Kit and method for increasing content of terpenoids in grapes

Abstract

The invention relates to the technical field of gene engineering, in particular to a kit and a method for increasing the content of terpenoids in grapes. According to the method, the kit is adopted, the grape transcription factor MYB1R1 is transferred into grapes and overexpressed, and the content of terpenoids in grape fruits is increased. According to the method disclosed by the invention, the MYB1R1 gene is over-expressed in grapes, so that grape plants with higher terpenoids content compared with wild types can be obtained, and the content of partial terpenoids in grape peels can be effectively increased, so that grape germplasm with rich flower fragrance can be cultivated; the method plays a positive role in improving the flower and fruit fragrance of varieties, cultivating new varieties and accelerating the breeding process.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular, to a kit and a method for increasing the content of terpene compounds in grapes. Background Art

[0002] Terpenoids are mainly divided into monoterpenes (such as linalool, geraniol, nerol, limonene), sesquiterpenes and diterpenes. Among them, monoterpenes are the most important in wine and have relatively high contents in varieties such as Muscat, Riesling, and Gewürztraminer. Their existing forms include free state (directly volatilizing to contribute to aroma) and bound state (bound to glycosides, requiring enzymatic hydrolysis or acid hydrolysis to release).

[0003] Terpenoids have an important impact on the aroma of wine, and different terpenoids have different aroma characteristics. For example, geraniol and nerol provide mild, sweet rose and orange flower aromas, linalool can provide a heavier rosewood odor, limonene contributes citrus and lemon flavors, and some terpenoids may also bring mint or spicy aromas. In addition, terpenoids also have a synergistic effect with compounds such as esters and thiols, which can enhance the aroma complexity. For example, the combination of terpenoids and thiols can highlight the passion fruit aroma of Sauvignon Blanc.

[0004] Too few terpenoids will bring many negative impacts, such as monotonous aroma, lack of varietal characteristics, and decreased flavor complexity. For example, terpenoids (such as linalool, geraniol) are the iconic aroma sources of varieties such as Muscat, Italian Riesling, and Marselan. If the terpenoid content is insufficient, the wines brewed from them will lose the varietal-specific floral aromas (rose, orange flower), fruity aromas (citrus, lychee) and herbal aromas, resulting in a single 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 spatio-temporal conditions, and thus affect the synthesis and accumulation of plant secondary metabolites.

[0006] Currently, there is little research on transcription factors related to the synthesis of terpenoids in grapes, and the specific regulatory mechanism of the MYB-related family in the metabolism of terpenoids in grape fruits is still unclear. Exploring and identifying key transcription factors that regulate the synthesis of terpenoids in grape fruits is of great significance for analyzing the molecular mechanism of grape fruit aroma formation. In addition, how to use these transcription factors to improve the aroma quality of grape fruits and the flavor of wine still needs 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 technical problems is as follows:

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

[0010] On the basis of the above technical solution, the present invention can also be improved as follows.

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

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

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

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

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

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

[0017] Further, it also includes the backbone plasmid of the recombinant vector as described above and the engineered bacterium 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 using the grape transcription factor MYB1R1 as described above.

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

[0020] Further, the terpenoid substances include bound linalool and trans-β-ocimene.

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

[0022] The method of the present invention can obtain grape plants containing higher levels of terpenoids compared to wild-type plants by overexpressing the MYB1R1 gene in grapes, effectively increasing the content of some terpenoid compounds in the grape peel, thereby enabling the cultivation of grape germplasms with strong floral scents, which has a positive effect on enhancing the floral and fruity scents of varieties, cultivating new varieties, and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the agarose gel electrophoresis pattern of the grape transcription factor MYB1R1 of the present invention;

[0024] Figure 2 It is the comparison diagram of the relative expression levels of the MYB1R1 gene in the grape peel transiently transformed with MYB1R1 and the grape peel without any transformation treatment in Experimental Example 1 for the grape transcription factor MYB1R1 of the present invention;

[0025] Figure 3 It is the comparison diagram of the terpenoid content in the grape peel transiently transformed with MYB1R1 and the grape peel without any transformation treatment in Experimental Example 2 for the grape transcription factor MYB1R1 of the present invention, Figure 3 wherein a is conjugated trans-β-ocimene, Figure 3 and b is conjugated linalool, * indicates P < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0026] The principles and features of the present invention will be described below in conjunction with 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] For the grape transcription factor MYB1R1 of the present invention, the nucleotide sequence of the coding region encoding the grape transcription factor MYB1R1 is shown as 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 the present invention belongs to the MYB_related class of transcription factors, which is involved in the regulation of terpenoid metabolism in grapes. By overexpressing the MYB1R1 gene in grapes, the content of some terpenoid compounds in the grape peel can be effectively increased, thereby enabling the cultivation of grape germplasms with strong floral scents.

[0029] Specifically, the cDNA of the gene encoding MYB1R1 is 903 bp long and encodes 300 amino acids, and the amino acid sequence is shown as 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 the upstream primer shown as SEQ ID NO.3 and the downstream primer shown as SEQ ID NO.4.

[0031] Preferably, the kit further includes a backbone plasmid and an engineered bacterium for transformation.

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

[0033] Preferably, other conventional reagents may also be included in the kit.

[0034] The method for increasing the content of terpenoid compounds in grapes according to the present invention uses a kit to transfer and overexpress the grape transcription factor MYB1R1 into grapes to increase the content of terpenoid substances in grape fruits.

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

[0036] Specifically, a recombinant vector containing the MYB1R1 nucleotide sequence can be constructed, then the recombinant vector is transferred into an engineered bacterium, and the engineered bacterium is used to infect the cells of grape fruits or peels, so that the grape transcription factor MYB1R1 is overexpressed, thereby increasing the content of terpenoid substances.

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

[0038] (1) Obtain the DNA of the grape transcription factor MYB1R1 by cloning. The specific experimental process is as follows:

[0039] Use the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) to reverse-transcribe the RNA of grape peels (extracted using an RNA extraction kit) into cDNA. The specific systems for each step are shown in Table 1 - Table 3.

[0040] Table 1 The reverse transcription system and reaction conditions for the first step

[0041]

[0042] Table 2 The reverse transcription system and reaction conditions for the second step

[0043]

[0044] Table 3 The reverse transcription system and reaction conditions for the third step

[0045]

[0046] Use SnapGene to design specific primers, and use PCR to amplify the full-length cDNA of MYB1R1. The PCR system is shown in Table 4, the primer sequences are shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0047] Table 4 PCR Amplification Reaction System and Reaction Program

[0048]

[0049]

[0050] Table 5 Primer Sequences

[0051]

[0052] Table 6 PCR Reaction Program

[0053]

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

[0055] In a certain embodiment of the present invention, the electrophoresis results are as Figure 1 shown. Figure 1 The three bands in are three sets of parallel experiments. By comparing with the Marker, it can be seen that the DNA lengths of the three bands are all about 903 bp, indicating that it is MYB1R1 and the cloning is successful.

[0056] After completing the electrophoresis detection, the Hipure Gel Pure DNA Mini Kit agarose gel DNA recovery kit was used to recover the target fragment from the gel.

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

[0058] The plasmid of the overexpression vector pCAMBIA1300 was extracted, and the restriction enzymes SacI and BamHI were selected for double digestion of pCAMBIA1300. After digestion, the linearized pCAMBIA1300 vector was obtained by purification.

[0059] The One Step Cloning Kit C112 was used to ligate the gel recovery product with the linearized vector pCAMBIA1300. The ligation system is shown in Table 7.

[0060] Table 7 Ligation Reaction System and Reaction Conditions

[0061]

[0062] Transform the ligation system into DH5α competent cells. Thaw the cloning competent cells on ice. Take 10 μL of the recombinant product and add it to 50 μL of the competent cells. Gently flick the tube wall to mix and then place it on ice for 30 min. Heat shock at 42 °C for 45 sec, and then incubate on ice for 5 min. Under sterile conditions, add 800 μL of antibiotic-free LB medium. Place it in a shaker at 37 °C and 220 r / min for 1 h of recovery. Centrifuge at 4000×g for 5 min, discard 500 μL of the supernatant, resuspend the cell pellet with the remaining medium, and then pipette 100 μL to spread on a solid medium containing kanamycin. Incubate it upside down in a 37 °C incubator for 16 h. When the colonies grow to about 2 mm in diameter, use a pipette tip to pick a single colony, pipette it up and down twice to dissolve it in 10 μL of LB, and then take 1 μL as a template. Use the system in Table 8 for colony PCR identification. Select the positive clone for sequencing, and save some of the bacterial liquid by mixing it with glycerol at a ratio of 1:1 and storing it at -80 °C.

[0063] Table 8 Colony PCR System

[0064]

[0065] Extract the plasmid from the positive clone with correct sequencing to finally obtain the recombinant plasmid pCAMBIA1300-MYB1R1.

[0066] (3) Construction of recombinant bacteria.

[0067] Use the freeze-thaw method for Agrobacterium transformation. Thaw the Agrobacterium competent GV3101 on ice. After thawing, take 10 μL of the recombinant plasmid and add it to 100 μL of the competent cells. Gently pipette up and down to mix, and then place it on ice for 5 min, quick-freeze in liquid nitrogen for 5 min, incubate in a 37 °C water bath for 5 min, and then incubate on ice for 5 min. In a laminar flow hood, add 900 μL of antibiotic-free liquid LB medium, mix well, and culture it at 28 °C and 200 r / min for 3 h. Centrifuge at 4000×g for 1 min to collect the bacteria, leave about 100 μL of the supernatant, gently pipette up and down to resuspend the bacterial pellet, and spread it on an LB + Rif (50 μg / mL) + Kan (50 μg / mL) solid medium. After air-drying, incubate it upside down at 28 °C in the dark for 3 d.

[0068] Pick a single GV3101 colony into 4 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium, shake the bacteria at 28 °C and 200 r / min for 16 h, and perform PCR to detect positive clones. Pipette 150 μL of the bacterial liquid of the positive clone and inoculate it into 4 mL of the above double-antibiotic LB liquid medium, culture it at 28 °C and 200 r / min for 5 - 8 h until the OD 600 reaches 0.6 - 0.8. Take 1 mL of this bacterial liquid, add an equal volume of sterilized glycerol, quick-freeze in liquid nitrogen, and store it at -80 °C.

[0069] (4) Obtain MYB1R1 transiently transformed grape fruits.

[0070] Add 100 - 200 μL of the above-mentioned Agrobacterium liquid containing the target gene and the GV3101 empty vector liquid into 4 - 5 mL of LB + Rif (50 μg / mL) + Kan (50 μg / mL) liquid medium respectively, and shake the bacteria at 28 °C for 16 h. Pour all the activated bacterial liquid into 40 mL of LB (Kan + Rif) medium for subculture. The second-generation culture is cultured at 28 °C and 220 rpm for 6 - 7 h until OD 600 reaches 1 - 1.2. Centrifuge the second-generation culture at 4000×g for 5 min and resuspend it 1:1 in the Agrobacterium infection buffer. Centrifuge and discard the supernatant 3 times to wash the bacterial cells. Resuspend the precipitate of the second-generation culture in the Agrobacterium infection buffer again until OD 600 is 0.9, and let it stand in the dark at room temperature for 3 h. The formulation of the Agrobacterium infection buffer is shown in Table 9, and the pH value of the Agrobacterium infection buffer needs to be adjusted to 5.2 with KOH.

[0071] Puncture each grape fruit 30 times with a syringe, soak it in the infection solution, and vacuum for 15 min. Shake it constantly during this period to promote the infection solution to enter the fruit interior. Take out the fruit, culture it for 3 days and 3 nights under natural rhythm, sample the obtained grape fruits, freeze them in liquid nitrogen, and then store them in a -80 °C refrigerator. The formulation of the infection solution is shown in Table 9, and the pH value is adjusted to 5 with KOH when in use.

[0072] Table 9 Formulation of the infection solution

[0073] Component Addition amount MES 2.13g <![CDATA[MgCl2·6H2O]]> 2.03g + Add acetosyringone As before use 200 μM Sterile water 1L

[0074] Through the above specific steps, the MYB1R1 gene can be successfully transferred into grapes and overexpressed. After the above treatment, grape plants containing higher levels of terpene substances compared to wild type can be obtained, which has a positive effect on improving the floral and fruity aroma of the variety, cultivating new varieties, and accelerating the breeding process.

[0075] The transcription factor for regulating terpene synthesis in grapes of the present invention is of great significance for analyzing the molecular mechanism of grape fruit aroma formation and improving the aroma quality of grape fruits by genetic engineering means.

[0076] The present invention is specifically described below through examples. Unless otherwise specified, the experimental materials, experimental instruments, and reagents used in the following examples are all obtained through conventional channels.

[0077] Example 1 Relative expression analysis of genes in MYB1R1 transiently transformed grape peels

[0078] The experimental process of this example is as follows:

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

[0080] (1) Genomic DNA removal: Add 500 ng of RNA to an RNase-Free tube. After adding 4 μL of 4×gDNA wiper Mix, add RNase-ddH2O to a total liquid volume of 16 μL. Gently pipette to mix evenly, then place it in a PCR instrument and react at 42 °C for 2 min.

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

[0082] In this example, Ubiquitin was used as the internal reference gene for fluorescence quantitative PCR. After adding reagents according to the reaction system in Table 10, the reaction was carried out, and three parallel replicates were set. The qRT-PCR reaction program was: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s, for 39 cycles; melting curve analysis from 65 °C to 95 °C. The primer sequences are shown in Table 11.

[0083] Table 10 Fluorescence quantitative PCR reaction system

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

[0085] Table 11 Primer sequences

[0086]

[0087] The results of fluorescence quantitative PCR were as Figure 2 shown. In the figure, "OE-MYB1R1" represents the transient transformation of MYB1R1 in grape pericarp in this invention, and "CK" represents grape pericarp without any transformation treatment. It can be Figure 2 seen that in the overexpressing plants, the expression level of MYB1R1 increased by 1.93 times.

[0088] Experimental Example 2 Extraction and detection of terpenoid components in transiently transformed grape pericarp with MYB1R1

[0089] In this example, the contents of terpenoids in the grape peel with transient transformation of MYB1R1 (OE-MYB1R1) and the grape peel without any transformation treatment (CK) were extracted and compared to verify the regulatory effect of MYB1R1 overexpression on the content of terpenoids.

[0090] The process for extracting terpenoids in this example is as follows:

[0091] After the grape peel was taken, it was immediately frozen with liquid nitrogen, put into a powder grinder pre-cooled with liquid nitrogen for powdering, and stored at -80°C. 1 g of the powder was added with 5 mL of citric acid / phosphate buffer (0.2 mol / L, pH = 5.0), soaked at 4°C for 16 h, centrifuged at 4°C and 5000×g for 15 min, and the supernatant was taken for the subsequent extraction and detection of aroma substances.

[0092] The bound terpenoid compounds in the grape peel were extracted by solid phase extraction (SPE). The specific process is as follows:

[0093] The Cleanert PEP-SP (150 mg / 6 mL) extraction column was activated with 10 mL of anhydrous methanol and 10 mL of pure water respectively to remove the impurities in the extraction column; then 2 mL of the above supernatant was added, and it was eluted successively with 5 mL of pure water and 5 mL of dichloromethane to remove polar compounds such as pigments, small molecular weight sugars, acids, and free non-polar aroma compounds; finally, 20 mL of anhydrous methanol was used to elute the glycoside-bound aroma substances into a test tube, and the flow rate was maintained at 2 mL / min during the whole elution process to obtain a methanol solution of glycoside aroma substances.

[0094] The methanol solution containing glycoside aroma substances collected was evaporated to dryness on a rotary evaporator at 30°C, and then re-dissolved with 10 mL of citric acid / phosphate buffer solution (0.2 mol / L, pH = 5.0). After re-dissolution, 100 μL of glycosidase AR2000 (100 g / L) was added and enzymatically hydrolyzed in a constant temperature incubator at 37°C for 16 h; after the enzymatic hydrolysis was completed, the pH of the enzymatic hydrolysate was adjusted to 3.0 with citric acid. The enzymatic hydrolysate was used for the detection of glycoside-bound aroma.

[0095] The free and bound terpenoid compounds in the grape peel were detected by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS) coupling method. The specific process is as follows:

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

[0097] The HS-SPME pretreatment was completed using a CTC CombiPAL multi-functional autosampler (CTC Analytic, Zwingen, Switzerland). After equilibration at 40 °C for 30 min, the activated polydimethylsiloxane / carboxen / 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 by shaking at 500 r / min for 30 min. After extraction, the extraction head was inserted into the GC injection port at 250 °C, and thermal desorption was performed without splitting for 8 min. The carrier gas was helium (purity > 99.999%), and the flow rate was 1 mL / min. Programmed temperature rise: hold at 50 °C for 1 min; increase to 220 °C at a rate of 3 °C / min and hold for 5 min. The mass spectrometry interface temperature was 250 °C, the mass spectrometry ion source was an electron ionization (EI) source, the ion energy was 70 eV, and the mass scanning range was 29 - 350 u.

[0098] Qualitative analysis was carried out based on the retention index and mass spectrometry information of the compound under the same chromatographic conditions. For aroma substances without standard samples, semi-qualitative analysis was performed using the retention index of the compound under similar chromatographic conditions reported in the literature and the comparison results of the NIST05 standard spectral library (NIST Chemistry WebBook. http: / / webbook.nist.gov / chemistry / ); for aroma substances whose retention indices under similar chromatographic conditions were not reported in the literature, semi-qualitative analysis was carried out according to the comparison results of the NIST05 standard spectral library.

[0099] The quantification of aroma substances was analyzed by the corresponding standard curves: for aroma substances with standard samples, the standard curves of the corresponding standard samples were directly used for quantification, and for aroma substances without standard samples, the standard curves of standard samples with similar chemical structures were used for semi-quantification. The content of terpenoids was expressed as ng / g.

[0100] The detection results of terpenoid components in the grape pericarp of the OE-MYB1R1 and CK groups are as Figure 3 shown. ByFigure 3 It can be seen that in the grape peel with overexpression of the transcription factor MYB1R1, the contents of bound trans-β-ocimene and bound linalool both increased significantly. The content of bound linalool increased by 2.01 times compared with the control group CK, indicating that MYB1R1 is involved in the regulation of terpene metabolism in grapes.

[0101] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A grape transcription factor MYB1R1, characterized in that, The nucleotide sequence encoding the grape transcription factor MYB1R1 is shown in SEQ ID NO.

1.

2. The grape transcription factor MYB1R1 according to claim 1, characterized in that, The amino acid sequence of the grape transcription factor MYB1R1 is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector includes the nucleotide sequence encoding the coding region of the grape transcription factor MYB1R1 as described in claim 1.

4. A recombinant vector according to claim 3, wherein The backbone plasmid of the recombinant vector is pCAMBIA1300.

5. An engineered bacterium, characterized in that, The engineered bacterium contains the recombinant vector as described in claim 3 or 4.

6. A kit for increasing the content of terpenoid compounds in grapes, characterized in that, The kit includes the grape transcription factor MYB1R1 as described in claim 1 or 2.

7. A kit for increasing the content of terpenoid compounds in grapes according to claim 6, characterized in that, It also includes an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.

4.

8. A method for increasing the content of terpenoid compounds in grapes, characterized in that, The kit as described in claim 6 or 7 is used to regulate the content of terpenoids in grapes.

9. A method for increasing the content of terpenoid compounds in grapes according to claim 8, characterized in that, The method includes the following steps: using the kit, transferring the grape transcription factor MYB1R1 into grapes and overexpressing it to increase the content of terpenoids in grape fruits.

10. A method for increasing the content of terpenoid compounds in grapes according to claim 9, characterized in that, The terpenoids include bound linalool and trans-β-ocimene.

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