Application of interaction between VvERF105 and VvSWEET15 promoter in reducing sugar accumulation in fruits

Abstract

The application discloses application of VvERF105 and VvSWEET15 promoter interaction in reducing sugar accumulation in fruits, and belongs to the technical field of biotechnology, and particularly relates to application of VvERF105 and VvSWEET15 promoter interaction in reducing sugar accumulation in fruits.The protein VvERF105 can interact with the VvSWEET15 promoter, the expression amount of the VvERF105 is regulated, the contents of grape glucose and fructose in grape fruits are negatively regulated, and the application prospect is good in the field of plant breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of VvERF105 and VvSWEET15 promoter interaction in reducing sugar accumulation in fruits. BACKGROUND

[0002] Sugar plays a crucial role in plant growth, development and metabolism, and the accumulation and distribution of sugar mainly depend on the functional regulation of sugar transport proteins. Sugar transport proteins regulate the distribution of sugar in plant tissues through transmembrane transport, and thus their expression and activity directly affect the sugar accumulation level in fruits. In many plants, excessive or insufficient sugar accumulation can lead to reduced fruit quality and affect agricultural production. Therefore, in-depth study of transcription factors regulating the expression of sugar transport proteins and their interaction mechanisms has important application prospects for regulating sugar accumulation and improving crop quality.

[0003] Currently, there are few studies on transcription factors regulating fruit sugar accumulation by binding to the promoters of sugar transport proteins. In grape, VvMSA positively regulates the promoter activity of VvHT1, promoting the accumulation of hexose in grape fruits. In addition, the R2R3 type MYB96 transcription factor directly binds to the promoter of STP13, activates its expression, and promotes sugar absorption in plants to improve their tolerance to environmental stress. In watermelon, the transcription factor SUSIWM1 promotes the accumulation of sucrose, glucose and fructose in the vacuoles of fruit cells by positively regulating the ClTST2 gene. In rice, the OsDOF11 transcription factor enhances the expression of OsSUT1, OsSWEET11 and OsSWEET14 by binding to their promoter regions, thereby affecting sugar transport in rice. The Osdof11 mutant exhibits dwarfism, reduced tillering, insensitivity to sucrose-mediated root growth inhibition, and reduced sugar accumulation in leaves and sucrose flow in phloem. The ABA-responsive transcription factor OsbZIP72 can also bind to the promoter regions of OsSWEET13 and OsSWEET15, activating the expression of these genes under drought stress. In cotton, the transcription factor GhMYB212 promotes the expression of GhSWEET12 by binding to its promoter region, thereby regulating the supply of carbon sources required for cotton fiber elongation.

[0004] VvERF105 is a transcription factor belonging to the AP2 / ERF transcription factor family, which is widely involved in the response of plants to environmental stress and the regulation of growth and development. Transcription factors regulate the transcription level of target genes by binding to the promoter of the target genes, thereby affecting the expression of downstream genes. Existing studies have shown that ERF-type transcription factors play a key role in plant stress resistance regulation and metabolic regulation. However, the function of VvERF105 in the regulation of sugar accumulation is not clear at present, and further research is needed on its regulatory targets and mechanism of action.

[0005] VvSWEET15 is an important sugar transporter belonging to the SWEET family. Members of the SWEET family are widely involved in the transmembrane transport of sugars such as sucrose and glucose, and have been shown to play a role in various plant organs, especially affecting sugar distribution and accumulation during fruit development. The expression level directly determines the transport efficiency of sugar in fruit cells, thereby affecting the sugar content in fruit.

[0006] In fruit crops such as grapes, excessive sugar accumulation can affect fruit flavor, texture and storage properties, therefore, controlling fruit sugar accumulation has important economic and application value. In particular, there is no in-depth report on the interaction between transcription factors and the promoter of VvSWEET15. Therefore, studying the interaction between VvERF105 and the promoter of VvSWEET15 can not only elucidate the expression regulation mechanism of VvSWEET15 gene, but also provide a theoretical basis for regulating fruit sugar accumulation through genetic engineering.

[0007] By exploring the regulatory effect of VvERF105 on the activity of VvSWEET15 promoter, it is expected to reveal the new function of ERF-type transcription factors in the process of sugar accumulation, and provide an effective molecular regulation tool for regulating sugar accumulation in fruit. This research not only has important significance in the scientific aspect, but also has wide application prospect in agricultural production. SUMMARY

[0008] The technical problem solved by the present application is how to regulate sugar accumulation in plant fruits, especially grapes.

[0009] In order to solve the above problems, the present application provides related applications of a protein, a substance for regulating the expression of the coding gene of the protein, or a substance for regulating the activity or content of the protein.

[0010] The protein, the substance for regulating the expression of the coding gene of the protein, or the substance for regulating the activity or content of the protein provided by the present application is applied in any one of the following:

[0011] 1) in regulating sugar accumulation in plant fruits;

[0012] 2) use in the manufacture of a product for modulating sugar accumulation in a fruit of a plant;

[0013] 3) use in the breeding of a plant with altered sugar accumulation in a fruit;

[0014] 4) use in the manufacture of a product for breeding a plant with altered sugar accumulation in a fruit;

[0015] 5) use in plant breeding;

[0016] The protein is any one of the following proteins:

[0017] a1) a protein having an amino acid sequence of SEQ ID No. 1;

[0018] a2) a protein having the amino acid sequence shown in SEQ ID No. 1 with substitution and / or deletion and / or addition of one or several amino acid residues and having the same function;

[0019] a3) a protein having an amino acid sequence defined in any one of a1) or (a2) with 80% or more identity and having the same function;

[0020] a4) a fusion protein obtained by linking a terminal tag to a protein defined in any one of a1) to (a3).

[0021] In the above protein, the protein tag refers to a polypeptide or protein fused and expressed with a target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0022] In the above protein, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage of the NCBI homepage. For example, the identity (%) of a pair of amino acid sequences can be obtained by searching the identity in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and then calculating the identity of the amino acid sequence.

[0023] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0024] Of the proteins mentioned above, SEQ ID No. 1 consists of 277 amino acid residues. It is named VvERF105 protein, and its encoding gene is the VvERF105 gene.

[0025] In the above applications, the protein is derived from grapes (Vitis vinifera L.).

[0026] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein VvERF105.

[0027] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0028] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.

[0029] In this article, the enhancement, increase or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0030] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0031] In this invention, the activity of the VvSWEET15 promoter can be regulated by controlling the activity and / or content of the protein VvERF105, thereby regulating the glucose and fructose content in grape berries through interaction with the VvSWEET15 promoter.

[0032] In the above applications, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein can be a biological material related to the protein described above, and the biological material can be any of the following:

[0033] c1) The nucleic acid molecule that encodes the protein described above;

[0034] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0035] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0036] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0037] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0038] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0039] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0040] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0041] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0042] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0043] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0044] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0045] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0046] e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0047] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0048] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3;

[0049] d2) The coding sequence is the DNA molecule shown in SEQ ID No. 2;

[0050] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0051] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0052] In the above applications, the nucleic acid molecule described in e1) can be a DNA molecule with a nucleotide sequence shown in SEQ ID No. 3.

[0053] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0054] The vectors described herein are well-known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vector plasmids such as pET28a(+), pGreenII-0800-LUC, pGADT7, pGAD-Rec2, pHIS2, pHB, pHB-HA, and pRI101.

[0055] In this paper, the *Escherichia coli* strains used for plasmid propagation and prokaryotic protein expression were Trans1-T1 (TransGen) and BL21 (TransGen); the *Agrobacterium tumefaciens* strains used for plant genetic transformation were GV3101 and GV3101 (pSoup) (Zhuang Meng); and the yeast strain used for one-hybridization was Y187 (Zhuang Meng).

[0056] Recombinant expression vectors containing the VvERF105 gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0057] When constructing a recombinant plant expression vector using the VvERF105 gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0058] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0059] The present invention also provides a method for altering sugar accumulation in plant fruits, the method comprising the following steps M or P:

[0060] Step M is to enhance, increase or upregulate the activity and / or content of the proteins mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the proteins mentioned above, so as to reduce sugar accumulation in plant fruits.

[0061] The method includes step P, which is to inhibit or reduce or silence the activity and / or content of the aforementioned protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned protein, in order to increase sugar accumulation in plant fruits.

[0062] In the above method, reducing the expression level and / or activity of the gene encoding the protein VvERF105 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown techniques to reduce or inactivate the gene encoding the protein VvERF105 in the genome of the target plant.

[0063] The present invention also provides a method for cultivating plants with increased fruit sugar accumulation, comprising inhibiting, reducing or silencing the expression level of the gene encoding the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a plant with increased fruit sugar accumulation, wherein the glucose and fructose content of the plant with increased fruit sugar accumulation is higher than that of the target plant.

[0064] In one specific embodiment, inhibiting, reducing, or silencing the expression of the gene encoding the protein described above in the plant includes introducing a substance, expression cassette, or recombinant vector that inhibits the expression of the nucleic acid molecules described above into the target plant to obtain a plant with high fruit sugar accumulation.

[0065] The present invention also provides a method for cultivating fruits with reduced sugar accumulation, comprising inhibiting, reducing or silencing the expression level of the gene encoding the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a plant with reduced fruit sugar accumulation, wherein the glucose and fructose content is lower than that of the target plant.

[0066] In one specific embodiment, inhibiting, reducing, or silencing the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a plant with reduced sugar accumulation in the fruit.

[0067] In this article, the purpose of breeding includes cultivating plants with increased sugar accumulation in fruits; the purpose of breeding also includes cultivating plants with decreased sugar accumulation in fruits.

[0068] Compared to the target plant, the plants with increased fruit sugar accumulation had higher glucose and fructose content. Conversely, the plants with decreased fruit sugar accumulation had lower glucose and fructose content compared to the target plant.

[0069] In this article, the grapes mentioned may refer to the Chardonnay grape variety.

[0070] In this article, the recombinant microorganisms may be Agrobacterium tumefaciens strains GV3101 and GV3101(pSoup).

[0071] The proteins and / or the biological materials mentioned above are also within the scope of protection claimed in this invention.

[0072] In the above applications or methods, the plant may be any of the following:

[0073] N1) Monocotyledonous or dicotyledonous plants;

[0074] N2) Vitales plants;

[0075] N3) Vitaceae plants;

[0076] N4) Vitis genus plants;

[0077] N5) grapes.

[0078] This invention relates to the interaction between the VvERF105 transcription factor and the VvSWEET15 promoter, with the primary aim of reducing sugar accumulation in fruits. By studying the binding site of VvERF105 to the VvSWEET15 promoter, the inhibitory effect on VvSWEET15 gene expression was clarified, thereby reducing the expression level of the sugar transporter VvSWEET15 and inhibiting excessive sugar accumulation in fruit cells. This invention uses genetic engineering techniques to regulate the expression of VvERF105 or its binding efficiency to reduce sugar accumulation in fruits and improve fruit quality, with particularly significant effects in fruits such as grapes.

[0079] Advantages of this invention compared to existing technologies:

[0080] 1) Precise regulation of sugar accumulation: Through the specific interaction between the VvERF105 and VvSWEET15 promoters, this invention can achieve precise regulation of sugar accumulation in fruits, avoiding the non-specific effects on sugar metabolism in traditional methods, thereby optimizing fruit flavor and quality.

[0081] 2) Genetic engineering methods are more operable: Compared with other methods that rely on exogenous hormones or complex environmental regulation, this invention regulates VvERF105 expression through genetic modification or transgenic methods, which is simple to operate and has stable effects, and is suitable for large-scale production;

[0082] 3) Wide range of applications: This technology is not limited to sugar regulation in grapes, but can also be applied to other fruits or crops. Through the interaction regulation of corresponding ERF transcription factors and SWEET transporter proteins, the sugar accumulation of different crops can be managed and optimized.

[0083] 4) Improve fruit storage and transportation performance: By reducing sugar accumulation, this invention can effectively extend the storage period of fruit, improve the quality preservation performance during transportation, and solve the problem of fruit spoilage caused by excessive sugar content. Attached Figure Description

[0084] Figure 1 To facilitate the construction and screening of single-hybrid libraries.

[0085] Figure 2 For rapid clone identification using PCR analysis and 2% agarose gel electrophoresis, 269 true positive interactions were distinguished from 8800 potential positive interactions. When a single band was observed on the gel, the AD / library cDNA insert could be sequenced. Arabic numerals indicate clone numbers.

[0086] Figure 3 Candidate genes were screened for yeast one-hybrid culture. Co-transformation with the AD-53m and pHIS2-S15 promoters served as a positive control, while co-transformation with empty vectors AD-53m and pHIS2, and empty vector AD and pHIS2-S15 promoters served as negative controls. Cloning sites were transferred to SD (-Leu-Trp) medium to ensure experimental reliability, as yeast growth is directly related to interactions, thus eliminating human error.

[0087] Figure 4 To verify the interaction between the VvERF105 and VvSWEET15 promoters in yeast monohybrids. The VvSWEET15 promoter sequence was amplified from Chardonnay fruit DNA. Co-transformation with AD-53m and pHIS2-S15 promoters served as a positive control, while co-transformation with AD-53m and empty pHIS2 served as a negative control.

[0088] Figure 5Experimental results on the regulation of VvSWEET15 promoter transcriptional activity by VvERF105. A) Structural analysis of the reporter and effector constructs; B) Detection of LUC fluorescence in tobacco leaves infected with the reporter and effector using a dual-fluorescent reporter system. The left figure shows that VvERF105 has repressor activity; the experimental group is abbreviated as ProVvSWEET15:LUC+VvERF105, and the control group is abbreviated as ProVvSWEET15:LUC+Empty vector; C) Relative LUC activity analysis of reporter and effector binding. Tobacco leaves were co-transfected with the reporter vector (ProVvSWEET15:LUC) and the effector empty vector (pHB-EV) as a negative control. Data are presented as mean ± SD, with 15 biological replicates. In all statistical figures, * indicates significant differences between the experimental and control groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Significant differences were determined using Student's t-test or Welch's t-test.

[0089] Figure 6 This section presents the test results for potential VvERF105 binding elements on the VvSWEET15 promoter. A. Schematic diagram of the distribution of potential VvERF105 binding elements on the VvSWEET15 promoter. Purple bars represent the location of MYB binding elements on the promoter, and red bars represent the location of LTR elements. B. EMSA assay to determine whether VvERF105 binds to the DREcore element. C and D. EMSA assay to determine whether VvERF105 binds to the MYB element. E. EMSA assay to determine whether VvERF105 binds to the LTR element. Arrows indicate the blocking band containing the probe and VvERF105. Candidate elements are highlighted: DREcore is marked in blue, MYB in purple, and LTR in red. Hot probes are biotin-labeled portions of the VvSWEET15 promoter sequence, while cold probes, as competing probes, are unlabeled portions of the VvSWEET15 promoter sequence. The core element of a cold probe is mutated to create a mutant probe, which is highlighted in green. Mutation probes are used to identify mutant probes; free probes are used to identify free probes.

[0090] Figure 7Experimental results to detect the binding of VvERF105 to the LTR element of the VvSWEET15 promoter. A. Schematic diagram of the distribution of potential VvERF105 binding elements on the VvSWEET15 promoter. Red bars represent the positions of LTR binding elements on the promoter; B. EMSA assay to identify whether VvERF105 binds to the LTR element. Arrows indicate the blocking band containing the probe and VvERF105. Candidate elements are highlighted: LTR elements are marked in red, NAC elements in blue, and mutant elements in green. The hot probe is a biotin-labeled portion of the VvSWEET15 promoter sequence, while the cold probe, as a competing probe, is an unlabeled portion of the VvSWEET15 promoter sequence. The core element in the cold probe is mutated and used as a mutant probe; the mutated core element is highlighted in green; C. ChIP-qPCR assay. Cross-linked chromatin samples were obtained from Kyoho fruit transiently overexpressing VvERF105-HA, with Kyoho fruit transiently overexpressing an empty vector tagged with HA serving as a negative control. Each transiently transformed fruit was considered an independent biological replicate, with a total of three biological replicates. Data are presented as mean ± standard deviation (SD). In all statistical figures, * indicates significant differences between the experimental and control groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Significant differences were assessed using Student's t-test or Welch's t-test.

[0091] Figure 8To verify the experimental results of VvERF105 inhibiting sugar accumulation in grape fruits. A. Phenotypes of two different transgenic grape fruits. These are randomly selected transiently transformed positive fruits; VvERF105-OE and VvERF105-RNAi fruits showed no significant changes compared to the control. White boxes indicate the locations of the needle holes where Agrobacterium-mediated bacterial solution was injected. B. Relative expression levels of VvERF105 and VvSWEET15 and sugar content in VvERF105-OE transiently transformed fruits. Six biological replicates; data are mean ± SD. C. Relative expression levels of VvERF105 and VvSWEET15 and sugar content in VvERF105-RNAi transiently transformed fruits. Six biological replicates; data are mean ± SD. D. Western blot analysis of VvERF105 protein content at different developmental stages, using VvRubisco as an internal control. EV was used as a control for transformation with the empty vector. The red numbers represent ratios, indicating the proportion of the gray value of the target protein band to the gray value of the control protein band (the control band ratio is set to 1.0). 10-15 grapes were randomly picked from a single bunch to form one independent biological replicate, for a total of three replicates. The mean and standard deviation were calculated. In all statistical graphs, * indicates a significant difference between the experimental and control groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Significant differences were assessed using Student's t-test or Welch's t-test. Detailed Implementation

[0092] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0093] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0094] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0095] The Kyoho grape (V. labrusca x V. vinifera) and Kyoho grape used in the following examples are described in: Masahiko Yamada, Akihiko Sato. Advances in table grape breeding in Japan, Breeding Science, 2016, Volume 66, Issue 1, Pages 34-45, Released on J-STAGE March 29, 2016, Online ISSN 1347-3735, Print ISSN 1344-7610, https: / / doi.org / 10.1270 / jsbbs.66.34. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0096] The Chardonnay used in the following examples is described in: D'Onofrio C, Scalabrelli G. Chardonnay[M] / / Italian Vitis Database (ISSN 2282-006X). 2015. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and may not be used for any other purpose.

[0097] The Jingxiangyu described in the following examples is described in: Fan Peige, Yang Meirong, Wang Lijun, Li Shengchen, Wu Benhong, Li Liansheng & Li Shaohua. (2009). A new high-quality early-maturing grape variety 'Jingxiangyu'. Fruit Growers' Friend (02), 13. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0098] In the following examples, Agrobacterium GV3101 was purchased from Zhuangmeng Biotechnology, catalog number ZC141; GV3101 (pSoup) was purchased from Zhuangmeng Biotechnology, catalog number ZC1406; yeast strain Y187 was purchased from Zhuangmeng Biotechnology, catalog number ZK283; and pHIS2-bait was purchased from Zhuangmeng Biotechnology, catalog number ZK971.

[0099] The pHB carrier used in the following examples is described in: Gao Zhen, Li Qin, Li Jing, Chen Yujin, Luo Meng, Li Hui, Wang Jiyuan, Wu Yusen, Duan Shuyan, Wang Lei, Song Shiren, Xu Wenping, Zhang Caixi, Wang Shiping, Ma Chao. Characterization of the ABA Receptor VlPYL1 That Regulates Anthocyanin Accumulation in Grape Berry Skin. Frontiers in Plant Science. 2018(9), DOI=10.3389 / fpls.2018.00592). This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.

[0100] The tobacco (Nicotiana benthamiana) used in the following embodiments is described in: Clemente T. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana) [J]. Agrobacterium protocols, 2006: 143-154. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0101] The pGADT7-Rec2-cDNA library in the following examples was constructed using the Clontech yeast one-hybrid screening kit (Clontech, Cat. No. 630304, PT3529-1).

[0102] Some of the experimental methods in the following examples are as follows:

[0103] I. Grape genomic DNA extraction

[0104] DNA was extracted from grape cells, leaves, and fruits using a DNA extraction kit (DP304-02, TIANGEN) with a centrifugal adsorption column that specifically binds to DNA and a unique buffer system, and then stored at -40°C for long-term preservation.

[0105] II. Total RNA extraction from grapes, various tissues and organs, and tobacco leaves

[0106] Total RNA was extracted from grapes, various tissues and organs, and tobacco leaves using the HiPure HP Plant RNA Mini Kit (R4165-02, Magen). Specific methods were described in the kit's instruction manual.

[0107] III. First-strand cDNA synthesis

[0108] The Novozymes HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-02) can be used to remove residual genomic DNA from the RNA template and synthesize first-strand cDNA. For specific methods, please refer to the kit instructions.

[0109] IV. RT-PCR

[0110] The Novozymes HiScript II One Step RT-PCR Kit (Dye Plus) (P612-01) was used. Gene-specific primers (GSP) were used, and the reverse transcription and PCR reactions were completed in one tube, eliminating the need for additional tube opening / pipetting operations. This improved the detection throughput and reduced the risk of contamination. The detection sensitivity can reach 1 pg of total RNA. For specific methods, please refer to the kit instructions.

[0111] Using total RNA from grape, tomato tissues and organs, and tobacco leaves as templates, gene-specific primers were synthesized from the Actin1, VvSWEET15, VvERF105, and VvNAC72 gene-specific RT-PCR detection fragments, respectively, and then the kit instructions were followed.

[0112] V. Quantitative Real-Time PCR

[0113] The Novozymes Biotechnology High Specific Dye Quantitative PCR Detection Kit was selected. Use qPCR SYBRGreen Master Mix (Without ROX) (Q121-02), and then refer to the kit instructions.

[0114] Actin1 was used as an internal control for grapes, Actin7 for tomatoes, and Actin97 for tobacco to standardize expression levels. All gene expression detections involved in this paper were performed in more than 6 biological replicates, and at least 3 representative replicates were selected for the final results presentation.

[0115] VI. The methods for determining the sugar content of various tissues in grapes, tomatoes, and tobacco leaves are as follows:

[0116] High-performance liquid chromatography (HPLC) was used to determine the sugar content and composition of samples. A differential refractive index detector was employed, allowing for direct measurement; the procedure was simple and the sensitivity was acceptable.

[0117] (1) Sample preparation: The sample was ground into a fine powder in liquid nitrogen. 0.2-0.3 g of the sample was placed in a 2 mL centrifuge tube, and 1 mL of ultrapure water was added. The sample was mixed every 30 minutes at room temperature, and allowed to stand for 2-3 hours to fully dissolve the sugar. The sample was then centrifuged at 10,000 g for 5 minutes, and the supernatant was carefully collected into a new 2 mL centrifuge tube. (Note: If necessary, the precipitate can be repeatedly extracted. Repeat the above steps several times to ensure complete extraction of the sugar from the tissue). The sample was then filtered through a 0.22 μm filter into a vial. The sugar content was determined using high-performance liquid chromatography (see below), with units of mg·g. -1 FW.

[0118] (2) A series of gradient dilutions of known concentrations were prepared using high-purity standard samples of glucose, fructose and sucrose. The peak times of these three sugars were determined by HPLC, and standard curves of glucose, fructose and sucrose were plotted by peak area for quantitative analysis.

[0119] (3) Determination of sugar content by high performance liquid chromatography: The sugar content of the sample to be tested was determined by high performance liquid chromatography with an autosampler, column oven, differential detector and chromatography workstation in cyclic operation.

[0120] The chromatographic conditions are as follows:

[0121] Column: Sugar-Pak™ II column packed by Water.

[0122] Detector: Differential detector

[0123] Detector temperature: 35℃

[0124] Mobile phase: ultrapure water

[0125] Column temperature: 80℃

[0126] Flow rate: 0.6 mL·min -1

[0127] Time: 15min

[0128] Injection volume: 10 μL

[0129] VII. Electrophoretic Mobility Detection (EMSA)

[0130] The Thermo Scientific LightShift Chemiluminescent EMSA Kit uses a non-isotopic method to detect DNA-protein interactions. Biotin-terminated DNA containing the target binding site is incubated with a nuclear extract or purified factor. The reaction is then subjected to gel electrophoresis on a non-denaturing polyacrylamide gel and transferred to a nylon membrane. Biotin-terminated DNA is detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. Refer to the kit instructions (Pierce, 89880) for specific methodological details.

[0131] VIII. Chromatin Immunoprecipitation (ChIP)

[0132] Using EpiQuik TM Plant ChIP Kit (P-2014, USA) - Refer to the kit instructions for specific methods.

[0133] Example 1: Yeast one-hybrid library screening and yeast one-hybrid screening

[0134] 1. Yeast one-hybrid screening library

[0135] The VvSWEET15 gene number is VIT_01s0146g00260. The VvSWEET15 promoter sequence (2024 bp, specific nucleotide sequence is SEQ ID No. 4) in Chardonnay was amplified by PCR, and the cis-regulatory elements on the VvSWEET15 gene promoter were predicted using PlantCARE (Table 1) as follows:

[0136] Table 1. Information on cis-regulatory elements of the VvSWEET15 gene promoter

[0137]

[0138]

[0139]

[0140] Furthermore, these elements (218 bp, nucleotide sequence SEQ ID No. 5) were artificially synthesized in tandem to construct a yeast one-hybrid bait vector. Simultaneously, a cDNA yeast one-hybrid library containing different developmental stages of Chardonnay, Kyoho, and Jingxiangyu fruits was constructed. Proteins interacting with the bait element vector were screened.

[0141] 1) Bait sequences for synthetic yeast one-hybrid screening libraries

[0142] The key elements were tandemly repeated to synthesize the following VvSWEET15 yeast one-hybrid bait sequence:

[0143] VvSWEET15 decoy sequence (218bp):

[0144]

[0145] 2) Detection of bait carrier self-activation

[0146] The minimum inhibitory concentration of 3AT (Coolaber, SL0930) to suppress the self-activation background generated by the bait was determined. The bait vector was transformed into Y187 strain and plated on yeast-deficient medium (Coolaber, PM2281-10 x 0.5L, abbreviated as SD / -Trp-His) with different 3AT concentration gradients (0mM, 20mM, 30mM, 40mM, 60mM, 100mM) for one week.

[0147] The pGAD53m (Forscience, HG-VJC1223) vector produces a fusion protein of the transcription factor GAL4 activation domain and the tumor suppressor protein p53. The p53HIS vector contains the target DNA sequence of the tumor suppressor protein p53, located in the promoter region of the vector reporter gene HIS3. The pHIS2 vector (Zhuang Meng, ZK971) is an empty vector containing only the HIS3 reporter gene. All three vectors were provided by Shanghai Haike Biotechnology Co., Ltd. along with the constructed library. pGAD53m and p53HIS were co-transformed into strain Y187 and grown at different 3AT concentration gradients (0m...). SD / -Trp-Leu-His yeast auxotrophic medium (Coolaber, PM2152-10*0.5L) at concentrations of 0mM, 20mM, 30mM, 40mM, 60mM, and 100mM served as positive controls. Y187 strain co-transformed with pGAD53m and pHIS2 and grown on SD / -Trp-Leu-His yeast auxotrophic medium (Coolaber, PM2152-10*0.5L) at different 3AT concentration gradients (0mM, 20mM, 30mM, 40mM, 60mM, and 100mM) served as negative controls (Table 2). All cultures were incubated at 30℃ for one week. By comparing the growth differences with the negative controls, the optimal 3AT concentration for inhibiting bait carrier self-activation was determined.

[0148] Table 2. Yeast self-activation verification

[0149] Reaction AD plasmid BD plasmid Transformation plate Detection plate Detection content 1 - pHIS2-bait SD-T SD-TH+3AT Self-activation detection 2 pGAD53m pHIS2 SD-TL SD-TLH+3AT Negative control 3 pGAD53m p53HIS SD-LH SD-TLH+3AT Positive control

[0150] 3) Single hybridization screening library

[0151] See the yeast one-hybrid screening library process. Figure 1The specific steps were performed according to the Clontech manual (Clontech, Cat. No. 630304, PT3529-1). The cis-regulatory element sequence of the VvSWEET15 promoter (Table 1), whether single copy or tandem repeat, was cloned upstream of the HIS3 reporter gene and located in the pHIS2 vector. A cDNA library of grape berries was created using SMART cDNA synthesis technology. A highly complex pre-transformed cDNA library was constructed through recombination between the cDNA library and the linear prey vector pGADT7-Rec2. The bait vector and prey vector were co-transformed into strain Y187. True positive interactions were detected in colonies that could grow on minimal medium of SD / -His / -Leu / -Trp / +3AT. Reliable positive clones were selected through 3-4 rounds of repeated streak screening. Figure 2 Sequencing was performed to obtain cDNA sequences of Chardonnay, Kyoho, and Jingxiangyu fruits at different developmental stages that interacted with the VvSWEET15 bait sequence. These cDNA sequences were compared with the grape database (http: / / plants.ensembl.org / Vitis_vinifera / Info / Index) to identify proteins in the library that interacted with the bait element. The selected genes were sequenced and annotated in Table 3 below.

[0152] Table 3. Sequencing and annotation information of selected genes

[0153]

[0154]

[0155]

[0156]

[0157] Yeast one-hybrid screening of candidate genes was performed. Co-transformation with the AD-53m and pHIS2-S15 promoter served as a positive control, while co-transformation with the empty vector pHIS2 and the empty vector AD and pHIS2-S15 promoter served as negative controls. Cloning sites were transferred to SD (-Leu-Trp) medium to ensure experimental reliability; the amount of yeast growth was determined by interactions, not by human error.

[0158] The results are as follows Figure 3 As shown: Five candidate genes interact with the VvSWEET15 promoter (2024 bp). Compared with the negative control and the control transformed with an empty prey vector-promoter vector, positive clones containing five candidate genes (VvTSO1, VvDPP6, VvSPFH, VvCML, VvERF105) showed stronger interactions at each 3AT selection pressure than the control group.

[0159] 2. The VvSWEET15 Promoter (2024 bp) was ligated into the pHIS2 vector, and point-to-point yeast single-hybrid technology was used to verify whether the VvSWEET15 promoter interacts with the candidate target protein. 3AT was used as a competitive inhibitor of yeast HIS3 protein synthesis to suppress the leakage expression of the HIS3 gene. The specific method is as follows:

[0160] a. Promoter vector cloning: The VvSWEET15Promoter (2024bp) amplified from Chardonnay fruit was constructed into the yeast one-hybrid bait vector pHIS2 through the restriction sites at both ends of the sequence. The CDS sequence of the candidate interacting protein VvERF105 was ligated into the pGADT7-Rec2 vector (Zhuang Meng, ZK977).

[0161] The structure of the promoter vector pHIS2-VvSWEET15Promoter is described as follows: The recombinant vector is obtained by replacing the small fragment between the EcoRI restriction endonuclease recognition site and the SacI enzyme recognition site of the starting vector pHIS2 with the VvSWEET15 promoter DNA sequence (nucleotide sequence is SEQ ID No. 4), while keeping the other sequences of the starting vector pHIS2 unchanged.

[0162] The structure of the interacting protein vector pGADT7-Rec2-VvERF105 is described as follows: The recombinant vector is obtained by single digestion of the recognition site of the restriction endonuclease Sma I of the starting vector pGADT7-Rec2, ligating the cDNA sequence of VvERF105 into it, and keeping the other sequences of the starting vector pGADT7-Rec2 unchanged.

[0163] b. Detection of self-activation of pHIS2-VvSWEET15 Promoter carrier: The method is the same as the detection of self-activation of cis-trans element decoy carrier in step 2) above.

[0164] c. Prepare competent yeast cells according to the Clontech method for preparing competent yeast cells (Cat. No. 630439).

[0165] d. Conversion: The conversion method shall be in accordance with the Clontech manual (Cat. No. 630439).

[0166] e. After transformation, suspend the bacterial cells in 200 μL of sterile water for each transformation, mix gently as much as possible, and then spread the serially diluted cells onto SD / -Trp-Leu defective selection medium.

[0167] f. Incubate at a constant temperature of 30℃ for 3-4 days.

[0168] g. Identify true positive clones using the Clontech Yeast Colony PCR Identification Kit Advantage 2 PCR Polymerase Mix Set (Cat. No. 639201) and the Yeast Plasmid Identification Kit Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433).

[0169] h. Use a pipette tip to pick up a small amount of large, round, positive yeast monoclonal colonies and place them in the culture medium. Incubate overnight until OD500. 600 =0.6-0.7, take an appropriate amount of culture medium to adjust the OD of the bacterial solution. 600 =0.6, then take an appropriate amount of bacterial culture, serially dilute it and spot it on SD / -Trp-Leu, SD / -Trp-Leu-His defective selection medium.

[0170] i. Observe the camera's photo records after 2-4 days.

[0171] Yeast point-to-point verification revealed that the VvERF105 and VvSWEET15 full-length (S15p, 2024bp) promoters interact significantly. Figure 4 ).

[0172] The coding sequence (CDS) of the VvERF105 gene in the grape variety Chardonnay is SEQ ID No. 2, encoding the VvERF105 protein with the amino acid sequence shown in SEQ ID No. 1. The gene encoding the VvERF105 protein in the genomic DNA of grape VvERF105 is shown in SEQ ID No. 3 of the sequence listing.

[0173] Example 2: Analysis of potential VvSWEET15 promoter binding sites for VvERF105

[0174] To analyze the potential VvSWEET15 promoter binding site of VvERF105, the fusion protein VvERF105-His was purified, and a biotinylated S15p fragment containing the reported VvERF105 binding element / motif was used as a hot probe. The corresponding unlabeled S15p fragment was used as a cold probe.

[0175] For details on the preparation steps of the target protein, please refer to section VII above, Electrophoretic Mobility Detection (EMSA).

[0176] Analysis revealed that the VvSWEET15 promoter sequence, in addition to the DREcore element (-71), also contains two elements with GCC motifs (MYB: CCGTTG and LTR: CCGAAA). The MYB recognition element contains two copies (-1371, -987) in the VvSWEET15 promoter. Figure 6 (A), but EMSA testing showed that VvERF105 does not bind to the MYB recognition element ( Figure 6 (BD). The LTR element also contains two copies (-858, -355) in the VvSWEET15 promoter ( Figure 7 In experiment A), VvERF105 and the S15p fragment containing an LTR binding element showed a gel-blocking band, and with the gradual increase of the concentration of the competing probe, the blocking band weakened or even disappeared. However, the addition of a mutant probe resulted in a gel-blocking band, indicating that VvERF105 binds to the LTR element in vitro. Figure 6 E, Figure 7 (B)

[0177] To further verify the EMSA experimental results, a ChIP-qPCR experiment was performed.

[0178] The pHB-35S-HA and pHB-35S-VvERF105-HA vectors were constructed, and the specific vector information is as follows:

[0179] 1) pHB-35S-HA is a recombinant vector modified from pHB vector. The structure of pHB-35S-HA is described as follows: The small fragment between the restriction endonuclease BamHI recognition site and the XbaI recognition site of the starting vector pHB-35S is replaced with the HA-tag cDNA sequence (5'-TACCCTTACGATGTTCCAGATTACGCCGGCTGA-3'), while keeping the other sequences of the starting vector pHB-35S unchanged.

[0180] 2) pHB-35S-VvERF105-HA is a recombinant vector modified from pHB-35S-HA. The structure of pHB-35S-VvERF105-HA is described as follows: The small fragment between the HindIII restriction endonuclease recognition site and the BamHI enzyme recognition site of the starting vector pHB-35S-HA is replaced with the CDS sequence of VvERF105 (nucleotide sequence is SEQ ID No. 2), while keeping the other sequences of the starting vector pHB-35S-HA unchanged.

[0181] The construct was transiently transformed into Kyoho fruit. Chromatin was extracted from the transiently transformed fruit for co-immunoprecipitation (ChIP) experiments. The VvERF105-HA-binding DNA complex was enriched using magnetic beads conjugated with HA antibody. The enriched promoter fragment was obtained by decrosslinking and purification. Probes (p1, p2, p3, p4) were designed near the VvERF105 binding element. qPCR detection was performed, and Kyoho fruit transformed with the pHB-35S-HA construct was used as a control.

[0182] qPCR detection

[0183] qPCR detection was performed using multiple pairs of quantitative primers designed near the target element. The primer sequences are as follows: ERF105-P1-F: 5'-GTATTGCGCTCCGGTTGT-3'; ERF105-P1-R: 5'-GCAAGACAACGGTGCAATT-3'; ERF105-P2-F: 5'-TGGTCTCCACCCAGCATT-3'; ERF105-P1-R: 5'-TTCGGCAATGCTGGGT-3'; ERF105-P3-F: 5'-CCTTACGCTAGACAGCTTTGC-3'; ERF105-P1-R: 5'-CCCGAAACCTGACACCC-3'; ERF105-P4-F: 5'-CCCACCATTTCCAAGCG-3'; ERF105-P1-R: 5'-AAGGGAAAGAAGACGACAGG-3'. qPCR test results as follows Figure 7 As shown in Figure C, VvERF105-HA was found to be associated with the VvSWEET15 promoter segments (red p2 and p3) containing LTR elements. This further confirms that VvERF105 binds to the LTR element (CCGAAA) on the VvSWEET15 promoter. Figure 7 (B)

[0184] Example 3: Dual-luciferase dual-reporter gene transient expression assay

[0185] By linking the VvSWEET15 promoter upstream of the LUC reporter gene, ProVvSWEET15:LUC was constructed as the reporter, and 35S:VvERF105 was constructed as the effector using the 35S promoter. The reporter and effector were co-transfected into tobacco leaves.

[0186] 1. Construction of the pGreenⅡ0800-VvSWEET15promoter-LUC recombinant vector

[0187] The VvSWEET15 promoter was ligated into the pGreenⅡ-0800-LUC vector (Shanghai Zeye Biotechnology, ZY8124) containing the firefly luciferase gene and the kidney luciferase gene, i.e., pGreenⅡ0800-VvSWEET15promoter-LUC.

[0188] The structure of pGreenⅡ0800-VvSWEET15promoter-LUC is described as follows: The recombinant vector is obtained by replacing the small fragment between the HindIII restriction endonuclease recognition site and the BamHI enzyme recognition site of the starting vector pGreenⅡ-0800-LUC with the genomic DNA sequence of the VvSWEET15 promoter (nucleotide sequence is SEQ ID No. 4), while keeping the other sequences of the starting vector pGreenⅡ-0800-LUC unchanged.

[0189] 2. Construction of the 35S:VvERF105 recombinant vector

[0190] VvERF105 was ligated into a pHB vector without any reporter gene, i.e., pHB-35S-VvERF105 vector (abbreviated as 35S:VvERF105).

[0191] The structure of 35S:VvERF105 is described as follows: The recombinant vector is obtained by replacing the small fragment between the restriction endonuclease BamHI recognition site and the XbaI recognition site of the starting vector pHB with the cDNA sequence of VvERF105 (nucleotide sequence is SEQ ID No. 2), while keeping the other sequences of the starting vector pHB unchanged.

[0192] 3. Agrobacterium GV3101 (pSoup) transformation

[0193] After transforming the recombinant vector pGreenⅡ0800-VvSWEET15promoter-LUC into Escherichia coli, positive clones were selected, and the plasmids of the positive clones were transformed into Agrobacterium GV3101 (pSoup) to obtain recombinant Agrobacterium GV3101 / VvSWEET15promoter-LUC, which was stored at -40℃ for later use.

[0194] 4. Instantaneous transformation of tobacco

[0195] The process of instantaneous infection of 5-week-old tobacco leaves is as follows:

[0196] a. Agrobacterium culture

[0197] (1) Agrobacterium GV3101 / VvSWEET15promoter-LUC containing the target transformant was streaked onto LB solid medium (50 mg / L Kana, 50 mg / L Rfp) and incubated upside down at 28°C for 2 days.

[0198] (2) Select the positive clones of Agrobacterium GV3101 / VvSWEET15promoter-LUC that have been activated and transformed into the target gene and incubate them overnight at 28°C and 200rpm in 1mL LB liquid medium (50mg / L Kana, 50mg / L Rfp).

[0199] (3) Transfer 0.1 mL of overnight culture to 5 mL of LB liquid medium (50 mg / L Kana, 50 mg / L Rfp), and incubate overnight at 28°C and 200 rpm until OD. 600 =0.8-1.0.

[0200] (4) Discard the supernatant, suspend the Agrobacterium cells in 5 mL of suspension (10 mM MES-KOH, pH 5.2; 10 mM MgCl2; 100 μM Macetosyringone) and adjust the OD of the suspension. 600 =1, let stand at room temperature for more than 3 hours.

[0201] b. Agrobacterium GV3101 / VvSWEET15promoter-LUC vacuum injection into tobacco leaves

[0202] (1) Mix the suspension well, use a 1mL syringe to draw up the infection solution, inject it from the back of the leaf and avoid the midrib. The leaf can be clearly seen to be wetted. Incubate at room temperature in the dark for 1 day, and then incubate under normal light for 2 days.

[0203] (2) Three days after injection, the leaf was cut off directly, and the substrate of dual-luciferase was sprayed at the injection site. The fluorescence was observed using a chemiluminescence analyzer.

[0204] 5. Luciferase assay

[0205] (1) Tissue lysis: Use a 1cm pore size punch to punch holes at the site of Agrobacterium infection, place the tube, freeze in liquid nitrogen, and break into powder using a tissue homogenizer under freezing conditions. Immediately add 500μL of 1×PLB, mix quickly, and let stand for 5min.

[0206] (2) Centrifuge at 8000 rpm for 5 min. Take 10 μL of the supernatant and add 90 μL of sterile water (1:10) and mix well for later use.

[0207] (3) Use Measure LUC activity using a 20 / 20 photometer (Promega). Take 10 μL of the diluent, add 50 μL of LARⅡ, pipette 2-3 times, and immediately place it in the photometer to record the firefly luciferase LUC activity.

[0208] (4) After adding 60 μL of Stop&Glo reagent, mix by pipetting 2-3 times and immediately put it back into the instrument to record the REN activity of sea cucumber luciferase.

[0209] (5) Finally, record the RLU1 (firefly luciferase activity), RLU2 (renaeus luciferase activity), and Ratio (RLU1 / RLU2) values.

[0210] The results show that ( Figure 5 VvERF105 acts as a transcriptional repressor, downregulating LUC expression.

[0211] Example 4: Construction of VvERF105 overexpression and interference vector

[0212] Using grape fruit cDNA as a template, the target gene CDS sequence was amplified using the high-fidelity enzyme KOD-Plus-Neo. After purification and recovery by agarose gel electrophoresis, the target fragment was seamlessly ligated into the target expression vectors pHB and pRI101 (TaKaRa, Code No. 3262) using a homologous recombination kit. The successfully constructed vectors were verified by sequencing, and the constructed bacterial culture was stored at -80°C for long-term preservation.

[0213] A. The construction method of the overexpression vector pHB is as follows:

[0214] The recombinant vector was obtained by replacing the small fragment between the restriction endonuclease BamHI recognition site and the XbaI recognition site of the starting vector pHB with the cDNA sequence of VvERF105 (nucleotide sequence is SEQ ID No. 2), while keeping the other sequences of the starting vector pHB unchanged.

[0215] B. The construction method of the RNAi vector pRI101 is as follows:

[0216] The interference fragments from VvERF105 are as follows:

[0217]

[0218]

[0219] The structure of the VvERF105-RNAi vector is described as follows: The small fragment between the restriction endonuclease NdeI recognition site and the EcoRI recognition site of the starting vector pRI101 is replaced with the fragment of SEQ ID No. 6, and the interference fragment of VvERF105 is reverse-linked to the starting vector pRI101 while keeping other sequences unchanged to obtain the recombinant vector.

[0220] Example 5: Application of VvERF105 in inhibiting hexose accumulation in fruit

[0221] To study the functions of VvSWEET15 and VvERF105 during grape ripening, transient transgenic experiments were conducted on grape berries for three consecutive years to overexpress or inhibit VvSWEET15 and VvERF105.

[0222] Plants to be tested: wild-type Kyoho, overexpressing plants EV-OE, VvERF105-OE; EV-RNAi, VvERF105RNAi.

[0223] This experiment is conducted two weeks before the Kyoho grapes begin to ripen. In 2020, transient EV-OE and VvERF105-OE experiments were carried out; in 2021 and 2022, RNAi experiments of EV-RNAi and VvERF105 RNAi were carried out.

[0224] The specific experimental procedure is as follows:

[0225] 1. Every year, thinning of flowers and fruits is carried out on the Kyoho grape variety to ensure that the growth of the experimental fruits is basically consistent;

[0226] 2. Select activated Agrobacterium GV3101 cells transformed with EV-OE, VvERF105-OE, EV-RNAi, and VvERF105 RNAi vectors respectively and incubate them overnight at 28 degrees Celsius and 200 rpm in 1 mL LB liquid medium (50 mg / L Kana, 50 mg / L Rfp).

[0227] 3. Transfer 0.5 mL of overnight culture to 5 mL of LB liquid medium (50 mg / L Kana, 50 mg / L Rfp) at 28°C and 200 rpm to OD. 600 It is 0.7;

[0228] 4. Add the above 5 mL of bacterial culture to 100 mL of LB liquid medium (50 mg / L Kana, 50 mg / L Rfp) at 28°C and 200 rpm until the OD value is reached. 600 It is 0.8-1;

[0229] 5. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and suspend the Agrobacterium cells in 80 mL of suspension (10 mM MES-KOH, pH 5.2; 10 mM MgCl2; 100 μM acetosyringone) and adjust the OD of the suspension. 600 =1, let stand at room temperature for 3 hours;

[0230] 6. Before using the bacterial suspension, vortex or pipette the suspended bacterial cells. Use a 1mL sterile syringe to take 1mL of the suspension and continuously inject the bacterial suspension from the grape stem end or stalk end. Insert the needle about 0.5 cm and slowly inject the recombinant Agrobacterium suspension GV3101 into the pulp to ensure that the solution slowly penetrates into the pulp. Typically, a significant amount of suspension will leak out, so the injection process can be repeated 2-3 times to ensure that at least 0.1 mL of suspension penetrates into the flesh (successfully injected fruits are noticeably firmer than uninjected fruits). Each suspension containing EV-OE, VvERF105-OE, EV-RNAi, or VvERF105 RNAi vector was injected into 3-4 bunches of grapes (approximately 80-100 fruits), with each fruit serving as an independent biological replicate. To explore the higher transformation efficiency of different transient transgenic experiments after several days of Agrobacterium GV3101 infection, dynamic sampling was performed (i.e., OE / RNAi: fruits were collected at 3, 5, 7, and 9 days after infection), ensuring 8-15 fruit replicates for each sampling. This experiment ultimately sampled 38-42 fruits (replicas) for overexpression and 31-61 fruits for RNAi experiments for further experimental research.

[0231] The results show that ( Figure 8 (AC): The fruit phenotypes of VvERF105-OE and VvERF105-RNAi were not significantly different from those of the control (EV). In VvERF105-OE fruit, the VvSWEET15 transcript was downregulated by 0.36-fold, with glucose and fructose contents decreasing by 33% and 28.7%, respectively. However, in VvERF-RNAi, it was upregulated by 1.65-fold, with glucose and fructose contents significantly increasing by 19.1% and 18.8%, respectively.

[0232] Example 6: Detection of VvERF105 protein content in grape berries after total protein extraction and overexpression / RNAi.

[0233] Total protein was extracted from Chardonnay grape berries at 32, 42, 52, 63, 72, 92, 106, 119, and 134 days post-flowering, as well as from berries containing VvSWEET15, VvERF105, transient overexpression of Kyoho grapes, and RNAi, obtained in Example 2. The total protein extraction assay was performed entirely according to the Plant Total Protein Extraction Kit (PE0230-1KT, Sigma-Aldrich). Proteins were detected by SDS-PAGE gel electrophoresis and Western blotting.

[0234] Western blot

[0235] 1) After SDS-PAGE, the protein gel was transferred for 7 minutes using Trans-Bio Turbo Min PVDF Transfer Packs (1704156, Bio-Rad) and a Trans-Blot Turbo (Bio-Rad) rapid semi-dry transfer instrument.

[0236] 2) After the transfer is complete, remove the upper and lower filter papers of the PVDF membrane and place the PVDF membrane in TTBS (50mM Tris-HCl pH 7.4, 150mM NaCl, 0.05% Tween-20) containing 5% skim milk powder for 1 hour.

[0237] 3) Incubate with primary antibody: Discard the blocking solution, add TTBS containing 1% skim milk powder (add primary antibody according to the dilution ratio of the target gene antibody), and incubate overnight at 4°C.

[0238] 4) Discard the incubation solution and rinse with TTBS solution 4 times for 5 minutes each time.

[0239] 5) Incubate with secondary antibody: Add TTBS containing 1% skim milk powder (1:10000 for secondary antibody) and incubate at room temperature for 2 hours.

[0240] 6) Discard the incubation solution and rinse with TTBS solution 4 times for 5 minutes each time.

[0241] 7) Using the high-sensitivity chemiluminescence detection kit eECL Western Blot Kit, mix chemiluminescence solutions A and B in equal proportions, drop them evenly onto the PVDF membrane, and develop the membrane using a chemiluminescence analyzer (Tanon).

[0242] Western blot analysis showed that ( Figure 8(D): Before the onset of ripening (72 days after flowering), the VvERF105 protein content showed the opposite trend to the VvSWEET15 protein content. That is, when the VvERF105 protein content was high during the developmental period, the VvSWEET15 protein content was low. This also indicates that VvERF105 inhibits VvSWEET15 expression, resulting in a generally low sugar accumulation level in the fruit before the onset of ripening.

[0243] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a protein or a substance modulating the expression of a gene encoding said protein or a substance modulating the activity or content of said protein in any one of the following: 1) for modulating sugar accumulation in a plant fruit; 2) for the manufacture of a product for modulating sugar accumulation in a plant fruit; 3) for breeding a plant with altered sugar accumulation in a fruit; 4) for the manufacture of a product for breeding a plant with altered sugar accumulation in a fruit; 5) for sugar accumulation breeding in a plant fruit; said protein being any one of the following: a1) a protein having the amino acid sequence of SEQ ID No. 1 ; a2) a fusion protein obtained by linking a tag to the end of a protein as defined in a1); said plant being a grape.

2. Use according to claim 1, characterized in that, said protein being of grape origin.

3. Use according to claim 1 or 2, characterized in that, the substance modulating the expression of a gene or the substance modulating the activity or content of said protein being a biological material associated with said protein in the use according to claim 1 or 2, said biological material being any one of the following: c1) a nucleic acid molecule encoding said protein; c2) an expression cassette comprising the nucleic acid molecule of c1); c3) a recombinant vector comprising the nucleic acid molecule of c1) or the expression cassette of c2); c4) a recombinant microorganism comprising the nucleic acid molecule of c1) or the expression cassette of c2) or the recombinant vector of c3); c5) a transgenic plant cell line comprising the nucleic acid molecule of c1) or the expression cassette of c2); c6) a transgenic plant tissue comprising the nucleic acid molecule of c1) or the expression cassette of c2); c7) a transgenic plant organ comprising the nucleic acid molecule of c1) or the expression cassette of c2); e1) a nucleic acid molecule inhibiting or reducing or silencing the expression of a gene encoding said protein; e2) an expression cassette comprising the nucleic acid molecule of e1); e3) a recombinant vector comprising the nucleic acid molecule of e1) or the expression cassette of e2); e4) a recombinant microorganism comprising the nucleic acid molecule of e1) or the expression cassette of e2) or the recombinant vector of e3); e5) a transgenic plant cell line comprising the nucleic acid molecule of e1) or the expression cassette of e2); e6) a transgenic plant tissue comprising the nucleic acid molecule of e1) or the expression cassette of e2); e7) a transgenic plant organ comprising the nucleic acid molecule of e1) or the expression cassette of e2).

4. Use according to claim 3, characterized in that: c1) the nucleic acid molecule being any one of the following DNA molecules, d1) a DNA molecule having the nucleotide sequence of SEQ ID No. 3; d2) a DNA molecule having the coding sequence of SEQ ID No. 2; d3) a DNA molecule having 90% or more identity with the nucleotide sequence as defined in d1) or d2) and encoding the protein according to claim 1. d4) DNA molecules hybridizing with the nucleotide sequences defined in d1) or d2) under stringent conditions and encoding a protein as described in claim 1.

5. A method of altering sugar accumulation in a plant fruit, comprising, The method comprises step M or P: The step P is to enhance, increase or up-regulate the activity and / or content of the protein as described in claim 1 or 2 in the plant of interest, or / and, to enhance, increase or up-regulate the expression level of the gene encoding the protein as described in claim 1 or 2, to reduce the sugar accumulation in the fruit of the plant; The step M is to inhibit or reduce or silence the activity and / or content of the protein as described in claim 1 or 2 in the plant of interest, or / and, to inhibit or reduce or down-regulate the expression level of the gene encoding the protein as described in claim 1 or 2, to increase the sugar accumulation in the fruit of the plant; The plant is grape.

6. A method of breeding a plant with increased sugar accumulation in the fruit, characterized in that, The plant of interest is grape. The step M is to inhibit or reduce or silence the activity and / or content of the protein as described in claim 1 or 2 in the plant of interest, or / and, to inhibit or reduce or down-regulate the expression level of the gene encoding the protein as described in claim 1 or 2, to increase the sugar accumulation in the fruit of the plant; 7. The method of claim 6, wherein, The plant is grape.

8. A method of breeding a plant with reduced sugar accumulation in the fruit, characterized in that, The step M is to inhibit or reduce or silence the activity and / or content of the protein as described in claim 1 or 2 in the plant of interest, or / and, to inhibit or reduce or down-regulate the expression level of the gene encoding the protein as described in claim 1 or 2, to increase the sugar accumulation in the fruit of the plant; The plant of interest is grape. The step M is to inhibit or reduce or silence the activity and / or content of the protein as described in claim 1 or 2 in the plant of interest, or / and, to inhibit or reduce or down-regulate the expression level of the gene encoding the protein as described in claim 1 or 2, to increase the sugar accumulation in the fruit of the plant;

Images