A method for increasing resveratrol content in vitis thunbergii cells by gene editing and a constructed vitis thunbergii chs2 mutant cell line

Abstract

The present application belongs to the field of plant genetic engineering, and particularly relates to a method for increasing the content of resveratrol in Vitis davidii Foex by gene editing and a constructed Vitis davidii CHS2 mutant cell line. The chalcone synthase gene CHS2 of Vitis davidii is edited by CRISPR / Cas9 method to construct the Vitis davidii CHS2 mutant cell line. The present application can down-regulate the expression of the key gene of the target metabolic pathway and the accumulation of the metabolic product, indirectly promote the biosynthesis of resveratrol in the competitive metabolic pathway, and increase the content of resveratrol by 2-4 times. The present application solves the problems of high cost, limited plant resources, long growth cycle, seasonal change, uncontrollable environmental factors and the like in the synthesis of natural plant resveratrol. Meanwhile, the present application solves the problems of impurity pollution, toxic substance residue, high safety risk for eating and medicine, and difficult industrial waste discharge and treatment in chemical synthesis. The present application produces resveratrol by plant cell metabolic factory, and is low in cost, high in efficiency, safe and harmless, and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering. Specifically, it relates to inhibiting the expression of key genes in target metabolic pathways through plant gene editing, causing the target genes to lose or partially lose their function, thereby indirectly promoting the expression of key genes in competing metabolic pathways and increasing the accumulation of competing metabolites. More specifically, it relates to a method for increasing resveratrol content in grapevine cells through CRISPR / Cas9-based CHS2 gene editing. Background Technology

[0002] Resveratrol, a stilbene compound also known as 3,4',5-trihydroxystilbene, is an active non-flavonoid polyphenol with antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protective effects. Its production currently mainly involves plant extraction and chemical synthesis. Since resveratrol is found in a limited number of plants, plant extraction methods primarily utilize grapes, Polygonum cuspidatum, Veratrum nigrum, and peanuts. While some research has been conducted domestically and internationally on the extraction of resveratrol from natural plants, and products extracted from natural plants are safe and reliable, their extraction efficiency is low, costs are high, and they are limited by plant resources, growth cycles, seasonal variations, and environmental factors. Besides natural plant extraction, chemical synthesis is also used in industrial applications. Chemical synthesis is efficient and cost-controllable, but the synthesized products may contain impurities and toxic residues, posing high safety risks for food and pharmaceutical use. Furthermore, industrial waste discharge and treatment are difficult, resulting in low ecological and social benefits. Therefore, exploring more economical, ecological, and efficient synthesis methods is a crucial issue for the research and application of resveratrol products.

[0003] With the development of biotechnology, plant in vitro culture can be carried out in an artificially created, controllable culture environment. Based on cell engineering, the synthesis and production of active substances can be achieved year-round, unaffected by weather or seasons, thus avoiding the difficulties faced by natural plant extraction and chemical synthesis. The application of genetic engineering in genetic improvement allows for the regulation of specific metabolites, enabling the production of plant cell secondary metabolites. The biosynthetic pathways of flavonoids and resveratrol both originate from phenylpropane metabolism. 4-CoA and malonyl-CoA enter the flavonoid and resveratrol synthesis pathways respectively under the catalysis of chalcone synthase (CHS) and stilbene synthase (STS). CHS and STS compete for common substrates to participate in their respective metabolic processes. Given the competitive relationship between the biosynthetic pathways of flavonoids and resveratrol, this invention uses CRISPR / Cas9 gene editing (including gene knockout) on grape thorn cells to cause the CHS gene to lose or partially lose its function, thereby reducing the competition of the CHS gene for substrates in the grape thorn mutant cell line, indirectly promoting the metabolic process of competitive resveratrol, and thus increasing the resveratrol content in the grape thorn mutant cell line.

[0004] This invention provides a new research perspective for the biosynthesis of secondary metabolites through gene editing. The combination of genetic engineering and in vitro cell culture will further enable the production, development, and utilization of natural active substances such as resveratrol through plant metabolic factories. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for increasing the resveratrol content in grape thorn cells through gene editing and to construct a grape thorn CHS2 mutant cell line.

[0006] This invention is achieved through the following technical solution:

[0007] The present invention discloses a method for increasing the resveratrol content in grape thorn cells through gene editing. The method involves using the CRISPR / Cas9 method to edit the chalcone synthase gene CHS2 of grape thorn, thereby constructing a grape thorn CHS2 mutant cell line, which increases the resveratrol content by 2 to 4 times.

[0008] The CHS2 sequence is characterized by base insertions or base deletions.

[0009] In the *Vitis thunbergii* CHS2 mutant cell line, the expression of flavonoids and their key genes for biosynthesis was inhibited, while the expression of key genes for resveratrol biosynthesis was promoted. The contents of total flavonoids, anthocyanins, and proanthocyanidins were significantly reduced, while the contents of resveratrol were significantly increased by 2 to 4 times.

[0010] The specific steps include: CHS2 target site design, gene editing expression vector construction, genetic transformation, cell line culture screening, and gene editing identification.

[0011] Furthermore, the target site of the CHS2 gene sequence has a PAM structure (-NGG), the sgRNA sequence has high GC (above 40%), and sequences with consecutive pairings of more than 7 bp are avoided. The designed target sequence is located at the front of the second exon.

[0012] Further, the expression vector needs to include Cas9 and one or more sgRNA sequences. The steps include: constructing an sgRNA expression cassette using overlapping PCR, performing two rounds of PCR. In the first round, two reactions are performed to amplify the sgRNA expression cassette fragments merged into two fragments. In the second round, position-specific primers are used to amplify the expression cassette product. The sgRNA expression cassette is ligated to the CRISPR / Cas9 vector using the Golden Gatecloning method, with variable-temperature cycling for the enzyme digestion-ligation reaction: 37℃ for 10 min, 10℃ for 5 min, 20℃ for 5 min, 4 cycles; then 37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min, 10 cycles; and finally 37℃ for 5 min. The ligation product is transformed into E. coli, and bacteria are selected for PCR verification and sequencing identification. The recombinant expression vector with correct sequencing is extracted as a plasmid for subsequent genetic transformation.

[0013] Furthermore, the genetic transformation includes the following steps: transforming the constructed gene-editing expression vector with Agrobacterium; after positive screening, Agrobacterium is first subjected to PCR detection; after positive detection, plasmid is extracted and sent for sequencing; transforming Grapevine callus tissue with Agrobacterium-mediated transformation; after infecting Grapevine callus tissue with Agrobacterium resuspension for 8 min and co-culturing for 3 days, screening is performed on antibiotic-resistant medium to obtain positive callus tissue.

[0014] Furthermore, the gene editing identification process includes the following steps: using positive callus tissue selected from resistant culture medium as material, synthesizing primers approximately 200-600 bp apart from the target site for PCR amplification, and directly sequencing the PCR product. A double peak in the sequencing at the target site is a preliminary indication of successful targeting. Simultaneously, the PCR product is recovered, ligated into a cloning vector, transformed into *E. coli*, and positive single clones are selected for PCR detection and sequencing. Successfully edited CHS2 genes show base insertion, deletion, or substitution at the target site.

[0015] The present invention also provides a CHS2 mutant cell line of Grape thorn constructed by the method described above.

[0016] Significant advantages of this invention:

[0017] (1) By editing the key gene CHS2 in the flavonoid metabolism pathway, the CHS2 gene loses or partially loses its function, which indirectly promotes the metabolic process of the competitive resveratrol metabolism pathway, increases the synthesis and accumulation of resveratrol, and provides a new perspective for the production, development and utilization of natural active substances such as resveratrol through plant metabolic factories.

[0018] (2) This invention solves the problems of high cost, limited plant resources, long growth cycle, seasonal changes and uncontrollable environmental factors in the synthesis of natural plant resveratrol; at the same time, it solves the problems of impurity pollution, toxic substance residue, high food and medicine safety risks, and difficulty in industrial waste discharge and treatment in chemical synthesis; resveratrol is produced through plant cell metabolic factories, which has the advantages of low cost, high efficiency, safety and harmlessness, environmental friendliness and year-round uninterrupted production. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 The colony PCR identification of the CHS2 gene structure, target site, and sgRNA expression cassette construction is shown. (a) CHS2 gene structure and gene editing target site; (b) Colony PCR identification of the sgRNA expression cassette construction.

[0021] Figure 2 The following images show the CHS2 mutant cell line of *Vitis thornii* and the characteristics of the CHS2 sequence mutation. (a) Phenotype of the wild-type cell line (WT) of *Vitis thornii*; (b) Phenotype of the mutant cell line (MT) of *Vitis thornii*; (c) PCR detection of the CHS2 gene editing target mutation; (d) DNA sequencing verification of the CHS2 gene editing target mutation; (e) CHS2 gene editing target sequence mutation information; (f) Protein translation characteristics of the CHS2 mutant sequence.

[0022] Figure 3 This study presents a quantitative expression analysis of key genes involved in the biosynthesis of flavonoids and resveratrol.

[0023] Figure 4 The heatmap shows the bioaccumulation clusters of flavonoids and resveratrol.

[0024] Figure 5 The growth characteristics, flavonoid and stilbene content of *Vitis thornii* cell lines at different culture times are shown. (a) Wild-type and mutant *Vitis thornii* cell lines at different culture stages; (b) Determination of total flavonoids, anthocyanins, proanthocyanidins, resveratrol and spruce glycosides. Detailed Implementation

[0025] A method for increasing resveratrol content in grape aralia cells through gene editing is described in detail below with reference to specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention in any way.

[0026] I. Construction of CRISPR / Cas9-based gene editing vector for CHS2 in Grape thorn

[0027] 1. CRISPR / Cas9 gene editing CHS2 target site design: Target sites for the CHS2 gene were screened and designed based on the structural characteristics of PAM (NGG). The CHS2 gene of *Vitis piraceae* (GenBank accession number: OL906400) was used as the target sequence for CRISPR / Cas9 gene editing. The CRISPR / Cas9 gene editing method employed the pYLCRISPR / Cas9PUbi-H gene editing vector system.

[0028] 2. sgRNA Expression Kit Construction: The sgRNA expression kit was constructed using overlapping PCR. Two rounds of nested PCR were performed. The first round of PCR consisted of two reactions using primers UF / CHS2AtU3dT1-R and CHS2gRT1-F / gR-R. The second round used position-specific primers Pps-GGL / Pgs-GGR to amplify the expression kit product. Primers UF, gR-R, Pps-GGL, and Pgs-GGR were...

[0029] The universal primers for the pYLCRISPR / Cas9PUbi-H system and the primers used for constructing the expression cassette are shown in Table 1.

[0030] Table 1 Primer Information

[0031]

[0032] 3. Ligation of the target sgRNA expression cassette with the pYLCRISPR / Cas9 vector

[0033] Enzyme digestion and ligation reactions were performed using the Golden Gate cloning method. The reaction system was as follows: 1.5 μL 10×CutSmartBuffer, 1.5 μL 10 mM ATP, 1 μL Cas9 plasmid (60-80 ng), 0.5 μL sgRNA expression cassette product (10-15 ng), 0.5 μL Bsa I-HF, 0.3 μL T4 DNA ligase, and 9.7 μL water to a final volume of 15 μL. Enzyme digestion and ligation were performed using a variable temperature cycling method: 37℃ for 10 min, 10℃ for 5 min, 20℃ for 5 min, for 4 cycles; then 37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min, for 10 cycles; finally, 37℃ for 5 min. The target sequence was located at the front of the second exon (…). Figure 1 a) The constructed vector was analyzed by PCR and electrophoresis using an sgRNA expression cassette. (See attached image) Figure 1 b) The size of the target band matched the expectation and was finally verified by sequencing.

[0034] Genetic transformation and knockout detection of Grape bifidum cells

[0035] 1. Culture medium:

[0036] Agrobacterium resuspension: MS + acetylsuccinone (AS) 20 μM, with added 3.0% sucrose, pH 5.8;

[0037] Co-culture medium: MS medium with 100 μM acetylsyringone (AS) supplemented with 3.0% sucrose and 0.7% agar, pH 5.8;

[0038] Resistance selection medium: MS + 2,4-D 1.0 mg / L + hygromycin (HPT) 30 mg / L + termethin (Tim) 100 mg / L, supplemented with 3.0% sucrose and 0.7% agar, pH 5.8.

[0039] 2. Genetic transformation: The recombinant plasmid was transformed with Agrobacterium. The Agrobacterium was verified by PCR detection and sequencing. The Agrobacterium (OD) cultured overnight with shaking was then transformed. 600 After centrifugation (to a concentration of 0.8-1.0), resuspend and adjust OD. 600 Approximately 0.6, 20-day-old Gracilaria callus tissue was inoculated in resuspension for 8 minutes, filtered dry, and then co-cultured on co-culture medium for 2-3 days. After washing, the bacteria were inoculated onto resistant medium.

[0040] 3. Resistance screening and knockout detection: Transformed *Vitis thunbergii* callus was screened for hygromycin resistance. The phenotypes of wild-type and mutant cell lines are shown in the figure. Figure 2 The mutant cell line obtained in a / 2b was yellowish-green. After approximately 25 days of culture, pale red cells were found on the surface of the callus, while the wild-type cell line remained red during culture. The color of the cell lines changed significantly before and after CHS2 gene editing. DNA was extracted from both wild-type and mutant cell lines. Primers were designed 566 bp apart from the target site. PCR amplification was performed using the DNA as a template. Nested PCR primers were further designed, and the PCR products were directly sequenced. The mutation characteristics were analyzed based on the sequencing results. The results showed a bimodal pattern near the target sequence. Figure 2 d) indicates a mutation near the gene editing target site. The PCR product was further recovered and purified, ligated into a pLB cloning vector, transformed into *E. coli*, and positive single clones were selected for PCR detection. Figure 2 c) The results showed that some products exhibited a high-low distribution of electrophoretic bands, suggesting changes in electrophoretic migration caused by insertion or deletion fragments. Further sequencing was performed to verify the mutation characteristics; the sequencing results are as follows... Figure 2 The results showed that the mutated sequences mainly consisted of deletions and insertions, including four deletion types with deletion bases of 1, 3, 4, and 4 bp, and three insertion types with insertion bases of 1, 103, and 200 bp. Six of the seven mutant genotypes caused premature termination of CHS2 protein translation. Figure 2f), which can further lead to the loss or weakening of CHS2 gene function. The PCR primers and sequencing primers used for mutation detection are shown in Table 1.

[0041] Quantitative expression of key genes in the synthesis of three flavonoids and resveratrol

[0042] 1. RNA Extraction and cDNA Synthesis: RNA was extracted from *Grapevine* cells using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit and PrimeScript was employed. TM cDNA synthesis was performed using the RT reagent Kit with gDNA Eraser.

[0043] 2. Quantitative Real-Time PCR: Based on key gene information in the biosynthetic pathways of flavonoids and resveratrol, primers for quantitative real-time PCR were further designed. Primer information is shown in Table 2. The quantitative real-time PCR experiment was performed according to TB. Premix Ex Taq TM The method was performed, and amplification was carried out on the ROCHE480 platform.

[0044] Table 2 Primer information for quantitative real-time PCR

[0045]

[0046] 3. Data Analysis: Using α-Tubulin as an internal reference gene, through 2 -△△ct Perform gene transcription level calculations.

[0047] 4. Quantitative gene expression characterization analysis: Quantitative PCR results are as follows Figure 3 As shown, the expression levels of six key genes in resveratrol biosynthesis, STS, were significantly upregulated in the mutant cell line, while the expression levels of key genes in anthocyanin and proanthocyanidin biosynthesis in flavonoid metabolism, including CHS, F3H, F3'H, DFR, FLS, LDOX, GT1, and LAR, were significantly downregulated. Therefore, CHS2 gene editing led to the downregulation of CHS2 and other genes related to flavonoid biosynthesis in the mutant cell line, indirectly promoting the upregulation of STS in the competitive metabolic pathway (resveratrol biosynthesis).

[0048] Four metabolome assays and analyses

[0049] 1. Sample Extraction: The collected wild-type and mutant *Grape argus* callus samples were stored at -80℃ and freeze-dried under vacuum using a freeze dryer. The samples were then further ground into powder using a grinder at 30 Hz for 1.5 min. 0.1 g of the powder was extracted in 1.2 mL of 70% ethanol for 3 h with vortexing every 30 min. After overnight storage at 4℃, the samples were centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous organic filter and stored in the dark for UPLC-MS / MS analysis.

[0050] 2. Detection system: The detection system consists of ultra-high performance liquid chromatography (SHIMADZUNexeraX2) and tandem mass spectrometry (Applied Biosystems QTRAP).

[0051] 3. Liquid phase conditions:

[0052] Chromatographic column: Agilent SB-C18 column (2.1 mm × 100 mm, 1.8 μm);

[0053] Mobile phase: Phase A is ultrapure water (containing 0.1% formic acid), and Phase B is acetonitrile (containing 0.1% formic acid);

[0054] Elution gradient: The proportion of phase B is 5% at 0.00 min, increases linearly to 95% within 9.00 min and is maintained at 95% for 1 min, and decreases to 5% between 10.00 and 11.10 min, and is equilibrated at 5% until 14 min.

[0055] Flow rate: 0.35 mL / min; column temperature: 40 °C; injection volume: 4 μL.

[0056] 4. Mass spectrometry conditions: A triple quadrupole linear ion trap mass spectrometer (Q TRAP)-AB6500 Q TRAPUPLC / MS / MS was used for scanning, and metabolite quantification was performed using the multiple reaction monitoring (MRM) mode of the triple quadrupole mass spectrometer.

[0057] 5. Analysis of the metabolic accumulation characteristics of flavonoids and stilbene compounds: Based on the differential expression fold (FC>1.5), p-value (p-value<0.05), and VIP value (VIP>1), a total of 310 differentially accumulated metabolites were detected in the *Vitis pilosa* mutant cell line compared with the wild-type cell line. Among them, 169 metabolites were upregulated and 141 metabolites were downregulated. Further clustering and comparative analysis of flavonoid and resveratrol metabolism-related metabolites in the target metabolic pathway was performed, and the results are as follows: Figure 4The results showed that, compared with the wild-type grape thorn cell line, the resveratrol derivative content in the mutant cell line MT was significantly increased, while the accumulation of flavonoids such as anthocyanins, proanthocyanidins, and quercetin was inhibited. Therefore, the CHS2 gene editing led to the inhibition of flavonoid metabolite biosynthesis, while the accumulation of resveratrol derivatives in the competitive resveratrol synthesis pathway was significantly promoted.

[0058] Determination of the content of five types of flavonoids and stilbene compounds

[0059] 1. Samples for determination: Wild-type Vitis thunbergii cell line WT and mutant cell line MT cultured for 20, 30, and 40 days were used as materials. Figure 5 a) The contents of total flavonoids, anthocyanins, proanthocyanidins, resveratrol and spruce glycosides were determined.

[0060] 2. Flavonoid Determination: Take an appropriate amount of fresh callus tissue, grind it into powder under liquid nitrogen, weigh 0.5g of powder sample into 4mL of 80% ethanol, and perform ultrasonic-assisted extraction at 800W ultrasonic power, 30℃ temperature, and 40min time (oscillation once every 10min). Centrifuge at 4000rpm for 10min, collect the supernatant, repeat the extraction once with the residue, combine the supernatants, use rutin as a standard to plot a standard curve, and determine the total flavonoid content using the sodium nitrite-aluminum nitrate colorimetric method.

[0061] 3. Anthocyanin determination: Take an appropriate amount of fresh callus tissue, grind it into powder under liquid nitrogen, weigh 0.5g of the powder sample and extract it in 4mL of 80% formic acid ethanol (2% formic acid) solution. Under the same ultrasonic conditions as for anthocyanin determination, centrifuge at 4000rpm for 10min, collect the supernatant, and repeat the extraction once with the residue. Combine the supernatants and prepare samples with gradient concentrations using cyanidin-3-glucoside as a standard. Measure the absorbance at wavelengths of 530, 620, and 650nm using an ELISA reader, and apply the Greey equation.

[0062] OD λ =(OD) 530 -OD 620 -0.1(OD) 650 -OD 620 Based on the absorbance and concentration, a standard curve was plotted, and the anthocyanin content was further calculated.

[0063] 4. Determination of proanthocyanidins: The extraction method of proanthocyanidins is the same as that of total flavonoids. Proanthocyanidins are used as a standard to prepare gradient concentrations. The absorbance is measured at 546 nm and determined by the n-butanol-hydrochloric acid method.

[0064] 5. Determination of resveratrol and spruce glycosides: Take an appropriate amount of fresh callus tissue, grind it into powder under liquid nitrogen, weigh 0.5g of sample, add 4mL of 80% methanol aqueous solution, extract by ultrasonication at 30℃ for 30min, centrifuge at 4000rpm for 15min at 320W power, collect the supernatant, repeat the extraction of the residue once, combine the supernatants, filter the supernatant through a 0.22μm microporous organic filter membrane and store it in the dark. Liquid chromatography conditions: Thermo Scientific UltiMate 3000 ultra-high performance liquid chromatograph, Thermo Scientific Acclaim 120 C18 column (4.6×250mm, 5μm, 120A), mobile phase A: methanol, B: ultrapure water, A:B ratio 40:60, injection volume 10μL, flow rate 0.8mL / min, column temperature 30℃, sample travel time 50min, detection wavelength 306nm, standard curves were plotted using trans-resveratrol and spruce glycoside standards.

[0065] 6. Accumulation characteristics of total flavonoids, anthocyanins, and proanthocyanidins at different culture stages: Metabolomics analysis showed that the accumulation of flavonoids in the CHS2 mutant cell line was inhibited, while the synthesis and accumulation of resveratrol, a competitive metabolic pathway, was promoted. To further verify the accumulation characteristics of these two types of compounds, the contents of total flavonoids, anthocyanins, and proanthocyanidins were determined. The results are as follows: Figure 5 b shows that the contents of total flavonoids, anthocyanins, and proanthocyanidins in the CHS2 mutant cell line (MT) of *Grapevine* were significantly lower than those in the wild-type (WT) cell line. The total flavonoid content in both WT and MT generally showed a decreasing-then-increasing accumulation pattern. Since a large number of intermediate products in total flavonoids provide substrates for downstream products, this was not a continuous accumulation process. At 40 days of culture, the total flavonoid contents in WT and MT were 594.5 and 406.2 μg / g (fresh samples), respectively. The accumulation pattern of anthocyanins and proanthocyanidins differed from that of total flavonoids. Their contents increased with increasing culture time. At 40 days of culture, both anthocyanins and proanthocyanidins in WT and MT reached their highest values. The anthocyanin contents in WT and MT were 62.7 and 34.3 μg / g (fresh samples), respectively, and the proanthocyanidin contents were 1225.3 and 497.2 μg / g (fresh samples), respectively. Proanthocyanidins were not detected at 20 days of culture. The levels of anthocyanins and proanthocyanidins in the CHS2 mutant cell line were significantly lower than those in the wild type.

[0066] 7. Accumulation characteristics of resveratrol and spruce glycosides: The accumulation levels of resveratrol and spruce glycosides in the *Vitis vinifera* cell line differ from those of flavonoids, anthocyanins, and proanthocyanidins. Figure 5Results showed that the contents of resveratrol and spruce glycosides in the mutant cell line MT were significantly higher than those in the wild-type cell line WT. After 20 days of culture, the resveratrol contents in WT and MT were 1.03 and 4.22 μg / g (fresh samples), respectively. At this point, the resveratrol content in MT was four times that in WT. With prolonged culture time, the resveratrol content in MT did not significantly increase, while the resveratrol content in WT showed an upward trend. After 40 days of culture, the resveratrol contents in WT and MT were 2.23 and 4.61 μg / g (fresh samples), respectively. At this point, the resveratrol content in the mutant cell line was twice that in the wild-type cell line. The accumulation of spruce glycosides (resveratrol derivatives) in both WT and MT showed an increasing pattern. After 40 days of culture, their contents reached 10.50 and 32.05 μg / g (fresh samples), respectively. The spruce glycoside content in the mutant cell line was three times that in the wild-type cell line.

[0067] In summary, CRISPR / Cas9 gene editing leads to mutations in the CHS2 gene, causing it to partially lose its function and weaken its ability to compete for substrates. This, in turn, causes a downregulation of the expression of key genes in flavonoid metabolism, indirectly promoting the expression of STS, a key gene in competitive resveratrol biosynthesis. Ultimately, this results in a decrease in the content of flavonoid compounds, while the content of resveratrol and its derivatives increases several times over.

Claims

1. A method for increasing resveratrol content in grapevine cells through gene editing, characterized in that: The CRISPR / Cas9 method was used to target the chalcone synthase gene of *Gnaphalium affine*. CHS2 Perform gene editing to make the CHS2 Gene mutation triggers CHS2 Premature termination of protein translation, constructing *Vitis thorn* CHS2 Mutant cell lines; The specific steps include: CHS2 target site design, gene editing expression vector construction, genetic transformation, cell line culture and screening, and gene editing identification; the target sequence of the CHS2 target site design is located at the front end of the second exon.

2. The method according to claim 1, characterized in that: The CHS2 Mutations in genes are characterized by base insertions or base deletions.

3. The method according to claim 1, characterized in that: The prickly grape CHS2 In mutant cell lines, the expression of flavonoids and their key genes for biosynthesis was inhibited, while the expression of key genes for resveratrol biosynthesis was promoted. The contents of total flavonoids, anthocyanins, and proanthocyanidins were significantly reduced, while the contents of resveratrol were significantly increased by 2 to 4 times.

4. The method according to claim 1, characterized in that: When constructing the gene editing expression vector, the expression vector must include: Cas9 Gene sequence, and one or more sgRNA sequences, the steps include: (1) An sgRNA expression cassette was constructed using the overlapping PCR method; (2) The sgRNA expression cassette was ligated to the CRISPR / Cas9 vector using the Golden Gate cloning method; (3) The ligation product was transformed into E. coli, and bacteria were selected for PCR verification and sequencing identification. The recombinant expression vector with correct sequencing was extracted for plasmid extraction and used for subsequent genetic transformation.

5. The method according to claim 1, characterized in that: The genetic transformation and cell line culture and screening steps include: transforming the constructed gene editing expression vector with Agrobacterium, screening Agrobacterium for positive results, first performing PCR detection, extracting plasmids after positive detection, and sending samples for sequencing; transforming Grape thorn callus tissue with Agrobacterium-mediated transformation, infecting Grape thorn callus tissue with Agrobacterium resuspension for 8 min and co-culturing for 3 days, and then screening on resistant medium to obtain positive callus tissue.

6. The method according to claim 1, characterized in that: The gene editing identification process includes the following steps: using positive callus tissue selected from resistant culture medium as material, synthesizing primers at a distance of 200-600 bp on both sides of the target site for PCR amplification, directly sequencing the PCR product, and preliminarily determining successful targeting if the sequencing at the target site produces a double peak; simultaneously, recovering the PCR product, ligating it into a cloning vector, transforming it into E. coli, picking positive single clones for PCR detection and sequencing, and confirming successful gene editing. CHS2 Genes may insert or delete bases at the target site.

Images