Method for constructing anthocyanin plant cell factory by using VdUFGT gene overexpression

Abstract

The invention belongs to the field of plant gene engineering, and particularly relates to a method for constructing an anthocyanin plant cell factory by using VdUFGT overexpression, a cloned vitis davidii VdUFGT gene is subjected to autogenetic transformation to obtain a VdUFGT high-expression and anthocyanin high-yield cell line, and the cell line remarkably improves the expression of anthocyanin biosynthesis key genes, so that the yield of anthocyanin is improved. And in combination with cell suspension culture, a high-yield anthocyanin plant cell factory is constructed. According to the method, the anthocyanin yield can be increased by 8.5 times and reaches 3.17%, the anthocyanin production period can be shortened to 12 days, the method has the advantages of being low in cost, short in period, high in efficiency, environmentally friendly and the like, and large-scale annual uninterrupted production of natural anthocyanin can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and specifically relates to a method for VxDV A method for constructing anthocyanin plant cell factories by gene overexpression. Background Art

[0002] Plant secondary metabolites are small molecule organic compounds produced by secondary metabolism and are one of the most important active substances in plants. Among them, anthocyanins belong to flavonoid compounds. Anthocyanins can combine with sugars with glycosidic bonds to form anthocyanins, which can exist more stably in various organs of plants, and then show color characteristics such as red, purple, and blue. Anthocyanins not only play an important role in plant growth and life activities, but also play an important role in human health. Anthocyanins have strong antioxidant, anti-tumor, anti-aging, anti-inflammatory, and vision improvement effects. Natural anthocyanins from plants are safe and non-toxic, and are widely used in the food and cosmetics industries.

[0003] Anthocyanins are present in the roots, stems, leaves, flowers and fruits of plants. The main source of utilization is currently extracted from plants, among which plant fruits are one of the most important ways to obtain anthocyanins. However, with the continuous increase in the cost of fruit planting, the selling price of fresh fruits is also rising, which seriously hinders the development and utilization of fruit anthocyanins. In addition, the utilization of plant-derived natural products faces mixed sources, different qualities, seasonality, and great environmental impact. There are also problems such as complex pre-extraction treatment, cumbersome steps, waste of resources, and environmental damage. In view of this, this application uses genetic engineering to construct anthocyanin high-yield cell lines, and further studies anthocyanin production based on cell engineering plant factories. This method is artificially controllable, not affected by seasonal environment, can be produced all year round, and is eco-friendly.

[0004] The synthesis of plant anthocyanins first synthesizes phenylalanine through the shikimic acid pathway, then synthesizes 4-coumaroyl-CoA through the phenylpropanoid pathway, and then enters the flavonoid and anthocyanidin metabolic pathway. The synthesis of flavonoids to anthocyanidins undergoes the action of multiple key enzymes, including UFGT It has the function of catalyzing anthocyanidins to form anthocyanidins, thereby making anthocyanidins more stably present in the vacuoles of plant cells.

[0005] The inventors used thorn grape cell UFGT By overexpressing the gene, we intend to construct a high-yield anthocyanin-producing Vitis thunbergii cell line, and produce anthocyanins in a plant factory by combining genetic engineering and cell engineering. This will seek an efficient alternative solution for the development and utilization of plant natural products to solve the current problems of high cost, low efficiency, and environmental and seasonal influences in the development and utilization of plant natural products. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a VxDV The method of constructing anthocyanin plant cell factory by gene overexpression realizes the efficient production of anthocyanins in plant cell factories. It has the advantages of low cost, short cycle, high efficiency, and environmental friendliness. It can produce all year round without being affected by seasonal environment.

[0007] The present invention is achieved in that: A use VxDV A method for constructing anthocyanin plant cell factories by gene overexpression, using VxDV Genes were used for autologous genetic transformation of thorny grape cells, making VxDV Gene overexpression, construction VxDV Overexpression of Vitis thornyii cell lines promotes anthocyanin accumulation and realizes anthocyanin production in cell factories.

[0008] Further, including UFGT The expression of several key genes for anthocyanin synthesis, including the flavonoids, was upregulated, and the accumulation of various flavonoid metabolites was significantly upregulated, among which the contents of anthocyanins and flavonoids were significantly increased.

[0009] Furthermore, the specific steps include VxDV Construction of gene plant expression vectors, genetic transformation, screening and identification of overexpression cell lines, and anthocyanin production in cell factories.

[0010] Furthermore, the VxDV The gene comes from Vitis thornysis and is constructed by enzyme ligation or homologous recombination VdUFGT-GFP Plant expression vector; specifically: VxDV Gene thorn grape, the target gene with the termination codon removed was constructed into pBI121 by restriction digestion-ligation or homologous recombination, and the construction was verified by colony PCR and sequencing. VdUFGT-GFP Recombinant plasmid.

[0011] Furthermore, the genetic transformation adopts the Agrobacterium-mediated method to transform the thorny grape cells, the Agrobacterium infection time of the thorny grape cells is 10-12 minutes, the total culture is 60-72 hours, and the screening is carried out in the resistance culture medium; Furthermore, the overexpression cell line was screened for resistance, and UFGT-GFP transformation was identified by fluorescence signal detection and DNA-PCR. UFGT The expression level of GFP was significantly increased, promoting the synthesis and accumulation of anthocyanins; specifically, the resistant callus cell line was first detected by a portable fluorescent excitation light source for GFP green fluorescence, and then DNA was extracted as a template and identified by PCR to obtain UFGT-GFP positive cell lines; the proliferation coefficient of the obtained positive cell lines was counted and the anthocyanin content was determined to screen for high anthocyanin production. VxDV Overexpressing cell lines.

[0012] Verification by transcriptomics, metabolomics, fluorescence quantitative PCR and anthocyanin content determination UFGT Expression accumulation characteristics of key genes and products in overexpressing cell lines.

[0013] Furthermore, in order to realize mass production of anthocyanin cell factories, an anthocyanin cell factory was constructed; the anthocyanin cell factory was carried out using liquid suspension cell culture, large-scale cell expansion and efficient synthesis of anthocyanins, the culture medium was MS basic medium, supplemented with 1.0 mg / L 2,4-D, the culture temperature was 25±2°C, and the rotation speed was 120-150 rpm.

[0014] The present invention has the following advantages: (1) The present invention realizes the efficient production of anthocyanins in plant cell factories. Overexpression of key genes for anthocyanin synthesis in Vitis thornyii cell lines UFGT The expression level was significantly improved, which promoted the expression of upstream genes and the accumulation of anthocyanin synthesis. A high-yield anthocyanin cell line was screened and obtained, and an anthocyanin synthesis cell factory was constructed by combining liquid suspension cell culture.

[0015] (2) The present invention solves the problems of waste of plant resources, difficulty in material pretreatment, high cost, long growth cycle, and great influence of seasons and environment in the extraction of anthocyanins from natural plants. The present invention can increase the anthocyanin yield by 8.5 times, reaching 3.17%. The anthocyanin production cycle can be shortened to 12 days. It has the advantages of low cost, short cycle, high efficiency, and environmental friendliness. It can be produced all year round without being affected by seasonal environment, providing a new perspective for the development and utilization of natural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0017] Figure 1 The thorn grape of the present invention VxDV Schematic diagram of gene cloning and vector construction; A: VxDV Gene amplification electrophoresis; B: Colony PCR verification of gene cloning; C: VdUFGT-GFP Schematic diagram of vector construction; D: pBI121 expression vector and VxDV Enzyme electrophoresis pattern; E: VdUFGT-GFP Colony PCR verification of vector construction results.

[0018] Figure 2 The Vitis thornyii cell of the present invention VxDVSchematic diagram of genetic transformation, cell proliferation and metabolite determination; A: genetic transformation process of Vitis thornyii cells; B: fluorescence detection of Vitis thornyii transformed cell lines; C: ecological status of positive cell lines; D: subcellular localization observation of positive cells; E: DNA-PCR electrophoresis detection map of transformed cell lines; F: cell proliferation coefficient; G: anthocyanin content; H: total flavonoids content.

[0019] Figure 3 For the present invention VxDV Schematic diagram of liquid culture of overexpression cell lines and analysis of gene differential expression; A: liquid suspension cell culture of wild-type and overexpression cell lines; B: fluorescence microscopy observation of overexpression cell lines; C: statistical analysis of differentially expressed genes; D: volcano plot of differentially expressed genes; E: KEGG enrichment analysis of differentially expressed genes; F: KEGG enrichment analysis of differentially upregulated genes.

[0020] Figure 4 Schematic diagram of quantitative expression analysis of key genes for anthocyanin synthesis of the present invention; wherein, A: anthocyanin synthesis pathway and key gene expression characteristics; B: anthocyanin synthesis key gene expression heat map.

[0021] Figure 5 For the present invention VxDV Schematic diagram of differentially accumulated metabolites analysis in overexpression cell lines; A: statistics of differentially accumulated metabolites; B: KEGG enrichment analysis of differentially accumulated metabolites; C: statistics of differential flavonoid metabolites; D: clustering heat map of differential flavonoid metabolites.

[0022] Figure 6 For the present invention VxDV Schematic diagram of liquid culture and content determination of overexpression cell lines; A: liquid culture of overexpression cell lines at different culture stages; B: microscopic observation of cell lines at different culture stages; C, D, E: anthocyanin, total flavonoids and proanthocyanidin contents at different culture stages.

[0023] Figure 7 The figure is a schematic diagram of the production and application of the anthocyanin plant cell factory of the present invention; wherein A, B, C, D, E, F, G: cell culture, collection, anthocyanin extraction, purification and drying; H: anthocyanin content; I: color reaction of anthocyanin solution at different pH values. DETAILED DESCRIPTION

[0024] The present invention relates to a method of utilizing VxDV The method of overexpression to construct anthocyanin cell factory of Vitis thornysis is described below in combination with the attached Figure 1-7The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0025] 1. Thorn Grape VxDV Cloning and vector construction (1) VxDV clone The cell line materials of Vitis thornii that had grown for 20 days were collected and the total RNA extraction kit of polysaccharide and polyphenol plants was used. The RNA that passed the integrity test, quality and concentration test was used for cDNA synthesis, and the EasyScript® One-Step gDNA Removal and cDNASynthesis SuperMix (AE311-02) kit of Beijing Quanshijin Biotechnology Co., Ltd. was used for reverse transcription. Using the cDNA of Vitis thornii as a template and the reference genome sequence information of Vitis thornii as the basis, PCR primers were designed for Vitis thornii. UFGT For gene cloning, the amplified product was recovered and connected to the pLB cloning vector, transformed into E. coli, and then electrophoresed and sequenced for verification. The primers for gene cloning and vector construction are shown in Table 1.

[0026] Table 1 VxDV Primer sequences for cloning and vector construction

[0027] Using Vitis thornyii cDNA as template UFGT Gene cloning, PCR amplification results are as follows Figure 1 As shown in A, the target product fragment is consistent with the expectation, about 1500 bp. After further recovery and purification, vector ligation and colony PCR, the electrophoresis results are shown in Figure 1 B, The PCR-positive clones were sequenced, and the results showed that the cloned sequence was 1469 bp in length, containing an open reading frame of 1371 bp, encoding 456 amino acids.

[0028] The cloned VxDV The gene sequence was constructed into the pBI121-GFP plant expression vector by homologous recombination. Bam HI enzyme and X I enzyme was used to double-digest the pBI121-GFP vector to obtain a linear vector, and the homology arm sequence of the vector was added. VxDV Specific primers are shown in Table 1. VxDV The cloned sequence was used as a template for PCR amplification, and the vector was connected and transformed according to the instructions. The constructed vector was verified by PCR and sequencing. The extracted plasmid was transformed into Agrobacterium GV3101, and the grape was UFGT Overexpression was carried out by Agrobacterium-mediated genetic transformation. The resuspended bacterial solution was infected for 10-12 min, and the co-culture was carried out for about 60 h after the filter paper was dried. The resuspended bacterial solution was MS+3% sucrose+10 μM AS, pH5.8, the co-culture medium was MS+10 μM AS+3% sucrose+0.6% agar powder, pH5.8, and the resistance screening medium was MS+ 1.0 mg / L 2,4-D + 50 mg / L Kan + 100 mg / L Tim + 0.6% agar powder + 3.0% sucrose, pH5.8.

[0029] like Figure 1 As shown in C, the target gene was constructed into the pBI121 plant expression vector to obtain the UFGT-GFP fusion expression recombinant plasmid, and the vector was double-digested and homology arm sequences were added. UFGT Electrophoresis of gene amplified fragments Figure 1 D, the results of the homologous recombination vector colony PCR are shown in Figure 1 E, and was finally verified by sequencing.

[0030] UFGT The results of genetic transformation are shown in Figure 2 A. Most of the cells of V. thornii cells after co-culture turned brown under the selection pressure of kanamycin, but some resistant callus tissues still grew. The resistant cells were able to grow and proliferate normally when transferred to the new resistant medium, and showed green fluorescent protein under fluorescent light, indicating that positive transformed cells were screened. Four positive cell lines were screened as follows Figure 2 B. When the subculture time is short, the cell viability is strong and the fluorescence intensity is high. As the culture time increases, some cells age and the fluorescence weakens or disappears. Subcellular observation shows that there is a significant fluorescence signal in the cytoplasm ( Figure 2 D), indicating VxDV Localized in the cytoplasm. The transformed cell lines were observed and photographed with the help of a portable fluorescent excitation light source LUYOR-3415. Cell lines with green fluorescent characteristics were initially judged as transformation-positive cell lines. Subsequently, genomic DNA was extracted and PCR and sequencing were performed for verification ( Figure 2 E), the positive cell lines were further screened for 2-3 generations, the concentrations of antibiotics and antibacterial agents in the culture medium were successively reduced, and finally the positive V. thornysis cells were transferred to MS solid medium containing 1.0 mg / L 2,4-D for subculture.

[0031] (2) UFGTEffects of overexpression on cell growth and accumulation of secondary metabolites in Vitis thornyii The phenotypic observation and growth determination of each transgenic cell line of Vitis thornii after 25 days of growth were carried out, and the contents of flavonoids, anthocyanins and proanthocyanidins were determined. Metabolites of Vitis thornii cells after 25 days of growth were determined. The growth status of wild-type cell lines and four transgenic positive cell lines was shown in Figure 2. Figure 2 C, different cell lines showed good growth status, proliferation coefficient ( Figure 2 F) The results showed that the proliferation coefficient of the vgt1 cell line was slightly lower than that of the other cell lines, but there was no significant difference in the proliferation coefficients among the five cell lines, all of which were above 23, indicating that UFGT Overexpression had no significant effect on the growth of thorny grape cells. The surface of the wild-type cell line and the transgenic cell line was red, but the red color of the transgenic cell line was deeper. The results of anthocyanin determination showed that ( Figure 2 G), the anthocyanin content of the overexpression cell lines was significantly higher than that of the wild-type cell line, among which the anthocyanin content of the vgt3 cell line reached 195.2 μg / g (FW), which was 4.2 times that of the wild-type cell line. However, the content of proanthocyanidins in the overexpression cell lines was significantly lower than that of the wild-type cell line ( Figure 2 H), the proanthocyanidin content of the wild-type cell line was 1262.2 μg / g (FW), and the proanthocyanidin content of the vgt3 cell line in the overexpression cell line was 965.93 μg / g (FW), UFGT Overexpression promoted and inhibited the accumulation of anthocyanins and proanthocyanidins, respectively, among which the vgt3 cell line had a stronger anthocyanin accumulation ability.

[0032] Transcriptome sequencing was commissioned to Beijing Biomike Biotechnology Co., Ltd. Since most grape cells failed to contact the culture medium under solid culture, grape cells could not obtain balanced nutrient supply, thus affecting the growth and metabolic activities of grape cells. Therefore, the thorny grape cells were further cultured in liquid culture. The growth status of wild-type and overexpression cell lines after 10 days of liquid culture is shown in Figure 3 A, At this time, wild-type cells appear light red, while vgt3 cells appear dark red. Observation of suspended cells under a fluorescence microscope shows that all cells show green fluorescence ( Figure 3 B), indicating UFGT The genes also have high activity. The results of transcriptome sequencing analysis of the two cell lines showed that there were 5113 differentially expressed genes in the two cell lines, of which 2509 were upregulated and 2604 were downregulated ( Figure 3 C, D). KEGG metabolic pathway enrichment display UFGT The pathways significantly enriched in the vgt3 overexpressing cell lines included pathways related to plant response to external conditions, signaling, and basic metabolism ( Figure 3 E), in addition to phenylpropanoid biosynthesis and flavonoid biosynthesis, therefore, UFGT Overexpression significantly affected the metabolism of phenylpropanoids and flavonoids in the target metabolic pathways. Further analysis revealed that among all metabolic pathways, the flavonoid biosynthesis pathway was the most significantly upregulated pathway ( Figure 3 F), in addition to phenylpropanoid and isoflavone metabolism, indicating UFGT Overexpression significantly promoted the target metabolism and significantly promoted the up-regulation of genes related to the target metabolic pathway. The clustering heat map results showed that most of the key genes for anthocyanin anthocyanin synthesis were up-regulated ( Figure 3 G), including CHS , CHI , F3H , F3'H , F3’ 5’H , DFR , UFGT etc., therefore UFGT Significantly promote the expression of key genes in anthocyanin synthesis.

[0033] Based on the transcriptome data and key genes of the target metabolic pathway, primers were designed for RT-qPCR experiments. UFGT Overexpression significantly promoted the expression of key genes for anthocyanin synthesis. RT-qPCR was further used to verify its expression pattern at different culture stages. The results showed that the gene expression characteristics were consistent with the transcriptome results. Compared with the wild-type cell line, the target genes of the vgt3 cell line were generally upregulated. UFGT The gene has strong expression activity in both cell lines, and both have the expression characteristics of first increasing and then decreasing ( Figure 4 ), the expression level in the vgt3 overexpressing cell line was more than 100 times that of the wild type. Among the 22 genes, 20 genes were up-regulated, and the genes with expression levels lower than the wild type included DFR (Vitvi03g01477) and ANR (Vitvi13g04712), but the difference in expression levels between the two was not as great as that of other members. The expression pattern of most of the key genes for anthocyanin synthesis showed a characteristic of first increasing and then decreasing, which was consistent with the overexpression of the target gene. UFGT Be consistent.

[0034] In order to improve the anthocyanin synthesis and accumulation efficiency of the Vitis thornyii cell line, the suspension cell culture was further carried out, and the suspension cells cultured for 25 days were used as materials. Beijing Biomark Biotechnology Co., Ltd. was commissioned to conduct metabolomics testing. UFGT Broad-target metabolomics analysis of the overexpressing cell lines showed that 1,175 metabolites were detected, of which 826 were differentially expressed, 441 were upregulated and 385 were downregulated ( Figure 5 A), KEGG metabolic pathway enrichment analysis showed that anthocyanin and flavonoid biosynthesis were significantly enriched pathways, and the metabolites involved included anthocyanins, proanthocyanidins, flavonoids, flavonols, chalcones, isoflavones, flavonols, flavanols, dihydroflavones, dihydroflavonols, etc. ( Figure 5 B, C), description UFGT Overexpression significantly affected the biosynthesis of anthocyanins and flavonoids. Further analysis of the relative content of flavonoid metabolites in different cell lines showed that the number of up-regulated metabolites of all types was higher than the number of down-regulated metabolites. The most differentially expressed metabolites were flavonoids, flavonols, and anthocyanins, which were 50, 34, and 33, respectively, and more than 90% of them were up-regulated metabolites ( Figure 5 D), UFGT Overexpression significantly promoted the synthesis and accumulation of flavonoid metabolites.

[0035] Transcriptome analysis showed UFGT Overexpression promotes the synthesis and accumulation of flavonoids. To further verify the accumulation characteristics of metabolites in overexpressed Vitis thornyii cells at different culture stages, the contents of anthocyanins, flavonoids and proanthocyanidins were determined. The growth status of Vitis thornyii cells at different culture stages is shown in Figure 2. Figure 6 A, When the culture was carried out for 8 days, the cells of V. thornii in the culture bottle were light red, and microscopic observation showed that some cells were light red, while other cells were not colored; as the culture time prolonged, the color of the transgenic V. thornii cells deepened at 10 days, and microscopic observation showed that most cells were colored ( Figure 6 B), and the color deepened; when it was cultured for 12 days, it turned dark red, at which time the accumulation of anthocyanins visible to the naked eye reached a high level, almost all cells were colored, and the color deepened again. The metabolite determination at the three culture stages showed that the content of anthocyanins, flavonoids and proanthocyanidins in the transgenic cell lines was significantly higher than that in the wild type. The anthocyanin content of the wild type cell line was between 14.3-29.4 μg / g (FW), but the transgenic vgt3 cell line was as high as 248.7-364.3 μg / g (FW), which was 10-19 times that of the wild type ( Figure 6 C). The flavonoid content in the two cell lines gradually increased with the extension of culture time, reaching the highest value at 12 days, which was 1116.1 and 4578.8 μg / g (FW), respectively. The flavonoid content in the vgt3 cell line was 4 times that of the wild-type cell line ( Figure 6 D). The accumulation pattern of proanthocyanidins in the two cell lines was first increased and then decreased. The accumulation levels reached the peak after 10 days of culture, which were 154.2 and 5325.2 μg / g (FW), respectively. At this time, vgt3 was 34 times that of the wild type ( Figure 6E). Therefore, vgt3 transgenic cells significantly increase the accumulation of metabolites such as anthocyanins, and in liquid culture, this accumulation has an amplification effect, laying a material foundation for the construction of anthocyanin plant cell factories.

[0036] 7. Based on UFGT Production and application of anthocyanin plant cell factories Liquid culture medium effectively promoted the accumulation of metabolites, and the high-yield cell line vgt3 was further applied to plant cell factories to produce natural products. Figure 7 As shown in Figure 2, a small grape cell factory for anthocyanin production was constructed. Grape cells were cultured in a triangular flask ( Figure 7 A), washing and collection ( Figure 7 B), extract ( Figure 7 C). The wild type anthocyanin concentration was 0.48 mg / g (DW), while the vgt3 cell line was 4.11 mg / g (DW). Anthocyanin increased 8.5 times ( Figure 7 H). Purification of anthocyanin solution using AB-8 macroporous resin ( Figure 7 D, E). Finally, the liquid is concentrated ( Figure 7 F) and vacuum freeze-dried to obtain anthocyanin purple powder ( Figure 7 G). Anthocyanin production of 0.50% and 3.17% was obtained in WT and vgt3 cell lines, respectively ( Figure 7 H). 10 mg of anthocyanin powder was further dissolved in 100 mL of ddH2O to obtain a 0.1 mg / mL anthocyanin solution. Anthocyanin solutions exhibited various color phenomena at different pH values ​​( Figure 7 I). When the pH values ​​are 1, 3, 5, 7, 9 and 11, the solution appears red, pink, light pink, purple, blue and green respectively. Therefore, different pH values ​​affect the color development effect of anthocyanins and have good activity.

[0037] In summary, the present invention clones the thorny grape VxDV Genes undergo autologous genetic transformation to obtain VxDV A high-expression and high-yield anthocyanin cell line, which significantly increases the expression of key genes in anthocyanin biosynthesis, thereby promoting the accumulation of anthocyanin and other metabolites, and combines cell suspension culture to construct a high-yield anthocyanin plant cell factory. UFGT Grape cell factories using genetic engineering and cell suspension culture can significantly increase anthocyanin production by 8.5 times to 3.17%. The anthocyanin production cycle can be shortened to 12 days. It has the advantages of low cost, short cycle, high efficiency, and environmental friendliness. It can realize large-scale, uninterrupted production of natural anthocyanins throughout the year and can be produced all year round without being affected by seasonal environment, providing a new perspective for the development and utilization of natural products.

[0038] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene, characterized in that: The VdUFGT gene was used for autologous genetic transformation of Vitis thornyii cells to overexpress the VdUFGT gene, construct a Vitis thornyii cell line overexpressing V. thornyii, promote the accumulation of anthocyanins, and realize the production of anthocyanins in cell factories.

2. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 1, characterized in that: Several key genes for anthocyanin synthesis, including the UFGT gene, were upregulated, and the accumulation of various flavonoid metabolites was significantly upregulated.

3. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 1, characterized in that: The specific steps include construction of VdUFGT gene plant expression vector, genetic transformation, screening and identification of overexpression cell lines, and anthocyanin production in cell factories.

4. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 3, characterized in that: The VdUFGT gene is derived from Vitis thunbergii, and a VdUFGT-GFP plant expression vector is constructed by enzyme cutting, connection or homologous recombination.

5. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 3, characterized in that: The genetic transformation adopts the Agrobacterium-mediated method, the Agrobacterium infection time of the thorn grape cells is 10-12 minutes, the total culture is 60-72 hours, and the screening is carried out in the resistance culture medium.

6. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 3, characterized in that: The overexpression cell line is screened for resistance, and UFGT-GFP transformation is identified by fluorescence signal detection and DNA-PCR method to obtain a UFGT-GFP positive cell line; the proliferation coefficient of the obtained positive cell line is counted and the anthocyanin content is determined to screen and obtain a VdUFGT overexpression cell line with high anthocyanin production.

7. The method for constructing anthocyanin plant cell factory by overexpressing VdUFGT gene according to claim 3, characterized in that: The anthocyanin cell factory adopts liquid suspension cell culture, large-scale cell expansion and efficient synthesis of anthocyanins. The culture medium is MS basic culture medium supplemented with 1.0 mg / L 2,4-D. The culture temperature is 25±2° C. and the rotation speed is 120-150 rpm.

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