Myb transcription factor for changing color of pentalipidum and application thereof

Abstract

The application discloses an MYB transcription factor for changing the color of Paphiopedilum callosum and application of the MYB transcription factor. The nucleotide sequence of the transcription factor PpMYB1 is shown as SEQ ID NO. 1. The PpMYB1 gene is cloned from the petal of Paphiopedilum callosum, and is stably transformed into tobacco through an agrobacterium-mediated leaf disc method. The results show that overexpression of the PpMYB1 gene in tobacco makes the color of tobacco flowers deepen. The PpMYB1 gene is transformed into the petal of Paphiopedilum callosum through an agrobacterium-mediated transient expression technology. The results show that overexpression of the PpMYB1 gene in the petal of Paphiopedilum callosum makes the white petal present purplish red. It is proved that the PpMYB1 gene of Paphiopedilum callosum has the function of regulating the synthesis of anthocyanin of flowers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering, and particularly relates to cloning of an R2R3 MYB transcription factor for changing the flower color of Phalaenopsis pulcherrima and application thereof. BACKGROUND

[0002] Phalaenopsis pulcherrima (Lindl.) J.J.Sm. is also known as Dorilan, which originally belongs to Doritaenopsis, and is now merged into Phalaenopsis. It is a species endemic to East Asia, and is originally from Hainan of China and Southeast Asia. Phalaenopsis pulcherrima has beautiful flower type, and the flower color includes pink, purple red, white and orange yellow, etc. It has strong stress resistance, and particularly, the flowering period is summer and autumn, which is different from most Phalaenopsis species that bloom in spring. Therefore, it has high ornamental value and application prospect, and is an important parent for Phalaenopsis hybridization and breeding.

[0003] Anthocyanidin is one of the most key factors affecting the flower color of ornamental plants, and also plays an important role in the growth and development of plants. It can protect plants from strong light, ultraviolet and low temperature, and improve the resistance of plants.

[0004] The biosynthesis of anthocyanidin is mainly controlled by two types of genes, namely structural genes and regulatory genes. The structural genes encode enzymes directly involved in the synthesis of anthocyanidin, which includes two important gene groups: the upstream gene group (CHS, CHI, F3H, F3'H) and the downstream gene group (F3'5'H, DFR, ANS, LAR, ANR, UFGT). The regulatory genes mainly include three types of transcription factors: MYB, bHLH and WD40, which can form an MBW complex to bind to the promoter of the structural gene for precise regulation. Among them, the MYB transcription factor plays an important role in regulating the synthesis of anthocyanidin. It can increase or decrease the accumulation of anthocyanidin in flowers by promoting or inhibiting the expression of structural genes in the anthocyanidin synthesis pathway, thereby ultimately determining the color of flowers.

[0005] In recent years, although the research on MYB transcription factors in Orchidaceae plants is increasing, it is still far from enough, and it is still necessary to continue to supplement and expand the resources of MYB transcription factors that can promote the synthesis of anthocyanidin in Orchidaceae plants. At present, there is no research report on the MYB transcription factor for regulating the synthesis of anthocyanidin in Phalaenopsis pulcherrima. Therefore, identifying the MYB transcription factor involved in the regulation of anthocyanidin biosynthesis in Phalaenopsis pulcherrima has important significance for elucidating the regulation mechanism of anthocyanidin synthesis in Phalaenopsis pulcherrima, and can be applied to the genetic engineering improvement of other plants. It has important significance for the molecular breeding improvement of the flower color of Phalaenopsis pulcherrima in the future. SUMMARY

[0006] The purpose of the present application is to provide an R2R3 MYB transcription factor PpMYB1 for promoting the formation of flower color of Phalaenopsis pulcherrima.

[0007] The amino acid sequence of the transcription factor PpMYB1 protein of the present application is shown in SEQ ID NO. 2.

[0008] The present application also provides a transcription factor PpMYB1 encoding the transcription factor PpMYB1 protein.

[0009] Preferably, the nucleotide sequence of the transcription factor PpMYB1 is shown in SEQ ID NO. 1.

[0010] The present application also provides a plant expression vector containing the transcription factor PpMYB1.

[0011] The plant expression vector is pCAMBIA1300-GFP and pSuper1300-GFP.

[0012] The present application also provides an engineered bacterium containing the above-mentioned expression vector, for example, Agrobacterium, which can be Agrobacterium tumefaciens GV3101 and EHA105.

[0013] The present application also provides the use of the PpMYB1 gene, the plant expression vector or the engineered bacterium in regulating the synthesis of anthocyanin in plants.

[0014] The regulation of the synthesis of anthocyanin in plants is to change the color of flowers, and the plants can be various plants, such as Paphiopedilum, tobacco, etc.

[0015] The present application also provides a method for regulating the synthesis of anthocyanin in plants, characterized in that:

[0016] The PpMYB1 gene is introduced into a recipient plant to obtain a transgenic plant.

[0017] Or the PpMYB1 gene is transiently overexpressed in the petals of Paphiopedilum.

[0018] The PpMYB1 gene is introduced into a recipient plant by a method of Agrobacterium-mediated transformation of a plant expression vector containing the PpMYB1 gene into plant tissue, and the transformed plant tissue is cultivated into a plant.

[0019] The present application has the following beneficial effects:

[0020] (1) The PpMYB1 gene is cloned from the petals of Paphiopedilum, and is stably transformed into tobacco by Agrobacterium-mediated leaf disc method, and the results show that the overexpression of the PpMYB1 gene in tobacco makes the color of tobacco deeper; the PpMYB1 gene is transformed into the petals of Paphiopedilum by Agrobacterium-mediated transient expression technology, and the results show that the overexpression of the PpMYB1 gene in the petals of Paphiopedilum makes the white petals present purple red. Both of them show that the PpMYB1 gene of Paphiopedilum has the function of regulating the synthesis of anthocyanin in flowers.

[0021] (2) The PpMYB1 gene of Orchidia rubra provided by this invention can serve as an excellent gene resource, enriching the transgenic material library of purple flower traits in orchids and providing strong support for molecular breeding of purple flower traits in orchids in the future. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of PpMYB1 coding region amplification provided by the present invention, wherein lane 1 is 2000 Mark Ker, and lanes 2 and 3 are amplified PpMYB1 coding region bands.

[0023] Figure 2 This is a comparison diagram of the amino acid sequences of PpMYB1 with MYBs proteins from other species, provided by the present invention.

[0024] Figure 3 This is a phenotypic diagram showing transient overexpression in *Cymbidium goeringii* petals using the present invention. In the left image, the left side shows the phenotype after injection of the empty vector, and the right side shows the phenotype after injection of the PpMYB1 vector. The right image shows the anthocyanin content determination after injection of the empty vector and the PpMYB1-carrying bacterial suspension.

[0025] Figure 4 This is a phenotypic diagram of the flowers of transgenic tobacco plants. The left side shows the empty vector transgenic plant, and the right side shows the transgenic plant overexpressing PpMYB1.

[0026] Figure 5 This image shows the anthocyanin content in the petals of PpMYB1 transgenic tobacco plants.

[0027] Figure 6 This is a graph showing the expression levels of genes related to anthocyanin synthesis in the corolla of PpMYB1 transgenic tobacco. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments and accompanying drawings. However, the embodiments of the present invention include, but are not limited to, the embodiments described below. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are all conventional means well known to those skilled in the art. In the embodiment section of the present invention, the cloning, functional characteristics, and applications of the PpMYB1 gene are described.

[0029] In this embodiment, the plant material, *Cymbidium goeringii*, was grown in the resource nursery of the South China Botanical Garden, Chinese Academy of Sciences. The nursery has temperature control facilities, with a temperature range of 15-32℃ and a humidity range of 70-85%.

[0030] Example 1: Cloning of the PpMYB1 gene in Orychophragmus violaceus ( Figure 1 ,2 )

[0031] By comparing transcriptomics methods, a MYB-type transcription factor gene whose expression was upregulated during flower coloring in purple-flowered varieties compared to white varieties was named PpMYB1. Total RNA (ribonucleic acid) was extracted from fresh purple flowers and reverse transcribed into cDNA. The coding region sequence of PpMYB1 was obtained by PCR amplification using specific primer pairs PpMYB1-F; 5'-3':atgggaaggaatccgagctgttcgaa and PpMYB1-R; 5'-3':ttagaactgtttcaacagctccccttcca. The PCR reaction volume was 50 μL, consisting of: 25 μL of 2×Phanta Flash Master Mix (Dye Plus), 2 μL each of forward and reverse primers, 1 μL of cDNA, and 20 μL of ddH2O. The PCR program was: 98℃ for 30 s, followed by 35 cycles of 98℃ for 10 s, 60℃ for 5 s, and 72℃ for 15 s. The amplified fragments were subjected to electrophoresis, gel recovery, and ligation into a pClone007 Versatile Simple Vector using the pClone007 Versatile Simple Vector Kit from Qingke Biotechnology. Positive clones were sequenced to determine their complete coding region sequence. The nucleotide sequence of PpMYB1 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0032] Example 2: Transient expression of the PpMYB1 gene in flowers of Orchidonia quinquefolia ( Figure 3 )

[0033] Preparation of recombinant plasmids containing the PpMYB1 gene and recombinant genetically engineered bacteria.

[0034] Using the plasmid that was correctly sequenced in Example 1 as a template, specific primer pairs PS1300-PpMYB1-F; 5'-3' were used. acatttaaatactag taccggatccactagt atgggaaggaatccgagctg (underlined portion is the upstream vector terminal homologous sequence, slanted text portion is the SpeⅠ restriction site) and PS1300-PpMYB1-R; 5'-3': Figure 3 The sequence gaactgtttcaacagctcc (underlined portion is the downstream vector terminal homologous sequence, slanted text portion is the SpeⅠ restriction site) was used for PCR amplification. The PCR reaction system and procedure were consistent with those in Example 1. After recovering the PCR product, a TSINGKE TSV-S3 was used. The Seamless Cloning Kit was used to ligate the pSuper1300-GFP vector, which had been digested with SpeI, into a homologous recombination vector. Sequencing verification was performed to obtain a recombinant plasmid containing the PpMYB1 gene. This plasmid was then introduced into Agrobacterium tumefaciens strain EHA105. Single colonies were picked, shaken, and screened by PCR to identify positive transformants, thus obtaining engineered bacteria containing the PpMYB1 overexpression vector pSuper1300-GFP-PpMYB1.

[0035] 100 μL of Agrobacterium containing the empty vector pSuper1300-GFP and 100 μL of Agrobacterium containing pSuper1300-GFP-PpMYB1 were respectively placed in 10 mL of LB broth containing kanamycin and rifampin antibiotics, and cultured at 28 °C and 180 rpm for about 24 h until the bacterial culture reached OD. 600 The value is 0.6 to 0.8. The formulation of the LB liquid culture medium containing kanamycin and rifampicin antibiotics is: 10 μL 50 mg / mL kanamycin + 10 μL 25 mg / mL rifampicin antibiotic + 10 mL LB liquid culture medium.

[0036] Centrifuge the cultured Agrobacterium tumefaciens at 5000 rpm for 5 minutes at room temperature, removing as much supernatant as possible; add invasion staining buffer to the bacterial pellet to reduce OD. 600 The concentration was adjusted to 0.5, and the mixture was incubated at 27℃ for 3 hours. After incubation, the solution was injected into the petals of flowering white five-lipped orchids. The infection buffer was prepared as follows: 1.9524 g of MES was dissolved in 10 mL of sterile water to obtain an MES solution; 0.196 g of acetylsuccinone (AS) was dissolved in 10 mL of AS to obtain an AS solution; 2.03 g of MgCl2 was dissolved in 10 mL of sterile water to obtain a MgCl2 solution; 100 mL of MS medium was mixed with 1 mL of MES solution, 1 mL of MgCl2 solution, and 100 μL of LAS solution to obtain the infection buffer. After injection, the mixture was placed in an artificial climate chamber for incubation under the following conditions: 24℃, 16 h light, 8 h darkness, 70% humidity, for 2–4 days. Phenotypic changes were observed, and samples were taken and photographed as needed.

[0037] like Figure 4 As shown, after injection of Agrobacterium superoxide dismutase pSuper1300-GFP-PpMYB1, the petals and sepals of the white Cymbidium goeringii turned purplish-red. This result indicates that PpMYB1 can alter the flower color of the Cymbidium goeringii, an orchid.

[0038] Example 3: Stable expression of the PpMYB1 gene from *Cymbidium goeringii* in tobacco ( gcttgatatcgaattc cagcgaattatctaga , 5 6)

[0039] Preparation of recombinant plasmids containing the PpMYB1 gene and recombinant genetically engineered bacteria. Using the correctly sequenced plasmid from Example 1 as a template, specific primer pairs PC1300-PpMYB1-F; 5'-3' were used. Figure 4 atgggaaggaatccgagctg (underlined portion is the upstream vector terminal homologous sequence, slanted text portion is the EcoRI restriction site) and PC1300-PpMYB1-F; 5'-3': Figure 5 The underlined portion of gaactgtttcaacagctccc represents the downstream vector terminal homologous sequence, and the slanted text portion represents the XbaI restriction site. PCR amplification was performed using the same PCR reaction system and procedure as in Example 1. After recovering the PCR product, a TSINGKE TSV-S3... The Seamless Cloning Kit uses homologous recombination to ligate the pGreen35S-GFP vector, which has been double-digested with EcoRI and XbaI. Sequencing verification yields a recombinant plasmid containing the PpMYB1 gene.

[0040] Using the flue-cured tobacco variety 'NC89' as the transgenic recipient material, the steps for seed disinfection and obtaining sterile seedlings are as follows:

[0041] (1) Take NC89 seeds and place them in a 2mL centrifuge tube. After removing impurities, wash them 2-3 times with sterile water.

[0042] (2) Soak in 75% alcohol for 30 seconds, then rinse twice with sterile water to remove residual alcohol.

[0043] (3) Disinfect with 5% sodium hypochlorite for 15 minutes, then rinse twice with sterile water to remove residual sodium hypochlorite.

[0044] (4) Add an appropriate amount of sterile water, seal the centrifuge tube with sealing film, and vernalize it in a 4℃ refrigerator for 3 days.

[0045] (5) After vernalization, the seeds were dried with sterile filter paper in a clean bench and then sown onto 1 / 2 MS solid plates and placed in a 26℃ light incubator for normal growth.

[0046] (6) Select the germinated seedlings and inoculate them into a new 1 / 2 MS solid culture medium. Culture them for about 1 month to serve as sterile seedlings for subsequent Agrobacterium infection.

[0047] Preparation of Agrobacterium infection solution

[0048] The constructed plasmid was introduced into Agrobacterium strain EHA105. Single colonies were picked and cultured, and positive transformants were screened by PCR to obtain engineered bacteria containing the overexpression vector pGreen35S-GFP-PpMYB1 of PpMYB1. 50 μL of the bacterial culture was transferred to an Erlenmeyer flask, and 50 mL of liquid culture medium containing antibiotics (50 μg / mL kanamycin + 20 μg / mL rifampin) was added. The culture was then further amplified by shaking until the bacterial culture reached OD500. 600 The OD value reached between 0.6 and 0.8. After centrifugation at 5000 rpm for 5 min, the bacterial cells were collected, the supernatant was discarded, and the bacteria were resuspended in 20 mL of sterile MS medium. After centrifugation at 5000 rpm for 5 min, the bacterial cells were collected, the supernatant was discarded, and an appropriate amount of tobacco infection solution was added to adjust the OD value of the bacterial suspension. 600 The value is adjusted to between 0.6 and 0.8. After standing at room temperature in the dark for 2-3 hours, it can be used for the next step of conversion.

[0049] Leaf disc method for tobacco conversion

[0050] (1) Transformation of tobacco leaf discs: Select sterile tobacco seedlings that have been cultured for about one month, use sterile scissors to cut off the main veins of the leaves, and cut the leaf discs on both sides into 0.5*0.5cm pieces. 2 The cubes were then immediately transferred to a sterile petri dish containing Agrobacterium infection solution. Gently stirred, the mixture was incubated for approximately 10-15 minutes. The incubator was then removed, blotted dry with sterile filter paper, and transferred to a co-culture medium (1L of co-culture medium contains 4.4g MS powder, 30g sucrose, 6g agar, 2mg 6-BA, and 0.2mg NAA, pH adjusted to 5.8). The leaves were placed face up and co-cultured in the dark for 3 days.

[0051] (2) Cleaning and screening culture: The leaf discs that have completed co-culture need to be sterilized. The leaf discs are cleaned 4-6 times with sterile water containing 400 mg / L of termethin, and then cleaned once with sterile water. After drying with sterile filter paper, they are inoculated into tobacco screening medium. 1L of screening medium contains 4.4g MS powder, 30g sucrose, 6g agar, 2mg 6-BA, 0.2mg NAA, 2mg Basta and 400mg termethin. The pH is adjusted to 5.8.

[0052] (3) Rooting culture: When the adventitious shoots that survived in the screening medium grew to more than 3 cm, they were cut off and inoculated into the rooting medium (1 L of rooting medium contains 4.4 g MS powder, 30 g sucrose, 6 g agar, 2 mg Basta, and pH adjusted to 5.8). After rooting, the seedlings were hardened off and transplanted into a light incubator. The transplanting substrate was peat moss:vermiculite = 3:1.

[0053] Experimental results are as follows Figure 6As shown, in transgenic tobacco flowers carrying PpMYB1 overexpression, the corolla, anthers, filaments, styles, ovaries, and sepals were all redder than those in tobacco plants with an empty vector. Furthermore, the expression levels of downstream structural genes related to anthocyanin synthesis, NtDFR, NtANS, and NtUFGT, were significantly upregulated in tobacco. This indicates that overexpression of the transcription factor PpMYB1 in tobacco promotes anthocyanin accumulation, resulting in the purplish-red color of the tobacco flowers. ​ The attached figure shows the anthocyanin content in the petals of PpMYB1 transgenic tobacco. ​ The attached figure shows the expression level analysis of anthocyanin synthesis-related genes in the corolla of PpMYB1 transgenic tobacco.

[0054] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0055] SEQ ID NO.1

[0056] atgggaaggaatccgagctgttcgaaggaagggctcaacaagggagcatggactgccgtggaagacaagctcttgattaaatacgtcaatgctcatggagaaggcaaatggacaacggtgcctcatatagccgggctgaaaaggtcggggaagagctgtcgacttcggtggttaaactacctgaggccaaacgtgaaacgtgggaacttttctgaggaggaaatcgacctcataatcaggctgcacaaactgcttggcaatagatggtctctgatcgctgggaggataccaggcagaactgacaacgaaataaagaactactggaacacaaccttggccaaaaagacaacctcaaagcaaccatttcaaaagccaccagctccaaatcaagcactgccatccccatcatcctctctatcacctccagccacaaataacaaaacattgaatcgacccaagcccctaaggtgcagccaattgggttttctagcaggagaacataatgcaacagtggcttctgggttgccaaatgacaacaccaaagccagcaccattgctgaagaagaacagcttcaggaggaggaggagaatgtagtctggaattgcaggtttgatgatgaaaatgcagtgtttgctgatgaggttttgatggagttcaatggaatgcaggattttgagagctggatgctgaatgatgatggttgttaccttcctgatgatgagaacataaattggctaacttctttgtttgatatggaaggggagctgttgaaacagttctaa

[0057] SEQ ID NO.2

[0058] MGRNPSCSKEGLNKGAWTAVEDKLLIKYVNAHGEGKWTTVPHIAGLKRSGKSCRLRWLNYLRPNVKRGNFSEEEIDLIIRLHKLLGNRWSLIAGRIPGRTDNEIKNYWNTTLAKKTTSKQPFQKPPAPNQALPSPSSSLSPPATNNKTLNRPKPLRCSQLGFLAGEHNATVASGLPNDNTKASTIAEEEQLQEEEENVVWNCRFDDENAVFADEVLMEFNGMQDFESWMLNDDGCYLPDDENINWLTSLFDMEGELLKQF。

Claims

1. The transcription factor PpMYB1 protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the transcription factor PpMYB1 protein as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A plant expression vector containing the gene described in claim 2 or 3.

5. The plant expression vector according to claim 4, characterized in that, The plant expression vectors mentioned are pCAMBIA1300-GFP and pSuper1300-GFP.

6. Engineered bacteria containing the plant expression vector as described in claim 4 or 5.

7. The engineered bacteria according to claim 6, characterized in that, The engineered bacteria mentioned is Agrobacterium.

8. The application of the gene of claim 2 or 3, the plant expression vector of claim 4 or 5, or the engineered bacteria of claim 6 or 7 in promoting anthocyanin accumulation and changing plant flower color, wherein the plant is *Cymbidium goeringii* or *Tobacco*.

9. A method for regulating anthocyanin synthesis in plants, characterized in that: The gene described in claim 2 or 3 is transiently overexpressed in the petals of *Cymbidium goeringii*, and the transformed plant tissue is cultured into a plant.

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