A kit and a method for increasing the content of terpenoids in grapes

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a kit and a method for increasing the content of terpenoid compounds in grape. The method uses the kit to transfer grape transcription factor MYB1R1 into grape and overexpress the same to increase the content of terpenoid compounds in grape fruits. The method can obtain grape plants with higher content of terpenoid compounds compared with wild type by overexpressing MYB1R1 gene in grape, effectively increase the content of part of terpenoid compounds in grape pericarp, and thus can cultivate grape germplasm with strong flower fragrance, and has a positive effect on improving flower and fruit fragrance of varieties, cultivating new varieties and accelerating the breeding process.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a kit and a method for increasing the content of terpenoid compounds in grapes. Background Technology

[0002] Terpenes are mainly classified into monoterpenes (such as linalool, geraniol, nerol, and limonene), sesquiterpenes, and diterpenes. Monoterpenes are the most important in wine and are found in high concentrations in varieties such as Muscat, Riesling, and Gewürztraminer. They exist in two forms: free (directly volatilized and contributing to aroma) and bound (bound to glycosides and requiring enzymatic or acidic hydrolysis for release).

[0003] Terpenes have a significant impact on the aroma of wine, with different terpenes possessing distinct aroma characteristics. For example, geraniol and nerol contribute mild, sweet rose and orange blossom aromas, linalool provides a heavier rosewood scent, limonene contributes citrus and lemon notes, and some terpenes may also impart minty or spicy notes. Furthermore, terpenes exhibit synergistic effects with compounds such as esters and thiols, enhancing aroma complexity. For instance, the combination of terpenes and thiols can accentuate the passion fruit aroma of Sauvignon Blanc.

[0004] Insufficient terpenes can have many negative effects, such as monotonous aromas, loss of varietal characteristics, and reduced flavor complexity. For example, terpenes (such as linalool and geraniol) are the signature aroma sources of varieties like Muscat, Riesling, and Marselan. If the terpenes content is insufficient, the wines made from them will lose the varietal's characteristic floral (rose, orange blossom), fruity (citrus, lychee), and herbaceous notes, resulting in a monotonous aroma.

[0005] Transcription factors are a class of protein molecules that can specifically recognize and bind to specific gene sequences, regulate the expression level of target genes under specific spatiotemporal conditions, and thus affect the synthesis and accumulation of plant secondary metabolites.

[0006] Currently, research on transcription factors related to terpene synthesis in grapes is limited, and the specific regulatory mechanisms of the MYB-related family in terpene metabolism in grape berries remain unclear. Identifying and recognizing key transcription factors regulating terpene synthesis in grape berries is crucial for elucidating the molecular mechanisms of grape aroma formation. Furthermore, how to utilize these transcription factors to improve grape aroma quality and wine flavor requires further exploration. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a kit and a method for increasing the content of terpenoid compounds in grapes.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] The present invention provides a grape transcription factor MYB1R1, and the coding region nucleotide sequence of the grape transcription factor MYB1R1 is shown in SEQ ID NO.1.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, the amino acid sequence of the grape transcription factor MYB1R1 is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant vector comprising the coding region nucleotide sequence for encoding the grape transcription factor MYB1R1 as described above.

[0013] Furthermore, the backbone plasmid of the recombinant vector is pCAMBIA1300.

[0014] The present invention also provides an engineered bacterium containing the recombinant vector as described above.

[0015] The present invention also provides a kit for increasing the content of terpenoids in grapes, the kit comprising the grape transcription factor MYB1R1 as described above.

[0016] Furthermore, it also includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0017] Furthermore, it also includes the backbone plasmid of the recombinant vector as described above and the engineered bacteria used for transformation as described above.

[0018] The present invention also provides a method for increasing the content of terpenoid compounds in grapes, which is regulated by the grape transcription factor MYB1R1 as described above.

[0019] Furthermore, the method includes the following steps: transferring the grape transcription factor MYB1R1 into grapes and overexpressing it to increase the content of terpenoids in grape fruits.

[0020] Furthermore, the terpenoids include bound linalool and trans-β-ocimene.

[0021] The beneficial effects of this invention are as follows:

[0022] The method of this invention, by overexpressing the MYB1R1 gene in grapes, can obtain grape plants with higher terpene content compared to wild types. It can effectively increase the content of some terpene compounds in grape skins, thereby cultivating grape germplasm with rich floral aromas. This has a positive effect on improving the floral and fruit aromas of varieties, cultivating new varieties, and accelerating the breeding process. Attached Figure Description

[0023] Figure 1 This is an agarose gel electrophoresis image of the grape transcription factor MYB1R1 of the present invention;

[0024] Figure 2 The image shows a comparison of the relative expression levels of the MYB1R1 gene in grape skins transiently transformed with MYB1R1 and in grape skins without any transformation treatment, which is the grape transcription factor MYB1R1 of the present invention.

[0025] Figure 3 The grape transcription factor MYB1R1 of this invention is shown in the comparison chart of terpene content in grape skins transiently transformed with MYB1R1 and grape skins without any transformation treatment in Experimental Example 2. Figure 3 In this context, 'a' represents the bound state of trans-β-ocimene. Figure 3 In the figure, b represents bound linalool, and * indicates P < 0.05. Detailed Implementation

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0027] The grape transcription factor MYB1R1 of the present invention has a coding region nucleotide sequence as shown in SEQ ID NO.1. This grape transcription factor MYB1R1 can be used to regulate the content of terpenoids in grapes.

[0028] The grape transcription factor MYB1R1 of this invention belongs to the MYB-related class of transcription factors. It participates in the regulation of terpene metabolism in grapes. By overexpressing the MYB1R1 gene in grapes, the content of some terpene compounds in grape skins can be effectively increased, thereby enabling the cultivation of grape varieties with rich floral aromas.

[0029] Specifically, the gene encoding MYB1R1 has a cDNA length of 903 bp and encodes 300 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2.

[0030] The kit of the present invention for increasing the content of terpenoid compounds in grapes includes the grape transcription factor MYB1R1 as described above; it also includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0031] Preferably, the kit also includes a backbone plasmid and engineered bacteria for transformation.

[0032] Further preferred, the backbone plasmid is pCAMBIA1300, and the engineered bacteria are Escherichia coli and Agrobacterium.

[0033] Preferably, the kit may also include other conventional reagents.

[0034] The present invention provides a method for increasing the content of terpenoids in grapes, which uses a kit to transfer the grape transcription factor MYB1R1 into grapes and overexpress it, thereby increasing the content of terpenoids in grape fruits.

[0035] Preferably, the terpenoids include bound linalool and trans-β-ocimene.

[0036] Specifically, a recombinant vector containing the MYB1R1 nucleotide sequence can be constructed, and then the recombinant vector can be transferred into engineered bacteria. The engineered bacteria can then be used to infect the cells of grape fruits or peels, thereby overexpressing the grape transcription factor MYB1R1 and increasing the content of terpenoids.

[0037] In one embodiment of the present invention, the specific steps are as follows:

[0038] (1) DNA of grape transcription factor MYB1R1 was obtained by cloning. The specific experimental procedure is as follows:

[0039] The RNA from grape skins (extracted using an RNA extraction kit) was reverse-engineered into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The specific systems for each step are shown in Tables 1-3.

[0040] Table 1. Step-1 reverse transcription system and reaction conditions

[0041]

[0042] Table 2. Second step reverse transcription system and reaction conditions

[0043]

[0044] Table 3. Reverse transcription system and reaction conditions for step 3

[0045]

[0046] Specific primers were designed using SnapGene, and the full-length cDNA of MYB1R1 was amplified by PCR. The PCR system is shown in Table 4, the primer sequences are shown in Table 5, and the PCR reaction extent is shown in Table 6.

[0047] Table 4. PCR amplification reaction system and procedure

[0048]

[0049]

[0050] Table 5 Primer sequences

[0051]

[0052] Table 6 PCR reaction procedures

[0053]

[0054] The target fragment was detected by 1% agarose gel electrophoresis.

[0055] In one embodiment of the present invention, the electrophoresis result is as follows: Figure 1 As shown. Figure 1 The three bands in the image represent three parallel experiments. By comparing the markers, it can be seen that the DNA length of all three bands is around 903 bp, indicating that it is MYB1R1 and the cloning was successful.

[0056] After electrophoresis, the target fragment was recovered using the Hipure Gel Pure DNA Mini Kit agarose gel DNA recovery kit.

[0057] (2) Construction of recombinant vector (overexpression vector) pCAMBIA1300-MYB1R1.

[0058] The plasmid of the overexpression vector pCAMBIA1300 was extracted, and pCAMBIA1300 was double-digested with restriction enzymes SacI and BamHI. After digestion, the pCAMBIA1300 vector was purified to obtain a linearized vector.

[0059] The gel recovery products were ligated to the linearization vector pCAMBIA1300 using the One Step Cloning Kit C112. The ligation system is shown in Table 7.

[0060] Table 7. Connection Reaction System and Reaction Conditions

[0061]

[0062] The ligation system was transformed into DH5α competent cells. The cloned competent cells were thawed on ice. 10 μL of the recombinant product was added to 50 μL of competent cells, and the mixture was gently tapped against the tube wall to mix. The cells were then placed on ice for 30 min, heat-shocked at 42°C for 45 sec, and incubated on ice for another 5 min. Under aseptic conditions, 800 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 220 rpm for 1 h. After centrifugation at 4000 × g for 5 min, 500 μL of supernatant was discarded. The cells were resuspended in the remaining medium, and 100 μL was spread onto a solid medium containing kanamycin. The cells were incubated upside down at 37°C for 16 h. When colonies reached approximately 2 mm in diameter, a single colony was dipped into a pipette tip, gently pipetted twice, and dissolved in 10 μL of LB medium. Another 1 μL was then used as a template. The system described in Table 8 was used for bacterial PCR identification. Positive clones were selected for sequencing, and a portion of the bacterial culture was mixed with glycerol in a 1:1 ratio and stored at -80°C.

[0063] Table 8 Bacterial PCR System

[0064]

[0065] Plasmids from the correctly sequenced positive clones were extracted, and the recombinant plasmid pCAMBIA1300-MYB1R1 was finally obtained.

[0066] (3) Construction of recombinant bacteria.

[0067] Agrobacterium transformation was performed using the freeze-thaw method. Competent Agrobacterium GV3101 cells were thawed on ice. After thawing, 10 μL of recombinant plasmid was added to 100 μL of competent cells, and the mixture was gently pipetted to mix. The cells were then sequentially placed on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, incubated in a 37°C water bath for 5 min, and then in an ice bath for 5 min. In a clean bench, 900 μL of antibiotic-free liquid LB medium was added, mixed thoroughly, and incubated at 28°C with shaking at 200 rpm for 3 h. The cells were harvested by centrifugation at 4000×g for one minute. Approximately 100 μL of the supernatant was collected, gently resuspended, and spread onto LB + Rif (50 μg / mL) + Kan (50 μg / mL) solid medium. After drying, the medium was inverted and incubated in the dark at 28°C for 3 days.

[0068] Select a single colony of GV3101 and inoculate it into 4 mL of LB broth + Rif (50 μg / mL) + Kan (50 μg / mL). Incubate at 28°C and 200 rpm for 16 h. Perform PCR detection on positive clones. Inoculate 150 μL of the bacterial culture from the positive clone into 4 mL of the above-mentioned double-antibiotic LB broth and incubate at 28°C and 200 rpm for 5-8 h until OD (Organic Dioxide) is reached. 600 The concentration is 0.6-0.8. Take 1 mL of the bacterial culture, add an equal volume of sterilized glycerol, quick-freeze in liquid nitrogen, and store at -80℃.

[0069] (4) Obtain MYB1R1 instant conversion grape fruit.

[0070] Add 100-200 μL of the Agrobacterium tumefaciens bacterial suspension containing the target gene and the GV3101 empty vector bacterial suspension to 4-5 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium, respectively, and incubate at 28°C for 16 h with shaking. Transfer the activated bacterial suspension to 40 mL of LB (Kan + Rif) medium for subculture. Incubate the second-generation culture at 28°C and 220 rpm for 6-7 h until OD (Organic Demand) is reached. 600 To achieve an OD of 1-1.2, centrifuge the second-generation culture at 4000×g for 5 min and resuspend it 1:1 in Agrobacterium infection buffer. Centrifuge three times to discard the supernatant and wash the bacterial cells. Resuspend the second-generation culture pellet again in Agrobacterium infection buffer to an OD of 1-1.2. 600 The pH was set to 0.9 and allowed to stand at room temperature in the dark for 3 hours. The preparation ratio of the Agrobacterium infection buffer is shown in Table 9. The pH of the Agrobacterium infection buffer needs to be adjusted to 5.2 using KOH.

[0071] Each grape berry was punctured with 30 holes using a syringe and immersed in the infection solution. A vacuum was applied for 15 minutes, during which the berries were continuously agitated to ensure the infection solution penetrated the berries. The berries were then removed and cultured naturally for 3 days and 3 nights. Samples of the resulting grapes were then frozen in liquid nitrogen and stored at -80°C. The preparation of the infection solution is shown in Table 9. The pH was adjusted to 5 using KOH before use.

[0072] Table 9. Infection solution ratio

[0073] Components Added amount MES 2.13g <![CDATA[MgCl2·6H2O]]> 2.03g Add acetylsuccione (As) before use. 200μM sterile water 1L

[0074] Through the specific steps described above, the MYB1R1 gene can be successfully transferred into grapes and overexpressed. This treatment yields grapevines with higher terpene content compared to wild-type grapes, which plays a positive role in enhancing the aroma of flowers and fruits, cultivating new varieties, and accelerating the breeding process.

[0075] The transcription factor of this invention that regulates the synthesis of grape terpenoids is of great significance for elucidating the molecular mechanism of grape fruit aroma formation and for improving the aroma quality of grape fruit using genetic engineering techniques.

[0076] The present invention will be specifically described below through examples. Unless otherwise specified, the experimental materials, instruments and reagents used in the following examples were obtained through conventional means.

[0077] Example 1: Relative Gene Expression Analysis of MYB1R1 Transiently Transformed Grape Skin

[0078] The experimental procedure in this embodiment is as follows:

[0079] RNA (extracted using an RNA extraction kit) was reverse-engineered into cDNA using the HiScript IIQ RT SuperMix for qPCR + gDNA wiper (R223) kit for qRT-PCR. The specific steps are as follows:

[0080] (1) Genomic DNA removal: Add 500 ng of RNA to an RNase-Free tube, add 4 μL of 4×g DNA wiperMix, then add RNase-ddH2O to a total liquid volume of 16 μL. Gently mix with a pipette and place in a PCR instrument. React at 42℃ for 2 min.

[0081] (2) Prepare the first strand cDNA synthesis reaction solution: Add 4 μL of 5×HiScript II qRTSuperMix II to the above system, place it in a PCR instrument, react at 55℃ for 15 min, react at 85℃ for 5 s, terminate the reaction, and store the product at -20℃ for later use.

[0082] In this embodiment, Ubiquitin was used as an internal reference gene for quantitative real-time PCR. Reagents were added according to the reaction system in Table 10, and the reaction was performed in triplicate. The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, 39 cycles; melting curve analysis was performed from 65℃ to 95℃. Primer sequences are shown in Table 11.

[0083] Table 10. Real-time PCR reaction system

[0084] system cDNA template 1μL upstream primer 0.2μL Downstream primer 0.2μL 2×ChamQ Universal SYBR qPCR Master Mix 5μL <![CDATA[ddH2O]]> 3.6μL

[0085] Table 11 Primer sequences

[0086]

[0087] The results of quantitative real-time PCR are as follows Figure 2 As shown in the figure, "OE-MYB1R1" represents the instantaneous conversion of grape skins using the MYB1R1 method of this invention, and "CK" represents grape skins without any conversion treatment. Figure 2 It can be seen that the expression level of MYB1R1 increased by 1.93 times in the overexpressing plants.

[0088] Example 2: Extraction and Detection of Terpenoids from Grape Peels Transformed by MYB1R1

[0089] In this embodiment, the contents of terpenoids were extracted from grape skins transiently transformed with MYB1R1 (OE-MYB1R1) and grape skins without any transformation treatment (CK) and compared to verify the regulatory effect of MYB1R1 overexpression on the content of terpenoids.

[0090] The extraction process of terpenoids in this embodiment is as follows:

[0091] Immediately after peeling the grapes, they were frozen with liquid nitrogen, ground into powder using a grinder pre-cooled with liquid nitrogen, and stored at -80℃. 1g of powder was added to 5mL of citric acid / phosphate buffer (0.2mol / L, pH=5.0), soaked at 4℃ for 16h, centrifuged at 5000×g for 15min at 4℃, and the supernatant was used for subsequent extraction and detection of aroma substances.

[0092] Solid phase extraction (SPE) was used to extract bound terpenoids from grape peels. The specific process is as follows:

[0093] The Cleanert PEP-SP (150mg / 6mL) extraction column was activated with 10mL of anhydrous methanol and 10mL of purified water to remove impurities. Then, 2mL of the supernatant was added, and the column was eluted with 5mL of purified water and 5mL of dichloromethane to remove pigments, low molecular weight sugars, acids and other polar compounds, as well as free nonpolar aroma compounds. Finally, glycoside-bound aroma substances were eluted with 20mL of anhydrous methanol into a test tube. The flow rate was maintained at 2mL / min throughout the elution process to obtain a methanol solution of glycoside aroma substances.

[0094] The collected methanol solution containing glycosidic aroma compounds was evaporated to dryness using a rotary evaporator at 30℃, and then reconstituted with 10 mL of citric acid / phosphate buffer solution (0.2 mol / L, pH = 5.0). After reconstitution, 100 μL of glycosidase AR2000 (100 g / L) was added, and the solution was incubated at 37℃ for 16 h. After enzymatic hydrolysis, the pH of the hydrolysate was adjusted to 3.0 with citric acid. The hydrolysate was used for the detection of glycosidic aroma compounds.

[0095] Free and bound terpenoids in grape peels were detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS). The specific procedure was as follows:

[0096] The GC-MS instrument used was an Agilent 6890 gas chromatograph equipped with an Agilent 5975 mass spectrometer. The capillary column used was a polar HP-INNOWAX column (60m × 0.25mm × 0.25μm, J&W Scientific, Folsom, CA, USA). 5 mL of fruit peel extract or enzymatic hydrolysate and 1.0 g of sodium chloride were added to a 20 mL gas chromatograph sample vial, along with 10 μL of internal standard (4-methyl-2-pentanol, 1.0 g / L). The vial was then sealed with a cap fitted with a polytetrafluoroethylene septum.

[0097] HS-SPME sample preparation was performed using a CTC CombiPAL multi-functional autosampler (CTC Analytic, Zwingen, Switzerland). After equilibration at 40°C for 30 min, an activated polydimethylsiloxane / carbon sieve / divinylbenzene (DVB / CAR / PDMS) 50 / 30 μm extraction head (Supelco, Bellefonte, PA, USA) was inserted into the headspace of the sample vial, and the sample was extracted with shaking at 500 rpm for 30 min. After extraction, the extraction head was inserted into the GC inlet at 250°C, and splitless injection thermal desorption was performed for 8 min. The carrier gas was helium (purity >99.999%) at a flow rate of 1 mL / min. The temperature program was: 50°C held for 1 min; then increased to 220°C at a rate of 3°C / min and held for 5 min. The mass spectrometry interface temperature is 250℃, the mass spectrometry ion source is an electron ionization (EI) source with an ion energy of 70eV, and the mass scan range is 29-350u.

[0098] Qualitative analysis was performed based on the retention index and mass spectrometry information of the compound under the same chromatographic conditions. For aroma compounds without standards, semi-qualitative analysis was performed using the retention index of the compound under similar chromatographic conditions reported in the literature and the comparison results with the NIST05 standard spectral library (NIST Chemistry WebBook, http: / / webbook.nist.gov / chemistry / ). For aroma compounds for which the retention index under similar chromatographic conditions was not reported in the literature, semi-qualitative analysis was performed based on the comparison results with the NIST05 standard spectral library.

[0099] The quantification of aroma compounds is performed using corresponding standard curves: for aroma compounds with available standards, quantification is directly performed using the standard curve of the corresponding standard; for aroma compounds without available standards, semi-quantification is performed using the standard curve of a standard with a similar chemical structure. The content of terpenoids is expressed as ng / g.

[0100] The results of terpenoid content detection in grape peels of OE-MYB1R1 and CK groups are as follows: Figure 3 As shown. By Figure 3 It was found that the contents of bound trans-β-ocimene and bound linalool were significantly increased in grape skins overexpressing the transcription factor MYB1R1. The content of bound linalool was 2.01 times higher than that of the control group CK, indicating that MYB1R1 is involved in the regulation of terpene metabolism in grapes.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for increasing the content of terpenoid compounds in grapes, characterized in that, grape transcription factor MYB1R1 Transfected into grapes and overexpressed, it increases the content of terpenoids in grape berries; The terpenoid compounds are bound linalool and bound trans- β -Ocimene; The grape transcription factor MYB1R1 The coding region nucleotide sequence is shown in SEQ ID NO.

1.

2. The method for increasing the content of terpenoid compounds in grapes according to claim 1, characterized in that, Used to amplify the grape transcription factor MYB1R1 The upstream primer, as shown in SEQ ID NO.3, is used to amplify the grape transcription factor. MYB1R1 The downstream primer is shown in SEQ ID NO.4.

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