A method for regulating anthocyanin synthesis in plants

Abstract

This invention discloses a method for regulating the synthesis of anthocyanins in plants, relating to the field of plant genetic engineering technology. The method involves editing the MYB123 gene sequence to relieve the inhibitory function of miR858, thereby obtaining MYB123. mut Gene; MYB123 mut Genes regulate the synthesis of anthocyanins in plants by modulating the expression of key genes in the anthocyanin pathway. The nucleotide sequence of the MYB123 gene is shown in SEQ ID NO.1. The nucleotide sequence of miR858 is shown in SEQ ID NO.3. This invention constructs an expression vector for the MYB123 gene after miR858 inhibition is relieved; transforms the constructed expression vector into tomato or Arabidopsis thaliana; and cultivates, screens, and obtains transgenic tomato or Arabidopsis thaliana plants with significantly increased anthocyanin content.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a method for regulating the synthesis of anthocyanins in plants. Background Technology

[0002] Blackberries are small berry-bearing fruit trees belonging to the genus *Rubus* in the family Rosaceae, specifically the subgenus *Eubatus*. The fruit is an aggregate fruit, which does not easily separate from the receptacle after ripening, forming a solid whole. As a newly emerging third-generation fruit, blackberries possess nutritional, health, and medicinal value, making them widely popular among consumers. Furthermore, blackberries are rich in vitamins, terpenes, and polyphenolic compounds, exhibiting significant antioxidant capabilities and being a primary source of research and utilization. Related studies have shown that total phenolic compounds isolated from blackberries have anti-tumor effects, flavonoids have anti-inflammatory, lipid-lowering, and blood pressure-lowering effects, and anthocyanins can also treat cardiovascular diseases.

[0003] Anthocyanins are a class of compounds formed by the glycosidic bond between anthocyanins and sugars. They are widely found in the cell sap of plant flowers, fruits, stems, leaves, and root organs, giving them various colors from red and purplish-red to blue. Anthocyanins belong to the flavonoid family—a group of compounds based on flavonoid nuclei that exhibit red coloration. Due to their unique functionalities, they are used to scavenge free radicals, promote lutein production, and have anti-tumor, anti-cancer, anti-inflammatory, lipid peroxidation and platelet aggregation inhibition, diabetes prevention, weight loss, and vision protection effects. As a natural pigment, anthocyanins are safe, non-toxic, and have many health benefits for the human body, and have been applied in the food, health product, cosmetic, and pharmaceutical industries.

[0004] miRNAs occupy a central position in gene expression regulation within plants, modulating transcription factors. These transcription factors effectively regulate the co-expression of related genes throughout the entire biosynthetic pathway, thereby promoting anthocyanin synthesis and accumulation. Currently, the regulatory network by which miRNAs regulate their target genes in anthocyanin synthesis in blackberries remains a blank. Therefore, clarifying the interaction between blackberry miRNAs and their target genes, and elucidating the mechanism by which blackberry miRNAs regulate anthocyanin biosynthesis, is of practical significance. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for regulating the synthesis of anthocyanins in plants, so as to promote the synthesis and accumulation of anthocyanins in plants.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for regulating the synthesis of anthocyanins in plants involves editing the MYB123 gene sequence to relieve the inhibitory function of miR858; the uninhibited MYB123 gene regulates the synthesis of anthocyanins in plants by controlling the expression of key genes in the anthocyanin pathway.

[0008] The nucleotide sequence of the MYB123 gene is shown in SEQ ID NO.1.

[0009] The nucleotide sequence of miR858 is shown in SEQ ID NO.3.

[0010] The anthocyanins include one or more of the following: delphinidin, pelargonidin, malvidin, petunidin-3-O-(6-O-malonyl-β-D-glucoside), petunidin-3-(6-Op-p-coumaryl)-glucoside, petunidin-3,5-O-diglucoside, cyanidin-3,5-O-diglucoside, cyanidin-3-O-moribiglycoside, and cyanidin-3-O-xyloside.

[0011] The key genes of the anthocyanin pathway include one or more of the following genes: SlPAL, SlC4H, SlCHS, SlF3H, SlF3'H, SlF3'5'H, SlCHI, SlFLS, SlCHS, and SlDRF.

[0012] The method described herein includes the following specific steps:

[0013] 1) Construct an expression vector for the MYB123 gene after miR858 inhibition was lifted;

[0014] 2) Transform the constructed expression vector into tomato or Arabidopsis thaliana;

[0015] 3) Cultivate, screen, and obtain transgenic tomato or Arabidopsis plants with significantly increased anthocyanin content.

[0016] Application of miR858 in regulating plant fruit growth.

[0017] Application of miR858 in regulating the soluble solids content, titratable acid content, and total phenol content of plant fruits.

[0018] Application of miR858 in regulating total antioxidant capacity in plants.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) The method for regulating anthocyanin synthesis in plants disclosed in this invention involves editing the MYB123 gene sequence to relieve the inhibitory function of miR858; the uninhibited MYB123 gene regulates the synthesis of anthocyanins in plants by controlling the expression of key genes in the anthocyanin pathway. The nucleotide sequence of the MYB123 gene is shown in SEQ ID NO.1. The nucleotide sequence of miR858 is shown in SEQ ID NO.3.

[0021] 2) Compared to wild-type (WT) Arabidopsis thaliana, the miR858 transgenic Arabidopsis line had a higher germination rate and remained stable after 14 days, but germination occurred later. Compared to wild-type (WT) Arabidopsis thaliana, the MYB123 transgenic Arabidopsis thaliana had a lower germination rate. Compared to wild-type (WT) Arabidopsis thaliana, the MYB123M transgenic Arabidopsis thaliana had a lower germination rate.

[0022] 3) The SPAD values ​​of transgenic Arabidopsis thaliana were significantly higher than those of WT. Compared with the MYB123 transgenic line, the MYB123M transgenic line had a higher SPAD value. Compared with WT, the leaves of transgenic Arabidopsis thaliana were darker, and the leaves of the MYB123M transgenic line were even darker.

[0023] 4) The protein, soluble sugar, total phenolic, and flavonoid contents of the miR858 transgenic Arabidopsis thaliana were significantly lower than those of the WT (whole-rooted) transgenic. In contrast, the protein, soluble sugar, total phenolic, and flavonoid contents of the MYB123 and MYB123M transgenic Arabidopsis thaliana were significantly higher than those of the WT. Specifically, the MYB123M transgenic Arabidopsis thaliana had a generally higher total phenolic content, and its flavonoid content was slightly higher than that of the MYB123 plants.

[0024] 5) The POD activity of MYB123M transgenic Arabidopsis thaliana was significantly higher than that of WT, while the POD activity of miR858 and MYB123 transgenic Arabidopsis thaliana was not significantly different from that of WT. The SOD activity of MYB123 and MYB123M transgenic Arabidopsis thaliana was higher than that of WT, while the SOD activity of miR858 transgenic Arabidopsis thaliana was significantly lower than that of WT. Compared with WT, the GSH content of miR858 transgenic Arabidopsis thaliana was lower, while the GSH content of MYB123 and MYB123M transgenic Arabidopsis thaliana was higher than that of WT. The total antioxidant capacity of MYB123 and MYB123M transgenic Arabidopsis thaliana was significantly higher than that of WT, while the total antioxidant capacity of miR858 transgenic Arabidopsis thaliana was lower than that of WT.

[0025] 6) Compared with WT, MYB123M transgenic tomatoes showed significantly increased fruit weight, longitudinal diameter, and transverse diameter, but decreased firmness. MYB123M transgenic fruits were larger at different growth stages, with fewer seeds; mature fruits were redder and larger, with fewer seeds and larger seeds. The soluble solids content, titratable acid content, anthocyanin content, total phenolic content, and total antioxidant capacity of MYB123M transgenic fruits were significantly higher than those of CK.

[0026] 7) In the S2, S3, and S4 stages, the expression levels of SlPAL, SlC4H, SlCHS, SlF3H, and SlDRF genes in the fruits of the MYB123M-1 strain were higher than those in the CK strain. In the S2, S3, and S4 stages, the expression levels of SlCHI and SlDRF in the fruits of the MYB123M-3 strain were higher than those in the CK strain, while the expression levels of SlF3H, SlF3'H, SlF3'5'H, and SlFLS were higher than those in the CK strain in the S2, S3, and S5 stages. In the S2, S4, and S5 stages, the expression levels of SlC4H, SlCHS, and SlF3'H genes in the MYB123M-4 strain were higher than those in the CK strain. This indirectly indicates that MYB123M can upregulate key structural genes in the anthocyanin pathway.

[0027] 8) The anthocyanin content in mature MYB123M transgenic tomatoes was generally higher than that in the control (CK) group. The contents of delphinidin, pelargonidin, malvidin, petunidin-3-O-(6-O-malonyl-β-D-glucoside), petunidin-3-(6-Op-coumaroyl)-glucoside, petunidin-3,5-O-diglucoside, cyanidin-3,5-O-diglucoside, cyanidin-3-O-moribiglycoside, and cyanidin-3-O-xyloside in mature MYB123M transgenic tomatoes were all higher than those in the control (CK) group. In conclusion, the contents of most anthocyanin components in MYB123M transgenic tomatoes were higher than those in the control (CK) group. Attached Figure Description

[0028] Figure 1 The expression levels of the RuMYB123 gene in blackberry at different developmental stages (a) and different tissue parts (b) of blackberry fruit (S1 is the green fruit stage; S2 is the green-red fruit stage; S3 is the red fruit stage; S4 is the red-purple fruit stage; S5 is the purple fruit stage).

[0029] Figure 2 The spectrum of the pGreenII 0800-62SK-MIR858a vector;

[0030] Figure 3 This is a graph showing the germination rate of T2 generation transgenic Arabidopsis thaliana.

[0031] Figure 4A comparison of SPAD values ​​(A) and leaves (B) of T2 generation transgenic Arabidopsis thaliana.

[0032] Figure 5 The graph shows the determination of major components in transgenic Arabidopsis thaliana (different lowercase letters indicate significant differences between different lines with the same gene, with a difference level of P < 0.05; different uppercase letters indicate significant differences between all different lines, with a difference level of P < 0.05).

[0033] Figure 6 The graph shows the determination of the main antioxidant indicators in transgenic Arabidopsis thaliana (different lowercase letters indicate significant differences between different lines with the same gene, with a difference level of P < 0.05; different uppercase letters indicate significant differences among all different lines, with a difference level of P < 0.05).

[0034] Figure 7 This is a graph showing the expression level analysis of the MYB123M gene in the leaves of transgenic tomato plants.

[0035] Figure 8 Phenotypic and cross-sectional images of fruits at different stages of transgenic tomato plants (S1: green fruit stage; S2: yellow fruit stage; S3: orange fruit stage; S4: orange-red fruit stage; S5: red fruit stage);

[0036] Figure 9 A graph showing the main physiological indicators of MYB123M transgenic tomato fruits;

[0037] Figure 10 This is a graph showing the expression levels of anthocyanin pathway genes in MYB123M transgenic tomato fruits.

[0038] Figure 11 Cluster diagram of anthocyanins in mature fruits of MYB123M transgenic tomatoes. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0040] The blackberries used in this application were harvested from the blackberry experimental base in Baima Town, Lishui District, Nanjing City, Jiangsu Province. They were frozen in liquid nitrogen immediately after being freshly picked and then stored in a -80°C freezer for RNA extraction.

[0041] The Arabidopsis thaliana used in this application was cultivated in the laboratory of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province. Freshly harvested Arabidopsis thaliana leaves were immediately frozen in liquid nitrogen and stored in a -80°C freezer for RNA extraction and physiological index determination. Seeds from mature Arabidopsis thaliana pods were also collected for the next sowing.

[0042] The tomatoes used in this application were grown in the nursery of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province. Tomato leaves and fruits at different stages, including green, yellow, orange, orange-red and red fruits, were harvested and immediately frozen in liquid nitrogen, and then stored in a -80℃ freezer for RNA extraction and subsequent physiological index determination.

[0043] Example 1

[0044] 1. Total RNA extraction and cDNA acquisition

[0045] Total RNA was extracted from blackberry fruits using the BioTeke Plant Total RNA Extraction Kit. Using the extracted total RNA as a template, reverse transcription was performed using the PrimeScript RT Master Mix (Perfect Real Time) reverse transcription kit to obtain cDNA.

[0046] 2. Blackberry MYB123 gene cloning

[0047] Primers were designed using Oligo 7.0 software based on the blackberry genome sequence, and its open reading frame (ORF) sequence was cloned using the high-fidelity PCR enzyme PrimeSTAR Max DNAPolymerase. The primer sequences are shown below:

[0048] RuMYB123 ORF-F: 5'-ATGGGTAGAGGTCCTCGG-3',

[0049] RuMYB123 ORF-R: 5'-ACTCAGCATGACATTTTAGATCATT-3'.

[0050] The target fragment was extracted and purified using a rapid agarose gel DNA recovery kit (Wuxi Biotech Biotechnology Co., Ltd.). Then, the purified product was ligated to a vector using the pClone007 Blunt Vector Kit (Nanjing Qingke Biotechnology Co., Ltd.), and the mixture was transferred into competent *E. coli* cells. After a brief recovery period, the cells were plated on ampicillin-resistant medium and incubated overnight at 37°C. Single colonies were then selected. PCR validation of the bacterial culture was performed using a 2×T5 Super PCRMix (Colony) (Nanjing Qingke Biotechnology Co., Ltd.). Positive bacterial cultures were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing validation.

[0051] The final sequencing yielded the nucleotide sequence of the blackberry MYB123 gene, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0052] 3. Analysis of the expression pattern of the MYB123 gene in blackberries

[0053] The gene expression level of MYB123 in blackberries was analyzed by RT-qPCR. Primer sequences are shown below:

[0054] MYB123 qRT-F: 5'-ACCGTCATTCCACAGCAGCAC-3',

[0055] MYB123 qRT-R: 5'-CAGCTTTGACCAAGTCGTCGT-3'.

[0056] Total RNA was extracted from blackberry fruits at different developmental stages (S1: green fruit stage; S2: green-red fruit stage; S3: red fruit stage; S4: red-purple fruit stage; S5: purple fruit stage) and from different parts using the Biotech RNA extraction kit. cDNA was then reverse transcribed using reverse transcription reagents from Shanghai Pudi Biotechnology Co., Ltd. Gene expression levels at different fruit developmental stages were detected by qRT-PCR. Sequence-specific primers and internal control genes were used for each gene. The qRT-PCR amplification system consisted of 15 μL of cDNA, 17.5 μL of 2X SYBR Green Pro Taq HS Oremix (ROX Pius), 0.6 μL of each primer, and 5.3 μL of ddH2O. The amplification program was: 95℃ for 30 s, 95℃ for 5 s, 60℃ for 30 s, 72℃ for 15 s, Melt for 6 s, for a total of 45 cycles. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0057] The results are as follows Figure 1 As shown, the expression level of the MYB123 gene was significantly higher in the S2 and S3 stages of fruit than in other stages. Figure 1 a), but in comparison of expression levels in different tissues, the expression level in fruit was not high, while the expression level of MYB123 in roots and flowers was significantly higher than in other tissues (a). Figure 1 b).

[0058] Example 2

[0059] 1. Target site elimination

[0060] Since the MYB123 nucleic acid sequence (3'-AAGTAACAGACAAGCGGGT-5') can be complementary to the miR858 sequence (5'-TTCGTTGTCTGTTCGACCT-3'), the open reading frame of the MYB123 gene, which is synonymous with the target site elimination, can be obtained using multi-site mutation technology, and used as the resistance target gene for subsequent transgenic materials.

[0061] Specifically, large primer PCR was used to perform multi-site mutations in the MRE region of the target gene MYB123, preventing the mature miR858 from complementary pairing with MYB123 without altering the amino acid sequence. Based on codon degeneracy, the 10-11 nt core matching region of the MRE was completely mutated, introducing as many mutation sites as possible, but avoiding polymorphic structures exceeding 3 bases. At least 10 nt of complementary bases were retained on the flanking regions of the MRE to ensure successful PCR. After one round of reaction and a second round of nested PCR, cloning and sequencing confirmed the successful point mutation, yielding MYB123. mut Specifically, it involves mutating only the MYB123 sequence (5'-CCTGGGCGAACAGACAATGAAA-3') to MYB123. mut The (5'-CCGGGCAGGACCGATAATGAGA-3') sequence. MYB123 and MYB123 mut The nucleotide sequences differ, but the amino acid sequences are exactly the same, and the function of the target gene MYB is not altered.

[0062] The nucleotide sequence of the MYB123 gene is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. MYB123 mut The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.4.

[0063] 3. Dual-luciferase reporter gene assay

[0064] The target site recognized by miR858a was fused into the 3'UTR region of the RLUC fluorescent reporter gene, and then co-transfected with miR858a to construct pGreenII-62-SK-miR858, pGreenII 800-miRNA-MYB123-LUC, and pGreenII 800-miRNA-MYB123. mut -LUC transient expression vector was detected using a dual-luciferase reporter gene assay kit (Vazyme).

[0065] 1) Construction of pGreenII-62-SK-miR858 transient expression vector

[0066] The precursor sequence of miR858 (SEQ ID NO.5) was obtained using a gene synthesis method.

[0067] The miR858 precursor sequence was recovered by gel extraction using 1.5% agarose gel electrophoresis and then ligated into the pGreenII-62-SK vector by enzyme digestion (BsaI / Eco31I).

[0068] The linearized vector and the recovered fragment digestion product were mixed and purified using a PCR purification kit for the next ligation reaction. The ligation product was then transformed into competent cells. 5-10 μL of the ligation product was transformed into competent *E. coli* cells and then into kanamycin-resistant agar plates. The cells were incubated at 37°C for 12 hours, followed by plaque PCR identification. Ten plaques were picked and simultaneously inoculated into 1.5 mL EP tubes for PCR identification. The primer sequences for pGreenII-62-SK-miR858 identification are shown below:

[0069] 62SK-F: 5'-tctccactgacgtaagggatg-3';

[0070] 62SK-R: 5'-caacacatgagcgaaaccc-3'.

[0071] The target band is a fragment of approximately 322 bp. Take 100 μL of bacterial culture corresponding to 1-3 positive bands for sequencing, and inoculate the remaining 400 μL of bacterial culture into 5-10 mL of kanamycin-resistant LB broth. Shake the tubes and wait for the sequencing results. Extract the plasmid from the tube corresponding to the correctly sequenced band. The pGreenII-62-SK-miR858 vector map is shown below. Figure 2 As shown.

[0072] 2) Construct pGreenII 0800-miRNA-MYB123-LUC and pGreenII 0800-miRNA-MYB123 mut -LUC transient expression vector

[0073] Using homologous recombination and the Golden Gate seamless cloning method, MYB123 and MYB123 were cloned respectively. mut The sequence was constructed in the pGreenII 0800-miRNA expression vector, with the target gene sequence ligated between LUC and 35S, and ligated using NcoI restriction enzyme digestion. After successful sequencing, the plasmid was extracted. For specific methods, refer to step 1) for the steps related to constructing the pGreenII-62-SK-miR858 transient expression vector.

[0074] 3) Protoplast preparation and transformation

[0075] Arabidopsis seedlings were cultured at around 25℃ for 25-30 days. Leaf strips were cut into 0.5-1 mm wide pieces and 5-10 mL of enzymatic hydrolysis solution (1.5% Cellulase R10, 0.75% Macerozyme R10, 0.6 M Mannitol and 10 mM MES (pH 5.7), adjusted to pH 5.8, and finally ddH2O was added to 100 mL) were added. The tissue was completely soaked and incubated at 24℃ for 4 h. After filtration through a 40 μm filter, the tissue was centrifuged at 300 rpm for 3 min and the supernatant was discarded. The tissue was washed twice with 10 mL of pre-cooled W5 solution, centrifuged at 300 rpm for 3 min, centrifuged at room temperature, and the supernatant was discarded to obtain Arabidopsis leaf mesophyll protoplasts. Take 100 μL of protoplasts, add 500 μL of MMG solution to suspend them, and then examine them under a microscope: approximately 20-40 protoplasts per field of view under 40x magnification. Take 100 μL of protoplast suspension + 10 μL of plasmid DNA from the transient expression vector (purified plasmid of 500 ng or more), and add 110 μL of PEG solution equal to the sum of the volumes of DNA and protoplasts. Gently mix and incubate at room temperature for 30 min. Terminate the reaction by diluting with 1 mL of W5. Centrifuge at 300 rpm for 3 min to collect the protoplasts and discard the supernatant. Wash 1-2 times with 1 mL of W5. Finally, add 1 mL of W5 solution and incubate in the dark at 28°C for 18-24 h.

[0076] 3) Fluorescence value detection

[0077] Cell lysis: Collect protoplasts by centrifugation, add 100 μL of 1×Cell Lysis Buffer, incubate at room temperature for 5 min with standing or shaking, pipette and aspirate the cell lysis product into a 1.5 mL centrifuge tube, centrifuge at 12000 g for 2 min at room temperature, and collect the supernatant for subsequent detection. Firefly luciferase reaction assay: Add 100 μL of Luciferase Substrate equilibrated to room temperature to a detection tube or microplate, carefully aspirate 20 μL of cell lysis supernatant into the detection tube or microplate well, mix rapidly, and immediately detect Firefly luciferase reporter gene activity using a fluorescence detector. Renilla luciferase reaction assay: Add 100 μL of freshly prepared Renilla substrate working solution to the above reaction solution, mix rapidly, and immediately detect Renilla luciferase reporter gene activity using a fluorescence detector. When using Renilla luciferase as an internal control, the value obtained by measuring firefly luciferase is divided by the value obtained by measuring Renilla luciferase, and the experimental result is Luc / Ren.

[0078] The results are shown in Table 1. The LUC / Ren ratio in the experimental group was significantly lower than that in the control group, indicating that the miRNA can bind to the target gene and inhibit the expression of the MYB123 gene, as evidenced by the decreased fluorescence ratio. In contrast, the fluorescence intensity remained essentially unchanged in the control group, indicating that the miRNA did not bind to the target gene. In conclusion, these results further confirm that MYB123 is a target gene of miR858.

[0079] Table 1. Results of dual-luciferase reporter gene assay for miR858-MYB123

[0080]

[0081]

[0082] Example 3

[0083] 1. Clone MYB123 mut

[0084] Primers were designed using Oligo 7.0 software (Table 2), and MYB123 was cloned using TakaRa's high-speed, high-fidelity PCR enzyme PrimeSTAR Max DNAPolymerase. mut Open reading frame (ORF) sequence. Primer sequences are shown below:

[0085] MYB123 mut -F:5'-CATTTCATTTGGAGAGGACAGGGTACCATGGGTAGAG GTCCTCGG-3',

[0086] MYB123 mut -R: 5'-CGCATATCTCATTAAAGCAGGGAATTCACTCAGCATGA CATTTAGATCATT-3'.

[0087] The PCR reaction system consisted of: 25 μL PrimeSTAR Max enzyme, 1 μL front primer, 1 μL back primer, 1 μL cDNA, and 2 μL ddH2O.

[0088] The PCR reaction program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 15 s, 35 cycles; 72℃ for 3 min; 8℃ for permanent incubation.

[0089] MYB123 was analyzed by 1.5% agarose gel electrophoresis. mutThe ORF sequence fragments were excised under UV light and purified using a BioTeKe rapid agarose gel DNA recovery kit. The purified product was then ligated to a vector using the TSINGKE pClone007 Blunt Vector Kit and transformed into competent *E. coli* cells. After a brief recovery period, the cells were plated on ampicillin-resistant medium and incubated overnight at 37°C. Single colonies were then selected. Colony PCR was performed using TSINGKE 2×T5 Super PCR Mix (Colony), and positive colonies were sent to TSINGKE Biotechnology Co., Ltd. for sequencing verification.

[0090] The final sequencing yielded Blackberry MYB123. mut The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.4.

[0091] 3. Construction of the MYB123 gene expression vector and transformation of Agrobacterium tumefaciens

[0092] The plasmid returned from the company was used to amplify the MYB123 gene using TakaRa's high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase. The 50 μL PCR system consisted of: 25 μL PrimeSTAR Max enzyme, 1 μL each of the front and rear primers, 1 μL cDNA, and 22 μL ddH2O. The PCR program was: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 15 s, 35 cycles; 72℃ for 3 min; and incubation at 8℃. The MYB123 sequence fragment was excised by 1.5% agarose gel electrophoresis under UV light and purified using the BioTeKe rapid agarose gel DNA recovery kit. The DNA concentration was then determined. A vector was constructed using TakaRa's 10×Buffer enzyme. The 50 μL PCR system consisted of: 5 μL 10×Buffer enzyme, 1 μL each of the front and rear rapid digestion enzymes, DNA ≤1 μg, and ddH2O. The PCR program was: 37℃ for 3 h. The vector fragments were excised by 1.5% agarose gel electrophoresis under UV light and purified using the BioTeKe Rapid Agarose Gel DNA Recovery Kit. The vector concentration was then determined. Ligation was performed using 2x Uniclone Seamless Cloning Mix enzyme. The 10 μL reaction mixture consisted of: 5 μL 2x Uniclone Seamless Cloning Mix, X μL vector (50-200 ng), Y μL gene fragment (10-200 ng), and 5-X-Y μL ddH2O. The reaction program was 50℃ for 30 min. The recombinant product was transformed into E. coli competent cells (Efficom 5α Chemically Competent Cell, TSINGKE Biotechnology). After a brief recovery period, the cells were plated on kanamycin-resistant medium and incubated overnight at 37℃. Single colonies were then selected. Colony PCR was performed using TSINGKE 2×T5 Super PCR Mix (Colony). Positive colonies were sent to TSINGKE Biotechnology Co., Ltd. for sequencing verification.

[0093] The plasmid returned by the company was transformed into Agrobacterium competent cells (Efficom GV3101 Chemically Competent Cell, Jinsheng Biotechnology). After a brief recovery period, the cells were plated on media resistant to kanamycin and rifampin, and incubated overnight at 28°C. Single colonies were then selected. PCR validation of the bacterial culture was performed using TSINGKE's 2×T5 Super PCR Mix (Colony). Positive bacterial cultures were sent to TSINGKE Biotechnology Co., Ltd. for sequencing validation.

[0094] 4. Construction of miR858 gene expression vector and transformation of Agrobacterium tumefaciens

[0095] The miR858 precursor plasmid returned by the company was used to amplify the miR858 gene using TakaRa's high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase. The 50 μL PCR system consisted of: 25 μL PrimeSTAR Max enzyme, 1 μL each of the front and rear primers, 1 μL cDNA, and 22 μL ddH2O. The PCR reaction program was: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 15 s, 35 cycles; 72℃ for 3 min; and incubation at 8℃. The miR858 precursor sequence fragment was excised by 1.5% agarose gel electrophoresis under UV light and purified using the BioTeKe rapid agarose gel DNA recovery kit. The DNA concentration was then determined. A vector was constructed using TakaRa's 10×Buffer enzyme. The 50 μL PCR system consisted of: 5 μL 10×Buffer enzyme, 1 μL each of the front and rear rapid digestion enzymes, DNA ≤ 1 μg, and ddH2O. PCR reaction procedure: 37℃ for 3 hours. The vector fragment was excised by 1.5% agarose gel electrophoresis under UV light and purified using the BioTeKe rapid agarose gel DNA recovery kit. The vector concentration was then determined. Ligation was performed using 2xUniclone Seamless Cloning Mix enzyme. The 10μL reaction mixture consisted of: 5μL 2xUniclone Seamless Cloning Mix, XμL vector (50-200ng), YμL gene fragment (10-200ng), and 5-X-YμL ddH2O. Reaction procedure: 50℃ for 30 minutes. The recombinant product was transformed into E. coli competent cells (Efficom 5α Chemically Competent Cell, Jinsha Biotechnology). After a brief recovery period, the cells were plated on kanamycin-resistant medium and incubated overnight at 37℃. Single colonies were then selected. TSINGKE's 2×T5 Super PCR Mix (Colony) was used for bacterial culture PCR verification. Positive bacterial cultures were sent to TSINGKE Biotechnology Co., Ltd. for sequencing verification.

[0096] The plasmid returned by the company was transformed into Agrobacterium competent cells (Efficom GV3101 Chemically Competent Cell, Jinsheng Biotechnology). After a brief recovery period, the cells were plated on media resistant to kanamycin and rifampin, and incubated at 28°C for 2 days. Single colonies were then selected. PCR validation of the bacterial culture was performed using TSINGKE's 2×T5 Super PCR Mix (Colony). Positive bacterial cultures were sent to TSINGKE Biotechnology Co., Ltd. for sequencing validation.

[0097] 5. Construct MYB123 mut(MYB123M) gene expression vector (disabling miR858 regulation) and transformation of Agrobacterium.

[0098] Return the company to MYB123 mut The miR858 gene was amplified using TakaRa's high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase. The 50 μL PCR system consisted of: 25 μL PrimeSTAR Max enzyme, 1 μL each of the primers, 1 μL cDNA, and 22 μL ddH2O. The PCR program was: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 15 s, 35 cycles; 72℃ for 3 min; and incubation at 8℃. The miR858 gene was amplified by 1.5% agarose gel electrophoresis. mut The electrophoretic fragments of the target DNA sequence were excised under UV light and purified using the BioTeKe Rapid Agarose Gel DNA Recovery Kit. DNA concentration was determined. A vector was constructed using TakaRa's 10×Buffer enzyme. The 50 μL PCR system consisted of: 5 μL 10×Buffer enzyme, 1 μL each of upper and lower fast digestion enzymes, DNA ≤ 1 μg, and ddH2O. The PCR reaction program was 37℃ for 3 h. The vector fragments were excised under UV light using 1.5% agarose gel electrophoresis and purified using the BioTeKe Rapid Agarose Gel DNA Recovery Kit. Vector concentration was determined. Ligation was performed using 2xUniclone Seamless Cloning Mix enzyme. The 10 μL reaction system consisted of: 5 μL 2xUniclone Seamless Cloning Mix, X μL vector (50-200 ng), Y μL gene fragment (10-200 ng), and 5-X-Y μL ddH2O. Reaction procedure: 50℃ for 30 min. The recombinant product was transferred into E. coli competent cells (Efficom 5α Chemically Competent Cell, Jinsheng Biotechnology). After a brief recovery period, the cells were plated on kanamycin-resistant medium and incubated overnight at 37℃. Single colonies were then selected. Colony PCR was performed using TSINGKE's 2×T5 Super PCR Mix (Colony). Positive colonies were sent to TSINGKE Biotechnology Co., Ltd. for sequencing verification.

[0099] The plasmid returned by the company was transformed into Agrobacterium competent cells (Efficom GV3101 Chemically Competent Cell, Jinsheng Biotechnology). After a brief recovery period, the cells were plated on media resistant to kanamycin and rifampin, and incubated at 28°C for 2 days. Single colonies were then selected. PCR validation of the bacterial culture was performed using TSINGKE's 2×T5 Super PCR Mix (Colony). Positive bacterial cultures were sent to TSINGKE Biotechnology Co., Ltd. for sequencing validation.

[0100] Example 4

[0101] 1. Genetic transformation of Arabidopsis thaliana

[0102] miR858 and its target genes MYB123 and MYB123 mut (MYB123M) (disabling miR858 regulation) was genetically transformed into Arabidopsis thaliana, with wild-type (WT) Arabidopsis thaliana as a control. Preliminary screening was performed using kanamycin, and the germination rate of the T2 generation Arabidopsis thaliana was statistically analyzed. Details are as follows:

[0103] Arabidopsis seeds were suspended in 0.05% agarose and placed in the dark at 4°C for 3 days to break dormancy. Then, they were placed in culture pots. Two weeks before sowing, the pots were covered with plastic wrap to maintain humidity. When the inflorescences reached 2-3 cm in length, they were cut off to encourage more inflorescence growth at the base. Transformation could then be performed 6-8 days later. Before transformation, flowers that had developed pods were removed to improve the transformation rate. Agrobacterium tumefaciens containing the prepared transformant gene was cultured (OD600 = 0.8-1.0). The bacterial suspension was centrifuged at 4000 rpm for 10 min at room temperature, and the bacterial cells were collected. The cells were resuspended in a freshly prepared 5% sucrose solution, and 0.02% Silwet was added to the resuspended solution and mixed immediately. Each inflorescence was soaked for 15 seconds. After soaking, the Arabidopsis seeds were wrapped in plastic wrap and placed in the dark at room temperature for 24 hours. The plastic wrap was then removed, and the plants were returned to the culture room for normal growth until the pods matured. Seeds were collected, and positive plants were selected.

[0104] The results are as follows Figure 3 As shown, compared with wild-type (WT) Arabidopsis, the miR858 transgenic Arabidopsis line had a higher germination rate and remained stable after 14 days, but the germination time was later. Figure 3 a). Compared with wild-type (WT) Arabidopsis thaliana, the MYB123 transgenic Arabidopsis thaliana has a lower germination rate ( Figure 3 b). Compared with wild-type (WT) Arabidopsis, the MYB123M transgenic Arabidopsis has a lower germination rate ( Figure 3 c).

[0105] 2. Determination of physiological indicators in transgenic Arabidopsis thaliana

[0106] 1) SPAD value measurement

[0107] The SPAD values ​​of transgenic Arabidopsis leaf tissues were determined using a SPAD instrument (TYS-4N type, Zhejiang Top Cloud Agriculture Technology Co., Ltd.).

[0108] The results are as follows Figure 4 As shown, the SPAD values ​​of transgenic Arabidopsis thaliana were significantly higher than those of WT. The SPAD value of the MYB123M transgenic line was even higher than that of the WT line. The leaves of the transgenic Arabidopsis thaliana were darker than those of the WT line, and the leaves of the MYB123M transgenic line were also darker.

[0109] 2) Determination of the content of main components

[0110] The protein content in the leaves of transgenic lines was detected using a plant protein assay kit (A045-2); the soluble sugar content (SSC) in the leaves of transgenic lines was determined using the anthrone colorimetric method, and the results are expressed as milligrams per gram of fresh weight (mg / g FW); the total phenol content in the leaves of transgenic lines was determined using a plant total phenol assay kit (A143-1-1); and the flavonoid content in the leaves of transgenic lines was determined using a flavonoid assay kit (A142-1-1).

[0111] The results are as follows Figure 5 As shown, the protein, soluble sugar, total phenolic, and flavonoid contents of the miR858 transgenic Arabidopsis thaliana were significantly lower than those of the WT group. In contrast, the protein, soluble sugar, total phenolic, and flavonoid contents of the MYB123 and MYB123M transgenic Arabidopsis thaliana were all significantly higher than those of the WT group. Among them, the MYB123M transgenic Arabidopsis thaliana had a higher overall total phenolic content. Figure 5 c) and the flavonoid content of MYB123M is slightly higher than that of MYB123 plants.

[0112] 3) Determination of key antioxidant indicators

[0113] The total superoxide dismutase (SOD) content in the leaves of transgenic plants was determined using the hydroxylamine method. SOD content was expressed in FW (U / g), where U was defined as the amount of SOD present in 1 mL of reaction solution at which 50% SOD inhibition was achieved per gram of tissue. The peroxidase (POD) and glutathione (GSH) contents in the leaves of transgenic plants were determined using a peroxidase assay kit (A084-3-1) and a glutathione assay kit (A006-1-1) (Nanjing Jiancheng Bioengineering Institute). The total antioxidant capacity of the leaves of transgenic plants was determined using the FRAP method and a total antioxidant capacity (T-AOC) assay kit (A015-3-1).

[0114] The results are as follows Figure 6As shown, the POD activity of MYB123M transgenic Arabidopsis thaliana was significantly higher than that of WT, while the POD activity of miR858 and MYB123 transgenic Arabidopsis thaliana was not significantly different from that of WT. Figure 5 a). The SOD activity of MYB123 and MYB123M transgenic Arabidopsis thaliana was higher than that of WT, while the SOD activity of miR858 transgenic Arabidopsis thaliana was significantly lower than that of WT. Figure 5 b). Compared to WT, the GSH content in miR858 transgenic Arabidopsis thaliana was decreased, while the GSH content in MYB123 and MYB123M transgenic Arabidopsis thaliana was higher than that in WT. Figure 5 c). The total antioxidant capacity of MYB123 and MYB123M transgenic Arabidopsis thaliana was significantly higher than that of WT, while the total antioxidant capacity of miR858 transgenic Arabidopsis thaliana was lower than that of WT. Figure 5 d).

[0115] In summary, the total phenols and flavonoids, as well as active components such as SOD and total antioxidant capacity, of miR858 transgenic Arabidopsis thaliana decreased compared with WT, while the total phenols and flavonoids, as well as SOD, GSH and total antioxidant capacity of MYB123 and MYB123M transgenic Arabidopsis thaliana increased significantly.

[0116] Example 4

[0117] SPAD values ​​and nitrogen content were determined using a SPAD instrument (TYS-4N type, Zhejiang Top Cloud-Agri Technology Co., Ltd.). The longitudinal diameter (length from the base to the apex) and transverse diameter (diameter at the widest point of the fruit) of the fruit were measured using vernier calipers. The weight of a single tomato fruit was measured using a balance accurate to 0.001 grams. Fruit firmness was determined using a KM-5 type hardness tester. Soluble solids were determined using a PAL-1 saccharimeter. Soluble sugar content (SSC) was determined using the anthrone colorimetric method, and the results are expressed in milligrams per gram of fresh weight (mg / g FW). Titratable acid content (TA) was determined by sodium hydroxide titration; the sugar-acid ratio = soluble sugar / titratable acid.

[0118] The plant protein detection kit (A045-2), flavonoid detection kit (A142-1-1), and total plant phenols detection kit (A143-1-1) from Nanjing Jiancheng Biotechnology Research Institute were used for determination. Anthocyanin content was determined according to the Li method. Fruit samples were ground into powder under liquid nitrogen, 1 g was weighed, added to the extraction solution (50% ethanol containing 0.1% formic acid), shaken well, and treated with ultrasound at 60 Hz for 20 min at 35℃. After centrifugation at 5000 rpm for 5 min, 0.3 mL of the supernatant was collected, 2.7 mL of pH=1 PBS was added, shaken well, and allowed to stand at room temperature in the dark for 20 min. The absorbance was measured at 510 nm, and the anthocyanin content (FW) (mg / g) was calculated. Total superoxide dismutase (SOD) content was determined using the hydroxylamine method. The unit of SOD content is FW (U / g), where U is defined as the amount of SOD corresponding to a 50% SOD inhibition rate per gram of tissue in 1 mL of reaction solution, which is one unit of SOD activity (U). The contents of peroxidase (POD) and glutathione (GSH) were determined using the peroxidase kit (A084-3-1) and glutathione assay kit (A006-1-1) from Nanjing Jiancheng Bioengineering Institute. The total antioxidant capacity of leaves and fruits of transgenic plants was determined using the FRAP method and the total antioxidant capacity (T-AOC) assay kit (A015-3-1).

[0119] 1. Analysis of gene expression levels in MYB123M transgenic tomatoes

[0120] MYB123M was genetically transformed into tomatoes to construct MYB123M transgenic tomato plants. Total RNA was extracted from tomato fruits at five developmental stages (S1: green fruit stage; S2: yellow fruit stage; S3: orange fruit stage; S4: orange-red fruit stage; S5: red fruit stage) using a Biotech RNA extraction kit, and cDNA was reverse transcribed using reverse transcription reagents from Shanghai Pudi Biotechnology Co., Ltd. The expression levels of the gene at different fruit developmental stages were detected by qRT-PCR. Sequence-specific primers and internal reference genes for each gene are shown in Table 2. The qRT-PCR amplification system was 15 μL, containing 1 μL of cDNA and 2X... Green ProTaq HS Oremix (ROX pius)* 17.5 μL, primers 0.6 μL each, ddH2O 5.3 μL. Amplification program: 95℃ 30s, 95℃ 5s, 60℃ 30s, 72℃ 15s, Melt 6s, for a total of 45 cycles. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0121] The results are as follows Figure 7 As shown, transgenic tomato lines with high gene expression levels in leaves were selected, namely MYB123M-1, MYB123M-3, and MYB123M-4.

[0122] 2. The appearance indicators of the fruits of the three selected transgenic lines were measured.

[0123] The results are shown in Table 2. Compared with WT, the MYB123M transgenic tomato fruit weight, longitudinal diameter and transverse diameter were significantly increased, while the firmness was reduced.

[0124] The results are as follows Figure 8 As shown, compared with CK, the MYB123M transgenic fruits at different stages were larger and had fewer seeds; the mature fruits were redder and larger, with fewer seeds and larger seeds.

[0125] Table 2. Determination of appearance indicators of transgenic tomato fruits

[0126] MYB123M-1 6.89±0.46Aa 20.69±1.82ABa 24.19±1.51Aa 5.24±0.27Ab MYB123M-3 6.51±0.21ABa 20.95±0.47ABa 23.16±0.05ABa 4.94±0.11Ab MYB123M-4 7.10±0.57Aa 22.7±1.87Aa 24.02±1.71Aa 5.60±1.01Aa CK(WT) 5.65±0.18Ca 19.57±0.66Ba 22.31±0.26ABa 5.79±0.79Aa

[0127] Note: Different lowercase letters represent significant differences at different time periods, and different uppercase letters represent significant differences between different strains. The difference level is P<0.05.

[0128] 3. Further determination of soluble solids, titratable acid, anthocyanins, total polyphenols and total antioxidant capacity of MYB123M transgenic tomato fruits.

[0129] The results are as follows Figure 9 As shown, the soluble solids content, titratable acid content, anthocyanin content, total phenol content, and total antioxidant capacity of MYB123M transgenic fruit were significantly higher than those of CK.

[0130] 4. The expression levels of key genes in the anthocyanin pathway of MYB123M transgenic tomato fruits were analyzed. Primer sequences are shown below:

[0131] SlPAL qRT-F: 5'-AACCTATCTCGTGGCTCTTT-3',

[0132] SlPAL qRT-R: 5'-TCTTTTTCGCTGAATCTTGC-3';

[0133] SlC4HqRT-F: 5'-CAACAGAAAGGAGAGATCAACGAG-3',

[0134] SlC4HqRT-R: 5'-CACAGCCTGAAGGTATGGAAGC-3';

[0135] Sl4CL qRT-F: 5'-ACACACAAAGGCTTAGTCACGA-3',

[0136] Sl4CL qRT-R:5'-AACAGAGGCAACACACACATCA-3';

[0137] SlCHS qRT-F:5'-TGGTCACCTGGAGGAGTATC-3',

[0138] SlCHS qRT-R:5'-GATCGTAGCTGGACCCTCTGC-3';

[0139] SlCHIqRT-F:5'-GTTTTTCACAAACCAACAGTTCTGAT-3',

[0140] SlCHIqRT-R: 5'-GAAGCAGTGCTCGATTCCATAAT-3';

[0141] SlF3HqRT-F:5'-CACACCGATCCAGGAACCAT-3',

[0142] SlF3HqRT-R:5'-GCCCACCAACTTGGTCTTGTA-3';

[0143] SlF3'HqRT-F:5'-GCACCAGAATGCACTTGC-3',

[0144] SlF3'HqRT-R:5'-CGTTAGTACCGTCGGCGAAT-3';

[0145] SlF3'5'HqRT-F:5'-GGCAATTGGACGAGATCCTG-3',

[0146] SlF3'5'HqRT-R:5'-AAGGAACCTCTCGGGAGTGAA-3';

[0147] SlFLSqRT-F:5'-GAGCATGAAGTTGGGCCAAT-3',

[0148] SlFLSqRT-R:5'-TGGTGGGTTGGCCTCATTAA-3';

[0149] SlDFR qRT-F:5'-TCCGAAGACGACAACGGTTT-3',

[0150] SlDFR qRT-R:5'-TGACAAGCCAAGAGCCGATAA-3';

[0151] SlANSqRT-F: 5'-GAACTAGCACTTGGCGTCGAA-3',

[0152] SlANSqRT-R:5'-TTGCAAGCCAGGCACCATA-3'.

[0153] The results are as follows Figure 10 As shown, the expression levels of SlPAL, SlC4H, SlCHS, SlF3H, and SlDRF genes in the fruits of MYB123M-1 were higher than those in the control (CK) at stages S2, S3, and S4. In the MYB123M-3 line, the expression levels of SlCHI and SlDRF were higher than those in the CK at stages S2, S3, and S4, while the expression levels of SlF3H, SlF3'H, SlF3'5'H, and SlFLS were higher than those in the CK at stages S2, S3, and S5. In the MYB123M-4 line, the expression levels of SlC4H, SlCHS, and SlF3'H genes were higher than those in the CK at stages S2, S4, and S5. This indirectly indicates that MYB123M can upregulate key structural genes in the anthocyanin pathway.

[0154] 5. Vacuum freeze-dry the mature MYB123M transgenic tomato fruits; grind them into powder using a ball mill (30Hz, 1.5min); then weigh 50mg of the powder and dissolve it in 500μL of extraction solution (50% methanol solution containing 0.1% hydrochloric acid); vortex for 5min, sonicate for 5min, centrifuge for 3min (12000r / min, 4℃), collect the supernatant, and repeat the operation once; combine the two supernatants, filter the sample through a microporous membrane (0.22μm pore size), and store it in a sample vial for LC-MS / MS analysis to determine the anthocyanin components of the MYB123M transgenic tomato fruits.

[0155] The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) (ExionLC™ AD, http: / / sciex.com.cn / ) and Tandem Mass Spectrometry (MSMS) (ExionLC™ AD, http: / / sciex.com.cn / ). 6500+, http: / / sciex.com.cn / ).

[0156] The main liquid chromatography conditions included: column: ACQUITY BEH C18 1.7μm, 2.1mm*100mm; mobile phase: phase A was ultrapure water (with 0.1% formic acid added), phase B was methanol (with 0.1% formic acid added); elution gradient: 0.00 min phase B ratio was 5%, 6.00 min increased to 50%, 12.00 min increased to 95%, held for 2 min, 14 min decreased to 5%, and equilibrated for 2 min; flow rate was 0.35 mL / min; column temperature was 40℃; injection volume was 2 μL.

[0157] The main mass spectrometry conditions included: an electrospray ionization (ESI) source at 550°C, a mass spectrometry voltage of 5500V in positive ion mode, and a curtain gas (CUR) pressure of 35 psi. In the Q-Trap6500+, each ion pair was scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0158] The results are as follows Figure 11 As shown, the anthocyanin content in mature MYB123M transgenic tomatoes was generally higher than that in the control (CK) group. Specifically, the contents of delphinidin, pelargonidin, malvidin, petunidin-3-O-(6-O-malonyl-β-D-glucoside), petunidin-3-(6-Op-p-coumaryl)-glucoside, petunidin-3,5-O-diglucoside, cyanidin-3,5-O-diglucoside, cyanidin-3-O-moribiglycoside, and cyanidin-3-O-xyloside in mature MYB123M transgenic tomatoes were all higher than those in the control (CK) group. In conclusion, the contents of most anthocyanin components in MYB123M transgenic tomatoes were higher than those in the control (CK) group.

[0159] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for increasing the anthocyanin content in Arabidopsis thaliana leaves or tomato fruits, characterized in that, Editing the MYB123 gene sequence to relieve the repressive function of miR858, thus obtaining MYB123. mut The MYB123 gene; its nucleotide sequence is shown in SEQ ID NO.1; the MYB123 gene... mut The nucleotide sequence of the gene is shown in SEQ ID NO.3; the specific steps include: 1) Construct MYB123 mut Gene expression vectors; 2) Transform the constructed expression vector into tomato or Arabidopsis thaliana; 3) Cultivate, screen, and obtain transgenic tomato or Arabidopsis plants with significantly increased anthocyanin content.

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