Genetic engineering application of VvUGT145 and VvUGT177 in grape G group UGT gene family

Abstract

The application belongs to the technical field of grape G group UGT gene family, and particularly relates to genetic engineering application of VvUGT145 and VvUGT177 grape G group UGT gene family. The VvUGT145 and VvUGT177 belong to grape VvUGT gene family G group, encode UDP-sugar transferase, specifically catalyze glycosylation of grape free linalool and other monoterpenes to generate bound monoterpenes glycosides. The VvUGT145 and VvUGT177 are specifically highly expressed in grape fruit pericarp from the fruit color changing period to the mature period, are functional genes for regulating accumulation of fruit terpenes aroma substances. The gene promoter regions all contain light response elements, abscisic acid and methyl jasmonate hormone response elements, and low temperature and drought stress response cis-elements, and the gene expression is up-regulated by exogenous light quality, ABA and methyl jasmonate.

Description

Technical Field

[0001] This invention belongs to the technical field of grape G group UGT gene family, and particularly relates to the genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family. Background Technology

[0002] Grape aroma is a core indicator determining the commercial quality and market value of table and wine grapes. Monoterpenes are key components of characteristic grape aromas. Free monoterpenes are easily volatilized and lost, while UDP-glycosyltransferases (UGTs) can catalyze the glycosylation of free linalool and other monoterpenes, generating stable bound monoterpene glycosides stored in fruit tissues, becoming precursors for aroma release during ripening. Existing research confirms that the grape genome contains a large family of UGT genes, but different subfamilies exhibit significant functional differentiation. Most studies focus only on the overall evolutionary characteristics of the family and have not yet precisely identified the key functional genes specifically involved in monoterpene glycoside synthesis during fruit ripening, making it difficult to support molecular breeding work for targeted improvement of grape aroma.

[0003] Current methods for improving grape quality largely rely on traditional hybridization and breeding, which have long breeding cycles and uncontrollable trait improvements. Gene editing and overexpression technologies can achieve precise regulation of target traits, but there is currently a lack of target genes specifically for regulating the accumulation of grape monoterpene glycosides and corresponding application schemes. Grape group G UGT is speculated to be involved in the monoterpene glycosylation process; however, there are significant differences in the physicochemical characteristics, spatiotemporal expression patterns, and evolutionary conservation among members within group G. Existing research has not distinguished the core functions of VvUGT145 and VvUGT177 in fruit aroma metabolism, nor has it developed an integrated application system based on molecular markers, transgenic vectors, and field cultivation regulation using these two genes. This hinders the targeted breeding of new high-aroma grape varieties and the improvement of grape fruit aroma quality and efficiency. Summary of the Invention

[0004] In view of the aforementioned problems, and in conjunction with the first aspect of the present invention, embodiments of the present invention provide the genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family, wherein VvUGT145 and VvUGT177 belong to the grape VvUGT gene family G group, encode UDP-glycosyltransferase, which specifically catalyzes the glycosylation of monoterpenes such as free linalool in grapes to generate bound monoterpene glycosides; VvUGT145 and VvUGT177 are specifically highly expressed in grape skins from the color change to maturity stage, and are functional genes that regulate the accumulation of terpenoid aroma substances in the fruit. The promoter regions of the genes all contain light-responsive elements, abscisic acid and methyl jasmonate hormone-responsive elements, and low temperature and drought stress-responsive cis-elements. Gene expression is upregulated by treatment with exogenous light quality, ABA, and methyl jasmonate. Among them, VvUGT145 exhibits cross-species collinearity with Arabidopsis thaliana AtUGT85A4 and AtUGT85A7, and its monoterpene glycosylation function is conserved in dicotyledonous plants.

[0005] Preferably, the genetic engineering application is used for grape molecular-assisted breeding, and the steps include: developing specific molecular markers using the VvUGT145 and VvUGT177 gene sequences, screening for highly expressed gene lines in grape hybrid offspring populations, and breeding new grape varieties with high monoterpene aroma.

[0006] Preferably, the genetic engineering application is gene overexpression modification: constructing a plant overexpression vector containing the complete coding sequence of VvUGT145 or VvUGT177, transforming grape callus / grape plants, and upregulating the expression level of the target gene.

[0007] Preferably, the genetic engineering application is gene editing-directed improvement: using CRISPR / Cas9 gene editing technology to knock down or remove endogenous VvUGT145 and VvUGT177 in grapes to reduce the content of bound monoterpenes in the fruit.

[0008] Preferably, the VvUGT145 is located on grape chromosome 18, encodes 473 amino acids, has an isoelectric point of 4.84, and is a stable acidic protein; The VvUGT177 has no clear chromosomal location, encodes 482 amino acids, has an isoelectric point of 6.14, and is an unstable acidic protein.

[0009] Preferably, the application also includes grape field cultivation regulation: by regulating light conditions and exogenously spraying abscisic acid or methyl jasmonate, the cis-responsive elements of the VvUGT145 and VvUGT177 promoters are activated, inducing endogenous gene expression and simultaneously improving the grape's resistance to low temperature and drought stress and the aroma quality of the fruit.

[0010] A plant overexpression vector for increasing the content of monoterpene glycosides in grapes, wherein the vector is inserted with a complete VvUGT145 or VvUGT177 coding sequence, which can specifically drive the efficient expression of the target gene in grape fruit tissue and catalyze the glycosylation of free monoterpenes to generate monoterpene glycosides.

[0011] A transgenic grape line with high monoterpene glycosides was obtained by transforming grapes with an overexpression vector. The expression levels of VvUGT145 or VvUGT177 in the line were significantly higher than those in the wild type, and the accumulation of monoterpene glycosides was increased during the fruit ripening stage.

[0012] Based on the above, VvUGT145 and VvUGT177 were identified for the first time as the core target genes catalyzing the synthesis of monoterpene glycosides during grape fruit ripening. Both genes are specifically highly expressed from color change to mature peel. Exogenous light, abscisic acid, and methyl jasmonic acid treatment can simultaneously activate the cis-response elements of the gene promoters, significantly increasing the accumulation of monoterpene glycosides such as linalool in the fruit. Among them, VvUGT145 has cross-species collinearity with homologous genes in Arabidopsis thaliana, and its monoterpene glycosylation function is highly conserved, ensuring the stability of gene function regulation. Compared with random screening of UGT family genes, targeted use of these two genes can significantly improve the accuracy and efficiency of grape aroma improvement.

[0013] Based on the multiple technical pathways provided by this invention, including molecular-assisted breeding, gene overexpression, CRISPR gene editing, and field exogenous regulation, it is possible to quickly screen high-aroma grape hybrids using molecular markers, shortening the breeding cycle. It is also possible to use transgenic vectors to directionally upregulate the expression of target genes, stably increasing the content of bound monoterpenes in the fruit. At the same time, exogenous hormones and light regulation methods can simultaneously improve fruit aroma and plant resistance to low temperature and drought without altering the grape genome, taking into account both grape fruit quality improvement and field adaptability. This solves the technical pain points of slow improvement of traditional breeding traits and difficulty in simultaneously improving quality and resistance to stress using conventional cultivation methods. Attached Figure Description

[0014] Figure 1 Chromosomal localization of the grape UGT gene; Figure 2 Phylogenetic tree of the VvUGT gene family; Figure 3 Phylogenetic relationships, conserved motifs, and gene structures of VvUGT family G group members; Figure 4 Analysis of cis-regulatory elements in the promoter subregion of the VvUGT family G group; Figure 5 Intraspecific collinearity analysis of the VvUGT gene; Note: Gray lines in the background represent all collinear blocks, while red lines highlight the copy pairs of the grape UGT gene. Figure 6 Interspecific collinearity analysis of UGT genes in grapes and Arabidopsis thaliana; Note: The gray lines in the background represent all collinear blocks, while the red lines indicate collinear UGT gene pairs between grapes and Arabidopsis, and the blue lines represent two gene pairs collinear with VvUGT145. Figure 7 Heatmap of VvUGT gene expression levels in different tissues in group G. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings.

[0016] Identification of the VvUGT gene in grapes; Grape protein sequences were downloaded from the Winberige database, while Arabidopsis UGT protein sequences were obtained from the CAZy database. Using Arabidopsis UGT proteins as the search sequence, BLAST homology alignment analysis was performed in the grape protein database. Simultaneously, the Hidden Markov Model (HMM) file corresponding to the UGT family domain PF00201 was downloaded from the Pfam database, and the hmmsearch program was used to search the grape protein data to screen for potential VvUGT members, with an E-value threshold of 1e-5. Subsequently, the candidate proteins obtained by both BLAST and HMM methods were further validated using the SMART tool, removing sequences lacking the complete PSPG motif. A total of 181 VvUGT genes were ultimately identified. Finally, the physicochemical properties of the VvUGT proteins were analyzed using the ExPASy online tool.

[0017] Phylogenetic, gene structure, conserved motifs and promoter analysis; The maize GRMZMG834303 and GRMZM2G07587 sequences were obtained from the EnsemblPlants database, and the rice Os07g14630 and Os07g30690 sequences were obtained from the National Rice Data Center (https: / / www.ricedata.cn / ). Eighteen UGT genes from Arabidopsis thaliana were also selected. Multiple alignment of the UGT protein sequences was performed using MUSCLE software, followed by the construction of a phylogenetic tree using the neighbor-joining method in MEGA 12 software, with a bootstrap replication factor of 1000. Based on the intron-exon structure information in the grape GFF3 annotation file, the VvUGT gene structure was visualized using TBtools software. A chromosome distribution map of the VvUGT gene was constructed using TBtools based on the chromosomal location information of the grape genome. Motifs of the VvUGT protein were analyzed using the MEME online platform, with a maximum of 10 motifs identified per sequence. Promoter sequences 2000 bp upstream of the start codon of each VvUGT gene were extracted from the grape genome. Their cis-acting elements were predicted using the PlantCARE database and visualized using TBtools software.

[0018] VvUGT chromosome localization and collinearity analysis; Based on grape genome location information, the chromosomal physical distribution map of the VvUGT gene was drawn using TBtools software. Tandem duplication and fragment duplication events in the grape UGT gene family were identified using MCScanX software with default parameters, and the collinearity between grape and Arabidopsis UGT genes was analyzed.

[0019] Analysis of tissue-specific expression patterns of VvUGT; The expression patterns of the VvUGT gene in different tissues were obtained from the Grape eFP Browser database (https: / / bar.utoronto.ca / efp_grape / cgi-bin / efpWeb.cgi), covering 12 tissues or developmental stages, including roots, stems, leaves, flowers, and fruits. All gene expression levels were expressed as FPKM values ​​and normalized to log2. Expression heatmaps were then created using TBtools software for visualization analysis.

[0020] Results and Analysis; Chromosomal localization of the grape UGT gene; 181 VvUGT genes were identified, based on their location on the chromosome ( Figure 1 These genes were named VvUGT1 to VvUGT181. Except for 10 members that were not located on chromosomes, the remaining genes are distributed on the 18 chromosomes of grape.

[0021] Phylogenetic analysis and physicochemical property analysis; A phylogenetic tree of the VvUGT gene family was constructed based on the UGT amino acid sequences of grape, Arabidopsis, maize, and rice. Figure 2 In this phylogenetic tree, UGT genes were divided into 16 phylogenetic groups (A-P) based on evolutionary relationships. It was found that 18 UGTs from Arabidopsis thaliana clustered in groups A-N, 2 UGTs from rice clustered in group P, and 2 UGTs from maize were located in groups O and P, respectively. Among these, group E (grape) had the most UGT members (56), followed by group L (32). Groups C and N had the fewest members (1 each), while group G had 16 members.

[0022] Physicochemical property analysis (Table 1) showed that the amino acid lengths encoded by the G group genes ranged from 420 aa (VvUGT17) to 1397 aa (VvUGT181), corresponding to molecular weights between 46.6 kDa and 156.5 kDa. The predicted isoelectric points (pI) ranged from 4.84 (VvUGT144) to 6.28 (VvUGT80), with all predicted pIs less than 7, indicating that all members of the G group are acidic proteins. Furthermore, the instability indices of VvUGT14, VvUGT15, VvUGT16, VvUGT17, VvUGT177, VvUGT179, and VvUGT181 were greater than 40, classifying them as unstable proteins, while the rest were classified as stable proteins. This difference in stability may affect their half-life and duration of function. Based on these physicochemical characteristics, it is speculated that members of this family may undertake diverse biological functions.

[0023] Table 1. Basic information and physicochemical properties of VvUGT;

[0024] Analysis of conserved motifs and gene structures in the grape UGT family; Analyzing gene structure can help us understand the evolutionary process of gene families. Therefore, this invention analyzes the gene structure of VvUGT members in group G (see...). Figure 3 Of these gene structures, 15 members contain untranslated regions (UTRs), while only VvUGT17 lacks a UTR and contains only coding sequences (CDS). The number of introns among the G group members is highly similar, with all genes containing at least one intron, VvUGT17 containing two introns, and VvUGT181 containing four introns.

[0025] Analysis of conserved motifs in the VvUGT gene using the MEME online platform identified nine conserved motifs. Motifs 1, 2, 3, 4, 5, 6, 7, and 8 appeared in most G group VvUGT sequences, with each motif appearing only once in most sequences. Notably, motif 4 was missing in the VvUGT16 and VvUGT17 sequences, while VvUGT14 contained two motifs 1. Furthermore, VvUGT181 exhibited some unique characteristics, including motif 9, and two repetitive motifs for each of motifs 1, 2, 3, 4, 5, 6, and 7.

[0026] Analysis of cis-acting elements of grape UGT family promoters; Cis-acting elements have a significant impact on the function of downstream genes. To further explore the promoter characteristics of the group G VvUGT gene, the upstream 2000bp region was analyzed (see...). Figure 4Analysis revealed that this region contains various cis-regulatory elements, primarily categorized into four main types: hormone response, abiotic stress response, light response, and tissue-specific development. Additionally, some universal cis-regulatory elements were also detected. Figure 3-4 As observed, all group G VvUGT genes contain light-responsive elements, suggesting that the transcriptional regulation of VvUGT genes may be strictly controlled by light signals, which is usually closely related to photosynthetic metabolism and the synthesis and accumulation of secondary metabolites (such as monoterpenes and flavonoids). Hormone-responsive elements include abscisic acid, salicylic acid, methyl jasmonate, gibberellin, and auxin-responsive elements, with abscisic acid and methyl jasmonate having the most abundant response elements. In addition, the promoter regions of group G VvUGT genes also contain elements related to stress responses such as drought induction, low temperature response, and anaerobic induction, as well as elements related to palisade tissue differentiation, endosperm expression, and root and seed-specific expression. These findings indicate that group G VvUGT genes may play an important role in plant responses to various environmental stimuli.

[0027] Intraspecific and interspecific collinearity analysis; To explore the expansion and evolutionary patterns of the VvUGT gene family, this invention analyzes gene duplication events in the grape genome. Figure 5 Collinearity analysis of the grape genome based on amino acid sequence homology using MCScanX detected 14 pairs of duplicated gene segments. These results suggest that some VvUGT genes may be generated by gene duplication and may have similar functions. However, no G-group VvUGT genes were present among these 14 pairs.

[0028] To further elucidate the evolutionary relationships of the VvUGT gene family, this invention performed collinearity analysis on the Arabidopsis and grape genomes. Figure 6 The results showed that 26 collinear UGT genes were identified between Arabidopsis and grape, involving 23 genes in Arabidopsis and 26 genes in grape. These VvUGTs were distributed on 12 grape chromosomes, namely chr2, chr3, chr4, chr6, chr8, chr13, chr14, chr15, chr16, chr17, chr18, and chr19. Among them, chr6 and chr18 had the most collinear genes, followed by chr3. One VvUGT gene, VvUGT145, belonging to group G, showed collinearity with AtUGT85A4 and AtUGT85A7 in Arabidopsis, indicating that VvUGT145 is likely a functionally conserved orthologous gene, originating from the same ancestral gene as AtUGT85A4 and AtUGT85A7.

[0029] Expression analysis of grape UGT gene in different tissues; The expression levels of the VvUGT gene in group G were analyzed in 12 different tissues, including roots, mature stems, young leaves, fruit-setting leaves, young flowers, fully developed flowers, full bloom flowers, fruit-setting pericarp, post-fruit-setting pericarp, pericarp at the color-changing stage, mid-mature pericarp, and mature pericarp, based on data from Grape eFP Browser. Figure 7 The results showed that VvUGT14 and VvUGT16 were highly expressed in roots, suggesting they may be related to root-specific developmental expression, secondary metabolite synthesis, or soil stress. VvUGT147 and VvUGT148 were highly expressed in roots and mature stems, potentially playing roles in plant growth, water metabolism, nutrient transport, and stress resistance. VvUGT15 was highly expressed only during full bloom, possibly related to glycosylation modification of floral volatiles or related hormones. VvUGT144, VvUGT145, and VvUGT177 were highly expressed during fruit ripening, likely closely related to fruit development, pigment synthesis, sugar accumulation, and the synthesis of fruit aroma compounds such as terpenes.

[0030] UGTs are key enzymes catalyzing the glycosylation of plant secondary metabolites, a process crucial for plant growth, development, metabolism, and stress responses. This invention references 181 VvUGT genes identified by Hou et al. VvUGT is a large gene family, fewer in number than apple (214) (Caputi et al., 2012), potato (295), and peanut (267), but more numerous than maize (147), wheat (179), and tea (178). Based on phylogenetic analysis, the 181 VvUGT gene family was divided into 16 subgroups (A-P). Group E has the most members, with 56, accounting for 31% of VvUGT genes. Group G has 16 members, accounting for 9% of VvUGT genes.

[0031] In this invention, a total of 14 fragment duplications and 103 tandem duplication events were found in the VvUGT gene. Figure 5 This explains why the VvUGT gene presents as an unevenly distributed cluster on the chromosome. Figure 3 Although there is no intraspecific collinearity among the VvUGT genes in group G, there is interspecific collinearity between VvUGT145 and AtUGT85A4 and AtUGT85A7 in Arabidopsis thaliana. Figure 6This means that these genes come from a common ancestor, and this "one-to-two" collinearity pattern suggests that a species-specific gene duplication event may have occurred in the Arabidopsis lineage, while in the grape lineage, the gene did not duplicate. Introns are key structural components of genes. Although intron sequences are removed in mature mRNA and do not participate in protein coding, structural variations such as deletions, insertions, or changes in site conservation are subject to significant selection pressure. These variations are crucial evidence for tracing gene duplication events, revealing functional innovations, and reconstructing the phylogenetic history of gene families. All 16 VvUGT genes in group G contain at least one intron.

[0032] Gene promoters determine gene function, and analyzing the functional characteristics of their cis-acting elements helps reveal transcriptional regulatory mechanisms and expression patterns. This invention analyzes the cis-acting elements of the first 2000 bp sequence of the VvUGT gene in group G (…). Figure 4The study found that the promoter region of the VvUGT gene contains various types of cis-elements, including light-responsive, hormone-responsive, stress-responsive, and developmental elements. The most widely distributed light-responsive elements are Box4 and G-Box, present in all VvUGT genes. In Stevia rebaudiana, its sweet component is steviol glycoside (SG). SrUGT85C2, SrUGT74G1, and SrUGT76G1 are UDP-glycosyltransferases involved in the SG synthesis pathway. The study showed that SG synthesis is affected by light quality. Furthermore, gibberellin (GA) shares a biosynthetic pathway with SG, and light can also upregulate GA biosynthesis, indicating that the promoters of these UGT genes also contain light-responsive elements. Therefore, we can speculate that the expression of the G group VvUGT gene is also regulated by light conditions and diurnal rhythms, and secondary metabolites may be synthesized in large quantities and stored through glycosylation during specific time periods of the day. ABA is a major signaling molecule involved in various physiological functions of plants. This invention discovered the ABA response element ABRE in the group G VvUGT gene, and its widespread distribution indicates that ABA is an important upstream signaling molecule for group G VvUGT gene expression. In tomato fruit, SlUGT75C1 alters ABA levels in both fruit and plant by converting active ABA into abscisic acid glucose ester (ABA-GE). SlUGT75C1 plays a crucial role in ABA-mediated tomato fruit ripening, seed germination, and drought response. Besides the abscisic acid (ABA) response element, the promoter regions of group G genes also contain cis-acting elements for responses to various plant hormones, such as salicylic acid (SA) and methyl jasmonate (MeJA). These hormones, as key signaling molecules, constitute the core signaling pathways regulating growth, development, and stress responses in plants. Among stress response elements, the low-temperature response element LTR was detected in three group G VvUGT members, with VvUGT15 containing two LTR elements, indicating that the VvUGT gene can participate in the plant's low-temperature stress response. In tea plants, the expression of CsUGT91Q2 is strongly induced by cold stress, specifically catalyzing the glycosylation of nerol. Nerol glucoside can scavenge reactive oxygen species, thus improving the cold resistance of tea plants. Similarly, CsUGT78A14 in tea plants is also induced by cold stress, playing an important role in cold stress by increasing the accumulation of flavonols and the ability to scavenge reactive oxygen species. Multiple stress-responsive elements in group G suggest that this group of genes may be regulatory targets for grapes in responding to multiple environmental stresses. Under relevant stress signals, related transcription factors can rapidly bind and initiate the expression of VvUGT, which participates in glycosylation to alter metabolite activity and participates in activities such as osmotic regulation and antioxidation.The promoters of this group of genes also contain MYB binding sites, indicating the type of key transcription factors regulated upstream. The MYB family is a core transcription factor in plants regulating secondary metabolism and stress responses, providing clues for elucidating the regulatory mechanism of the VvUGT gene in specific pathways. In summary, the G group genes are multifunctional in grape life activities, not only widely involved in the biosynthesis and modification of secondary metabolites, but also likely playing a crucial role in the stress resistance and temporal regulation of grape growth and development.

[0033] This invention further performed tissue expression analysis on genes in group G. Figure 7 The absence of VvUGT, which is highly expressed in all tissues, indicates that this group of genes exhibits tissue specificity, meaning they function in specific tissues. Three genes (VvUGT144, VvUGT145, and VvUGT177) are highly expressed during fruit ripening, suggesting that these genes are likely involved in the glycosylation of secondary metabolites in the fruit.

[0034] It should be noted that, in order to simplify the description of the present invention and thus help to understand one or more embodiments of the invention, multiple features may sometimes be grouped into one embodiment, drawing or description thereof in the foregoing description of the embodiments of the present invention.

[0035] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. The genetic engineering application of VvUGT145 and VvUGT177 in regulating the synthesis of monoterpene glycosides, characterized by: VvUGT145 and VvUGT177 belong to the grape VvUGT gene family G group, which encodes UDP-glycosyltransferase, which specifically catalyzes the glycosylation of monoterpenes such as free linalool in grapes to generate bound monoterpene glycosides. VvUGT145 and VvUGT177 are specifically highly expressed in grape skins from the color change to maturity stage, and are functional genes that regulate the accumulation of terpenoid aroma substances in the fruit. The promoter regions of the genes all contain light-responsive elements, abscisic acid and methyl jasmonate hormone-responsive elements, and low temperature and drought stress-responsive cis-elements. Gene expression is upregulated by treatment with exogenous light quality, ABA, and methyl jasmonate. Among them, VvUGT145 exhibits cross-species collinearity with Arabidopsis thaliana AtUGT85A4 and AtUGT85A7, and its monoterpene glycosylation function is conserved in dicotyledonous plants.

2. The genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family according to claim 1, characterized in that: The genetic engineering application is for molecular-assisted breeding of grapes. The steps include: developing specific molecular markers using the VvUGT145 and VvUGT177 gene sequences, screening for highly expressed gene lines in grape hybrid offspring populations, and breeding new grape varieties with high monoterpene aroma.

3. The genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family according to claim 2, characterized in that: The genetic engineering application is gene overexpression improvement: constructing plant overexpression vectors containing the complete coding sequences of VvUGT145 or VvUGT177, transforming grape callus / grape plants, and upregulating the expression level of the target gene.

4. The genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family according to claim 2, characterized in that: The genetic engineering application is gene editing-oriented improvement: using CRISPR / Cas9 gene editing technology to knock down or remove endogenous VvUGT145 and VvUGT177 in grapes to reduce the content of bound monoterpenes in the fruit.

5. The genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family according to claim 1, characterized in that: The VvUGT145 is located on grape chromosome 18, encodes 473 amino acids, has an isoelectric point of 4.84, and is a stable acidic protein. The VvUGT177 has no clear chromosomal location, encodes 482 amino acids, has an isoelectric point of 6.14, and is an unstable acidic protein.

6. The genetic engineering application of the VvUGT145 and VvUGT177 grape G group UGT gene family according to claim 5, characterized in that: The application also includes grape field cultivation regulation: by regulating light conditions and exogenously spraying abscisic acid or methyl jasmonate, the cis-response elements of the VvUGT145 and VvUGT177 promoters are activated, inducing endogenous gene expression and simultaneously improving the grape's resistance to low temperature and drought stress and the aroma quality of the fruit.

7. A plant overexpression vector for increasing the content of monoterpene glycosides in grapes, characterized in that: The vector contains a complete VvUGT145 or VvUGT177 coding sequence, which can specifically drive the efficient expression of the target gene in grape fruit tissue and catalyze the glycosylation of free monoterpenes to generate monoterpene glycosides.

8. A transgenic grape line high in monoterpene glycosides, characterized in that: The strain was obtained by transforming grapes with the overexpression vector described in claim 7. The expression levels of VvUGT145 or VvUGT177 in the strain were significantly higher than those in the wild type, and the accumulation of monoterpene glycosides was increased during the fruit ripening stage.

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