Application of overexpression of dir15 gene in promoting synthesis of anthocyanin in populus tomentosa

Abstract

The application discloses application of overexpression of a DIR15 gene in promoting synthesis of anthocyanin of populus tomentosa, and belongs to the technical field of plant genetic engineering, and overexpression of the DIR15 gene is achieved by constructing an overexpression vector of the DIR15 gene, transforming wild-type populus tomentosa by means of infection of agrobacterium tumefaciens, and obtaining transgenic plants of overexpression of the DIR15 gene, and then screening plants with higher expression from the transgenic plants by subsequent positive identification, and the transgenic plants of overexpression of the DIR15 gene are redder after high light treatment, and the total anthocyanin content is significantly increased, which indicates that the DIR15 can promote accumulation of anthocyanin. The cultivation technical method provided by the application provides a theoretical basis and excellent germplasm materials for a new variety of poplar with colorful leaves.

Description

Technical Field

[0001] This invention relates to the field of plant transgenic engineering technology, specifically to the application of overexpression of the DIR15 gene in promoting anthocyanin synthesis in Populus tomentosa. Background Technology

[0002] Colorful foliage forests are an important ecological landscape resource in my country. Colorful-leaved tree species are the core components of colorful foliage forest landscapes, and their leaves maintain a stable non-green color throughout the growing season, thus possessing ornamental value.

[0003] Currently, my country lacks superior varieties of colorful-leaved trees. Using molecular breeding technology to create new varieties of colorful-leaved trees is an effective way to quickly overcome the current bottleneck in the development of the colorful-leaved forest industry in our city. Poplar is a traditional tree used for greening and afforestation in my country. Furthermore, the completion of its whole genome sequencing and its well-developed genetic transformation system make it an ideal material for creating superior varieties of colorful-leaved trees through molecular breeding.

[0004] Anthocyanins are important flavonoids widely found in plants, playing crucial roles in plant growth, metabolism, and stress responses. During growth and development, anthocyanins contribute to the vibrant colors of flowers and fruits, attracting insect pollination and animal feeding, thus promoting seed dispersal. Studies have shown that the formation of transcription factor complexes in plants can enhance anthocyanin yield and stability. Three transcription factors, MYB, bHLH, and WD40, have been reported to be widely involved in anthocyanin regulation. These three transcription factors work together by forming an MBW transcriptional complex. This complex has been identified in various model species, including Arabidopsis thaliana, maize, petunia, sedge, and alfalfa.

[0005] Several MYB transcription factors have been reported to regulate anthocyanin synthesis in poplar. PtrMYB57 encodes an R2R3-MYB protein located in the cell nucleus, primarily expressed in mature leaves, and plays a negative regulatory role in anthocyanin and PA biosynthesis in poplar. Transcription factor MYB120 significantly promotes anthocyanin accumulation in poplar. Other regulatory proteins have also been found to participate in plant anthocyanin biosynthesis; these transcription factors play a promoting or inhibiting role in regulating the expression of genes involved in anthocyanin structural biosynthesis and determine anthocyanin accumulation in developmental and environmental regulation.

[0006] Extensive research exists on how structural genes and transcription factors regulate anthocyanins, but the presence of other functional genes regulating anthocyanin remains unclear. While the DIR gene has been analyzed in several species, its involvement in the regulation of anthocyanin biosynthesis in poplar is still unknown. Therefore, investigating whether DIR can regulate anthocyanin synthesis in poplar is crucial for refining the anthocyanin regulatory network, ultimately providing a theoretical basis and superior germplasm for the creation of new colorful-leaved poplar varieties. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide the application of overexpression of the DIR15 gene in promoting anthocyanin synthesis in Populus tomentosa.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] 1. Application of DIR15 gene overexpression in promoting anthocyanin synthesis in Populus tomentosa.

[0010] Preferably, the nucleotide sequence encoding the DIR15 gene is shown in SEQ ID NO.3.

[0011] The present invention preferably includes the following steps:

[0012] (1) The DIR15 gene was amplified by PCR, and the amplified gene fragment was ligated into a plant expression vector to construct a DIR15 overexpression vector.

[0013] (2) Transform the DIR15 overexpression vector constructed in step (1) into Agrobacterium competent cells to obtain Agrobacterium engineered bacteria;

[0014] (3) Using the Agrobacterium engineered bacteria constructed in step (2), the DIR15 gene was transferred into plants through Agrobacterium-mediated genetic transformation. The plants were then identified at the molecular level to obtain positive transgenic plants with upregulated expression of the DIR15 gene. Transgenic plants with increased anthocyanin content were then screened out.

[0015] Preferably, the plant expression vector in step (1) of this invention is pCXSN-FLAG.

[0016] Preferably, the Agrobacterium mentioned in step (1) of this invention is Agrobacterium GV3101 strain.

[0017] The beneficial effects of this invention are as follows: the application of DIR15 gene overexpression in promoting anthocyanin synthesis in Populus tomentosa. By constructing a DIR15 overexpression vector, wild-type Populus tomentosa was transformed using Agrobacterium tumefaciens infection to obtain DIR15-overexpressing transgenic plants. Subsequent positive identification screened plants with high expression levels, and after high-light treatment, their phenotype and total anthocyanin content were observed and measured. The results showed that after strong light treatment, compared with the wild type at the same stage, the transgenic plants overexpressing DIR15 were redder and had a significantly higher total anthocyanin content, indicating that DIR15 can promote anthocyanin accumulation. The breeding technology provided by this invention provides a theoretical basis and excellent germplasm materials for new varieties of colorful-leaved Populus tomentosa. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0019] Figure 1 This represents the expression level of the DIR gene under hormone or stress treatment.

[0020] Figure 2 The expression levels of the DIR gene in different tissues.

[0021] Figure 3 The expression level of the DIR gene in leaves under high light treatment.

[0022] Figure 4 The macroscopic phenotype of the DIR15 overexpressing strain;

[0023] A shows the phenotypic diagrams of wild-type and DIR15 overexpressing lines; B shows the phenotypic diagrams of stems and leaves of DIR15 overexpressing lines, with a scale bar of 20 cm; C shows the quantitative fluorescence identification of DIR15 expression level in DIR15 overexpressing lines; D shows the plant height statistics of wild-type and DIR15 overexpressing lines.

[0024] Figure 5 Phenotypic analysis of plants overexpressing DIR15 after high light treatment;

[0025] A shows the phenotypic diagrams of wild-type and DIR15 overexpression lines after high light treatment; C shows the leaf phenotypic diagrams of wild-type and DIR15 overexpression lines after high light treatment, with a scale bar of 5 cm; B shows the identification of DIR15 overexpression lines; D shows the determination of total anthocyanin content in wild-type and DIR15 overexpression lines.

[0026] Figure 6 To provide genetic validation of MYB120 regulation of DIR15;

[0027] A shows the phenotypic diagrams of wild-type and MYB120 overexpression lines after high light treatment; B shows the leaf phenotypic diagrams of super-wild-type and MYB120 overexpression lines after high light treatment. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0029] Example 1: Construction of Evolutionary Tree and Domain

[0030] The genotypes and sequence information of the DIR gene family were obtained using Phytozome v13 (US Department of Energy website, https: / / phytozome-next.jgi.doe.gov / pz / portal.htmL) and NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ), and then a phylogenetic tree was constructed using MEGA5 software. The amino acid sequences of the DIR family were obtained using the same method, and then plotted using TBtools.

[0031] Example 2: Transcriptome Data Analysis

[0032] Poplar DIR transcriptome data under hormone or stress treatment were obtained from Dr. Chaofeng Li, a postdoctoral researcher at the University of Tokyo, Japan. (Apoplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light, and was then plotted using TBtools.) Figure 1 ).like Figure 1 As shown, yellow represents the fold increase in DIR gene expression, with darker colors indicating higher fold increases; blue represents the fold decrease in DIR gene expression, with darker blue indicating higher fold decreases. The results showed that most DIR genes were downregulated under different stress and hormone treatments. Some DIR genes were upregulated under hormone and stress treatments; DIR11, DIR15, DIR12, DIR14, DIR19, DIR31, DIR39, and DIR27 showed significantly increased expression levels under stress and hormone treatments.

[0033] The expression levels of the DIR gene in different tissues are as follows: Figure 2As shown, total RNA was collected from young leaves, old leaves, young xylem and phloem of the 1st and 2nd internodes, old xylem and phloem of the 6th and 7th internodes, roots, and petioles of wild-type poplar trees for reverse transcription to obtain cDNA from various poplar tissues. The expression levels of the above eight DIR genes in poplar tissues were detected by qRT-PCR. DIR11, DIR15, DIR27, and DIR39 were all specifically expressed. Among them, DIR11 and DIR15 were specifically expressed in old leaves, while DIR27 and DIR39 were specifically expressed in roots.

[0034] The expression level of the DIR gene in leaves under high light (HL) treatment is as follows: Figure 3 As shown, the expression levels of DIR11 and DIR15, which are specifically expressed in older leaves, were examined under different temporal gradient high light (40,000 Lux) treatments. The expression levels of both DIR15 and DIR11 were significantly upregulated after 12 hours of high light treatment. As the treatment time increased, the plants gradually adapted to abiotic stress, and their expression levels decreased accordingly. Compared to DIR11, DIR15 showed a higher fold upregulation.

[0035] Example 3: Extraction and reverse transcription of total RNA from poplar trees to synthesize cDNA

[0036] 1. RNA extraction

[0037] Preliminary preparation: Before the experiment, place a pestle, a key, a small amount of silica, and a small amount of anhydrous ethanol in a mortar, then burn them to remove RNase. Use the BioFLux Polysaccharide and Polyphenol Plant Total RNA Extraction Kit and RNase-free EP tubes. RNA extraction should be performed according to the instructions of the BioFLux Polysaccharide and Polyphenol Plant Total RNA Extraction Kit.

[0038] According to the kit's instructions, the specific operating steps are as follows:

[0039] 1) Preparation of lysis buffer: Add 10 μL of β-mercaptoethanol to 490 μL of AG Lysis. The solution can be mixed and prepared according to the amount extracted and then dispensed.

[0040] 2) Add liquid nitrogen and plant material to a sterile mortar, grind quickly into powder, put into a 1.5 mL centrifuge tube containing 500 μL of lysis buffer, and shake vigorously until there are no obvious particles.

[0041] 3) After centrifuging at 12000 rpm for 5 min, aspirate 450 μL of the supernatant into a new 1.5 mL centrifuge tube pre-filled with 225 μL of anhydrous ethanol, and immediately vortex to mix.

[0042] 4) Transfer the mixture to an adsorption column (Spin column), centrifuge at 12000 rpm for 1 min, and then discard the liquid in the tube;

[0043] 5) Add 600 μL of PG Buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the tube;

[0044] 6) Add 500 μL Wash Buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the tube;

[0045] 7) Add 250 μL Wash Buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the tube;

[0046] 8) After centrifuging at 12000 rpm for 1 min, centrifuge the empty column once under the same conditions;

[0047] 9) Transfer the adsorption column to a new 1.5 mL RNase-Free centrifuge tube, add 50-100 μL of Elution Buffer directly above the adsorption membrane, let stand at room temperature for two minutes, and then centrifuge at 12000 rpm for 1 min to obtain total RNA. Double column pass can increase the concentration.

[0048] 10) Take 1-2 μL of RNA into 2 μL of Loading Buffer and run a gel to check the extraction quality.

[0049] 2. RNA reverse transcription

[0050] Perform the following procedures according to the instructions for the Takara PrimeScript™ RT reagent Kit with gDNAEraser (Perfect Real Time).

[0051] 1) Genomic DNA elimination reaction, as shown in Table 1.

[0052] Table 1

[0053]

[0054] After centrifugation and mixing, place the mixture in a PCR instrument at 42°C for 2 minutes.

[0055] 2) Reverse-transcription reaction, as shown in Table 2.

[0056] Table 2

[0057]

[0058] After centrifugation and mixing, place the mixture in a PCR instrument at 37°C for 15 min; then at 85°C for 5 sec, and store at -20°C.

[0059] Example 4: Construction of plant expression vectors

[0060] 1. Amplification of gene fragments

[0061] The gene sequence cloned in this paper was based on the genome sequencing of *Populus hairy fruit* (http: / / energy.gov / genome.jgi-psf.org / poplar). Forward and reverse primers were designed according to the reference sequence, as follows:

[0062] F-OE-DIR15: 5′-ATGGGTAAGCTTCCTTCCATG-3′ (SEQ ID NO. 1);

[0063] R-OE-DIR15: 5'-CTAGCTTAGATAAACATTG-3' (SEQ ID NO. 2).

[0064] Then, using Populus tomentosa cDNA as a template and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers, PCR amplification was performed using PrimeSTAR high-fidelity enzyme. The reaction system is shown in Table 3 below:

[0065] Table 3

[0066]

[0067] Amplification reaction conditions: 98℃, 1 min 30 sec pre-denaturation; 98℃, 15 sec denaturation; 56℃, 20 sec annealing; 72℃, 30 sec extension; 36 cycles; 72℃, 10 min complete extension; 4℃, 5 min storage. Products were detected by 1%-2% agarose gel electrophoresis. After detection, gel blocks containing the target fragment were recovered using a gel recovery kit. The gel recovery procedure is described in the BioFLux gel recovery instructions.

[0068] 1) Locate the band containing the target fragment under UV light, cut off the gel block, weigh it, and place it into a 2mL EP tube;

[0069] 2) Add extraction buffer to the EP tube at a weight ratio of 1:3, place it in a water bath at 65°C, and heat until the adhesive is completely dissolved;

[0070] 3) Transfer the liquid in the EP tube to the Spin column, let it stand for two minutes, then centrifuge at 6000 rpm for 1 minute and discard the liquid in the tube.

[0071] 4) Add 500 μL of extraction buffer to the spin column, centrifuge at 6000 rpm for 1 min, and discard the liquid in the tube;

[0072] 5) Add 650 μL Wash Buffer, incubate at 12000 rpm for 1 min, then discard the liquid in the tube;

[0073] 6) After repeating the centrifugation for 1 min, place the Spin column into a new 1.5 mL EP tube, add 20 μL of preheated Elution Buffer to the column, let stand for 2 minutes, then centrifuge at 12000 rpm for 1 min.

[0074] 2. TA connection between fragment and carrier

[0075] The plant expression vector used in this invention is pCXSN-FLAG, and TA ligation is employed to ligate the gel-recovered target fragment to the target vector. The nucleotide sequence of the target fragment (DIR15) is shown in SEQ ID NO.3.

[0076] 1) First, add "A" to the target fragment for glue recovery, as shown in Table 4 below:

[0077] Table 4

[0078]

[0079] After mixing, place in a PCR instrument and incubate at 72℃ for 50 min, 4℃ for 10 min, and store at -20℃.

[0080] 2) Enzymatic digestion preparation of the vector backbone, the reaction system is shown in Table 5 below:

[0081] Table 5

[0082]

[0083] The TA ligation vector was digested using the XcmI single enzyme digestion method. After mixing the digested vector, it was placed in a metal bath at 37°C for 4 hours. After digestion, it was recovered by gel extraction and stored at -20°C.

[0084] 3) TA ligation was performed using the Solution I kit from Takara. The reaction system is shown in Table 6 below:

[0085] Table 6

[0086]

[0087] After mixing, place in a 16℃ metal bath for 8 hours, and then perform E. coli DH5α competent cell transformation.

[0088] Example 5: Preparation and transformation of Escherichia coli (DH5α) competent cells

[0089] 1. Preparation of competent Escherichia coli (DH5α) cells

[0090] 1) Streak the DH5α strain culture onto LB solid medium without resistance, and incubate it upside down overnight in a 37°C incubator;

[0091] 2) Pick a single clone from the culture medium, mix it in LB liquid medium without resistance, and incubate it overnight in a shaker at 37°C to make a live culture;

[0092] 3) Add the live liquid to fresh LB liquid medium at a volume ratio of 1:100 and incubate at 37°C in a shaker until the OD value is 0.4;

[0093] 4) Remove the culture medium and place it on ice. Incubate on ice for 1 hour, then centrifuge at 5000 rpm for 10 minutes at low temperature. Discard the supernatant.

[0094] 5) Add 40 mL of pre-cooled 0.1 mol / L CaCl2, resuspend the bacterial cells, place on ice, incubate on ice for 15 min, centrifuge at low temperature and low speed at 5000 rpm for 10 min, and discard the supernatant;

[0095] 6) Add 1-3 mL of pre-cooled CaCl2 glycerol solution, gently resuspend the bacterial cells, dispense 100 μL into 1.5 mL EP tubes, freeze in liquid nitrogen, and store at -80 °C.

[0096] 2. Escherichia coli transformation

[0097] 1) The ligation product prepared in Example 4 was added to the prepared colonic competent cells and placed on ice for 25 min;

[0098] 2) Place the competent cells in a 42°C water bath for 1 min 30 sec, then on ice for 2 min, add 800 μL of LB liquid medium, and incubate at 37°C for 1 h;

[0099] 3) Centrifuge at 5000 rpm for 5 min. In a clean bench, discard a portion of the bacterial culture and resuspend the cells in the remaining culture. Spread the resuspended cells onto LB agar containing the corresponding antibiotic and incubate overnight at 37°C. Once single colonies have grown, identify positive clones.

[0100] 3. Colony PCR screening for positive clones and plasmid extraction

[0101] Single clones from the plate were picked and placed into PCR tubes as amplification templates for PCR amplification. The positive control used a gene gel-recovered fragment as a template, and the negative control used ddH2O as a template. The PCR system setup is shown in Table 7 below:

[0102] Table 7

[0103]

[0104]

[0105] The reaction conditions were as follows: 98℃, 1 min 30 sec; 98℃, 15 sec; 56℃, 20 sec; 72℃, 20 sec; for a total of 35 cycles; 72℃, 10 min; 16℃, 10 min for cooling and storage. The product was detected by electrophoresis, and colonies with bands of the same size as the positive control were considered positive clones.

[0106] Positive clones were picked and cultured overnight at 37°C in LB liquid medium containing the corresponding antibiotic. Plasmid extraction was then performed using the Biospin plasmid DNA mini-extraction kit, following these steps:

[0107] 1) Transfer the bacterial culture to a 2ml EP tube, centrifuge at 1200rpm for 1min, and discard the supernatant. Resuspend the bacterial cells in 250μL of resuspension buffer;

[0108] 2) Add 250 μL of Lysis Buffer and mix by inverting.

[0109] 3) Add 350 μL of Neutralization Buffer, invert and mix well. When white flocculent material appears, centrifuge at 12000 rpm for 10 min.

[0110] 4) Transfer the supernatant to a spin column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the receiving tube;

[0111] 5) Add 600 μL Wash Buffer to the Spin column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the receiving tube;

[0112] 6) Centrifuge the empty column for 1 min, transfer the spin column to a 1.5 ml EP tube, add 30 μL of preheated Elution Buffer, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, and store the plasmid at -20℃.

[0113] Example 6 Preparation of Agrobacterium (GV3101) competent cells and Agrobacterium transformation

[0114] 1. Preparation of Agrobacterium (GV3101) competent cells

[0115] 1) Streak GV3101 strain onto YEP solid medium containing rifampicin resistance, and incubate upside down in a 28°C incubator for 36 hours;

[0116] 2) Pick a single colony from the culture medium, mix it in YEP liquid medium containing rifampicin resistance, and culture it in a shaker at 28°C for 36 hours to make a live culture;

[0117] 3) Add the live liquid to fresh rifampicin-resistant YEP liquid medium at a volume ratio of 1:50, and incubate at 28°C in a shaker until the OD value is 0.4;

[0118] 4) Place on ice for 30 minutes, centrifuge at 5000 rpm for 10 minutes at low temperature, and discard the supernatant;

[0119] 5) Add 1-3 mL of pre-cooled CaCl2 solution containing 15% glycerol, dispense into 1.5 mL EP tubes (100 μL per tube), flash freeze in liquid nitrogen, and store at -80 °C.

[0120] 2. Freeze-thaw transformation of Agrobacterium

[0121] 1) Add 5 μL of the recombinant plasmid extracted in Example 5 to Agrobacterium competent cells and mix gently;

[0122] 2) Ice bath on ice for 5 min, quick freeze in liquid nitrogen for 2 min, and then heat shock in a 37℃ water bath for 5 min;

[0123] 3) Add 800 μL of YEP liquid medium containing rifampicin and incubate at 28°C in a shaker for 4 h;

[0124] 4) Centrifuge at 5000 rpm for 5 min, resuspend the bacterial cells in 100 μL of supernatant, discard part of the bacterial solution, resuspend the bacterial cells in the remaining bacterial solution, spread on YEP solid medium containing the corresponding antibiotic, and incubate upside down in an incubator at 28℃ for 2 days.

[0125] Example 7: Genetic transformation of Populus tomentosa

[0126] Table 8. WPM Culture Medium Preparation Formula (1L)

[0127]

[0128] All culture media were adjusted to pH 5.80-5.85 after adding WPM powder and 1 mL of calcium salt, and then agar was added. After autoclaving at 121°C for 20 min, hormones (NAA was added before adjusting the pH) were added.

[0129] The transformation material was wild-type poplar leaves, and the genetic transformation method mediated by Agrobacterium tumefaciens was used.

[0130] 1) Activation of Agrobacterium

[0131] Agrobacterium GV3101 containing the recombinant plasmid prepared in Example 6 was inoculated onto YEP solid medium (containing 50 mg / L Kan + 40 mg / L Rif) and cultured at 28°C for 1–2 days. Single colonies were picked and inoculated into YEP liquid medium containing 50 mg / L Kan and 40 mg / L Rif and cultured at 28°C with shaking until the OD600 reached 0.8–1.0. 200 μL of viable bacterial culture was transferred to fresh YEP medium and cultured at 28°C with shaking for 6–8 hours. When the OD600 reached 0.6–0.8, the bacterial cells were collected by centrifugation, resuspended in 30 mL of WPM liquid medium supplemented with AS, and cultured in a shaker at 28°C for 1–2 hours before transformation.

[0132] 2) Transformation of Agrobacterium

[0133] Sterile wild-type poplar material in tissue culture bottles was selected as the background for transformation. First, a suitable amount of tender green leaves were cut into small pieces with a sterile scalpel in a suitable amount of sterile water (ensuring smooth cuts to facilitate transformation and growth). A suitable amount of the cut leaves were transferred to the resuspended bacterial solution and incubated for 10 minutes, shaking once every 5 minutes to ensure that the cut wounds were fully in contact with the resuspended bacterial solution.

[0134] 3) Co-cultivation of Populus tomentosa

[0135] Carefully remove the infected leaf material with tweezers, absorb the bacterial solution, lay the leaf flat with the back side down on WPM co-culture medium, and incubate at room temperature (25°C) in the dark for 2 days.

[0136] 4) Selection and cultivation of Populus tomentosa

[0137] Two days later, the transformed leaves were transferred to WPM selective medium (placed in the appropriate resistance medium as required for the experiment) and cultured at room temperature (25°C) in the dark for 3 to 4 weeks, changing the medium every 5 days during the period (during the selective culture, it is necessary to observe the degree of change in the leaves more often).

[0138] 5) Sprouting culture of Populus tomentosa

[0139] When white, loose callus tissue appears around the leaves, transfer it to WPM budding medium (place it in the appropriate resistance medium as needed for the experiment) and induce budding on a light-controlled culture rack under conditions of 2000–10000 Lux and 25°C for about 4–5 weeks. During the budding culture period, it is still necessary to observe the changes in callus growth. If the callus does not grow or turns brown, the medium should be replaced in time.

[0140] 6) Rooting culture of Populus tomentosa

[0141] When adventitious buds emerge from the callus in the budding medium and are about 3-4 cm in length, they are cut off and transferred to WPM rooting medium. Under normal circumstances, the rooting time for wild-type callus is about 7 days, while the rooting time for resistant callus is slower.

[0142] 7) Transplanting of genetically modified white poplar

[0143] When the rooted seedlings grow to about 10cm and have a relatively well-developed root system, remove the seedlings, wash off the agar from the roots with water, mark them, and transplant them into the greenhouse for cultivation. (Note that the seedlings removed from the tissue culture bottle need a period of adaptation to the external environment, so they need to be sealed with plastic wrap. After they have adapted, slowly uncover the film.)

[0144] Example 8: Analysis of expression levels in transgenic plants

[0145] 1. Extraction of Poplar DNA using the CTAB method

[0146] 1) Take fresh leaves into a 2mL EP tube, add 2-3 small steel balls, 500μL of CTAB extraction solution and 90μL of β-mercaptoethanol, and break them in a tissue homogenizer for 30min.

[0147] 2) Place the 2mL EP tube in a 65℃ water bath and incubate for 45min, inverting and mixing once every 15min;

[0148] 3) Add 600 μL of chloroform:isoamyl alcohol in a ratio of 24:1, mix well, emulsify on a flat surface for 10 min, centrifuge at 12000 rpm for 10 min, and aspirate the supernatant into a new sterile EP tube.

[0149] 4) Add an equal volume of isopropanol, shake to mix, centrifuge at 12000 rpm for 15 min, and discard the supernatant;

[0150] 5) Wash once with 75% ethanol, centrifuge at 12000 rpm for 1 min, remove the supernatant, dry the remaining liquid until transparent, add 30 μL-50 μL of ddH2O containing RNase, dissolve and store at -20℃.

[0151] 2. Quantitative Real-Time PCR

[0152] The expression level of DIR15 overexpressing plants was identified. RNA was extracted from the light-treated material and reverse transcribed into cDNA. Quantitative real-time PCR was performed using Takara's quantitative enzyme.

[0153] qPCR-DIR15-F: 5′-GCTTGGTTGGCTTGCT-3′ (SEQ ID NO.4);

[0154] qPCR-DIR15-R: 5′-TTGGGAAATGGTATCTGG-3′ (SEQ ID NO. 5).

[0155] The reaction system is shown in Table 9 below:

[0156] Table 9

[0157]

[0158] Amplification was performed using a quantitative real-time gene amplification instrument. The amplification program was as follows:

[0159] Pre-denaturation at 95℃ for 3 min; 95℃, 30 sec, 60℃, 1 min, 60℃, 30 sec; 95℃, 15 sec, for 40 cycles.

[0160] The quantitative detection results are shown in Table 10 below:

[0161] Table 10

[0162] WT 0.0000134982 0.0000010788 L3 0.462123638 0.11628276 L4 0.271473561 0.02126734 L12 0.024164 0.001682 L14 0.022092 0.003211

[0163] (Note: The Mean value is the average of three repeated tests)

[0164] Analysis of the above quantitative detection results revealed that the transgenic plants L3 and L4 showed higher DIR15 expression levels, while there was no significant difference in plant height between wild-type and DIR15-overexpressing L3 and L4 lines. Figure 4 Transgenic plants with high DIR15 overexpression levels were selected and subjected to high light treatment, and the changes in leaf color were observed.

[0165] Example 9: Strong light treatment and total anthocyanin content of transgenic plants

[0166] 1. Highlighting

[0167] Tissue culture seedling treatment: Wild-type seedlings with uniform growth in rooting medium were selected and subjected to time gradient treatment in a 30,000 Lux light incubator for 6 h, 12 h, and 24 h, respectively. The leaves were then quick-frozen and stored at -80℃.

[0168] Soil-grown seedling treatment: Two-month-old soil-grown seedlings were placed under a light intensity of 40,000 Lux.

[0169] 2. Extraction of total anthocyanins

[0170] 1) Take 0.5g of sample, grind it into powder with liquid nitrogen, and then transfer it to a 15mL centrifuge tube;

[0171] 2) Add 5 mL of 1% acidified ethanol, mix well, and extract at 4°C overnight;

[0172] 3) Centrifuge at 12000 rpm for 1 min, take 200 μL of supernatant, and measure the absorbance of the sample liquid at 530 nm and 657 nm in a UV spectrophotometer. Measure each sample 3 times.

[0173] 4) Using the formula: Total anthocyanin content = (A 530 -A 657 The total anthocyanin content is calculated as *0.25) / M, where A 530 A 657 The absorbance values ​​at 530 nm and 657 nm represent the sample values, and M refers to the sample weight. The total anthocyanin content of each strain is shown in Table 11 below.

[0174] Table 11

[0175]

[0176] Based on the above data, it can be seen that after overexpressing DIR15, transgenic plants with high DIR15 expression levels were selected for high-light treatment, and the changes in leaf color were observed. The results are as follows: Figure 5 As shown, under high light treatment, the wild type showed significant anthocyanin accumulation, while the leaves of the transgenic plants were darker in color compared to the wild type. Figure 5 A, C). The determination of the total anthocyanin content of the plants showed ( Figure 5 Compared with the wild type, the total anthocyanin content of the transgenic plants was significantly increased, indicating that DIR15 promotes anthocyanin accumulation under high light treatment.

[0177] Example 10: Effects of transgenic plants on key genes in anthocyanin synthesis

[0178] 1. Yeast one-hybrid screening for upstream regulators of the DIR15 gene

[0179] The MYB120 transcription factor sequence shown in SEQ ID.6 was constructed into the pGADT7-AD plasmid using EcoRI and BamHI restriction sites to obtain the AD recombinant vector plasmid; the DIR15 gene promoter sequence shown in SEQ ID.7 was constructed into the pHIS2 plasmid using EcoRI and SacI restriction sites to obtain the pHIS2 recombinant vector plasmid.

[0180] 1) Draw lines with Y1H bacteria on YPDA solid medium and incubate upside down at 30°C for two days;

[0181] 2) Pick a single colony from the plate and incubate it in YPDA liquid medium for 2 days to prepare a live culture for later use;

[0182] 3) Take 1 mL of the live culture medium and incubate it in an Erlenmeyer flask containing 50 mL of YPDA liquid medium until OD. 600 It ranges from 0.4 to 0.6;

[0183] 4) Transfer the bacterial culture to a 1.5 mL EP tube, centrifuge at 12000 rpm for 1 min, repeat the bacterial collection once, discard the supernatant, add 1 mL ddH2O to resuspend the bacterial cells, centrifuge at 12000 rpm for 1 min, and discard the supernatant.

[0184] 5) Add 1 mL of 1×TE / LiAc to a 1.5 mL EP tube, resuspend the cells, mix well, and prepare competent yeast cells. Place on ice for later use.

[0185] 6) Take a new 1.5mL EP tube, add 5μL single-stranded salmon sperm DNA, 5μL AD recombinant vector plasmid, and 5μL pHIS2 recombinant vector plasmid, centrifuge at 12000rpm for 1min to mix, add 100μL of yeast competent cells, and mix gently.

[0186] 7) Add 500 μL PEG / LiAc, mix by inverting, and incubate at 30°C for 30 min, inverting and mixing once every 10 min;

[0187] 8) Add 20 μL of DMSO and mix gently. Incubate in a 42°C water bath for 15 min, inverting the container every 5 min to mix.

[0188] 9) Centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL ddH2O to resuspend, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 100 μL ddH2O to resuspend, spread on SD-deficient solid medium (SD / -L, leucine deficient), and incubate at 30℃ for 3 days.

[0189] 10) Pick a single clone and dilute it in 1 mL ddH2O. Take 2 μL of the bacterial culture and spot it onto SD triple-deficient solid medium containing different concentrations of 3AT (SD / -T / -L / -H, deficient in tryptophan, leucine and histidine). Incubate at 30℃ for 2-3 days.

[0190] Yeast single-hybrid experiments demonstrated that the MYB120 transcription factor can bind to the upstream promoter of the DIR15 gene.

[0191] 2. To identify the expression level of MYB120 overexpressing plants, RNA was extracted from the light-treated material and reverse transcribed into cDNA. Real-time PCR was performed using Takara's quantitative enzyme, with the following primers:

[0192] qMYB120-F: 5′-GGAAGAACAGCGAATG-3′ (SEQ ID NO. 8);

[0193] qMYB120-R: 5′-AATGCCGTGGTAGAAT-3 (SEQ ID NO. 9).

[0194] The quantitative detection results of the MYB120 gene in the MYB120 overexpression lines are shown in Table 12 below:

[0195] Table 12

[0196] WT 0.006547 0.000128 L8 0.02583 0.000633 L10 0.058225 0.008531

[0197] The quantitative detection results of the DIR15 gene in the MYB120 overexpression line are shown in Table 13 below:

[0198] Table 13

[0199] WT 0.0000569892 0.00000307 L8 0.000112183 0.0000213 L10 0.000197065 0.0000548

[0200] To further verify the regulatory role of MYB120 on DIR15 in poplar, transgenic plants overexpressing MYB120 were created. The expression level in the leaves of the overexpressing MYB120 lines was quantitatively detected, and positive lines with high expression levels, L8 and L10, were selected. The expression level of the DIR15 gene in the transgenic positive lines was then measured. The results showed that, compared with the wild type, the leaves of the MYB120 overexpressing plants were darker and anthocyanin accumulation was increased (see attached image). Figure 6 (A, B); Simultaneously, compared to the wild type, the expression level of DIR15 was significantly increased in the MYB120 overexpression lines, demonstrating that MYB120 can induce the transcription of the DIR15 gene in poplar. This indicates that MYB transcription factors can directly bind to DIR15 and regulate its transcriptional level, thereby regulating anthocyanin accumulation in plants. This further clarifies the position of the DIR15 gene in the anthocyanin regulatory network and improves the regulatory network of anthocyanin biosynthesis.

[0201] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of overexpression of the DIR15 gene in promoting anthocyanin synthesis in Populus tomentosa under strong light, characterized by: The nucleotide sequence encoding the DIR15 gene is shown in SEQ ID NO.3, and the intensity of the strong light is 40000 Lux.

2. The application according to claim 1, characterized in that: It includes the following steps: (1) The DIR15 gene shown in SEQ ID NO.3 was amplified by PCR, and the amplified gene fragment was ligated into a plant expression vector to construct a DIR15 overexpression vector. (2) Transform the DIR15 overexpression vector constructed in step (1) into Agrobacterium competent cells to obtain Agrobacterium engineered bacteria; (3) Using the Agrobacterium engineered bacteria constructed in step (2), the DIR15 gene was transferred into plants through Agrobacterium-mediated genetic transformation. The plants were then identified at the molecular level to obtain positive transgenic plants with upregulated expression of the DIR15 gene. Transgenic plants with increased anthocyanin content were then screened out.

3. The application according to claim 2, characterized in that: The plant expression vector mentioned in step (1) is pCXSN-FLAG.

4. The application according to claim 2, characterized in that: The Agrobacterium mentioned in step (2) is Agrobacterium GV3101 strain.

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