MYB transcription factor for changing color of cheilophila odorata and application of MYB transcription factor

Abstract

The invention discloses an MYB transcription factor capable of changing the color of cheilocheilus quinquefolius and application of the MYB transcription factor. The nucleotide sequence of the transcription factor PpMYB1 is as shown in SEQ ID NO. 1. According to the invention, the PpMYB1 gene is cloned from petals of Wucheilanthes, the PpMYB1 gene is stably transformed into tobacco through an agrobacterium tumefaciens-mediated leaf disc method, and the result shows that the flower color of the tobacco is deepened due to overexpression of the PpMYB1 gene in the tobacco; the PpMYB1 gene is transformed into the petals of the Cymbidium wucheilanthii by an agrobacterium-mediated transient expression technology, and the result shows that the PpMYB1 gene is over-expressed in the petals of the Cymbidium wucheilanthii, so that the white petals are purple The results show that the Wuliang orchid PpMYB1 gene has the function of regulating and controlling flower anthocyanin synthesis.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to the cloning of an R2R3 MYB transcription factor for changing the color of a five-lipped orchid and application thereof. Background Art

[0002] Phalaenopsis pulcherrima (Lindl.) JJSm., also known as Dolilan, originally belonged to the genus Dolilan and is now merged into the genus Phalaenopsis. It is endemic to East Asia and native to Hainan, my country and Southeast Asia. The five-lipped orchid has a beautiful flower shape, with flower colors including pink, purple, white and orange, and strong resistance to adversity. In particular, the flowering period is summer and autumn, which is different from the spring flowering of most Phalaenopsis species. It has high ornamental value and application prospects, and is an important parent for Phalaenopsis hybrid breeding.

[0003] Anthocyanin is one of the most critical factors affecting the flower color of ornamental plants. It also plays an important role in the growth and development of plants. It can protect plants from damage by strong light, ultraviolet rays and low temperatures, and improve plant resistance.

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

[0005] In recent years, although the research on MYB transcription factors in orchids has increased, it is still far from enough. It is necessary to continue to supplement and expand the resources of MYB transcription factors that can promote anthocyanin synthesis in orchids. At present, there is no research report on MYB transcription factors that regulate anthocyanin synthesis in five-lipped orchids. Therefore, the identification of MYB transcription factors involved in regulating the biosynthesis of anthocyanin in five-lipped orchids is of great significance for clarifying the regulatory mechanism of anthocyanin synthesis in five-lipped orchids, and can be applied to the genetic engineering improvement of other plants, which is of great significance for the molecular breeding improvement of the color of five-lipped orchids in the future. Summary of the invention

[0006] The object of the present invention is to provide a R2R3 MYB transcription factor PpMYB1 which promotes the color formation of five-lipped orchid.

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

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

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

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

[0011] The plant expression vectors are pCAMBIA1300-GFP and pSuper1300-GFP.

[0012] The present invention also provides an engineered bacterium containing the above expression vector, such as Agrobacterium, which may be Agrobacterium tumefaciens GV3101 and EHA105.

[0013] The present invention also provides the use of the PpMYB1 gene, plant expression vector or engineering bacteria in regulating the synthesis of plant anthocyanins.

[0014] The regulation of plant anthocyanin synthesis is to change the plant flower color, and the plant can be various plants, such as five-lipped orchid, tobacco, etc.

[0015] The present invention also provides a method for regulating the synthesis of plant anthocyanins, characterized in that:

[0016] Introducing the PpMYB1 gene into a recipient plant to obtain a transgenic plant;

[0017] Or transiently overexpress the PpMYB1 gene in the petals of the five-lipped orchid.

[0018] The method of introducing the PpMYB1 gene into the recipient plant comprises transforming the plant expression vector containing the PpMYB1 gene into plant tissues through the Agrobacterium-mediated method, and cultivating the transformed plant tissues into plants.

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

[0020] (1) The present invention cloned the PpMYB1 gene from the petals of the five-lipped orchid, and stably transformed it into tobacco through the Agrobacterium-mediated leaf disc method. The results showed that the overexpression of the PpMYB1 gene in tobacco deepened the color of the tobacco flowers; and the transient expression technology mediated by Agrobacterium-mediated was used to transform the petals of the five-lipped orchid. The results showed that the overexpression of the PpMYB1 gene in the petals of the five-lipped orchid made the white petals appear purple-red. Both indicate that the PpMYB1 gene of the five-lipped orchid has the function of regulating the synthesis of anthocyanins in flowers.

[0021] (2) The PpMYB1 gene of the five-lipped orchid provided by the present invention can be used as an excellent gene resource to enrich the transgenic material library of orchid plants with purple flower traits, and provide strong support for the molecular breeding of orchids with purple flower traits in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of amplifying the coding region of PpMYB1 provided by the present invention, wherein lane 1 is 2000MarKer, 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 and MYBs proteins of other species provided by the present invention.

[0024] Figure 3 The phenotype diagram of transient overexpression in the petals of five-lipped orchid using the present invention. The left side of the left figure is the phenotype of injection of empty vector, and the right side is the phenotype of injection of vector carrying PpMYB1. The right figure is a graph of anthocyanin content determination after injection of empty vector and bacterial solution carrying PpMYB1.

[0025] Figure 4 The flower phenotype of transgenic tobacco plants. The left side shows an empty-transgenic plant, and the right side shows a transgenic plant that overexpresses PpMYB1.

[0026] Figure 5 This is a diagram showing the determination of anthocyanin content in the petals of transgenic PpMYB1 tobacco.

[0027] Figure 6 This is an analysis of the expression levels of anthocyanin synthesis-related genes in the corolla of transgenic PpMYB1 gene tobacco. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with examples and drawings, but embodiments of the present invention include but are not limited to the following examples. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples section of the present invention, the cloning, functional characteristics and applications of the PpMYB1 gene are described.

[0029] The plant material, Cymbidium quinquefolium, in the embodiment was planted in the resource nursery of South China Botanical Garden, Chinese Academy of Sciences. The nursery has temperature control facilities, the temperature range is 15-32° C., and the humidity range is 70-85%.

[0030] Example 1: Cloning of the PpMYB1 gene of Cymbidium quinquefolium ( Figure 1 ,2 )

[0031] By comparative transcriptomics, a MYB transcription factor gene was found to be upregulated in the flower coloring process of purple flower varieties compared with white varieties, and was named PpMYB1. Total RNA (ribonucleic acid) was extracted from fresh purple flowers and reversely 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 system was 50 μL, and the components included: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, 2 μL of upstream and downstream primers, 1 μL of cDNA, and 20 μL of ddH2O. The PCR program was: 98℃30s, 35 cycles of 98℃10s, 60℃5s, and 72℃15s. The amplified fragments were subjected to electrophoresis, gel recovery, and connected to pClone007 Versatile Simple Vector using the pClone007 Versatile Simple Vector Kit from Qingke Biotechnology Co., Ltd. The positive clones were sequenced to determine the 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 PpMYB1 gene in flowers of Cymbidium quinquefolium ( Figure 3 )

[0033] Preparation of recombinant plasmid and recombinant genetic engineering bacteria containing PpMYB1 gene.

[0034] The plasmid sequenced correctly in Example 1 was used as a template and a specific primer pair PS1300-PpMYB1-F; 5'-3': acatttaaatactag atgggaaggaatccgagctg (the underlined part is the homologous sequence at the upstream vector end, and the italic text part is the SpeⅠ restriction site) and PS1300-PpMYB1-R; 5'-3': taccggatccactagt gaactgtttcaacagctcc (the underlined part is the downstream vector terminal homologous sequence, and the italic text part is the SpeⅠ restriction site), and PCR amplification was performed. The PCR reaction system and procedure were consistent with those in Example 1. After the PCR product was recovered, TSINGKE TSV-S3 The Seamless Cloning Kit was used to connect the vector to the pSuper1300-GFP vector digested with SpeⅠ by homologous recombination, and the vector was sent to the company for sequencing verification to obtain a recombinant plasmid containing the PpMYB1 gene. The plasmid was then introduced into the EHA105 Agrobacterium strain, and a single colony was picked to shake the bacteria and PCR screening was performed to identify the positive transformants, thereby obtaining an engineered bacterium containing the PpMYB1 overexpression vector pSuper1300-GFP-PpMYB1.

[0035] 100 μL of Agrobacterium containing pSuper1300-GFP empty vector and pSuper1300-GFP-PpMYB1 were respectively taken and placed into 10 mL of LB liquid medium containing kanamycin and rifampicin antibiotics, and cultured at 28°C and 180 rpm in a shaking incubator for about 24 h until the bacterial solution OD reached 0. 600 The value is 0.6 to 0.8. The formula of the LB liquid culture medium containing kanamycin and rifampicin antibiotics is: 10 μL 50 mg / mL kanamycin + 10 μL 25 mg / mL rifampicin antibiotics + 10 mL LB liquid culture medium.

[0036] Centrifuge the cultured Agrobacterium solution at 5000 rpm for 5 min at room temperature and remove the supernatant as much as possible. Add infection buffer to the bacterial pellet and adjust the OD 600 Adjust to 0.5, let stand in a 27°C incubator for 3 hours, and inject into the petals of the blooming white five-lipped orchid after standing. The preparation method of the infection buffer is as follows: dissolve 1.9524g of MES in 10mL of sterilized water to obtain a MES solution; dissolve 0.196g of acetosyringone (AS) in 10mL of AS to obtain an AS solution; dissolve 2.03g of MgCl2 in 10mL of sterilized water to obtain a MgCl2 solution; take 100mL of MS culture medium, add 1mL of MES solution, 1mL of MgCl2 solution and 100μLAS solution and mix well to obtain an infection buffer. After injection, place in an artificial climate box for culture, the culture conditions are: 24°C light for 16h, dark for 8h, humidity of 70%, culture for 2-4d, observe phenotypic changes, and take samples and take photos in time.

[0037] like Figure 3 As shown in the figure, after the injection of pSuper1300-GFP-PpMYB1, the petals and sepals of the white five-lipped orchid turned purple-red. This result shows that PpMYB1 can change the flower color of the orchid plant five-lipped orchid.

[0038] Example 3: Stable expression of the PpMYB1 gene of Cymbidium pentaphyllum in tobacco ( Figure 4 , 5 、6)

[0039] Preparation of recombinant plasmid and recombinant genetic engineering bacteria containing PpMYB1 gene. Using the correctly sequenced plasmid in Example 1 as a template, a specific primer pair PC1300-PpMYB1-F; 5'-3': gcttgatatcgaattc atgggaaggaatccgagctg (the underlined part is the homologous sequence at the upstream vector end, and the italic text part is the EcoRⅠ restriction site) and PC1300-PpMYB1-F; 5'-3': cagcgaattatctaga gaactgtttcaacagctcccThe underlined part is the downstream vector terminal homologous sequence, and the italic text part is the XbaⅠ restriction site), and PCR amplification was performed. The PCR reaction system and procedure were consistent with those in Example 1. After the PCR product was recovered, TSINGKE TSV-S3 The Seamless Cloning Kit was used to connect the plasmid to the pGreen35S-GFP vector that had been double-digested with EcoRⅠ and XbaⅠ by homologous recombination, and the recombinant plasmid containing the PpMYB1 gene was obtained after sequencing verification.

[0040] The flue-cured tobacco variety 'NC89' was used as the transgenic recipient material. The steps for seed sterilization and obtaining sterile seedlings were as follows:

[0041] (1) Place NC89 seeds in a 2 mL centrifuge tube, remove impurities, and wash with sterile water 2-3 times.

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

[0043] (3) Disinfect with 5% sodium hypochlorite for 15 minutes, then wash 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 freeze in a 4°C refrigerator for 3 days.

[0045] (5) The vernalized seeds were dried with sterile filter paper on a clean bench and sown on 1 / 2MS solid plates and placed in a 26°C light incubator for normal growth.

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

[0047] Preparation of Agrobacterium infection medium

[0048] The constructed plasmid was introduced into the EHA105 Agrobacterium strain, and a single colony was picked and shaken to screen positive transformants by PCR to obtain an engineered bacterium containing the PpMYB1 overexpression vector pGreen35S-GFP-PpMYB1. 50 μL of the bacterial solution was transferred to a conical flask and 50 mL of liquid culture medium containing antibiotics (50 μg / mL kanamycin + 20 μg / mL rifampicin) was added and continued to be shaken until the bacterial solution OD reached 0. 600 The value reached between 0.6 and 0.8. After centrifugation at 5000 rpm for 5 minutes, collect the bacteria, discard the supernatant and add 20 mL of sterilized MS medium to resuspend the bacteria. After centrifugation at 5000 rpm for 5 minutes, collect the bacteria, discard the supernatant and add an appropriate amount of tobacco infection liquid to adjust the OD of the bacterial solution. 600 The value is adjusted to between 0.6 and 0.8, and after standing at room temperature in the dark for 2-3 hours, it can be used for the next step of conversion.

[0049] Leaf disk method for tobacco transformation

[0050] (1) Transformation of tobacco leaf discs: Select sterile tobacco seedlings that have been cultured for about one month, cut the main vein of the leaves with sterilized scissors, and cut the leaf discs on both sides into 0.5*0.5cm 2 The cubes were immediately transferred to a sterile culture dish containing Agrobacterium infection solution, stirred gently, and infected for about 10-15 minutes. Then, the cubes were removed and the bacterial solution was dried with sterile filter paper, and then transferred to the co-cultivation medium (1L co-cultivation medium contains 4.4g MS powder, 30g sucrose, 6g agar, 2mg 6-BA and 0.2mg NAA, and the pH is adjusted to 5.8). The leaves were facing up and co-cultivated in a dark environment for 3 days.

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

[0052] (3) Rooting culture: When the adventitious buds that survived in the screening medium grew to more than 3 cm, they were cut 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 the pH is adjusted to 5.8). After rooting, the seedlings were transplanted into a light incubator, and the transplanting medium was peat soil: vermiculite = 3:1.

[0053] The experimental results are as follows Figure 4As shown in the figure, in the transgenic tobacco flowers carrying overexpression of PpMYB1, the corolla, anther, filament, style, ovary, sepals, etc. are all redder than those of the tobacco with no transfection, and the expression levels of the downstream structural genes NtDFR, NtANS and NtUFGT related to anthocyanin synthesis in tobacco are significantly upregulated. This indicates that the overexpression of transcription factor PpMYB1 in tobacco promotes the accumulation of anthocyanins, making the tobacco flowers appear purple-red. Figure 5 The attached figure shows the determination of anthocyanin content in petals of transgenic PpMYB1 tobacco. Figure 6 The attached figure is an analysis of the expression levels of anthocyanin synthesis-related genes in the corolla of transgenic PpMYB1 gene tobacco.

[0054] The above is a description of the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in 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 is characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. A transcription factor PpMYB1 encoding the transcription factor PpMYB1 protein according to claim 1.

3. The transcription factor PpMYB1 according to claim 2, characterized in that The nucleotide sequence of the transcription factor PpMYB1 is shown in SEQ ID NO.

1.

4. A plant expression vector containing the transcription factor PpMYB1 according to claim 2 or 3.

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

6. An engineered bacterium containing the plant expression vector according to claim 4 or 5.

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

8. Use of the transcription factor PpMYB1 according to claim 2 or 3, the plant expression vector according to claim 4 or 5, or the engineered bacteria according to claim 6 or 7 in regulating the synthesis of plant anthocyanins.

9. The use according to claim 8, characterized in that: The regulation of plant anthocyanin synthesis is to change the plant flower color. Preferably, the plant is Cymbidium or tobacco.

10. A method for regulating plant anthocyanin synthesis, characterized in that: Introducing the transcription factor PpMYB1 according to claim 2 or 3 into a recipient plant to obtain a transgenic plant; Or transiently overexpressing the PpMYB1 gene in the petals of the five-lipped orchid. Preferably, the introduction of the PpMYB1 gene into the recipient plant is carried out by transforming a plant expression vector containing the PpMYB1 gene into plant tissues through an Agrobacterium-mediated method, and cultivating the transformed plant tissues into plants.

Images