Nucleic acid molecules, proteins or biomaterials and their use in modulating anthocyanin content in plants of interest

Abstract

The application discloses a nucleic acid molecule, a protein or a biological material and application thereof in regulating anthocyanin content of a plant. In a first aspect, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO. 1. According to the embodiments of the application, at least the following beneficial effects are provided: a new functional gene EuPAP1-1 of Eucommia ulmoides that can induce plant anthocyanin accumulation is provided. The gene can provide an effective candidate gene for creating new plant germplasm resources in molecular breeding, and can also be used as a new visual marker reporter gene, and applied to a plant genetic transformation system and functional gene verification.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to nucleic acid molecules, proteins or biomaterials and their application in regulating the anthocyanin content of target plants. Background Technology

[0002] Anthocyanins, also known as anthocyanin pigments, are a class of water-soluble natural pigments widely found in plants and are among the main pigments contributing to the color of leaves, petals, and fruits. Anthocyanins are primarily stored in water-soluble form within the vacuoles of plant cells and come in a wide variety of types. Anthocyanins combine with sugars via glycosidic bonds to form anthocyanin glycosides. There are more than 20 types of anthocyanins, while the number of anthocyanin types can reach as high as 250. Anthocyanins exhibit different colors under different pH conditions; for example, they are red under acidic conditions, purple under neutral conditions, and blue under alkaline conditions. Therefore, by regulating the accumulation level of anthocyanins in plants, different colors can be produced in plant organs.

[0003] With the deepening of molecular biology research, studies have discovered that many key genes and transcription factors in the anthocyanin synthesis pathway regulate anthocyanin synthesis in plants, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3' hydroxylase gene (F3'H), dihydroflavonol 4-reductase (DFR), and colorless anthocyanin dioxygenase (LDOX). However, most research on these key genes and anthocyanins has focused on plants such as Arabidopsis thaliana, turnip, yam, mulberry, kale, and buckwheat; the relevant functional genes in Eucommia ulmoides have not yet been identified and applied. Summary of the Invention

[0004] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a nucleic acid molecule, protein, or biological material and its application in regulating the anthocyanin content of a target plant.

[0005] A first aspect of this application provides an isolated nucleic acid molecule comprising any one of the following nucleotide sequences (A1) to (A3):

[0006] A1) The nucleotide sequence shown in SEQ ID NO.1;

[0007] A2) A nucleotide sequence with equivalent function formed by substituting, deleting or adding at least one nucleotide from the nucleotide sequence shown in A1);

[0008] The nucleotide sequences shown in A3) and A1) have more than 90% sequence identity and are equivalent nucleotide sequences.

[0009] Sequence identity refers to the maximum percentage of nucleotides that make the target sequence identical to the candidate sequence through methods such as introducing gaps. Sequence identity can be obtained using common methods known in this art, such as software or algorithms like BLAST, BLAST-2, and ALIGN.

[0010] In some embodiments of this application, the nucleotide sequence of A3) has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the nucleotide sequence shown in A1).

[0011] A second aspect of this application provides a protein encoded by the aforementioned nucleic acid molecule.

[0012] In some embodiments of this application, the amino acid sequence of the protein includes any one of the following B1) to B3):

[0013] B1) The amino acid sequence as shown in SEQ ID NO.2;

[0014] B2) An amino acid sequence with equivalent function formed by substituting, deleting or adding at least one amino acid from the amino acid sequence shown in B1);

[0015] The amino acid sequences shown in B3) and B1) have more than 90% sequence identity and are amino acid sequences with equivalent functions.

[0016] In some embodiments of this application, the amino acid sequence of B3) has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in B1).

[0017] A third aspect of this application provides a biomaterial comprising any one of the following:

[0018] C1) An expression cassette containing the aforementioned nucleic acid molecules;

[0019] C2) A recombinant vector containing the aforementioned nucleic acid molecules or expression cassettes;

[0020] C3) Recombinant cells containing the aforementioned nucleic acid molecules, expression cassettes, or recombinant vectors.

[0021] An expression cassette is a construct containing regulatory elements necessary for the expression of the contained nucleic acid molecules in recombinant cells. Specific regulatory elements include promoters, polyadenylation sites, etc.

[0022] A recombinant vector is a molecule containing all the elements necessary for the replication, integration, amplification, and / or expression of the contained nucleic acid molecule in recombinant cells. These elements include, but are not limited to, promoters, polyadenylation sites, terminators, replication initiation sites, transcription initiation sequences, enhancers, selection elements, and reporter genes. For example, antibiotic resistance genes or selection marker genes (such as the ampicillin resistance gene AmpR, the thymidine kinase gene TK, etc.) and the coding sequence for the target protein can be inserted into the multiple cloning site (MCS). Available types of recombinant vectors include plasmids, bacteriophages, lentiviruses, adenoviruses, and adeno-associated viruses.

[0023] Recombinant cells are cells used to replicate, integrate, amplify, and / or express nucleic acid molecules. Recombinant cells include, but are not limited to, bacteria, fungi, insect cells, plant cells, and animal cells. Specifically, they can be competent cells used to mediate the transformation of nucleic acid molecules, or target plant cells transformed with the aforementioned nucleic acid molecules. In some embodiments, the recombinant cell is, for example, Agrobacterium, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes. In some embodiments, the plant cell is angiosperm cell, such as plant cells of plants in the Magnoliopsida class, or plant cells of plants in the Brassicales, Solanales, and Ficus orders, or plant cells of plants in the Brassicaceae, Solanaceae, and Eucommia families, or plant cells of plants in the genera Arabidopsis, Nicotiana, and Eucommia.

[0024] A fourth aspect of this application provides the use of nucleic acid molecules, proteins, or biological materials in regulating the anthocyanin content of a target plant.

[0025] In some embodiments of this application, the target plant includes angiosperms.

[0026] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0027] In some embodiments of this application, the target plants include plants from the orders Brassicales, Solanales, and Ficus orders.

[0028] In some embodiments of this application, the target plant includes plants of the Brassicaceae, Solanaceae, and Eucommiaceae families.

[0029] In some embodiments of this application, the target plant includes plants of the genera Arabidopsis, Nicotiana, and Eucommia.

[0030] In some embodiments of this application, anthocyanins in at least one part of the target plant, including buds, leaves, stems, flowers, fruits, and seeds, are regulated.

[0031] A fifth aspect of this application provides the use of a substance that regulates the expression level and / or activity of EuPAP1-1 in regulating anthocyanin accumulation in a target plant.

[0032] EuPAP1-1 has the nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence with equivalent function formed by substituting, deleting or adding at least one nucleotide to the nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence with more than 90% sequence identity and equivalent function to the nucleotide sequence shown in SEQ ID NO.1.

[0033] In some embodiments of this application, substances that upregulate the expression level and / or activity of EuPAP1-1 enhance anthocyanin accumulation in the target plant.

[0034] In some embodiments of this application, the substance that upregulates the expression level and / or activity of EuPAP1-1 includes any one of D1) to D5);

[0035] D1) Substances that specifically knock in or knock up the gene encoding EuPAP1-1;

[0036] D2) Substances that specifically increase the mRNA level of EuPAP1-1;

[0037] D3) Substances that specifically increase the expression level of EuPAP1-1 protein;

[0038] D4) Substances that specifically enhance the activity of EuPAP1-1 protein;

[0039] D5) includes any one of the carriers from D1) to D4).

[0040] In some embodiments of this application, the material that specifically knocks in or knocks up the EuPAP1-1 encoding gene includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0041] In some embodiments of this application, material specifically knocking in or knocking up the EuPAP1-1 coding gene is used to knock in the EuPAP1-1 coding gene in the target plant through direct gene transfer technology (such as gene gun method, protoplast method, liposome method, pollen tube channel method, electroporation method, PEG-mediated transformation method, etc.) or biologically mediated transformation method (such as Agrobacterium-mediated transformation method, virus-mediated transformation method, etc.).

[0042] In some embodiments of this application, the carrier is any one of an inorganic carrier, an organic carrier, or an inorganic-organic composite carrier.

[0043] In some embodiments of this application, the vector is at least one of lentivirus, adenovirus, and adeno-associated virus.

[0044] In some embodiments of this application, the target plant includes angiosperms.

[0045] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0046] In some embodiments of this application, the target plants include plants from the orders Brassicales, Solanales, and Ficus orders.

[0047] In some embodiments of this application, the target plant includes plants of the Brassicaceae, Solanaceae, and Eucommiaceae families.

[0048] In some embodiments of this application, the target plant includes plants of the genera Arabidopsis, Nicotiana, and Eucommia.

[0049] In some embodiments of this application, substances are used to regulate the accumulation of anthocyanins in at least one part of the target plant, including buds, leaves, stems, flowers, fruits, and seeds.

[0050] A sixth aspect of this application provides a method for adjusting anthocyanin accumulation in a target plant, comprising regulating the expression level and / or activity of EuPAP1-1 in the target plant.

[0051] In some embodiments of this application, the expression level and / or activity of EuPAP1-1 are upregulated or downregulated in the target plant. Substances that upregulate the expression level and / or activity of EuPAP1-1 can increase anthocyanin accumulation in the target plant, while substances that downregulate the expression level and / or activity of EuPAP1-1 can reduce anthocyanin accumulation in the target plant.

[0052] In some embodiments of this application, the substance that upregulates the expression level and / or activity of EuPAP1-1 includes any one of D1) to D5);

[0053] D1) Substances that specifically knock in or knock up the gene encoding EuPAP1-1;

[0054] D2) Substances that specifically increase the mRNA level of EuPAP1-1;

[0055] D3) Substances that specifically increase the expression level of EuPAP1-1 protein;

[0056] D4) Substances that specifically enhance the activity of EuPAP1-1 protein;

[0057] D5) includes any one of the carriers from D1) to D4).

[0058] In some embodiments of this application, the material that specifically knocks in or knocks up the EuPAP1-1 encoding gene includes at least one of the CRISPR / Cas, ZFN, TALEN, and Cre-LoxP gene editing systems.

[0059] In some embodiments of this application, material specifically knocking in or knocking up the EuPAP1-1 coding gene is used to knock in the EuPAP1-1 coding gene in the target plant through direct gene transfer technology (such as gene gun method, protoplast method, liposome method, pollen tube channel method, electroporation method, PEG-mediated transformation method, etc.) or biologically mediated transformation method (such as Agrobacterium-mediated transformation method, virus-mediated transformation method, etc.).

[0060] In some embodiments of this application, regulating the expression level and / or activity of EuPAP1-1 in a target plant includes obtaining new plants by inserting an exogenous EuPAP1-1 gene or a fragment thereof into the genome of the target plant through plant genetic transformation.

[0061] In some embodiments of this application, plant genetic transformation includes Agrobacterium-mediated transformation.

[0062] In some embodiments of this application, the carrier is any one of an inorganic carrier, an organic carrier, or an inorganic-organic composite carrier.

[0063] In some embodiments of this application, the vector is at least one of lentivirus, adenovirus, and adeno-associated virus.

[0064] In some embodiments of this application, the target plant includes angiosperms.

[0065] In some embodiments of this application, the target plant includes plants of the Magnoliopsida class.

[0066] In some embodiments of this application, the target plants include plants from the orders Brassicales, Solanales, and Ficus orders.

[0067] In some embodiments of this application, the target plant includes plants of the Brassicaceae, Solanaceae, and Eucommiaceae families.

[0068] In some embodiments of this application, the target plant includes plants of the genera Arabidopsis, Nicotiana, and Eucommia.

[0069] In some embodiments of this application, substances are used to regulate the accumulation of anthocyanins in at least one part of the target plant, including buds, leaves, stems, flowers, fruits, and seeds.

[0070] A seventh aspect of this application also provides a plant breeding method, including adjusting the anthocyanin accumulation of a target plant according to the aforementioned method.

[0071] According to the application of the embodiments of this application, at least the following beneficial effects are achieved:

[0072] This application provides a novel functional gene, EuPAP1-1, from Eucommia ulmoides that can induce anthocyanin accumulation in the plant. This gene can provide an effective candidate gene for creating new plant germplasm resources through molecular breeding, and can also serve as a new reporter gene for visual markers, applied to plant genetic transformation systems and functional gene verification.

[0073] Specifically, heterologous overexpression of this gene in Arabidopsis anthocyanin accumulation-deficient mutants significantly induced the expression of genes in the anthocyanin synthesis pathway in Arabidopsis, resulting in an anthocyanin-deficient phenotype in complementary mutants, particularly in high-expression lines where flowers, pods, and seeds exhibited a purple hue. Furthermore, overexpression of EuPAP1-1 in tobacco leaves significantly induced anthocyanin accumulation at the expression sites.

[0074] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0075] Figure 1 This document presents phenotypic analysis of green and reddened regenerated buds, and a phylogenetic tree constructed based on the MYB protein sequence, as described in the embodiments of this application. A shows phenotypic photographs of green and reddened regenerated buds, with a scale bar of 2 cm. B shows color photographs of the crude anthocyanin extract obtained from the green and reddened regenerated buds. C shows the anthocyanin content in the crude extract as detected by spectrophotometry; the content data are averages (n=3), and the error bars show positive and negative standard deviations. * indicates significant differences in the t-test results (**, p<0.01). D shows the NJ phylogenetic tree constructed from the MYB protein sequence of the Eucommia ulmoides subfamily 6 and other published protein sequences of subfamilies 4, 5, 6, 7, and 15 of other plants (bootstrap value 1000, Poisson model, 50% deletion of missing amino acids).

[0076] Figure 2 This represents the expression levels of EuPAP1-1 and EuPAP1-2 in green and reddened regenerated buds in the embodiments of this application. Data are averages (n=3), and error bars show positive and negative standard deviations. * indicates significant differences in the t-test results (**, P<0.01, ***, P<0.001).

[0077] Figure 3 This is the CDS sequence of the cloned EuPAP1-1 gene and the signal peak diagram of Sanger sequencing in the embodiments of this application.

[0078] Figure 4This is the CDS sequence of the cloned EuPAP1-2 gene and the signal peak diagram of Sanger sequencing in the embodiments of this application.

[0079] Figure 5 This is a vector map of the vector skeleton p33-5×FLAG in the embodiments of this application.

[0080] Figure 6 This is the vector map of the 35S::EuPAP1-1 overexpression vector in the embodiments of this application.

[0081] Figure 7 This is a vector map of the 35S::EuPAP1-2 overexpression vector in the embodiments of this application.

[0082] Figure 8 This document describes the anthocyanin accumulation at the expression sites induced by overexpression of the EuPAP1-1 gene in the embodiments of this application. A shows the anthocyanin accumulation phenotypes in different leaves of the experimental and control groups, with a scale bar of 2 cm. B shows photographs of crude anthocyanin extracts from leaves of the experimental and control groups in three independent replicates. C shows the corresponding anthocyanin amounts in the crude anthocyanin extracts of the experimental and control groups, with the origin representing the value of each biological replicate. The graph is plotted using the mean of each group's data (n=9), and the error bars show the positive and negative standard deviations. * indicates significant differences in the t-test results (***, P<0.001). D shows the relative expression levels of certain genes compared to the internal reference gene in the leaves of the experimental and control groups, with * indicating significant differences in the t-test results (***, P<0.001).

[0083] Figure 9 This application illustrates the effect of EuPAP1-1 gene overexpression on anthocyanin accumulation in Arabidopsis pap1-c mutant plants. Figure A shows the phenotypes of the Arabidopsis pap1-c mutant and its complementary transgenic plants. Figures 2# and 3# are two representative complementary plants. Red arrows indicate anthocyanin accumulation at the base of rosette leaves and in the veins of the overexpressing complementary line #3. The scale bar is 1 cm. B shows the expression level of EuPAP1-1 in the pap1-c mutant, rosette leaves #2 and #3. AtACTIN is the internal reference gene. nd indicates that no gene expression was detected. Different letters on the bar chart indicate that the data between different groups were significantly different in a one-way ANOVA (p<0.05). C shows the relative expression levels of Arabidopsis anthocyanin synthesis pathway genes AtCHS, AtCHI, AtF3H, AtDFR, AtANS, AtF3GT, AtGST, and transcription factor AtTT8 in the pap1-c mutant, rosette leaves #2 and #3, compared to the internal reference gene AtACTIN.

[0084] Figure 10This is the phenotype of a transgenic Arabidopsis thaliana line that highly expresses EuPAP1-1 in one embodiment of this application. A, B, and C represent the phenotypes of the flower, pod, and seed, respectively. Detailed Implementation

[0085] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0086] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0087] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number, and "approximately" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the stated number. The use of "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0088] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0089] Example 1: Extraction and Detection of Anthocyanins

[0090] refer to Figure 1 In the genetic transformation experiment of Eucommia ulmoides, a reddening phenotype (RRB) appeared in the tips and leaves of some regenerated buds in the later stage. Therefore, Eucommia ulmoides regenerated buds with the reddening phenotype and those with the conventional green phenotype (GRB) were taken as the experimental group and the control group, respectively, and anthocyanins were extracted according to the following steps:

[0091] (1) 200 mg tissue samples were collected from the regenerated buds of different phenotypes in the experimental group and the control group and placed in centrifuge tubes. After being quick-frozen in liquid nitrogen, the samples were thoroughly ground using a tissue grinder.

[0092] (2) After grinding, add 0.1% hydrochloric acid methanol solution (hydrochloric acid: methanol = 1:99) to the sample in the centrifuge tube, sonicate for 5 min, place in a 4℃ refrigerator for 3 hours, centrifuge at 12000 rpm (about 13400 g) for 10 min, collect the supernatant, which is the crude anthocyanin extract obtained.

[0093] (3) Add anthocyanin extract to the precipitate obtained by centrifugation at 12000 rpm in step (2), and repeat the extraction 4 times with 1.5 mL of anthocyanin extract. Collect the supernatant obtained after the 4 extractions into a new centrifuge tube and combine it with the crude anthocyanin extract in step (2).

[0094] The anthocyanin content in the crude anthocyanin extracts of the experimental and control groups was determined using a spectrophotometer. The specific steps are as follows:

[0095] 200 μL of crude anthocyanin extract was placed in a 96-well plate. The absorbance of the crude anthocyanin extract was detected at 530 nm and 657 nm using a SpectraMax ABS plus full-wavelength microplate reader (Meigu Molecular Instruments). The difference in anthocyanin content was calculated and compared according to the formula (A530-0.25×A657) / FW.

[0096] Where A530 is the absorbance value at 530nm, A657 is the absorbance value at 657nm, and FW is the fresh weight of the sample corresponding to the crude extract.

[0097] like Figure 1 As shown in Figure B, the color of the crude anthocyanin extract in the experimental group (RRS) is redder than that in the control group (GRS). Figure 1 The anthocyanin content extracted from the safflower regenerated buds in the experimental group was significantly higher than that from the conventional green regenerated buds.

[0098] Example 2: Screening of functional genes

[0099] Using the Arabidopsis thaliana AtPAP1 protein sequence as the query sequence, two similar sequences, EuPAP1-1 and EuPAP1-2, were found in the Eucommia ulmoides genome. Combined with published subfamily 4, 5, 6, 7, and 15 protein sequences from other plants, a phylogenetic tree was constructed as follows: Figure 1 As shown in Figure D, the results indicate that EuPAP1-1 and EuPAP1-2 belong to the R2R3-MYB transcription factor family 6. Therefore, the expression of these genes in Eucommia ulmoides was detected using the following steps:

[0100] Total RNA was extracted from tissue samples of regenerated shoots of Eucommia ulmoides with both reddish and conventional green phenotypes using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (DP452, Tiangen).

[0101] The quality of the extracted total RNA was determined using 1.2% agarose gel electrophoresis, and the concentration of the extracted total RNA was determined using Nanopore.

[0102] Take 1.0 μg of total RNA and use the HiScript III First Strand cDNA Synthesis Kit (R312, Novizan) to synthesize the first strand cDNA according to the reaction system and conditions recommended in the manufacturer's instructions for subsequent PCR.

[0103] Using 25 ng of cDNA transcribed from total RNA as a template, ArtiCanATM SYBR qPCR Mix (TSE501, qPCR) reagents were added according to the instructions, and the corresponding upstream and downstream primers, ROX Reference Dye II and qPCR Mix were added. The qRT-PCR reaction was performed on a QuantStudio 3 real-time quantitative PCR system (Applied Biosystems, Inc., USA), and the signal was collected.

[0104] The qRT-PCR reaction program was as follows: 95℃, 1 min; 95℃, 10 s, 60℃, 20 s, 40 cycles; 72℃, 5 min. The melting curve acquisition program used the instrument's default program. The EuEF1α gene was used as an internal reference gene in the corresponding species in the qRT-PCR reaction, and three biological replicates were performed. Primer details are shown in Table 1.

[0105] Table 1. Primer sequences for Eucommia ulmoides qRT-PCR

[0106] name sequence DEuPAP1-1-F ACCAGGAAGAACAGCAAACGA(SEQ ID NO.5) DEuPAP1-1-R GGGCCGGGTTTGGATTATGA(SEQ ID NO.6) DEuPAP1-2-F TGCAGGCTAAGGTGGTTGAA(SEQ ID NO.7) DEuPAP1-2-R CTCACGTCACTCCTGTCGG(SEQ ID NO.8) EuEF1α-F CCGAGCGTGAACGTGGTAT(SEQ ID NO.9) EuEF1α-R TAGTACTTGGTGGTTTGGAATTTCC(SEQ ID NO.10)

[0107] Test results as follows Figure 2 As shown in the figure, both genes in the red phenotype (Red SAM) and the green phenotype (Green SAM) showed significant upregulation. However, the expression levels of EuPAP1-1 and EuPAP1-2 in red regenerated shoots were significantly higher than those in green regenerated shoots. Furthermore, the expression abundance of EuPAP1-1 relative to the internal reference gene was 2–3 times that of EuPAP1-2 relative to the internal reference gene. Therefore, EuPAP1-1 and EuPAP1-2 were selected as candidate functional genes for further functional validation.

[0108] Example 3: Construction of overexpression vector

[0109] The CDS sequences of EuPAP1-1 and EuPAP1-2 were cloned from regenerated buds of Eucommia ulmoides with a reddening phenotype. The specific steps are as follows:

[0110] (1) Total RNA was extracted from the red regenerated buds of Eucommia ulmoides using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (DP452, Tiangen).

[0111] (2) The quality of the extracted total RNA was detected by 1.2% agarose gel electrophoresis, and the concentration of total RNA was determined by Nanopore.

[0112] (3) Take 2.0 μg of total RNA and use the HiScript III First Strand cDNA Synthesis Kit (R312, Novizan) to synthesize the first strand cDNA according to the reaction system and conditions recommended in the instructions for subsequent PCR.

[0113] (4) Take 2 μL of reverse-transcribed cDNA and use Phanta Max Super-Fidelity DNA Polymerase (P505, Novizan). Add the corresponding amounts of upstream and downstream primers, dNTP Mix, 2×Phanta Max Buffer, and DNA Polymerase to the reaction system recommended in the manufacturer's instructions. Then, clone and amplify the EuPAP1-1 CDS sequence on an ABI SimpliAmp thermal cycling PCR instrument according to the following reaction program: 95℃, 3 min; 95℃, 15 s, 56℃, 15 s, 72℃, 1 min, 32 cycles; 72℃, 5 min. Primer information for cloning and amplifying the EuPAP1-1 CDS sequence is shown in Table 2.

[0114] Table 2. Primer sequences used for gene cloning

[0115] name sequence cdsEuPAP1-1-F AGAGGATCTCGAGGCATGGGAAGTGAGGCAGCATC(SEQ ID NO.11) cdsEuPAP1-1.2-R CGTCTGTACACCTAGGTCACATCACCACTTGGTCGT(SEQ ID NO.12)

[0116] EuPAP1-1 CDS sequence reference Figure 3 The details are as follows:

[0117] ATGGGAAGTGAGGCAGCATCAGGACTGAGAAAAGGTGCGTGGACTGAACCAGAAGACAATCTTCTCAGGAAGTGCATGGAGAAATATGGAGAAGGGAAATGGAATCTAGTTCCTTTAAGAGCTGGGTTGAACAGATGCAGAAAGAGTTGCAGGCTAAGGTGGTTGAACTATCTCAGGCCTAATATAAAGAGAGGAAACTTCAACCCAGATGAAGTTGATCTCATTATCAGACTTCACAAGCTCTTAGGCAACAGATGGTCGTTAATTGCAGGTAGACTACCAGGAAGAACAGCAAACGACGTCAAAAACTATTTTAATACTCATCTCCTCAAGAAATTAGCAGCCGACAGGAGTGACGTGAGGCGTTCCGAACCCATGACGAAAACAACCATCATAAGACCCCAACCTCGGACCCTCTCGAGGAGCTCAACATGGGTGAAGGAAAGACCAAACTCTACCAGTGTTTTAACAAATCATAATCCAAACCCGGCCCCATCTCCGACACTGGAACCGGCGGTGGACGGCAGCGTTGAGTGGTTGAATAACTTGACCGTTGAGGATGAGATTATGGAGCAAGTAATAACGTGGCCCAAAAATGGCCTTTGGCCCGAGGAAACATGGTCAGCAACGAAAACGTACGATGATTCCTGCACGAGGGAAGACCCAAATGGATTGAGTGATGTGTTTAGTGAGGAACTTTGGGATATTCTCAAGAGTCCAGACGACCAAGTGGTGATGTGA(SEQ IDNO.1)。

[0118] The amino acid sequence of EuPAP1-1 protein is as follows:

[0119] MGSEAASGLRKGAWTEPEDNLLRKCMEKYGEGKWNLVPLRAGLNRCRKSCRLRWL

[0120] NYLRPNIKRGNFNPDEVDLIIRLHKLLGNRWSLIAGRLPGRTANDVKNYFNTHLLKKLAAD

[0121] RSDVRRSEPMTKTTIIRPQPRTLSRSSTWVKERPNSTSVLTNHNPNPAPSPTLEPAVDGSVE

[0122] WLNNLTVEDEIMEQVITWPKNGLWPEETWSATKTYDDSCTREDPNGLSDVFSEELWDILK

[0123] SPDDQVVM*(SEQ ID NO.2).

[0124] CDS sequence reference of EuPAP1-2 Figure 4 is as follows:

[0125] ATGGCTGCTACAGGATTGAGAAAGGGTGGATGGACTGAAGAAGAGGATATTCTTCTGAGAAATTGCATTCATAAATATGGAGAAGTAAAATGGCATCAAGTTCCTCATAGAGCAGGATTGAATAGGTGCCGGAAAAGTTGTAGATTGAGGTGGCTGAATTATCTTAGGCCAGATATAAAAAGAGGAAGTTTTGCACATGATGAAGTTGATCTCATTATCAGGCTTCACAAACTCTTGGGAAATAGATGGTCTTTAATTGCCGGTAGAATTCCGGGAAGAACAGCAAACGACGTTAAAAATTACTGGAACACCCACCTGCTGAAGAAATCAGTTGCTCTCAGTACAGAAGTTGGAAAATCTGAATCCAAGAATGAAATAACGAGGACTGCCGCCATAAAACCTCGACCTCGGACTTTCACAGCTTTGAAGCTGAAAAGCACTGTTGATCATCAGTGCTTGGGAGAGAAACCCATCGACCCATCTCCGGCGCCACTTCCGGCGGTGGATGACGGCGTTGAGTGGTGGGATAAATTGTCTTTTGACGATGAAATGATCATTGGCGATGGAATAATTTGGCCCGACTGGGGGTAA(SEQ IDNO.3).

[0126] The amino acid sequence of EuPAP1-2 is as follows:

[0127] MAATGLRKGGWTEEEDILLRNCIHKYGEVKWHQVPHRAGLNRCRKSCRLRWLNYL

[0128] RPDIKRGSFAHDEVDLIIRLHKLLGNRWSLIAGRIPGRTANDVKNYWNTHLLKKSVALSTE

[0129] VGKSESKNEITRTAAIKPRPRTFTALKLKSTVDHQCLGEKPIDPSPAPLPAVDDGVEWWDKL

[0130] SFDDEMIIGDGIIWPDWG* (SEQ ID NO. 4).

[0131] Construct the corresponding overexpression vector based on the cloned CDS sequence using the following method:

[0132] (1) The vector backbone was derived from the pCAMBIA33-5×FLAG vector, modified from the pCAMBIA3301 vector by the Plant Functional Gene Research Group of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. The vector map is shown in [link to vector map]. Figure 5 .

[0133] (2) The EuPAP1-1 gene overexpression vector was constructed using homologous recombination of terminal sequences. The specific method is as follows: Thermo Scientific was used. TM FastDigest TM The vector pCAMBIA33-5×FLAG plasmid was double-digested with restriction endonucleases BamHI and SmaI. The digestion system consisted of: 1 μg plasmid, 1 μL BamHI, 1 μL SmaI, 2 μL 10×FastDigest green buffer, and sterile ddH2O added to a final volume of 20 μL. The digestion was performed at 37°C for 45 minutes, followed by electrophoresis on a 1.5% agarose gel at 140V for 25 minutes.

[0134] (3) The linearized target fragment was recovered and purified using the FastPure Gel DNA Extraction Mini Kit (DC301, Novizan) according to the instructions to obtain a linearized vector backbone containing BamHI and SmaI sticky ends. The CDS sequence fragment of EuPAP1-1 with homologous end sequences amplified by cloning was also recovered and purified using the FastPure Gel DNA Extraction Mini Kit.

[0135] (4) Using the linearized vector backbone and the EuPAP1-1 CDS sequence with homologous terminal sequences recovered and purified in step (3), homologous recombination was performed using the ClonExpress II One Step Cloning Kit (C112, Novizan) according to the system and reaction conditions recommended in the manufacturer's instructions to construct the EuPAP1-1 gene overexpression vector 35S::EuPAP1-1. (Refer to...) Figure 6 .

[0136] (5) After the overexpression vector was constructed, it was sent to the sequencing department of Qingke for Sanger sequencing using primers Termi-R and M13-rev. The primer sequences are shown in Table 4.

[0137] Table 4. Sanger sequencing primer sequences

[0138] name sequence Termi-R AAGACCGGCAACAGGATTC(SEQ ID NO.13) M13rev CAGGAAACAGCTATGAC(SEQ ID NO.14)

[0139] Combination Figure 3 The sequence inserted into the 35S::EuPAP1-1 overexpression vector was sequenced and found to be correct, indicating that the 35S::EuPAP1-1 overexpression vector plasmid was successfully constructed.

[0140] Similarly, the overexpression vector 35S::EuPAP1-2 of EuPAP1-2 can be constructed using the above method. (See the chromatogram for reference.) Figure 7 Sequencing results for reference Figure 4 .

[0141] Example 4: Transformation of Agrobacterium tumefaciens

[0142] The 35S::EuPAP1-1 overexpression vector plasmid constructed in Example 3 was transformed into Agrobacterium tumefaciens AGL1 chemocompetent cells (AC1020S, Weidi Biotechnology) by heat shock method. For specific transformation methods, please refer to the instruction manual.

[0143] Transformed competent cells were revived at 28°C for 3 hours and cultured on solid LB medium containing 50 μg / ml kanamycin sulfate and 20 μg / ml rifampin for 2 days. Eight single clones were picked and colony PCR was performed using primers cdsEuPAP1-1-F and Termi-R to identify positive clones.

[0144] All positive clones were mixed and shaken in liquid LB medium containing 50 μg / ml kanamycin sulfate and 20 μg / ml rifampin. The mixture was then cultured on a constant temperature shaker at 28°C and 180 rpm until the OD600 value of the bacterial culture exceeded 1.2. The culture was then preserved as glycerol culture with a final concentration of 25% glycerol and stored at -80°C for later use.

[0145] Example 5: Genetic transformation of tobacco

[0146] Take 100 μl of AGL1 Agrobacterium tumefaciens transformed with the overexpression vector plasmid from Example 4 and activate it in 5 ml of liquid LB medium containing 50 μg / ml kanamycin sulfate and 20 μg / ml rifampin.

[0147] The activated bacterial culture was inoculated at a ratio of 1:200 of bacterial culture and resistant LB liquid medium and cultured on a constant temperature shaker at 28℃ and 180rpm until the OD600 value of the bacterial culture was 0.8.

[0148] After centrifuging the expanded bacterial culture at 5000 rpm for 10 minutes, the supernatant was discarded, and the bacterial clumps were resuspended in 10 ml of MMA buffer (100 μl of 1 M MES, 100 μl of 1 M MgCl2, 10 μl of 100 mM AS, and water to 10 ml) until the OD600 was 0.6.

[0149] After the resuspended bacterial suspension was allowed to stand in the dark at room temperature for 1 hour, it was injected into the tobacco leaves through the lower epidermis using a 1ml syringe (without the needle). Four to five healthy tobacco plants with approximately five leaves were injected into both the experimental and control groups. The leaves selected for both groups were located at the same growth node on the plant. The experimental group received Agrobacterium tumefaciens transformed with the overexpression vector, while the control group received Agrobacterium tumefaciens transformed with the empty pCAMBIA33-5×FLAG vector.

[0150] The results are as follows Figure 8 As shown in the figure, A represents the phenotype of anthocyanin accumulation in leaves 4 days after overexpression. It can be seen that the injection site of Agrobacterium in the control group (CK) is slightly yellowed, while the experimental group shows a large area of ​​red on the leaf surface, indicating that overexpression of EuPAP1-1 can significantly induce anthocyanin accumulation in the leaves at the expression site. Two subsequent independent replicate experiments also showed that overexpression of EuPAP1-1 induced anthocyanin accumulation in the leaves at the expression site.

[0151] In contrast, EuPAP1-2 was constructed using the same method as EuPAP1-1 above, with the overexpression vector 35S::EuPAP1-2, transformed into Agrobacterium tumefaciens, and genetically transformed. However, no anthocyanin accumulated in the overexpression site, and no large area of ​​red appeared on the leaf surface.

[0152] Referring to Example 1, anthocyanins were extracted from tobacco leaves of the experimental and control groups 4 days after overexpression. The color of the obtained anthocyanin extract was as follows: Figure 8 As shown in B, the quantitative detection results of anthocyanins are as follows: Figure 8 As can be seen from C, the anthocyanin content in the leaf parts overexpressing EuPAP1-1 was significantly higher than that in the control group.

[0153] For tobacco leaves from the experimental and control groups, following the method in Example 2, using internal reference genes as a baseline, the relative expression levels of genes NtCHS, NtCHI, NtF3H, NtDFR, NtANS, NtF3GT, and NtGST in the anthocyanin synthesis pathway of tobacco, as well as the bHLH transcription factor NtAN1 that regulates anthocyanins in tobacco, were detected by qRT-PCR. The primers used in the detection process are shown in Table 5 below:

[0154] Table 5. Primers used for tobacco qRT-PCR

[0155] name sequence NtCHS F TACTCCCGGATAGCGATGGT(SEQ ID NO.15) NtCHS R TTCCAATCCGAAAGGCCCAA(SEQ ID NO.16) NtCHI-F ACTGGCACTGGAAATGCTGT(SEQ ID NO.17) NtCHI-R AAACTGACGCGTCGGCATAG(SEQ ID NO.18) NtF3H-F ACCCTTGGACTGAAACGACA(SEQ ID NO.19) NtF3H-R ACACCAGACTTACGTGACCAT(SEQ ID NO.20) NtDFR-F ATAAGGACTTGTCCGTGGTGT(SEQ ID NO.21) NtDFR-R TCTGCAGTGCTTCGGGTA(SEQ ID NO.22) NtANS-F TGGTCAGCTTGAATGGGAGG(SEQ ID NO.23) NtANS-R GACCCAGACACCACCGTTAC(SEQ ID NO.24) NtUF3GT-F ATTCGATCCAACGAGGAAACA(SEQ ID NO.25) NtUF3GT-R ATAGATGGCATTGGCCCCTC(SEQ ID NO.26) NtGST-F ATGTGCCGACAAGCTAGAGAAA(SEQ ID NO.27) NtGST-R GGCCTGCTTGAAACCAATCATT(SEQ ID NO.28) NtAn1a-F TCGCCACTGTTGATTCCTCC(SEQ ID NO.29) NtAn1a-R CCTTCACTGTAGCTGTGCCA(SEQ ID NO.30) Nttubα3-F CCCTCGGGCTGTCTTTGTAG(SEQ ID NO.31) Nttubα3-R TGCATCTTCCTTGCCGCTTA(SEQ ID NO.32)

[0156] The results are as follows Figure 8 As shown in Figure D, these genes were all significantly upregulated. Therefore, it can be inferred that overexpression of the EuPAP1-1 gene from Eucommia ulmoides can significantly activate the expression levels of anthocyanin synthesis-related genes in tobacco, thereby inducing anthocyanin accumulation in tobacco.

[0157] Example 6: Recovery of anthocyanin deficiency in Arabidopsis pap1-c mutant

[0158] The Arabidopsis pap1-c (myb75-c) mutant is a loss-of-function mutant of the AtMYB75 / AtPAP1 gene, exhibiting a lack of anthocyanin accumulation under normal light conditions, and even under strong light, anthocyanin accumulation is significantly lower than in the wild type. In this embodiment, the EuPAP1-1 gene was overexpressed in this mutant to detect anthocyanin accumulation. The specific process is as follows:

[0159] Referring to Example 5, the transformed Agrobacterium tumefaciens culture was activated, then expanded, centrifuged, and the supernatant was discarded to collect the bacterial clumps. The bacterial clumps were resuspended in a 5 wt% sucrose solution containing 100 μM AS until the OD600 was 0.6.

[0160] The resuspended bacterial solution was left to stand in the dark at room temperature for 1 hour, then 0.02 v / v% Silwet L-77 solution was added and mixed well. Then, the Arabidopsis thaliana in full bloom was genetically transformed using the "flower-dipping method".

[0161] The transformed Arabidopsis thaliana was kept in the dark and moist for 16 hours, and then returned to normal light conditions for cultivation. The normal cultivation conditions were as follows: a photoperiod of 16 hours of light / 8 hours of darkness, corresponding temperatures of 22℃ / 20℃, 50%-60% relative humidity, timely watering of the plants, and nutrient solution irrigation every 2-3 weeks.

[0162] After 3-5 days of normal cultivation and the plant produces new flowers, repeat the above transformation process once. The entire transformation cycle involves 3-4 transformations.

[0163] The plant resistance gene in the 35S::EuPAP1-1 overexpression vector is the Bar gene. Therefore, in this example, resistance screening was carried out by spraying seedlings that had just grown true leaves with a final concentration of 20 mg / L glufosinate solution (CB2471, Coolabor).

[0164] Genomic DNA was extracted from the selected resistant plants using 2×CTAB, and PCR genotyping was performed using primers Termi-R and M13-rev to confirm the transgenic positive plants. A plasmid diluted 100-fold was used as a positive control. The PCR reagent used was 2×Taq PCR Mix (KT201, Tiangen), and the reaction program followed the reagent's recommended procedure. Phenotypic photographs and analysis were performed on the obtained transgenic positive plants.

[0165] Results Reference Figure 9 In transgenic plants A and B, the anthocyanin deficiency phenotype at the base of rosette leaves was completely complementary. Figure 10 For both A and B, as the expression level of the EuPAP1-1 gene increases, anthocyanin accumulation further increases, even leading to the appearance of purple veins in leaves. (Reference) Figure 9 In the B and C variants, the expression levels of Arabidopsis anthocyanin synthesis pathway genes AtCHS, AtF3H, AtDFR, AtANS, AtF3GT, AtGST, and transcription factor AtTT8 in complementary transgenic positive plants were significantly higher than those in the pap1-c mutant. Furthermore, the expression levels of these genes showed a certain positive correlation with the expression level of the EuPAP1-1 gene. The higher the expression level of the EuPAP1-1 gene, the higher the expression levels of Arabidopsis anthocyanin synthesis pathway genes and the transcription factor AtTT8 gene that regulates anthocyanin synthesis.

[0166] The above results indicate that overexpression of the EuPAP1-1 gene in *Eucommia ulmoides* complements the phenotype of its Arabidopsis homolog, the AtPAP1 mutant pap1-c. Furthermore, overexpression of the EuPAP1-1 gene can activate the expression levels of anthocyanin synthesis-related genes in *Arabidopsis thaliana*, thereby inducing anthocyanin accumulation. And referring to... Figure 10 In transgenic Arabidopsis lines with high expression of the EuPAP1-1 gene, large amounts of anthocyanins accumulate ectopically in flowers, pods, and seeds.

[0167] In summary, the EuPAP1-1 gene of Eucommia ulmoides can serve as a visualized molecular marker gene for efficiently inducing anthocyanin accumulation in different plants. In addition, this gene can also provide an effective candidate gene for the creation of plant germplasm resources using molecular breeding and can be applied in related research fields.

[0168] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

Claims

1. An isolated nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

2. The protein encoded by the nucleic acid molecule of claim 1.

3. A biomaterial, characterized in that, The biomaterial comprises any one of the following: C1) An expression cassette comprising the nucleic acid molecule of claim 1; C2) A recombinant vector comprising the nucleic acid molecule of claim 1, or the expression cassette of C1; C3) A recombinant cell comprising the nucleic acid molecule of claim 1, the expression cassette of C1), or the recombinant vector of C2), wherein the recombinant cell is Agrobacterium.

4. The use of the nucleic acid molecule of claim 1, the protein of claim 2, or the biomaterial of claim 3 in inducing anthocyanin accumulation in tobacco.

5. A method for inducing anthocyanin accumulation in tobacco, characterized in that, This includes knocking in the EuPAP1-1 coding gene in tobacco via Agrobacterium-mediated transformation, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. A plant breeding method, characterized in that, This includes inducing anthocyanin accumulation in tobacco using the method according to claim 5.

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