Application of VvMYC2 gene in promoting the synthesis of monoterpenoid components in Yangguangmei grape

Abstract

The present invention provides the use of the VvMYC2 gene to promote the synthesis of monoterpene components in Sunshine Rose grapes, belonging to the technical field of functional gene screening and application. The present invention discovers that VvMYC2 can promote the accumulation of the monoterpene component linalool by directly activating the expression of the monoterpene synthase gene VvTPS453. VvMYC2 is an important signal transduction factor for methyl jasmonate to promote monoterpene synthesis and plays a key regulatory role in the formation of aroma quality. By regulating the expression of the VvMYC2 gene, the expression of genes related to the aroma substance synthesis pathway can be regulated, thereby achieving the purpose of regulating the aroma quality of grape fruit.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional gene screening and application, and particularly relates to the application of the VvMYC2 gene in promoting the synthesis of monoterpene components of Sunshine Rose grapes. Background Art

[0002] Terpenoids are an important class of secondary metabolites in plants, classified as monoterpenes, diterpenes, and sesquiterpenes. They are not only closely related to pathogen defense and insect pollination during plant development, but also play a crucial role in influencing plant aroma quality. Terpenoid synthesis can be broadly divided into the 2-C-methyl-D-erythritol-4-phosphate pathway and the mevalonate pathway, controlled by multiple genes, including deoxy-D-xylose 5-phosphate synthase, reductoisomerase, and terpene synthase. Studies have shown that some transcription factors can bind to specific cis-acting elements on the promoters of target genes in the terpenoid biosynthesis pathway, regulating their expression and thus controlling terpenoid synthesis.

[0003] The MYC transcription factor belongs to the bHLH transcription factor family. It has a bHLH-MYC-N domain at its N-terminus and a helix-loop-helix (HLH) domain at its C-terminus. It plays a crucial role in regulating plant growth, development, and fruit quality. Currently, little research has been conducted on the function of MYC transcription factors in grapes.

[0004] Aroma is a key indicator of grape quality, with consumers often preferring grapes with a strong aroma. Monoterpenes, particularly linalool, are a key contributor to grape aroma and are closely associated with the formation of floral, fruity, and musky aromas. Currently, the discovery of aroma-regulating genes in grapes is still relatively limited. With the advancement of modern molecular design breeding techniques, fully identifying and utilizing functional genes has become a key development direction in modern grape aroma quality breeding. The discovery and utilization of aroma-regulating genes in grapes is a pressing need for the genetic improvement of grape quality traits and the efficient selection of high-quality grape varieties. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of the VvMYC2 gene in promoting the synthesis of monoterpene components in Sunshine Rose grapes. Through homologous functional verification, yeast one-hybrid, dual-luciferase and other experiments, its gene function and mechanism of action were elucidated. It was found that VvMYC2 can directly activate the expression of VvTPS453 and promote the synthesis of linalool, thereby improving the aromatic smell of Sunshine Rose grapes.

[0006] To achieve the above objectives, the present invention provides an application of the VvMYC2 gene in promoting the synthesis of monoterpene components of Sunshine Rose grapes, and the accumulation of monoterpene components of grapes in Sunshine Rose grapes is increased by overexpressing the VvMYC2 gene of Sunshine Rose grapes;

[0007] Wherein, the nucleotide sequence of the VvMYC2 gene is shown as SEQ ID NO.1.

[0008] The present invention also provides the use of a protein encoded by the VvMYC2 gene in promoting the synthesis of monoterpene components of Yangguangmei grapes. The amino acid sequence of the protein encoded by the VvMYC2 gene is shown in SEQ ID NO.2.

[0009] The present invention also provides the use of the recombinant vector of the VvMYC2 gene in promoting the synthesis of monoterpene components of Yangguangmei grapes.

[0010] The present invention also provides the use of the genetically engineered bacteria of the recombinant vector in promoting the synthesis of monoterpene components of Yangguangmei grapes.

[0011] The present invention also provides a method for improving the aroma of Sunshine Rose grapes, comprising introducing the VvMYC2 gene into Sunshine Rose grapes to overexpress the VvMYC2 gene of the Sunshine Rose grapes; the nucleotide sequence of the VvMYC2 gene is shown in SEQ ID NO.1.

[0012] Preferably, the method for improving the aroma of Sunshine Rose grapes comprises the following steps:

[0013] 1) introducing the VvMYC2 gene into an overexpression vector to construct a VvMYC2 gene overexpression vector;

[0014] 2) Transforming the VvMYC2 gene overexpression vector into Agrobacterium to construct a recombinant bacterium;

[0015] 3) inoculating the recombinant bacteria into Sunshine Rose grapes to introduce the VvMYC2 gene into the Sunshine Rose grapes.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discovered that VvMYC2 can promote the accumulation of the monoterpene component linalool by directly activating the expression of the monoterpene synthase gene VvTPS453. VvMYC2 is an important signal transduction factor for methyl jasmonate to promote monoterpene synthesis and plays a key regulatory role in the formation of aroma quality. By regulating the expression of the VvMYC2 gene, the expression of genes related to the aroma synthesis pathway can be controlled, thereby achieving the goal of regulating the aroma quality of grape fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Figure 1A shows the effect of transient transformation of grape berries with VvMYC2. WT represents the PRI101 empty vector control, OE-VvMYC2 represents the VvMYC2 overexpression vector, TRV represents the viral interference vector control, and TRV-VvMYC2 represents the VvMYC2 interference vector. CK indicates no treatment, and MeJA indicates methyl jasmonate treatment. Figure 1B shows the effect of transient transformation of grape berries with VvMYC2. The vertical axes of the two figures in the upper panel of 1B represent the linalool content and relative gene expression levels, respectively, indicating the changes in relevant indicators in each treatment group without MeJA addition. The vertical axes of the two figures in the lower panel of 1B represent the linalool content and relative gene expression levels, respectively, indicating the changes in relevant indicators in each treatment group with MeJA addition. Different letters indicate significant differences (P < 0.05).

[0020] Figure 2 Expression analysis of monoterpene synthase genes in grape berries transiently transformed with VvMYC2. Darker colors indicate higher gene expression levels. WT represents the PRI101 empty vector control, OE-VvMYC2 represents the VvMYC2 overexpression vector, TRV represents the viral interference vector control, and TRV-VvMYC2 represents the VvMYC2 interference vector. Groups with MeJA indicate changes in gene expression after MeJA addition.

[0021] Figure 3 Figure 3A shows the expression of monoterpene synthase genes in grape calli stably transformed with VvMYC2. 3A represents the expression of each gene in the absence of MeJA, and 3B represents the expression in the presence of MeJA. The horizontal axis represents different genes. EV represents the empty vector; OE-VvMYC2-1, OE-VvMYC2-2, and OE-VvMYC2-4 represent the three lines overexpressing VvMYC2; and RNAi-VvMYC2-1, RNAi-VvMYC2-2, and RNAi-VvMYC2-3 represent the three lines with knockdown of VvMYC2 expression. Different letters indicate significant differences (P < 0.05).

[0022] Figure 4Figure 4A shows the effect of transient transformation of grape berries with VvTPS453. WT represents the PRI101 empty vector control, OE-VvTPS represents the VvTPS overexpression vector, TRV represents the viral interference vector control, and TRV-VvTPS represents the VvTPS overexpression vector. CK indicates no treatment, and MeJA indicates methyl jasmonate treatment. Figure 4B shows the effect of transient transformation with VvTPS. The vertical axes of the two upper panels in Figure 4B represent the linalool content and relative gene expression levels, respectively, indicating changes in the relevant indicators in each treatment group without MeJA addition. The vertical axes of the two lower panels in Figure 4B represent the linalool content and relative gene expression levels, respectively, indicating changes in the relevant indicators in each treatment group with MeJA addition. Different letters indicate significant differences (P < 0.05).

[0023] Figure 5 Figure 5A shows the validation of VvTPS453 transcriptional activation by VvMYC2. Figure 5A shows analysis of the VvTPS promoter and the distribution of key elements in the yeast one-hybrid vector. Figure 5B shows the yeast one-hybrid validation results. Yeast strains were grown on SD / -Leu and SD / -Leu + ABA media for 3 days. The experiment was repeated three times, and representative images are shown. Figure 5C shows the results of the LUC activation assay. Figure 5D shows the enzyme activity analysis of the LUC activation assay. Different letters indicate significant differences (P < 0.05). DETAILED DESCRIPTION

[0024] The present invention will be described in detail below by way of specific examples. These examples are provided to provide a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0026] Unless otherwise indicated, the practice of the present invention will utilize conventional botanical techniques, microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics techniques readily apparent to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods employed in the present invention for DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and gene-edited plant production, in addition to the methods employed in the following examples, can all be accomplished using methods disclosed in the existing literature.

[0027] The inventors used homologous cloning technology and reverse transcription-polymerase chain reaction (RT-PCR) based on the Pinot Noir genome sequence. The first chain of cDNA synthesized by reverse transcription of total RNA from Sunshine Rose fruit was used as a template to amplify the aroma substance synthesis regulatory gene VvMYC2 in Sunshine Rose grapes. The complete open reading frame sequence of this gene is 1815bp in length and encodes 604 amino acids.

[0028]

[0029] The amino acid sequence of the protein encoded by the VvMYC2 gene is as follows:

[0030] Example 1 Construction of VvMYC2 expression vector

[0031] 1. Using homologous cloning technology, the first strand of cDNA synthesized by reverse transcription of RNA from the leaves of Vitis vinifera was used as a template to amplify the VvMYC2 sequence of the aroma synthesis gene of Vitis vinifera.

[0032] 2. Construct pRI-VvMYC2 overexpression vector, TRV-VvMYC2 interference expression vector, and RNAi-VvMYC2 inhibition expression vector.

[0033] The VvMYC2 gene cloning and MYC2 expression vector construction in this example can be carried out using conventional methods in the art.

[0034] Example 2 Effect of VvMYC2 on Grape Monoterpene Content and Correlation Analysis with Monoterpene Synthase Gene Expression

[0035] 1. The pRI-VvMYC2 overexpression vector and the TRV-VvMYC2 interference expression vector were respectively transferred into the wild-type Sunshine Rose grape fruit through Agrobacterium-mediated transfection. The fruit was then treated with control and methyl jasmonic acid (MeJA) for 5 days.

[0036] 2. Detect changes in monoterpene content and expression levels of related genes, such as Figure 1 shown.

[0037] according to Figure 1 Analysis revealed that changes in linalool content revealed that, under normal conditions, overexpression of VvMYC2 significantly increased linalool content in the fruit, whereas disruption of VvMYC2 expression significantly decreased linalool content. Furthermore, the results showed that VvMYC2 was significantly upregulated in OE-VvMYC2 grapes after MeJA application, while VvMYC2 expression remained significantly downregulated in TRV-VvMYC2 material after TRV disruption. Changes in linalool content revealed that MeJA treatment continued to significantly increase linalool content in overexpressing grapes, while TRV disruption of VvMYC2 significantly inhibited monoterpene accumulation in grape fruit after MeJA application. Comprehensive analysis of these results suggests that VvMYC2 can activate the monoterpene biosynthesis pathway, promoting linalool accumulation. Furthermore, VvMYC2 also transduces jasmonic acid signals to key genes regulating monoterpene accumulation.

[0038] 3. Detect the expression level of the monoterpene synthase gene VvTPS in the fruit after VvMYC2 transient transformation, such as Figure 2 shown.

[0039] according to Figure 2 Analysis revealed significant differences in the expression patterns of each gene under different treatment conditions. Cluster analysis of each gene, combined with the gene expression patterns under different treatment conditions, revealed that VvTPS050, VvTPS453, VvTPS18, VvTPS52, VvTPS36, and VvMYC2 clustered together, exhibiting significant correlation. This suggests that VvMYC2 may regulate monoterpene accumulation by influencing the expression of VvTPS050, VvTPS453, VvTPS18, VvTPS52, and VvTPS36.

[0040] Example 3 Analysis of the expression of monoterpene biosynthesis genes promoted by VvMYC2 in grape callus

[0041] In this example, to further reveal the correlation between VvMYC2 and VvTPS expression, the overexpression vector pRI-VvMYC2 and the RNAi-VvMYC2 inhibition expression vector were transformed into grape callus, and the expression changes of related genes were analyzed under control and MeJA treatment conditions. Figure 3 shown.

[0042] according to Figure 3 Analysis revealed that under normal conditions, the expression patterns of genes in callus tissue showed significant similarities among VvTPS453, VvTPS18, and VvMYC2. However, in MeJA-treated callus tissue, the expression patterns of VvTPS453 and VvMYC2 were similar, with a significant correlation between the two genes. Overall, a significant correlation exists between VvMYC2 and VvTPS453 in grape callus tissue, suggesting that VvTPS453 expression may be regulated by VvMYC2.

[0043] According to the results of transient transformation in fruits and stable transformation in grape callus, VvTPS453 may be regulated by VvMYC2, which directly acts on the monoterpene synthase gene.

[0044] Example 4 Analysis of VvTPS453 Promoting Monoterpenoid Accumulation in Grape Berries

[0045] 1. Construct the pRI-VvTPS453 overexpression vector and the TRV-VvTPS453 interference expression vector.

[0046] 2. This example demonstrates that VvTPS453 can directly promote the accumulation of linalool in grapes. The pRI-VvTPS453 overexpression vector and the TRV-VvTPS453 interference expression vector were introduced into wild-type Sunshine Rose grapes via Agrobacterium-mediated transfection. The fruits were then treated with either a control or MeJA treatment. Similar to the analysis in Example 1, by comparing changes in linalool content, it was determined that overexpression of VvTPS453 in grape fruit promoted the accumulation of the monoterpene component linalool. The analysis results are shown in Figure 2. Figure 4 shown.

[0047] according to Figure 4 Analysis showed that interfering with VvTPS453 expression after MeJA application also significantly inhibited the accumulation of monoterpenes. The results indicate that VvTPS453 catalyzes the production of linalool in fruit and plays an important role in the pathway of jasmonic acid signaling regulating monoterpene synthesis.

[0048] Example 5 VvMYC2 can directly bind to the promoter of VvTPS453 and activate its expression

[0049] In this example, yeast one-hybrid experiments were conducted to further verify whether VvMYC2 has the ability to directly regulate the expression of VvTPS453. Figure 5 A and B) and LUC dual luciferase assay (as shown in Figure 5 (as shown in C and D).

[0050] according to Figure 5 For analysis, the 986-787 bp sequence of the VvTPS453 promoter region was ligated into the reporter vector, and the CDS region of VvMYC2 was ligated into the effector vector, and the binding was verified in yeast, as shown in Figure 2 . Figure 5 B. The reporter gene-effect vector was able to grow normally on SD / -Leu medium supplemented with aurobacitin, demonstrating that VvMYC2 binds to the VvTPS453 promoter. LUC luciferase assay results showed that leaves co-transformed with the VvTPS453 promoter and VvMYC2 produced fluorescence, with the highest luciferase activity, indicating that VvMYC2 activates the VvTPS453 promoter.

[0051] Based on the above results, it can be concluded that VvMYC2 can positively regulate the synthesis of linalool by directly activating the expression of VvTPS453, and VvMYC2 can be used to regulate the synthesis of grape aroma substances.

[0052] As shown in Examples 1-5, VvMYC2 can promote the synthesis of linalool in grapes and can be used to regulate the aroma traits of grapes.

[0053] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. The application of VvMYC2 gene in promoting the synthesis of monoterpene components of Sunshine Rose grape is characterized by: The accumulation of grape monoterpenoid components in Sunshine Rose was improved by overexpressing the VvMYC2 gene. Wherein, the nucleotide sequence of the VvMYC2 gene is shown as SEQ ID NO.

1.

2. The application of the protein encoded by the VvMYC2 gene in promoting the synthesis of monoterpene components of Sunshine Rose grapes is characterized in that: The accumulation of grape monoterpene components in Yangguangmeigui grape is increased by overexpressing the protein encoded by the VvMYC2 gene of Yangguangmeigui grape. The amino acid sequence of the protein encoded by the VvMYC2 gene is shown in SEQ ID NO.

2.

3. Use of a recombinant vector containing the VvMYC2 gene according to claim 1 in promoting the synthesis of monoterpene components of Yangguangmei grapes, characterized in that: The accumulation of grape monoterpene components in Sunshine Rose was improved by overexpressing the VvMYC2 gene of Sunshine Rose grape.

4. Use of a genetically engineered bacterium containing the recombinant vector of claim 3 in promoting the synthesis of monoterpene components of Yangguangmei grapes, characterized in that: The accumulation of grape monoterpene components in Sunshine Rose was improved by overexpressing the VvMYC2 gene of Sunshine Rose grape.

5. A method for improving the aroma of Sunshine Rose grapes, characterized in that: The VvMYC2 gene was introduced into Sunshine Rose grapes to overexpress the VvMYC2 gene of the Sunshine Rose grapes; the nucleotide sequence of the VvMYC2 gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that The following steps are involved: 1) introducing the VvMYC2 gene into an overexpression vector to construct a VvMYC2 gene overexpression vector; 2) Transforming the VvMYC2 gene overexpression vector into Agrobacterium to construct a recombinant bacterium; 3) inoculating the recombinant bacteria into Sunshine Rose grapes to introduce the VvMYC2 gene into the Sunshine Rose grapes.

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