB90 gene;

[0014] (A2) a bioengineered bacterium containing the plant expression vector described in (A1);

[0015] (A3) A transgenic plant containing the plant expression vector described in (A1).

[0016] The biological material related to the Hemerocallis HfMYB90 gene is characterized by:

[0017] The plant expression vector is pCAMBIA1300-GFP.

[0018] The biological material related to the Hemerocallis HfMYB90 gene is characterized by:

[0019] The bioengineering bacteria is Agrobacterium tumefaciens GV3101 (Weidi, China).

[0020] Use of any one of the biological materials related to the Hemerocallis HfMYB90 gene in regulating plant anthocyanin synthesis;

[0021] The method for regulating plant anthocyanin synthesis is characterized by:

[0022] (B1) introducing the aforementioned Hemerocallis HfMYB90 gene into a recipient plant to obtain a transgenic plant;

[0023] (B2) overexpressing the Hemerocallis HfMYB90 gene in a recipient plant;

[0024] The method for introducing a plant expression vector into a recipient plant is characterized by:

[0025] The plant expression vector containing the Hemerocallis HfMYB90 gene is transformed into plant tissues through an Agrobacterium-mediated method, and the transformed plant tissues are cultivated into plants.

[0026] According to the technical solution of the present invention, it is characterized in that the recipient plant is common tobacco.

[0027] The present invention has the following beneficial effects:

[0028] (1) The present invention cloned the MYB90 gene from the petals of daylily, constructed the pCAMBIA1300-MYB90-GFP plant expression vector, transformed Agrobacterium tumefaciens GV3101 by the freeze-thaw method, and transformed the pCAMBIA1300-MYB90-GFP plant expression vector into tobacco using the Agrobacterium transformation method. The results showed that overexpression of the MYB90 gene in tobacco deepened the color of the tobacco flowers, indicating that the daylily MYB90 gene has the function of regulating the synthesis of anthocyanins in plants.

[0029] (2) The Hemerocallis HfMYB90 gene provided by the present invention can be used as an excellent gene resource and can be widely applied to the genetic breeding of Hemerocallis or other flower plants, which is of great significance for the breeding of ornamental plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The electrophoresis diagram of HfMYB90 gene cloning, where M stands for DL2000 Maker and 1-2 are cloned sequences.

[0032] Figure 2 This is the subcellular localization map of HfMYB90 protein.

[0033] Figure 3 This is the phenotypic diagram of HfMYB90-transfected tobacco, where WT is the wild-type tobacco plant and OE is the T3 generation of HfMYB90-transfected tobacco. The color of its corolla deepens, and its leaves and stems turn red. DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0035] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0037] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a gene MYB90 for regulating the synthesis of anthocyanins in daylilies and its application.

[0038] The relevant culture medium preparation method in the embodiment is as follows:

[0039] LB liquid medium (100 mL): 0.5 g yeast extract + 1 g tryptone + 1 g sodium chloride

[0040] LB solid medium (100 mL): 0.5 g yeast extract + 1 g tryptone + 1 g sodium chloride + 1.5 g agar

[0041] YEP liquid medium (100 mL): 1 g yeast extract + 1 g tryptone + 0.5 g sodium chloride

[0042] YEP solid medium (100 mL): 1 g yeast extract + 1 g tryptone + 0.5 g sodium chloride + 1.5 g agar

[0043] MS1 medium: MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L

[0044] MS2 medium: 1 / 2MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + Hyg 20 mg / L + Timentin 200 mg / L

[0045] MS3 medium: 1 / 2MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + Hyg 25 mg / L + Timentin 200 mg / L

[0046] MS4 medium: 1 / 2MS + NAA 0.1 mg / L + Hyg 25 mg / L + Timentin 200 mg / L

[0047] The specific experimental scheme of this embodiment is as follows:

[0048] Example 1: Gene cloning

[0049] 1. The specific primer sequences used for cloning the Hemerocallis HfMYB90 gene are as follows:

[0050] MYB90-F1:TCAATGGCTTTTCTTCAATGCATCG

[0051] MYB90-R1:CAGTGCTTAAAGTAGTTTTTCCC

[0052] Using daylily petal cDNA as a template, a 50 μL PCR reaction system was prepared, including 2 μL template cDNA, 25 μL 2× PCR buffer for KOD, 10 μL 2 mM dNTPs, 1 μL each of upstream and downstream primers, 1 μL KOD FX and 10 μL ddH2O. The sample addition operation was carried out on ice, and after addition, the mixture was mixed and centrifuged; the PCR reaction program was: 94°C for 2 min; 98°C for 10 s, 58°C for 30 s, 68°C for 90 s, 35 cycles; 68°C for 10 min. Amplified products were obtained by PCR amplification; the amplified products were ligated into the cloning vector pEASY-Blunt-Zero (Quanshijin, China), transformed into Escherichia coli DH5α, and plated on solid LB medium containing 100 mg / L Amp resistance. The culture was inverted in a 37°C incubator for 10-12 h. Monoclonal strains were selected for bacterial liquid PCR identification, and positive clones were expanded to 10 mL in LB liquid medium containing 100 mg / L Amp resistance before being sent to a biological company for sequencing.

[0053] The nucleotide sequence of the Hemerocallis HfMYB90 gene was obtained as follows:

[0054] ATGGCTTTTCTTCAATGCATCGAGAGCCAACCTCTGGGTGTTCGAAAAGGAGCTTGGAGCAGG GAGGAAGATGAGTTGCTAAAGAAGTGCATTGAGAGGTACGGAGAAGGGAAGTGGAGTTCAGTTCCCGGGAGGGCAGGATTGAAGAGGTGCAGGAAAAGTTGCCGACTGAGATGGCTGAACTATCTTTCACCTGACATTCATCGAGGGAAGTTCATGGAGGATGAGGTCGACCTCATTATCAGGCTTCATAAGCTATTAGGTAACAGGTGGTCCCTAATAGCAAGGCGGCTTCCAGGGCGCACGGCTAACGACATTAAGAACTTTTGGAACTCTCATTTGAATAAGAAAGTAGGTGCTCAACATGAGAGGAATCATGAACCCATTTCCAAAATCCAAGTAGTGAAGCCTAAGCCTCAGAGAGTCGTAGTGATTCAGACCAATTGGCCCATATGCCAAGAGAATAACAATAATAACAATGAAGCTGGTGGATCTCAGATCAACCAAAACCTCATCATGCACAAATCCAAACAACCAGTAGCTGATCAGATTATTAACGAAGAAGAACAATGGTTGAATGATTTAACCAGTACTGATCAAGCTGAGAATAACAATAATAACAATGAAGCTGGTGGATCTCAGATTAACCAAGACCTCATCATGCACAAATCCAAACAACCAGTAGCTGATCAGATTATTAACGAAGAAGAACAATGGTTGAATGATTTAACCAGTACTGATCAAGCTGAGAATGTGATGAACACTGAACAAGATTTCCACTTTCTCTTTGAAACTGAGGAAACACTGCAGGAAATGGTTGATGAAGAGCTGTTTTGGGGAAGGATTTGTGAATTGGAGAAACTACTTTAA

[0055] The ORF region of the Hemerocallis fulva HfMYB90 gene contains 870 bases, encoding 289 amino acids. The amino acid sequence is shown below:

[0056] MAFLQCIESQPLGVRKGAWSREEDELLKKCIERYGEGKWSSVPGRAGLKRCRKSCRLRWLNYLSPDIHRGKFMEDEVDLIIRLHKLLGNRWSLIARRLPGRTANDIKNFWNSHLNKKVGAQHERNHEPISKIQVVKPKPQRVVV IQTNWPICQENNNNNNEAGGSQINQNLIMHKSKQPVADQIINEEEQWLNDLTSTDQAENNNNNNEAGGSQINQDLIMHKSKQPVADQIINEEEQWLNDLTSTDQAENVMNTEQDFHFLFETEETLQEMVDEELFWGRICELEKLL

[0057] Example 2: Construction of plant expression vector

[0058] Using homologous recombination, we designed vector homology arm primers with Sal I and BamH I restriction sites, respectively. Using the MYB90 cloning plasmid connected to the cloning vector as a template, we amplified the Hemerocallis MYB90 gene with the pCAMBIA1300 vector homology arms and recovered the target fragment. The vector homology arm primer sequences are as follows:

[0059] MYB90-F2:ttgatacatatgcccgtcgacATGGGTCCTCTTCCCTCGG

[0060] MYB90-R2:cccttgctcaccatggatcc AAGTAGTTTCTCCAATTCACAAATC

[0061] The expression vector pCAMBIA1300-GFP plasmid was digested with Sal I and BamH I restriction endonucleases, and the vector fragment was recovered. The linearized vector was ligated with the MYB90 gene fragment with vector homology arms added, and the vector was transformed into Escherichia coli competent DH5α and spread on LB solid containing 100 mg / L Kana resistance. The culture was inverted in a 37°C incubator for 10-12 hours, and single clones were picked for bacterial liquid PCR identification. Positive clones were expanded to 10 mL in LB liquid medium containing 100 mg / L Kana resistance and then sent to a biological company for sequencing.

[0062] Example 3: Subcellular localization

[0063] The subcellular localization of MYB90 protein was detected by transient transformation of Nicotiana benthamiana. The specific method of subcellular localization is as follows:

[0064] Plasmid pCAMBIA1300-MYB90-GFP was transformed into Agrobacterium tumefaciens GV3101 (Weidi, China) by the freeze-thaw method. After obtaining positive clones, single colonies were picked and inoculated into LB liquid medium containing 50 mg / L Kana and 25 mg / L Rif, and cultured overnight in a shaker at 180 rpm at 28°C. 50 μL of fresh bacterial culture was inoculated into 10 mL LB liquid medium containing 50 mg / L Kana, 25 mg / L Rif, 10 mM MES, and 40 μM AS (acetosyringone). The culture was shaken at 28°C for 14 h and expanded to the logarithmic growth phase of the strain. The next day, the cells were collected by centrifugation at 4000 rpm for 10 min, the supernatant was discarded, the cells were resuspended in 10 mM MgCl2, and the OD value of each bacterial culture was adjusted using a spectrophotometer. 600 The concentration of AS was 0.6. All resuspended bacterial suspensions were added to a final concentration of 200 μM and allowed to stand at room temperature for 3 hours. The Nicotiana benthamiana plants were watered the day before injection and kept in low light conditions. Selected tobacco leaves in good growth condition were pierced on the underside of the leaf using the needle of a 1 mL syringe. The needle was removed and the mixed bacterial suspension was injected into the leaf from the underside. The injected tobacco leaves were incubated in the dark for 12 hours, then removed and incubated under normal light conditions for 3 days. The lower epidermis of the injected leaves was removed and placed on a glass slide to prepare a temporary water-mounted slide. GFP expression was observed using laser confocal microscopy.

[0065] Example 4: Genetic transformation of tobacco

[0066] 1. Cultivation of sterile tobacco seedlings.

[0067] Several wild-type tobacco seeds were placed in a sterile 1.5 mL centrifuge tube, disinfected with 75% ethanol for 1 min on a sterile workbench, rinsed three times with sterile water, then disinfected with 1% NaClO for 10 min, rinsed five times with sterile water, inoculated on MS medium, and cultured under light at 25°C to obtain tobacco sterile seedlings.

[0068] 2. Preparation of bioengineered bacteria.

[0069] Agrobacterium was transformed. The competent strain of Agrobacterium tumefaciens was GV3101 (Weidi, China). The transformation steps were referred to the instruction manual.

[0070] Pick a single colony from the transformation plate and transfer it to 10 mL of YEP liquid medium containing 50 mg / L Kana and 25 mg / L Rif, and culture it at 28°C, 180 rpm, and shake it for 12-16 h; then take 1 mL of the bacterial solution and add it to 50 mL of YEP (or LB) liquid medium containing 50 mg / L Kana and 25 mg / L Rif, and culture it at 28°C, 180 rpm, and shake it until the OD 600Reach 0.6-0.8; centrifuge the cultured bacterial solution at 4500rpm for 5 minutes, discard the supernatant, and resuspend the bacteria in an equal volume (50mL) of 1 / 2MS osmotic culture medium (pH5.8) under sterile conditions. Place the resuspended solution on ice and wait for use in infecting tobacco.

[0071] 3. Infect tobacco.

[0072] Pre-culture: Cut sterile tobacco seedling leaves into 1 cm × 1 cm cubes and place them on MS1 ​​solid culture medium (with the upper surface of the leaves facing up) for 2 days.

[0073] Infection. In a clean bench, pour the resuspended bacterial solution into a sterile conical flask. Take a tobacco leaf that has been pre-cultured for 2 days and place it in the bacterial solution. Soak for 4 minutes. Remove the leaf and place it on sterile filter paper to absorb the bacterial solution attached to the leaf surface. At the same time, set up a leaf disc that has not been infected with Agrobacterium as a negative control.

[0074] Co-cultivation: Inoculate infected tobacco leaves onto MS1 medium (with the upper surface of the leaves facing up), seal with parafilm, and co-cultivate at 28°C in the dark for 2 days.

[0075] Selective culture. The co-cultured leaves were inoculated on MS2 medium and cultured under light for Hyg resistance screening. Non-transgenic tobacco was also inoculated on the Hyg resistance medium as a control. Under normal circumstances, the leaves of non-transgenic tobacco will gradually die.

[0076] Subculture. After about 1-2 weeks, inoculate tobacco leaves onto MS3 medium for subculture.

[0077] Rooting. When adventitious buds about 1 cm long grow on the edge of the leaves, cut them off and transfer them to MS4 medium to induce rooting.

[0078] Transplant. When the seedlings have developed a well-developed root system, uncap and culture for 2 days to harden them. Add a small amount of sterile water to the culture bottle to keep the culture medium moist. Before transplanting, clean off the agar carried by the plant roots. Plant them in a small pot filled with sterilized soil (garden soil: vermiculite = 3:1) and place them at room temperature.

[0079] DNA of transgenic tobacco plants was extracted and PCR identification was performed using universal primers of overexpression vectors to obtain transgenic positive tobacco plants.

[0080] Result analysis.

[0081] 1. Subcellular localization analysis of Hemerocallis MYBL90 protein.

[0082] By transiently transforming Nicotiana benthamiana leaves, the distribution of GFP green fluorescence signals was observed under a laser confocal microscope. It was found that the green fluorescence signal of the empty vector was evenly distributed throughout the cell, while the green fluorescence signal of Hemerocallis MYB90 was localized in the cell nucleus ( Figure 2 ), indicating that Hemerocallis MYBL90 has the general characteristics of a transcription factor and belongs to a transcription factor protein that plays a transcriptional regulatory function in the cell nucleus and participates in specific biological processes.

[0083] 2. Identification of transgenic positive tobacco plants.

[0084] DNA was extracted from the three transgenic tobacco plants obtained and PCR identification was performed using universal primers for the vector. The results showed that all three transgenic tobacco lines contained the target band, indicating that the MYB90 gene had been successfully inserted into the tobacco genome. The corolla of the MYB90-transgenic tobacco was darker in color than that of the wild-type corolla, and the leaves and stems turned red ( Figure 3 ).

Claims

1. A gene MYB90 that regulates anthocyanin synthesis in daylily, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. A protein encoded by the gene MYB90 for anthocyanin synthesis in daylily according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. Use of the gene MYB90 for the synthesis of anthocyanins in daylilies according to claim 1 or the encoding gene according to claim 2 in promoting the synthesis of anthocyanins in daylilies.

4. The method for overexpressing the Hemerocallis MYB90 gene in a recipient plant according to claim 3, wherein: The daylily MYB90 gene is transformed into plant tissues through an Agrobacterium-mediated method, and the transformed plant tissues are cultivated into plants.

5. The method according to claim 4, characterized in that The recipient plant is common tobacco.

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