Application of VvDT41-2 in increasing content of grape flavonoid substances

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an application of VvDT41-2 in increasing the content of grape flavonoid substances, and the nucleotide sequence of the VvDT41-2 is shown as SEQ ID NO.3. The invention also relates to an application of the VvDT41-2 in increasing the content of grape flavonoid substances. On the basis of the VvDT41-2 gene, exploration is carried out from the molecular level, the content of flavonoid substances in grapes is increased, and then positive influences are generated on growth and development, disease resistance, color and luster, flavor, nutritive value and the like of the grapes. The strategy for increasing the content of the flavonoid substances in the grapes through the VvDT41-2 is not limited by time, space, financial resources and manpower; meanwhile, compared with conventional field treatment modes such as moisture, illumination, fertilization and hormone, the method is more stable and efficient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a method for improving the content of flavonoid substances in grape VvDT41-2 and application thereof in improving the content of flavonoid substances in grape. BACKGROUND

[0002] Flavonoids are the most abundant secondary metabolites in grape, and play a crucial role in plant growth and development, resistance to ultraviolet rays and pests and diseases, fruit color, flavor quality and nutritional value. The synthesis of flavonoids in grape is limited or insufficient, which will have a negative impact on the above processes.

[0003] Grape fruit contains various types of flavonoids, and the accumulation characteristics are specific to varieties, tissues and development. The metabolism of grape flavonoids is regulated by multiple structural genes, such as UDP-glucose: flavonoid-3-O-glucosyltransferase, and the expression of transcription factors also has a significant impact on the synthesis. However, existing research mainly focuses on MYB transcription factors, such as MYBA1. In addition, the synthesis is also affected by various biological and non-biological factors. The existing technology can improve the content of flavonoids in grape by moderately regulating environmental conditions such as water and light, or by adding fertilizers and hormones. However, it is limited in time, space, cost and labor to different degrees. Therefore, it is necessary to continue to provide a new strategy for improving the content of flavonoids in grape. SUMMARY

[0004] The purpose of the present application is to provide a method for improving the content of flavonoid substances in grape VvDT41-2 application thereof in improving the content of flavonoid substances in grape, for improving the content of anthocyanins and tannins in grape, and promoting grape coloration.

[0005] The technical solution adopted by the present application is as follows: The present application provides a method for improving the content of flavonoid substances in grape VvDT41-2 application thereof in improving the content of flavonoid substances in grape, the nucleotide sequence of the VvDT41-2 is shown in SEQ ID NO. 3.

[0006] Preferably, the flavonoid substances include at least one of anthocyanins and tannins.

[0007] Preferably, the recombinant overexpression vector is constructed to prepare overexpression plants to improve the content of flavonoid substances in grape. VvDT41-2

[0008] Preferably, the preparation method of the recombinant overexpression vector comprises the following steps: extracting grape RNA, reverse transcribing it into cDNA, and amplifying the cDNA as a template VvDT41-2 ; performing enzyme digestion on the expression vector;​ The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1. VvDT41-2 The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0009] Preferably, the expression vector is pRI101-eGFP. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0010] Preferably, when the expression vector is digested, the restriction endonuclease used is Xba I and BamH I. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0011] Preferably, the preparation method of the overexpression plant is as follows: The recombinant overexpression vector is transformed into Agrobacterium competent cells. The culture is expanded to prepare an infection solution. The infection solution is used to infect and culture the grape to obtain an overexpression plant. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0012] Preferably, the Agrobacterium competent cells are LBA4404. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0013] Preferably, the OD value of the infection solution is 0.8. 600 The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0014] Preferably, the culture expansion conditions are 30°C, 100 rpm for 24 h. The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

[0015] Compared with the prior art, the present application has the beneficial effects that the present application provides an application of increasing flavonoid content in grape, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1. VvDT41-2 The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1. VvDT41-2 The present application is based on the exploration of the gene from the molecular level to increase the content of flavonoids in grape, which has a positive impact on the growth and development, disease resistance, color, flavor and nutritional value of grape. VvDT41-2 The strategy for increasing the content of flavonoids in grape is not limited by time, space, financial resources and manpower, and is more stable and efficient than conventional field treatment methods such as water, light, fertilization and hormones. VvDT41-2 BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0016] BRIEF DESCRIPTION OF DRAWINGS Figure 1 BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0017] BRIEF DESCRIPTION OF DRAWINGS Figure 2 BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0018] BRIEF DESCRIPTION OF DRAWINGS Figure 3 BRIEF DESCRIPTION OF DRAWINGS VvDT41-2 BRIEF DESCRIPTION OF DRAWINGS Group BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0019] BRIEF DESCRIPTION OF DRAWINGS Figure 4for total flavonoid content analysis.

[0020] Figure 5 for total anthocyanin content analysis.

[0021] Figure 6 for total flavan-3-ol content analysis.

[0022] Figure 7 for total tannin content analysis. DETAILED DESCRIPTION

[0023] The application will be further described in the following specific examples, but the scope of the application is not limited thereto. The details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and these modifications or replacements all fall within the protection scope of the application.

[0024] In order to enable those skilled in the art to better understand the technical solutions of the application and implement the same, the application will be further described below in conjunction with specific examples. In the description of the application, if not specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0025] Example 1 VvDT41-2 The application in improving the content of flavonoids in grapes is as follows: 1. Subcellular localization.

[0026] 1.1. Total RNA extraction.

[0027] (1) Grapes samples were ground in liquid nitrogen with a mortar and pestle, and 1 mL of lysis buffer RL was added to each 100 mg of tissue. The volume of the tissue sample should not exceed 10% of the volume of the RL.

[0028] (2) The homogenized sample was mixed by vigorous shaking and incubated at 30°C for 5 min to completely dissociate the ribosomes.

[0029] (3) The sample was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was carefully transferred to a new RNase-free centrifuge tube.

[0030] (4) 0.2 mL of chloroform was added to each 1 mL of RL. The sample tube was tightly capped, vigorously shaken for 15 s, and incubated at room temperature for 3 min.

[0031] (5) The sample was centrifuged at 12000 rpm for 10 min at 4°C, and the aqueous phase was transferred to a new tube.

[0032] (6) 1 volume of 70% v / v ethanol was added and mixed by inversion.

[0033] (7) Add 500 μL of protein-free solution RE, centrifuge at 12000 rpm for 45 s, discard the waste liquid, and reinsert the adsorption column into the collection tube.

[0034] (8) Add 500 μL of protein-free solution RE, centrifuge at 12000 rpm for 45 s, discard the waste liquid.

[0035] (9) Add 700 μL of rinse solution RW, centrifuge at 12000 rpm for 60 s, discard the waste liquid.

[0036] (10) Add 500 μL of rinse solution RW, centrifuge at 12000 rpm for 60 s, discard the waste liquid. Then air spin for 1 min to remove as much ethanol as possible, which affects DNA digestion.

[0037] (11) Take out gDnase, 10x Buffer, and free water, and configure 20 μL of digestion solution according to a volume ratio of 1:1:8. Add the digestion solution to the adsorption column RA and incubate in a 42°C water bath for 5 min, then continue with steps (8) and (9) and then proceed to step (12).

[0038] (12) Place the adsorption column RA back into the empty collection tube and centrifuge at 12000 rpm for 2 min.

[0039] (13) Take out the adsorption column RA and place it in an RNase free centrifuge tube. Add 80 μL of RNase free water that has been previously heated in a 70°C water bath to the middle part of the adsorption membrane, and let it stand at room temperature for 2 min. Centrifuge at 12000 rpm for 1 min to obtain total RNA.

[0040] 1.2, cDNA reverse transcription.

[0041] (1) On the clean bench, take 1 mL of sterile water into an EP tube, add 2 μL of total RNA solution, mix well, and measure the light absorption values at 260 nm and 280 nm on a UV spectrophotometer to calculate the concentration of RNA and analyze its purity.

[0042] (2) In a DEPC-treated EP tube, add about 4 μg of total RNA and 1 μL of 0.5 μg / μL Oligo(dT)18 primer, mix carefully, and incubate at 70°C for 5 min, then immediately immerse in ice water.

[0043] (3) Add the following reagents in order: 5 μL of 5x M-MLV RT Buffer, 2 μL of dNTPmix (2.5 mM), 1 μL of 30 U / μL RNase inhibitor, 1 μL of M-MLV Reverse Transcriptase, and then add DEPC water to 25 μL.

[0044] (4) Mix carefully, centrifuge 5s at room temperature, collect all solutions to the bottom of the tube, and incubate at 37°C for 1h.

[0045] (5) Treat at 90°C for 5min, cool on ice, and obtain cDNA.

[0046] (6) For PCR amplification or storage at -20°C for standby. 1.3、 VvDT41-2 Cloning of the gene.

[0047] A pair of primers were synthesized for amplification of the gene using cDNA as a template. VvDT41-2 The primer sequence information is shown in Table 1.

[0048] Table 1 Primer sequence VvDT41-2 The cloning of the gene used 2xHieff PCR Master Mix high-fidelity enzyme premix produced by Shanghai Yisheng Biotechnology Co., Ltd. The gene cloning reaction system and PCR reaction program are shown in Table 2 and Table 3, and the amplification effect was detected by agarose gel electrophoresis technology.

[0049] Table 2 Gene cloning reaction system Table 3 PCR reaction program VvDT41-2 The nucleotide sequence information of the gene is shown as SEQ ID NO. 3; VvDT41-2 The amino acid sequence information of the gene is shown as SEQ ID NO. 4.

[0050] SEQ ID NO. 3:

[0051] SEQ ID NO. 4: MGSEEYQPLLLGLNSHARIPDLSSFAVEEFLAHKPVAVRWWPRLFGWESRLLWLLSGSSIVASIFNYMLSFVTLMFTGHLGALELAGASIASVGIQGLAYGIMLGMASAVQTVCGQAYGAKKYKAMGIICQRAIILHLGAAVLLTFLYWFSGPFLRAIGQSDSISAQGQIFARGLILQLYAFAISCPMQRFLQAQNIVNPLAYMAVGVFFLHVLLTWLVVYVLDYGLLGAALTLSFSWWILVVVIALYILLSPSCKETWTGFSSKAFKGMWPYFKLTVASAVMLCLEIWYNQGLVLISGLLSNPTISLDSISICMNYLNWDMQFMLGLSAATSVRVSNELGASHPKVAKLSVLVVNTNSIIISIFFSAIILIFKVGLSKLFTNDAEVIEAVSNLTPLLAISVFLNGIQPILSGVAIGSGWQAIVAYVNLATYYLIGLPIGCVLGFKTSLGVAGIWWGMIIGVLLQTVTLIILTARTDWNAEVSKAAERLRNSANVENLNLLEDV.

[0052] 1.4, Construction of plant expression vector.

[0053] (1) Plasmid extraction.

[0054] E. coli DH5a containing plant expression vector pRI101-eGFP was inoculated in LB liquid medium with a concentration of 50 μg / mL kanamycin, and cultured at 150 rpm and 37°C overnight. The plasmid was extracted using the kit "Plasmid Miniprep Purification Kit II" of Tiangen. The steps are as follows:

[0055] Take 10 mL of the cultured bacteria and centrifuge at 12000 g for 1 min. Discard the supernatant and add 200 μL of Solution I to the precipitate. Resuspend the cells by vortexing at high speed. Add 200 μL of Solution II and immediately invert the centrifuge tube 5 times. Add 200 μL of Solution III and immediately invert the centrifuge tube 5 times. Centrifuge the lysate at 12000 g for 5 min. Carefully transfer the supernatant to a filter column with a sleeve and centrifuge at 12000 g for 1 min. Discard the filtrate and add 500 μL of Wash solution A to the filter column and centrifuge at the same speed for 1 min. Discard the filtrate and add 700 μL of Wash solution B to the filter column and centrifuge at the same speed for 1 min. Discard the filtrate in the sleeve and centrifuge at 12000 g for 3 min to remove the ethanol completely. Place the filter column in a new centrifuge tube and add 60 μL of Elution Solution preheated at 65 °C to the center of the filter membrane. Allow it to stand at room temperature for 2 min. Centrifuge at 12000 g for 1 min. Store the filtrate at -20 °C. Use a NanoDrop 2000 spectrophotometer to detect the concentration of the plasmid: take 2 μL of the plasmid DNA sample and place it in the sample well. Observe and record the values of the concentration, A260 / A280 and A260 / A230.

[0056] (2) Linearization and purification of the plasmid.

[0057] Add 1 μg of the extracted circular plasmid pRI-101-eGFP to a PCR tube, add 5 mL of reaction Buffer and 1 μL of each of Xba I and BamH I, and finally add water to 50 μL. Incubate at 37 °C for 3 h. Xba I and BamH I are both purchased from BioLabs, and the enzyme activity of each of Xba I and BamH I is 20000 units / mL.

[0058] The reaction product was purified by gel recovery, and the kit was "TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0". First, the reaction product was mixed with the Loading Buffer and added to the 1% wt agarose gel loading well, and electrophoresis was performed at 120V for 40min. After electrophoresis, in the gel imaging system, the target fragment gel block was cut and placed in a 1.5mL centrifuge tube. 600μL of gel block dissolution Buffer GM was added to the centrifuge tube, mixed evenly, and the gel block was dissolved at 37°C for 30min. When the gel block was completely dissolved, the solution was transferred to a Spin Column pre-added with a sleeve, centrifuged at room temperature at 12000rpm for 1min, and the filtrate was discarded. 700μL of Buffer WB was added to the Spin Column, centrifuged at room temperature at 12000rpm for 30s, and the filtrate was discarded. Buffer WB was used for repeated washing once. The Spin Column was repositioned in the collection tube and centrifuged at 12000rpm for 2min to remove excess ethanol. The Spin Column was placed in a new centrifuge tube, 30μL of Elution Buffer was added to the center of the membrane, and it was left at room temperature for 1min. The linear plasmid was eluted by centrifugation at room temperature at 12000rpm for 2min. After detecting the linear plasmid, it was stored in the -20°C refrigerator for standby use.

[0059] (3) One-step cloning.

[0060] One-step cloning uses the NovoRec® plus One step PCR Cloning Kit produced by Suzhou Coastal Protein Biotechnology Co., Ltd. The specific operation steps are as follows: first, prepare the reaction system, add 5×CE IIBuffer 4μL, linearized cloning vector 200μL, insert fragment amplification product VvDT41-2 of cDNA 200μL, Exnase®II 2μL, and then add ddH2O to make up to 20μL volume. After the system is prepared, mix the components with a pipette by blowing up and down several times. After 37°C reaction for 30min, immediately place the reaction tube in an ice water bath for cooling for 5min.

[0061] Take 20μL of the cooled reaction solution and add it to 200μL of competent E. coli DH5α cell solution, mix by flicking several times, and place on ice for 30min. Heat shock at 42°C for 90s, and incubate in an ice water bath for 2min. Add 900μL of SOC medium, incubate at 37°C for 10min to fully recover. Shake the bacteria at 37°C at 150rpm for 45min. Take 100μL of bacterial solution and evenly spread it on a plate containing kanamycin. Incubate at 37°C overnight.

[0062] After overnight incubation, colonies grew, and then clone identification was performed. First, colony PCR verification was conducted. Then, double enzyme digestion verification was performed. Colonies that tested positive for colony PCR were inoculated into LB medium containing kanamycin and incubated overnight at 37°C and 150 rpm. The reaction product was mixed with loading buffer and added to the wells of a 1% wt agarose gel, and electrophoresis was performed at 120V for 30 min. The enzyme digestion products were observed using a gel imaging system. Finally, the samples that tested positive for double enzyme digestion were sent to the company for sequencing, and the successfully sequenced clones were preserved, thus obtaining the recombinant overexpression vector.

[0063] 1.5 Subcellular localization.

[0064] E. coli containing the recombinant overexpression vector were inoculated into LB broth containing kanamycin and incubated overnight at 37°C and 150 rpm. The plasmid was extracted and the constructed recombinant overexpression vector was recovered using agarose gel electrophoresis. The vector was then transformed into Agrobacterium tumefaciens (Agrobacterium tumefaciens) using electroporation. Rhizobium radiocbactor In LBA4404 Electro-Cells, competent cells were thawed on ice before use. On ice, 1 ng of recombinant overexpression vector was added to a 1.5 mL centrifuge tube containing 20 μL of competent cells and gently mixed. The electroporator was set to 25 μF, 200 Ω, and 2–2.5 kV. The competent cells and recombinant overexpression vector were transferred to a pre-chilled 0.1 cm electroporation cuvette, and the cuvette was placed in a Gene Pulser II for electroporation. The cuvette was removed, 1 mL of SOC medium was added, and the mixture was transferred to a round-bottom centrifuge tube and cultured at 30°C with shaking at 100 rpm for 1 h. 50 μL of cells were plated onto LB solid medium containing 50 μg / mL kanamycin and 100 μg / mL streptomycin. The cells were incubated upside down at 30°C for 48 h. After incubation, cell clone identification was performed; positive clones were identified as recombinant positive Agrobacterium.

[0065] Recombinant positive Agrobacterium was scraped from a solid culture dish using an inoculation loop and inoculated into 10 mL of YEB liquid medium. The culture was carried out at 170 rpm / min for 1 h. After centrifugation at 4000 rpm / min for 4 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in a 10 mM MgCl2 suspension containing 120 μM acetylsylcholine, and the OD was adjusted to approximately 0.6. Healthy tobacco plants were selected, and injections were made into the lower epidermis of tobacco leaves using a 1 mL syringe (without the syringe tip). The injection sites were then labeled. The injected tobacco plants were cultured under low light for 2 days. The tobacco leaves were then prepared into slides, and the subcellular localization was observed and photographed under a laser confocal microscope.

[0066] 1.6 Construction of overexpression plants.

[0067] The present application is carried out in the laboratory of the Northwest A&F University Wine College. On August 5, 2021, 'Cabernet Sauvignon' grape clusters were taken from the Yangling Caoxinzhuang Experimental Demonstration Base and quickly transported back to the laboratory. The clusters were sequentially disinfected with 70% v / v ethanol, 4% v / v sodium hypochlorite solution, and sterile water. Then, uniform-sized berries were cut off with sterile scissors, leaving about 3 mm of stem, and immediately placed in a culture dish containing 2% wt sucrose solution for use.

[0068] The above recombinant positive Agrobacterium was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 100 μg / mL streptomycin, and expanded at 30°C, 100 rpm for 24 h. At this time, the recombinant positive Agrobacterium was in the exponential growth phase, and the infection activity was best. The culture solution was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. 2 mL of 10 mM MgCl2 suspension containing 120 μM acetosyringone was added to resuspend the cells. The cells were washed once more with the suspension to completely remove the antibiotics to avoid affecting the subsequent infection test. After washing, the cells were resuspended with the suspension, and the OD 600 =0.8 of the bacterial solution was adjusted, i.e. the infection solution was obtained. After adding the infection solution to the culture dish, the disinfected grapes were placed in the culture dish, the resuspension solution was immersed in the stem, and the culture dish was transferred to the plant incubator, 20°C, 16 h light-8 h dark for culture, and the infection was carried out by stem absorption of the culture solution.

[0069] 1.7, Sample collection.

[0070] After starting the in vitro culture, samples were collected once a day until the end of the culture, and the last sample was collected from all in vitro fruits. Immediately after each collection, the fruits were quickly frozen with liquid nitrogen, and then stored in a -80°C refrigerator.

[0071] 2, Results and analysis.

[0072] 2.1, VvDT41-2 Cloning.

[0073] Figure 1 The agarose gel electrophoresis map of colony PCR amplification, VvDT41-2 1515 bp in length, indicating that the VvDT41-2 of the present application has been successfully cloned.

[0074] 2.2, Subcellular localization.

[0075] In order to determine the position of the above-mentioned gene to play a role, the present application first predicts the positioning site of the gene through the PSORT tool, and then uses the laser confocal microscope for subcellular localization analysis and verification. Figure 2 The subcellular localization result. PSORT tool: https: / / wolfpsort.hgc.jp / .

[0076] The PSORT prediction result shows VvDT41-2 Most likely to be located in the vacuole membrane or Golgi membrane, the encoded protein is most likely to be a vacuole membrane protein or an integral membrane protein; the subcellular localization result shows that there is only clear fluorescence around the cytoplasmic membrane and no fluorescence in the nucleus, indicating that VvDT41-2 The gene is most likely to be located in the vacuole membrane or Golgi membrane.

[0077] 2.3, Construction of overexpression plants.

[0078] (1) Phenotype observation.

[0079] After transient expression of the gene, VvDT41-2 The expression amount is significantly increased, indicating that the treatment of the application successfully leads to VvDT41-2 Overexpression of the gene in the isolated grape. Figure 3 For VvDT41-2 Phenotype observation of the isolated grape after transient expression of the gene. It can be known that Figure 3 The number of grapes starting to change color and the degree of color change in the treatment group are obviously higher than those in CK. VvDT41-2

[0080] (2) Flavonoid content analysis.

[0081] Flavonoids are an important class of plant secondary metabolites, including anthocyanins, flavanols, flavonols and other compounds. These substances gradually accumulate during grape fruit development, especially during the grape color change period, i.e. the stage when the fruit changes from green to red or purple. Anthocyanins are the main coloring substances in grape skin, and their types and contents directly affect the color of grape fruits. In addition to anthocyanins as the main coloring substances, other flavonoids such as flavonols and flavanols also have auxiliary effects on grape color change. Flavonols and flavanols can combine with anthocyanins to enhance the stability of anthocyanins, thereby improving the brightness of grape fruit color.

[0082] Through omics analysis, the application finds that fruit illumination has a significant impact on key genes and metabolites of the flavonoid pathway, and transcriptome verification analysis of the flavonoid pathway finds VvPAL , VvCHS , VvF3H1 , VvLAR1 , VvANR , VvFLS1 , VvGST1 , VvMATE1 , VvMYBPA1 , VvMYBPA2 , VvDT41-1 , VvDT41-2 , VvMYB61 , VvMYB86 which play a key regulatory role. The application further clones and functionally verifies VvDT41-2 The results are shown in Figures 4-7 ​In VvDT41-2 In the overexpressing grape, the total flavonoids did not change significantly; but VvDT41-2 After overexpression, the total anthocyanin content and total tannin content in grape were significantly improved, so it was speculated that it played a key regulatory role in the content change of flavonoid substances.

[0083] Each technical feature of the above-described embodiments can be combined arbitrarily, and to make the description concise, each technical feature in the above-described embodiments is not described in all possible combinations, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the description.

[0084] The above-described embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. VvDT41-2 Its application in increasing the flavonoid content of grapes is characterized by... The VvDT41-2 The nucleotide sequence is shown in SEQ ID NO.

3.

2. The application as described in claim 1, characterized in that, The flavonoids include at least one of anthocyanins and tannins.

3. The application as described in claim 1, characterized in that, By building VvDT41-2 The recombinant overexpression vector was used to prepare overexpression plants to increase the content of flavonoids in grapes.

4. The application as described in claim 3, characterized in that, The preparation method of the recombinant overexpression vector includes the following steps: RNA was extracted from grapes, reverse transcribed into cDNA, and then amplified using the cDNA as a template. VvDT41-2 ; The expression vector was digested with enzymes; Will VvDT41-2 The recombinant overexpression vector is ligated with the enzyme-digested expression vector to obtain the recombinant overexpression vector.

5. The application as described in claim 4, characterized in that, The expression vector is pRI101-eGFP.

6. The application as described in claim 5, characterized in that, The restriction endonucleases used for digesting the expression vector were XbaⅠ and BamHⅠ.

7. The application as described in claim 3, characterized in that, The method for preparing the overexpression plant is as follows: The recombinant overexpression vector was transformed into Agrobacterium competent cells; Expand the culture and prepare the infection solution; Grapes were infected with an inoculation solution and cultured to obtain overexpressing plants.

8. The application as described in claim 7, characterized in that, The Agrobacterium competent cells were LBA4404.

9. The application as described in claim 7, characterized in that, The OD of the infiltration solution 600 The value is 0.

8.

10. The application as described in claim 7, characterized in that, The conditions for the expanded culture are as follows: Incubate at 30°C and 100 rpm for 24 hours.

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