Product, method and application for regulating and controlling aroma of grape fruits

Abstract

According to the product, the method and the application for regulating and controlling the aroma of the grape fruits, two key enzyme genes VvDXS and VvHDS capable of regulating and controlling the aroma of the grape fruits are found through whole genome correlation analysis and transcriptomics analysis, and the grape fruits in the growing period are treated by using agrobacterium liquid containing a gene overexpression vector, so that the aroma of the grape fruits is regulated and controlled. Wherein the overexpression effect of the VvHDS gene is more obvious. The determination and analysis of the content of volatile terpene substances in grape fruits show that the two genes can obviously promote the synthesis of terpene compounds in the grape fruits and improve the aroma of the fruits, and the promotion effect of the VvHDS gene is more obvious, so that a basis is provided for regulating and controlling the aroma of the grape fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit cultivation, and in particular relates to a product, method and use for regulating the aroma of grape fruits. Background Art

[0002] Grapes have a long history as fresh fruits. With the improvement of living standards, people are paying more and more attention to the quality of fresh grapes. Grape quality evaluation includes many aspects, including the content of nutrients such as minerals, vitamins, amino acids, soluble solids, and aroma substances such as alcohols and esters that form flavors. Aroma is an important indicator for evaluating fruit quality. The fragrance brought by aroma substances affects consumers' preference to a certain extent. Therefore, studying grape aroma is not only of great significance to improving consumer preference and market acceptance, but also helps to optimize grape quality management.

[0003] Grapes can be divided into rose-scented, strawberry-scented, non-scented and mixed types according to the aroma type of the fruit. The main aroma substances of rose-scented grapes are terpenes, such as geraniol, linalool, nerol and linalool; the main characteristic aroma substances of strawberry-scented grapes are volatile aliphatic compounds, including ethyl acetate, ethyl butyrate and ethyl heptanoate; while the content of aroma characteristic substances of non-scented fruits is relatively low, resulting in a less obvious aroma. In addition, some grapes present a mixed characteristic of two or even more aroma types, and the aroma substances they contain are complex and diverse.

[0004] At present, the research on grape fruit aroma mainly detects the types and contents of volatile components in the fruit ripening process, analyzes the aroma characteristics of each period; or compares the differences between aroma substances of grape varieties with different aroma types. There are few studies on the synthesis and regulation of grape fruit aroma, and they mainly focus on the metabolism of terpenes in rose-scented grape fruits. For example, previous studies have found that the rose aroma of grape fruit is caused by the SNP mutation of the VvDXS gene. The mutation at this site causes the 284th amino acid to be converted from lysine to asparagine. The expression of the VvDXS gene is significantly associated with the accumulation of monoterpenes, but the specific regulation mode of the VvDXS gene is unknown, and whether other enzyme genes in the metabolic pathway of terpenes in grape fruit are involved in the regulation is unknown. Therefore, exploring the regulation mode of changes in grape fruit aroma components is of great significance for improving the flavor of grape fruit varieties and providing theoretical guidance for grape variety cultivation technology. Summary of the invention

[0005] In view of the existing technical problems, the present invention provides a product, method and use for regulating the aroma of grape fruit. Through whole genome association analysis and transcriptomics analysis, two key enzyme genes VvDXS and VvHDS that can regulate the aroma of grape fruit are found. By treating growing grape fruit with Agrobacterium liquid containing a gene overexpression vector, the synthesis of terpene compounds in grape fruit can be significantly increased and the fruit aroma can be improved, which provides a basis for realizing the regulation of grape fruit aroma.

[0006] In one aspect, the present invention provides a product for regulating the aroma of grape fruit, comprising a gene overexpression vector having a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

[0007] In another aspect, the present invention provides a method for regulating the aroma of grape fruit, using the product as described above to regulate the aroma of grape fruit.

[0008] The formation and accumulation of aroma compounds of different grape varieties are mainly affected by factors such as genotype differences, fruit structure and self-metabolism. The type and content of volatile aroma compounds play a decisive role in the typical aroma of grapes. For example, alcohols and aldehydes mainly show grassy aromas, and grape varieties with grassy aromas include "Moldova" and "Hutai No. 8"; esters show fruity aromas, and terpenes mainly show floral and rose aromas. Different varieties of grapes have different metabolic regulation pathways due to their different volatile aroma compounds.

[0009] Furthermore, the method comprises treating grape berries with a reagent containing a gene overexpression vector during their growth; the gene overexpression vector has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

[0010] Plant overexpression technology is a technology widely used in the field of biology. It introduces the target gene into the host cell or organism and uses appropriate promoters or regulatory elements to promote the overexpression of the target gene. By observing the effect of gene overexpression on the organism, the specific function and regulatory mechanism of the gene in the organism are revealed. By enhancing the expression level of specific genes, it can help researchers better understand how genes affect plant growth and development, metabolic pathways, and responses to the external environment, thereby providing an important theoretical basis and practical guidance for plant improvement and crop breeding.

[0011] In another aspect, the present invention provides a use of a gene for preparing an agent for improving fruit aroma, wherein the gene comprises VvDXS and / or VvHDS, and the gene has a nucleotide sequence as shown in Seq ID NO.3 and / or Seq ID NO.4.

[0012] Furthermore, the use is to improve the aroma of fruits by constructing an overexpression vector of the gene.

[0013] Furthermore, the overexpression vector of the gene has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

[0014] Furthermore, the gene overexpression vector improves the aroma of the fruit by promoting the synthesis of terpene compounds in the fruit.

[0015] Furthermore, the fruit includes grape fruit.

[0016] On the other hand, the present invention provides a use of a gene for preparing an agent for promoting the synthesis of terpene compounds in grape fruit, characterized in that the gene includes VvDXS and / or VvHDS, and the gene has a nucleotide sequence as shown in Seq ID NO.3 and / or Seq ID NO.4.

[0017] Furthermore, the synthesis of terpene compounds is promoted by constructing an overexpression vector of the gene, and the overexpression vector of the gene has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

[0018] The present invention has the following beneficial effects:

[0019] 1. It was found that constructing overexpression vectors of VvDXS and VvHDS genes can improve the synthesis of terpene compounds in grape fruits, thereby improving the synthesis and release of aroma substances in grape fruits, and the promoting effect of VvHDS gene is more significant;

[0020] 2. By overexpressing the aroma substance genes in grape fruits, the edible quality of grapes can be improved, providing a theoretical basis for the cultivation of rose-scented grape varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the aroma ratios of 133 portions of grapes in Example 1 are as follows;

[0022] Figure 2 This is a heat map of the volatile substance content in 133 grapes in Example 2;

[0023] Figure 3 is a stacked graph of volatile substance contents of 133 types of grapes in Example 2;

[0024] Figure 4 is a graph showing the volatile substance contents of six types of fragrant grapes in Example 2;

[0025] Figure 5The whole genome association analysis of the content of volatile terpenes in grape fruit in Example 3;

[0026] Figure 6 The KEGG pathway and GO enrichment annotation results of the grape volatile terpenes content in Example 3;

[0027] Figure 7 is the volatile terpene content of the mature fruits of Black Balado and Sunshine Rose in Example 3;

[0028] Figure 8 is the volatile terpene content and geraniol content of the 15 grape varieties in Example 3;

[0029] Fig. 9 The expression levels of VvG8H, VvDXS and VvHDS in 15 grape varieties in Example 3;

[0030] Fig.10 The correlation analysis between the terpene volatile substances and geraniol content and VvG8H, VvDXS and VvHDS in Example 3;

[0031] Fig.11 is the vector map of the 35S::VvDXS overexpression vector in Example 4;

[0032] Fig.12 is the vector map of the 35S::VvHDS overexpression vector in Example 4;

[0033] Fig.13 is the expression level of VvDXS and VvHDS in grape fruit after overexpression in Example 4;

[0034] Fig.14 The changes in the content of volatile terpenes in grape berries after overexpression of VvDXS and VvHDS in Example 4; DETAILED DESCRIPTION

[0035] Example 1: Grape fruit aroma classification

[0036] At the grape test base, ripe fruits of 133 grape varieties were collected and sensory evaluation was carried out through human olfactory identification. A total of 20 healthy and well-trained panelists (10 females and 10 males) were selected from volunteers working in horticulture, ranging in age from 18 to 35 years old. The sensory room has a uniform light source, no noise, and an ambient temperature between 23 and 25°C. The variety name is represented by a code, and the fresh samples are placed in the sensory room for 1 hour to reach the same temperature (23-25°C) and then evaluated. The intensity score of each aroma is based on a ten-point system, where 0-10 represents the aroma intensity, which is very low, low, medium, high and very high, respectively, and 0 means that the aroma is not perceived. The aroma types of common grapes mainly include rose aroma, grass aroma, strawberry aroma and no aroma. Before the evaluation, the assessors were clearly told to evaluate and record the intensity of the aroma type and signed an informed consent form for participation. After completing each sample, the assessors took a bite of unsalted crackers and drank a sip of water.

[0037] according to Figure 1 It can be seen that in addition to the four basic aroma types of no aroma, rose aroma, strawberry aroma and grass aroma, some grapes showed mixed characteristics of two aroma types. No aroma grapes accounted for 28.57% (38 types) of the total samples, rose aroma accounted for 21.80% (29 types), strawberry aroma accounted for 11.28% (15 types), and grass aroma accounted for 15.79% (21 types). In addition, strawberry and grass mixed aroma accounted for 12.78% (17 types), and strawberry and rose mixed aroma accounted for 9.77% (13 types).

[0038] Table 1 Aroma types and intensities of 133 grape varieties

[0039]

[0040]

[0041]

[0042] As can be seen from Table 1, the aroma intensity of strawberry and rose mixed grapes is the highest, followed by strawberry, strawberry and grassy, ​​and rose grapes. In contrast, the grassy grapes are more limited in perception and have a lower degree of aroma perception.

[0043] Example 2: Determination and analysis of aroma substances in grape fruit

[0044] The content of volatile substances was determined by SPME-GC-MS technique. 65 g of berry pulp was ground in liquid nitrogen, centrifuged and filtered to obtain a transparent juice. The juice was transferred to a 20 mL Agilent bottle (Agilent Technologies, Santa Clara, USA) equipped with a magnetic stir bar and 1.5 g of NaCl. 2-octanol was used as an internal standard to quantify the aroma components in the pulp. After the sample bottle was equilibrated at 50 °C for 10 min, it was inserted into a solid phase microextraction fiber extraction head (50 / 30 μM DVB / CAR / PDMS, Supelco) and the volatile aroma compounds were extracted at 50 °C for 30 min. It was then inserted into the injection port of a chromatograph (Agilent 8890 GC, Agilent Technologies) and resolved at 260 °C for 3 min without separation mode and equipped with a 5977 mass selective detector (Agilent Technologies). HP-INNOWAX column (30 mm × 0.25 mm × 0.25 μm, Agilent) was used; the temperature program was: 40 °C, 5 min, rising to 240 °C at a rate of 5 °C / min, and then rising to 260 °C at a rate of 20 °C / min for 5 min. The carrier gas was helium with a flow rate of 1 ml / min. The mass transfer line temperature and ionization source temperature were 260 °C and 230 °C, respectively. Electron bombardment mass spectrometer data were collected in the range of 20 to 400 m / z at an ionization voltage of 70 eV.

[0045] according to Figure 2 It can be seen that in the GC-MS analysis, a total of 88 volatile compounds were detected, including 7 types of substances such as 17 alcohols, 13 aldehydes, 13 esters, 24 terpenes, 8 acids, 4 ketones and 9 other components.

[0046] according to Figure 3 It can be seen that the total content of volatile compounds in each grape sample ranges from 16.18 μg kg -1 (18-51)~471.55μg·kg -1 (Giant Rose), while the volatile compound types ranged from 20 (18-27) to 63 (Hanxiang Honey). The volatile organic compound content of the six types of grapes was mainly aldehydes. In addition, in rose-scented and strawberry-rose mixed-scented grapes, the proportion of terpenes was relatively high, while in strawberry-scented and strawberry-grass mixed-scented grapes, the proportion of lipids was relatively high.

[0047] Table 2 17 volatile alcohols in 133 grape samples

[0048]

[0049] As shown in Table 2, the 17 alcohol volatile substances mainly present a grassy aroma, with a content ranging from 2.64 to 96.91 μg kg -1 The average value was 20.85 μg kg -1 , accounting for 1.59% to 66.00% of the total volatile compounds, with an average of 15.05% and a coefficient of variation of 77.93%. Hexanol and trans-2-hexen-1-ol were the main volatile substances, detected in 100% (133) and 94.73% (126) of grape varieties, respectively. Previous studies have shown that hexanol is described as floral, grassy and woody, while trans-2-hexen-1-ol is described as grassy. Therefore, it is inferred that hexanol and trans-2-hexen-1-ol are the components of alcohols that have a greater impact on the aroma of grapes.

[0050] Table 3 13 volatile lipids in 133 grape samples

[0051]

[0052]

[0053] According to Table 3, the 13 volatile lipids mainly showed fruity aroma, with a content ranging from 0 to 56.83 μg kg -1 The average value was 7.26 μg kg -1 , accounting for 0-26.87% of the total volatile compounds, with an average of 2.86% and a coefficient of variation of 207.80%, indicating that the volatile lipids vary greatly among varieties. Among lipids, the main volatile substances are ethyl caproate and ethyl butyrate, which were detected in 59.40% (79) and 69.17% (92) of grape varieties, respectively. Ethyl caproate was described as fruity, apple and pineapple, and ethyl butyrate was described as strawberry, apple, banana and pineapple.

[0054] Table 4 24 volatile terpenes in 133 grape samples

[0055]

[0056] Note: “-” indicates no results found

[0057] From the results in Table 4, we can see that the 24 terpene volatile substances mainly present floral and rose scents. The content of volatile substances is around 0.01 μg kg -1 ~289.33μg·kg -1 The average value was 17.10 μg kg -1, accounting for 0.01% to 61.36% of the total volatile compounds, with an average of 10.49% and a coefficient of variation of 208.17%, indicating that the content of terpene volatiles in the 133 grape varieties varies greatly. Among terpenes, the main volatile substances are linalool and geraniol, which are detected in 62.41% (83) and 57.14% (76) of the grape varieties, respectively. Linalool is described as citrus, floral, and grape, and geraniol is described as rose and citrus.

[0058] Table 5 8 volatile acids in 133 grape samples

[0059]

[0060] Note: “-” indicates no results found

[0061] According to the results in Table 5, the eight volatile acids mainly showed spoilage and grease smells. The content of the substances was between 0 and 15.36 μg kg -1 The average value was 3.33 μg kg -1 , accounting for 0-6.69% of the total volatile compounds, with an average of 1.45% and a coefficient of variation of 81.73%. The most abundant substance in the acids was caproic acid, which was detected in 83.45% (111) of the grape varieties and was described as sweet, rancid, cheesy and oily.

[0062] Table 6 Four volatile ketones in 133 grapes

[0063]

[0064] As shown in Table 6, the contents of the four volatile ketones ranged from 0 to 7.57 μg kg -1 The average value is 0.96 μg kg -1 , accounting for 0-3.29% of the total volatile compounds, with an average of 0.42% and a coefficient of variation of 113.14%. The highest ketone was β-damascena, which was detected in 51.13% (68) of the grapes and was described as floral, sweet and apple-flavored.

[0065] Table 7 9 other volatile substances in 133 grape samples

[0066]

[0067]

[0068] Note: “-” indicates no results found

[0069] From Table 7, we can see that the contents of 9 other volatile substances are around 0.24 μg·kg -1~7.57 μg kg -1 The average value was 3.38 μg kg -1 , accounting for 0.10% to 4.75% of the total volatile compounds, with an average of 1.47% and a coefficient of variation of 74.37%. The highest substance among the others was 2-ethylfuran, which was detected in 92.48% (123) of the grapes and was described as nutty, sweet and coffee-flavored.

[0070] Based on the GC-MS results, a heat map was drawn using the average content of volatile substances in each grape variety, such as Figure 4 . The results showed that compared with other grape aroma types, the content of volatile substances in strawberry-rose mixed aroma grapes was generally higher, such as linalool, geraniol, ethyl isovalerate and ethyl propionate. Each type of grape has its own unique volatile substances, such as ethyl caproate, ethyl caproate and ethyl propionate in strawberry aroma grapes, benzyl alcohol, heptanol, lauric acid in strawberry-grass mixed aroma, cis-3-hexen-1-ol, hexanol and caproic acid in grassy aroma, γ-terpinene, eucalyptol and diethylene glycol monoethyl ether in rose aroma.

[0071] Table 8 Key volatile substances of rose-scented grapes

[0072]

[0073] According to Table 8, rose-scented grapes have 22 characteristic aroma substances, of which alcohols account for 36.4% (8 substances), aldehydes account for 27.3% (6 substances), terpenes account for 18.2% (4 substances), and ketones account for 18.2% (4 substances). α-Citral, geraniol, linalool, nerol, and cis-citral VIP have higher scores, and it is speculated that these alcohol substances play a major role in contributing to the rose aroma of grapes.

[0074] Table 9 Key volatile substances of strawberry-scented grape

[0075]

[0076] According to Table 9, there are 12 characteristic aroma substances in strawberry-scented grapes, of which lipids account for 50% (6 species), terpenes account for 25% (3 species), aldehydes account for 16.7% (2 species), and alcohols account for 8.3% (1 species). Ethyl caproate, ethyl caproate, ethyl propionate, ethyl valerate, and ethyl heptanoate have higher VIP scores and are significantly different from the other aroma substances. They are all lipid substances, indicating that these substances are crucial to the strawberry aroma and play a major role in contributing to the strawberry aroma of grapes.

[0077] Table 10 Key volatile substances of Qingcaoxiang grape

[0078]

[0079] As shown in Table 10, there are 17 characteristic aroma substances in the grassy grape, of which alcohols account for 35.3% (6 species), acids account for 11.8% (2 species), terpenes account for 11.8% (2 species), aldehydes account for 11.8% (2 species), ketones account for (2 species), lipids account for (2 species), and others account for 5.9% (1 species). 1-Octene-3-ol, hexanol, cis-3-hexenol and isooctyl alcohol have higher VIP scores, which indicates that these alcohols play a major role in contributing to the grassy aroma of grapes.

[0080] Table 11 Key volatile substances of strawberry and rose mixed fragrant grapes

[0081]

[0082]

[0083] According to Table 11, there are 25 characteristic aroma substances in the strawberry and rose mixed aroma grapes, of which terpenes account for 52% (13 species), lipids account for 24% (6 species), alcohols account for 16% (4 species), aldehydes account for 4% (1 species), and ketones account for 4% (1 species). The substance with a higher VIP value is nerol, followed by geraniol, geranylgeraniol, ethyl caproate and ethyl caproate, etc., mainly terpenes and lipids, and it is speculated that these substances are the factors that lead to its mixed aroma.

[0084] Table 12 Key volatile substances of strawberry and grass mixed grape flavor

[0085]

[0086] According to Table 12, there are 18 characteristic aroma substances in the strawberry and grass mixed aroma grapes, of which lipids account for 27.8% (5 kinds), alcohols account for 22.2% (4 kinds), terpenes account for 16.7% (3 kinds), ketones account for 11.1% (2 kinds), aldehydes account for 11.1% (2 kinds), and acids account for 11.1% (2 kinds). The substances with higher VIP values ​​are ethyl hexanoate, ethyl valerate, ethyl hexanoate, ethyl propionate and ethyl heptanoate, etc. Unlike strawberry aroma grapes, strawberry and grass mixed aroma grapes contain more alcohol substances with higher VIP values, which may be the reason for the two aroma types of grapes.

[0087] Example 3: Screening for candidate genes regulating the formation of grape fruit aroma

[0088] (I) Genome-wide association analysis

[0089] Based on the GLM model, a genome-wide association analysis of the content of grape volatile terpenes was performed, and the SNP sites with -log10(P)>8 were taken as significant sites. The QQ plot shows ( Figure 5 -A), the observed value on the lower left is consistent with the expected value, and the observed value on the upper right is upward, indicating that the GLM model is suitable for this association study. From the Manhattan plot, we can see that ( Figure 5 -B), a total of 122 SNP loci were significantly associated with total terpene content in the GLM model.

[0090] 1 412 candidate genes were screened in the 100 kb interval upstream and downstream of the SNP site, and enriched by KEGG and GO ( Figure 6 ), we found pathways related to the biosynthesis of volatile terpenes, and found a total of 10 genes that may be related to the accumulation of volatile terpenes (Table 13).

[0091] Table 13 Genes related to the synthesis of volatile terpenes

[0092]

[0093] (II) Transcriptome data analysis

[0094] There is a significant difference in the content of volatile terpenes in the mature fruits of 'Sunshine Rose' (rose fragrance) and 'Black Balado' (no fragrance) ( Figure 7 ), the expression patterns of the above 10 candidate genes were analyzed by transcriptome data (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA926483 / ), among which 3 genes were significantly differentially expressed between the two varieties (Table 13), namely VIT_02s0109g00310 (log2(Y / H)=3.05), VIT_05s0020g02130 (log2(Y / H)=1.52) and VIT_06s0004g02900 (log2(Y / H)=1.49), which were annotated as geraniol-8-hydroxylase (G8H), 1-deoxy-D-xylulose-5-phosphate reductoisomerase ( ... synthase, DXS) and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS).

[0095] (III) Analysis of expression patterns of VvG8H, VvDXS and VvHDS genes

[0096] Fifteen grape varieties with large differences in the content of volatile terpenes ('Golden Queen', 'Giant Rose', 'Honey Light', 'Romantic Beauty', 'Qinlong Dasui', 'Royal', 'Madonna Ruby', 'Queen Nina', 'Golden Finger', 'Moldova', 'Xiayi Rose', 'Hanxiang Mi', 'Red Fuji', 'Zaocui Xiangti' and 'Deep Red Seedless') were selected to analyze the expression patterns of genes. Among them, the terpene content of 'Giant Rose', 'Zaocui Xiangti' and 'Madonna Ruby' was high, the terpene content of 'Golden Queen', 'Royal' and 'Qinlong Dasui' was medium, and the content of 'Golden Finger', 'Deep Red Seedless' and 'Queen Nina' was extremely low ( Figure 8 ). The geraniol content of high-terpene grapes such as 'Giant Rose', 'Early Cui Shanti' and 'Madonna Ruby' is also high, while the geraniol content of 8 grape varieties such as 'Golden Finger', 'Deep Red Seedless' and 'Queen Nina' is extremely low or even undetectable ( Figure 8 ).

[0097] The expression of VvG8H, VvDXS and VvHDS genes in 15 grape varieties was analyzed by real-time fluorescence quantitative PCR, with the gene expression level of 'Romantic Beauty' grape fruit as the control. The results showed that ( Fig. 9 ), VvG8H, VvDXS and VvHDS were expressed to varying degrees in 15 grape varieties, and were variety-specific. Among them, the expression level of VvG8H gene in 'Nina Queen' was the highest, followed by 'Golden Queen' and 'Golden Finger', which were significantly different from other varieties, and were 4.13, 2.4 and 2.3 times that of the control group, respectively, while the expression level in 'Royal' was the lowest, only 0.27 times that of the control group; the gene expression level of VvDXS was the highest in 'Giant Rose', followed by 'Mi Guang' and 'Madonna Ruby', which were 17.82 times, 9.46 times and 9.03 times that of the control group, respectively, which were significantly different from other varieties, while the expression levels in 'Qinlong Dasui', 'Red Fuji' and 'Nina Queen' were low, only about 0.25 times that of the control group; in 'Zao Cui Xiangti', the gene expression level of VvHDS was the highest, followed by 'Han Xiang Mi' and 'Golden Queen', which were 2.60 times, 2.40 times and 2.12 times that of the control group, respectively, which were significantly different from other varieties, while the gene expression level in 'Royal' was the lowest, only 0.14 times that of the control group.

[0098] The correlation analysis was performed between the expression levels of VvG8H, VvDXS and VvHDS genes and the contents of volatile terpenes and geraniol. Fig.10As shown in the figure, the content of volatile terpenes was significantly correlated with the content of geraniol, especially VvDXS was significantly positively correlated with the content of volatile terpenes and geraniol. VvHDS also had relatively high expression levels in some grape varieties with higher terpenes, such as 'Zaocui Xiangti', 'Mi Guang' and 'Jumeigui'. Therefore, it is speculated that VvDXS and VvHDS genes may be key genes for the synthesis of terpenes and the formation of aroma in grape fruits.

[0099] Example 4: Functional verification of VvDXS and VvHDS genes

[0100] (I) Construction of overexpression vectors of VvDXS and VvHDS genes

[0101] In this example, a 35S::VvDXS overexpression vector and a 35S::VvHDS overexpression vector were constructed.

[0102] The VvDXS overexpression vector was transformed from the pBWA(V)HS vector. The construction process was as follows: the VvDXS overexpression vector (Seq ID NO.1) was constructed by homologous recombination and golden gate seamless cloning method. The primers were:

[0103] VvDXS-F:ACGGCATGGACGAGCTCTAatggcgctgtgcaccctgagc(Seq ID NO.5)

[0104] VvDXS-R:TGAAGACAGAGCTAGTTACAcagcataatttccagcgct(Seq ID NO.6)

[0105] The vector map of 35S::VvDXS overexpression vector is shown in Fig.11 .

[0106] The 35S::VvHDS overexpression vector was transformed from the pBWA(V)HS vector. The construction process was as follows: the VvHDS gene overexpression vector (Seq ID NO.2) was constructed by homologous recombination and golden gate seamless cloning method. The primers were:

[0107] VvHDS-F:ACGGCATGGACGAGCTCTAatggcgaccggcagcgtgccgacca(Seq ID NO.7)

[0108] VvHDS-R:TGAAGACAGAGCTAGTTACAttcttccgccggcggatccacc(Seq ID NO.8)

[0109] The vector map of 35S::VvHDS overexpression vector is shown in Fig.12 .

[0110] (II) Analysis of VvDXS and VvHDS gene expression levels

[0111] The inflorescence of 'Golden Queen' (with a medium content of terpenes in the fruit) was immersed in 1 ml of Agrobacterium tumefaciens liquid containing 35S::VvDXS and 35S::VvHDS overexpression vectors for 5 minutes to construct VvDXS-OE and VvHDS-OE groups of grapes, which were compared with the empty vector group (35S::FLAG) and the blank control group (WT). Fig.13 ). The fruits were collected at maturity and the gene expression in each group of grape fruits was detected.

[0112] Depend on Fig.13 It can be seen that there was no significant difference in the expression levels of VvDXS and VvHDS between the wild group and the empty vector group, while the expression levels of VvDXS and VvHDS genes in the pulp of VvDXS-OE and VvHDS-OE 'Golden Queen' grapes were significantly increased, which were 23.65 times and 27.15 times that of the wild group and 22.74 times and 26.11 times that of the empty vector group, respectively, indicating that the positive test of the functional verification experiment was relatively successful, and the overexpression effect of the VvHDS gene was more obvious.

[0113] (III) Effects of VvDXS and VvHDS gene expression on grape berry aroma

[0114] Depend on Fig.14 It can be seen that the contents of volatile terpenes in the wild group and the empty group of 'Golden Queen' were 5.57 μg·kg -1 and 5.53 μg kg -1 , there was no significant difference; the content of volatile terpenes in the pulp of VvDXS-OE and VvHDS-OE'Golden Queen' grapes increased significantly, and the contents were 7.55μg / kg and 14.18μg / kg, respectively, which were 1.36 times and 2.55 times that of the wild group and 1.37 times and 2.56 times that of the empty group, respectively. This shows that overexpression of both genes can promote the synthesis of terpene compounds in grape fruits, but the promoting effect of overexpression of VvHDS gene is more significant. Therefore, VvHDS gene is preferred in this embodiment.

[0115] The application of the present invention is not limited thereto. For example, the scope of application of the present invention can be expanded according to the environmental protection aspect. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A product for regulating the aroma of grape fruit, characterized in that: It comprises a gene overexpression vector, wherein the gene overexpression vector has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.

2.

2. A method for regulating the aroma of grape fruit, characterized in that: The product as claimed in claim 1 is used to regulate the aroma of grape fruits.

3. The method according to claim 2, characterized in that The method comprises treating grape berries with a reagent containing a gene overexpression vector during their growth, wherein the gene overexpression vector has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.

2.

4. A use of a gene for preparing a reagent for improving the aroma of fruit, characterized in that: The gene includes VvDXS and / or VvHDS, and the gene has a nucleotide sequence as shown in Seq ID NO.3 and / or Seq ID NO.

4.

5. The use according to claim 4, characterized in that The aroma of the fruit is improved by constructing an overexpression vector of the gene.

6. The use according to claim 5, characterized in that The gene overexpression vector has the nucleotide sequence shown in Seq ID NO.1 and / or Seq ID NO.

2.

7. The use according to claim 6, characterized in that The gene overexpression vector improves the aroma of the fruit by promoting the synthesis of terpene compounds in the fruit.

8. The use according to claim 7, characterized in that The fruit includes grape fruit.

9. A use of a gene for preparing a reagent for promoting the synthesis of terpene compounds in grape fruit, characterized in that: The gene includes VvDXS and / or VvHDS, and the gene has a nucleotide sequence as shown in Seq ID NO.3 and / or Seq ID NO.

4.

10. The use according to claim 9, characterized in that The synthesis of terpene compounds is promoted by constructing an overexpression vector of the gene, and the overexpression vector of the gene has a nucleotide sequence as shown in Seq ID NO.1 and / or Seq ID NO.2.

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