Application of interaction of VvERF105 and VvSWEET15 promoters in reduction of fruit sugar accumulation

Abstract

The invention discloses an application of interaction of a VvERF105 promoter and a VvSWEET15 promoter in reduction of fruit sugar accumulation. The invention belongs to the technical field of biology, and particularly relates to application of interaction of VvERF105 and VvSWEET15 promoters in reduction of fruit sugar accumulation. The protein VvERF105 can interact with a VvSWEET15 promoter, the content of glucose and fructose in grape fruits is negatively regulated and controlled by regulating and controlling the expression quantity of the VvERF105, and the protein VvERF105 has an application prospect in the field of plant breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of interaction between VvERF105 and VvSWEET15 promoters in reducing sugar accumulation in fruits. Background Art

[0002] Sugar plays a vital role in plant growth, development and metabolism, and the accumulation and distribution of sugars mainly depend on the functional regulation of sugar transporters. Sugar transporters regulate the distribution of sugars in plant tissues by transporting them across cell membranes. Therefore, their expression and activity directly affect the accumulation level of sugars in fruits. In many plants, excessive or insufficient sugar accumulation may lead to a decline in fruit quality and affect agricultural production. Therefore, in-depth research on transcription factors that regulate the expression of sugar transporters and their interaction mechanisms has important application prospects for regulating sugar accumulation and improving crop quality.

[0003] At present, there are few studies on the regulation of fruit sugar accumulation by transcription factors by binding to the promoters of sugar transporter genes. In grapes, VvMSA has a positive regulatory effect on 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 and activates its expression, thereby improving the tolerance of plants to environmental stress by promoting the absorption of sugars by plants. In watermelon, the transcription factor SUSIWM1 promotes the accumulation of sucrose, glucose and fructose in the vacuoles of pulp cells by positively regulating the ClTST2 gene. In rice, the OsDOF11 transcription factor enhances the gene expression of OsSUT1, OsSWEET11 and OsSWEET14 by binding to their promoter regions, thereby affecting sugar transport in rice. The Osdof11 mutant exhibits short plants, reduced tillering, insensitivity to sucrose-mediated root growth inhibition, reduced leaf sugar accumulation, and decreased sucrose mobility in the 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 carbon source supply required for cotton fiber elongation.

[0004] VvERF105 is a transcription factor that belongs 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 target genes, thereby affecting the expression of downstream genes. Existing studies have shown that ERF transcription factors play a key role in the regulation of plant stress resistance and metabolic regulation. However, the function of VvERF105 in the regulation of sugar accumulation is still unclear, and further research is needed on its regulatory targets and its mechanism of action.

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

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

[0007] By exploring the regulatory effect of VvERF105 on the promoter activity of VvSWEET15, it is expected to reveal the new function of ERF transcription factors in the sugar accumulation process and provide an effective molecular regulatory tool for regulating sugar accumulation in fruit. This research is not only of great scientific significance, but also has broad application prospects in agricultural production. Summary of the invention

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

[0009] In order to solve the above problems, the present invention provides related applications of proteins, substances for regulating the expression of genes encoding the proteins, or substances for regulating the activity or content of the proteins.

[0010] Use of the protein provided by the present invention, a substance for regulating the expression of a gene encoding the protein, or a substance for regulating the activity or content of the protein in any of the following:

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

[0012] 2) Application in the preparation of products for regulating sugar accumulation in plant fruits;

[0013] 3) Application in breeding plants with altered fruit sugar accumulation;

[0014] 4) Use in the preparation of products for cultivating plants with altered fruit sugar accumulation;

[0015] 5) Application in plant breeding;

[0016] The protein is any of the following:

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

[0018] a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 after one or more amino acid residues are replaced and / or deleted and / or added;

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

[0020] a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to (a3).

[0021] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology 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-mentioned proteins, 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 page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

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

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

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

[0026] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein VvERF105.

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

[0028] In the present invention, the regulation may be up-regulation, enhancement or increase; the regulation may also be down-regulation, attenuation or reduction.

[0029] Herein, the enhancement, increase or upregulation of the expression level of the gene encoding the protein mentioned above in the recipient plant, or / and the enhancement, increase or upregulation of the activity and / or content of the gene encoding the above protein is achieved by introducing the gene encoding the above protein into the recipient plant.

[0030] Herein, the regulation of the expression of the coding gene of the protein may be inhibition, reduction or down-regulation of the coding gene expression. Inhibition, reduction or down-regulation of the coding gene expression may be achieved by gene knockout or gene silencing.

[0031] In the present invention, the activity of the VvSWEET15 promoter can be regulated by regulating the activity and / or content of the protein VvERF105, and the glucose and fructose content in grape fruit can be regulated by interacting with the VvSWEET15 promoter.

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

[0033] c1) a nucleic acid molecule encoding 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) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism 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 molecule 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) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene 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) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism 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 molecule 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 application, 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) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described above;

[0051] d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0052] In the above application, the nucleic acid molecule described in e1) may be a DNA molecule whose nucleotide sequence is shown as SEQ ID No.3.

[0053] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule 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, including but not limited to: plasmids, phages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors. Specifically, the vector plasmids may be pET28a(+), pGreenII-0800-LUC, pGADT7, pGAD-Rec2, pHIS2, pHB, pHB-HA, pRI101.

[0055] In this paper, the Escherichia coli used for plasmid propagation and protein prokaryotic expression are Trans1-T1 (TransGen) and BL21 (TransGen); the Agrobacterium tumefaciens strains used for plant genetic transformation are GV3101 and GV3101(pSoup) (Zhuangmeng); and the yeast (Saccharomyces cerevisiae) single hybrid strain is Y187 (Zhuangmeng).

[0056] The existing plant expression vector can be used to construct a recombinant expression vector containing the VvERF105 gene. The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as rouge synthase Nos gene), plant genes (such as soybean storage protein genes) 3' end transcription of the non-translated region has similar functions.

[0057] When using the VvERF105 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0058] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

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

[0060] The step M is to enhance, increase or up-regulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or up-regulate the expression of the gene encoding the protein mentioned above, so as to reduce the sugar accumulation in the plant fruit;

[0061] The method comprises step P, wherein step P is to inhibit, reduce or silence the activity and / or content of the protein mentioned above in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein mentioned above, so as to increase the sugar accumulation in the plant fruit.

[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 technology to reduce or inactivate the activity of 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 or reducing or silencing the expression level of the gene encoding the protein mentioned above in the target plant, and / or the activity and / or content of the protein to obtain plants with increased fruit sugar accumulation, wherein the glucose and fructose content of the plants with increased fruit sugar accumulation is higher than that of the target plant.

[0064] In a specific embodiment, the inhibiting, reducing or silencing the expression of the gene encoding the protein mentioned above in the plant comprises introducing a substance, an expression cassette or a recombinant vector that inhibits the expression of the nucleic acid molecule mentioned above into the target plant to obtain a high fructose accumulating plant.

[0065] The present invention also provides a method for cultivating a plant with reduced fruit sugar accumulation, comprising inhibiting or reducing or silencing the expression level of the gene encoding the protein mentioned 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 a specific embodiment, the inhibition, reduction or silencing of the expression of the gene encoding the protein described above in the plant comprises introducing into the target plant the nucleic acid molecule, expression cassette or recombinant vector for inhibiting, reducing or silencing the protein described above, to obtain a plant with reduced fruit sugar accumulation.

[0067] Herein, the breeding purpose includes cultivating plants with increased fruit sugar accumulation; the breeding purpose also includes cultivating plants with reduced fruit sugar accumulation.

[0068] The plant with increased fruit sugar accumulation has increased glucose and fructose content compared with the target plant. The plant with reduced fruit sugar accumulation has reduced glucose and fructose content compared with the target plant.

[0069] Herein, the grape may be the grape variety Chardonnay.

[0070] Herein, the recombinant microorganism may be Agrobacterium tumefaciens strains GV3101 and GV3101 (pSoup).

[0071] The proteins and / or biological materials mentioned above also fall within the scope of protection claimed by the present invention.

[0072] In the above application or method, the plant may be any of the following:

[0073] N1) monocotyledonous or dicotyledonous plants;

[0074] N2) plants of the order Vitales;

[0075] N3) Plants of the grape family;

[0076] N4) Grapes;

[0077] N5) Grapes.

[0078] The present invention relates to the interaction between the VvERF105 transcription factor and the VvSWEET15 promoter, with the main purpose of reducing the accumulation of sugar in fruits. By studying the binding sites of VvERF105 and VvSWEET15 promoters, it is clear that the inhibitory effect on the expression of the VvSWEET15 gene is clear, thereby reducing the expression level of the sugar transporter VvSWEET15 and inhibiting the excessive accumulation of sugar in fruit cells. The present invention uses genetic engineering means to regulate the expression of VvERF105 or its binding efficiency, so as to achieve the purpose of reducing sugar accumulation in fruits and improving fruit quality, especially in the application of fruits such as grapes, which has a significant effect.

[0079] The advantages of the present invention compared with the prior art are:

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

[0081] 2) Genetic engineering is more feasible: Compared with other methods that rely on exogenous hormones or complex environmental regulation, the present invention regulates the expression of VvERF105 through genetic improvement or transgenic means, 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 the regulation of sugar content in grapes, but can also be applied to other fruits or crops. Through the interaction and regulation of corresponding ERF transcription factors and SWEET transporters, the management and optimization of sugar accumulation in different crops can be achieved;

[0083] 4) Improving fruit storage and transportation performance: By reducing sugar accumulation, the present invention can effectively extend the storage period of fruits, improve the shelf life during transportation, and solve the problem of easy deterioration of fruits caused by excessive sugar content. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 For one-hybrid library construction and screening.

[0085] Figure 2 Rapid identification of clones for PCR analysis and 2% agarose gel electrophoresis distinguished 269 true positive interactions 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 Screen candidate genes for yeast one-hybrid. AD-53m and pHIS2-S15 promoter co-transformation was used as a positive control, and empty vector AD-53m and empty vector pHIS2, empty vector AD vector and pHIS2-S15 promoter co-transformation were used as negative controls. The cloning point was placed in SD (-Leu-Trp) medium to ensure the reliability of the experimental technology. The amount of yeast growth was caused by the interaction, eliminating human experimental errors.

[0087] Figure 4 The interaction between VvERF105 and VvSWEET15 promoter was verified by yeast one-hybrid assay. The VvSWEET15 promoter sequence was amplified from Chardonnay fruit DNA, and AD-53m and pHIS2-S15 promoter co-transformation was used as a positive control, and AD-53m and empty pHIS2 co-transformation was used as a negative control.

[0088] Figure 5The experimental results of VvERF105 regulating the transcriptional activity of the VvSWEET15 promoter. A. Analysis of the construct structure of the reporter and effector; B. The dual fluorescence reporter system detects the LUC fluorescence of tobacco leaves infected with the reporter and effector. The left figure shows that VvERF105 has inhibitory activity. The experimental group is referred to as ProVvSWEET15:LUC+VvERF105, and the control group is referred to as ProVvSWEET15:LUC+Empty vector; C. Analysis of the relative activity of LUC combined with the reporter and effector. The reporter vector (abbreviated as ProVvSWEET15:LUC) and the effector empty vector (abbreviated as pHB-EV) were co-transfected into tobacco leaves as negative controls. The data are mean ± sd, with a total of 15 biological replicates. In all statistical graphs, * indicates significant differences between the experimental group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Significant differences were analyzed using Student's two-tailed test or Welch's t two-tailed test.

[0089] Figure 6 Results of testing potential binding elements of VvERF105 on the VvSWEET15 promoter. A, schematic diagram of the distribution of potential VvERF105 binding elements on the VvSWEET15 promoter. Purple columns represent the location of MYB binding elements on the promoter, and red columns represent the location of LTR on the promoter; B, EMSA test to identify whether VvERF105 binds to the DREcore element; C and D, EMSA test to identify whether VvERF105 binds to the MYB element; E, EMSA test to identify whether VvERF105 binds to the LTR element. The arrow points to the blocking band containing the probe and VvERF105. Highlighting of candidate elements: DREcore is marked with blue font, MYB is marked with purple font, and LTR is marked with red font. The hot probe is a portion of the VvSWEET15 promoter sequence labeled with biotin, while the cold probe is a portion of the VvSWEET15 promoter sequence that is not labeled with biotin. The core element in the cold probe is mutated as a mutation probe, and the mutated core element is highlighted in green. Mutation Probe is a mutation probe; Free Probe is a free probe.

[0090] Figure 7The results of the experiment 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. The red column represents the location of the LTR binding element on the promoter; B. EMSA test to identify whether VvERF105 binds to the LTR element. The arrow points to the blocking band containing the probe and VvERF105. The candidate elements are highlighted: LTR elements are marked with red fonts, NAC elements are marked with blue fonts, and mutant elements are marked with green fonts. The hot probe is a part of the VvSWEET15 promoter sequence labeled with biotin, while the cold probe, as a competitive probe, is a part of the VvSWEET15 promoter sequence that is not labeled with biotin. The core element in the cold probe is mutated as a mutant probe, and the mutated core element is highlighted in green fonts; C. ChIP-qPCR test. The cross-linked chromatin samples were from Kyoho fruits transiently overexpressing VvERF105-HA, and Kyoho fruits transiently overexpressing an empty vector with an HA tag were used as negative controls. Each transiently transformed fruit was used as an independent biological replicate, with a total of 3 biological replicates. Data are mean ± sd. In all statistical graphs, * indicates significant differences between the experimental group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Significant differences were tested using Student's t two-tailed test or Welch's t two-tailed test.

[0091] Figure 8To verify the experimental results of VvERF105 inhibiting sugar accumulation in grape fruit. A. Phenotypes of two different transgenic grape fruits. Randomly selected transient transformation positive fruits, VvERF105-OE, VvERF105-RNAi fruits have no obvious changes compared with the control. The white box indicates the location of the needle hole for injecting Agrobacterium bacteria solution; B. Relative expression and sugar content of VvERF105 and VvSWEET15 in VvERF105-OE transient transformation fruit. 6 biological replicates, data are mean ± sd; C. Relative expression and sugar content of VvERF105 and VvSWEET15 in VvERF105-RNAi transient transformation fruit. 6 biological replicates, data are mean ± sd; D. Protein immunoblotting to detect VvERF105 protein content at different developmental stages, with VvRubisco as the internal reference control. EV is the control for transformation of empty vector. The red numbers are ratios, representing the ratio of the grayscale value of the target protein band to the grayscale value of the control protein band (the control band ratio is set to 1.0). 10-15 fruits were randomly picked from a single bunch of grapes as an independent biological replicate, with a total of 3 replicates. The mean and standard deviation were calculated. In all statistical graphs, * indicates a significant difference between the experimental group and the control group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), and the significant difference was tested using Student's t two-tailed test or Welch's t two-tailed test. DETAILED DESCRIPTION

[0092] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0093] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0094] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0095] Grapes Kyoho (American grape V. labrusca x European population V. vinifera) and Kyoho in the following examples have been recorded 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. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0096] The Chardonnay in the following examples has been recorded in: D'Onofrio C, Scalabrelli G. Chardonnay [M] / / Italian Vitis Database (ISSN 2282-006X). 2015. The public can obtain the biological material from the applicant. The biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

[0097] The Jingxiangyu in the following examples has been recorded in: Fan Peige, Yang Meirong, Wang Lijun, Li Shengchen, Wu Benhong, Li Liansheng & Li Shaohua. (2009). New high-quality early-maturing grape variety 'Jingxiangyu'. Friends of Fruit Farmers (02), 13. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

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

[0099] The pHB carrier in the following embodiment has been recorded 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). The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0100] The tobacco (Nicotiana benthamiana) in the following examples has been described in: Clemente T. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana) [J]. Agrobacterium protocols, 2006: 143-154. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

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

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

[0103] I. Extraction of grape genomic DNA

[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 in a -40°C refrigerator for a long term.

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

[0106] Total RNA from grapes, various tissues and organs, and tobacco leaves was extracted using the HiPure HP Plant RNA Mini Kit (R4165-02, Magen). The specific method is described in the kit instructions.

[0107] III. First-strand cDNA synthesis

[0108] The HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-02) from Novazon Biotech was used to remove the residual genomic DNA in the RNA template and synthesize the first-strand cDNA at the same time. The specific method is described in the instructions of the kit.

[0109] IV. RT-PCR

[0110] The Novozymes Biotech kit HiScript II One Step RT-PCR Kit (Dye Plus) (P612-01) was selected, and gene-specific primers (GSP) were used. Reverse transcription and PCR reactions were completed in one tube, without the need for additional tube opening / pipetting operations, which 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] The prepared total RNA of grape, tomato tissues and organs and tobacco leaves were used as templates, and the gene-specific primers were primers synthesized from Actin1, VvSWEET15, VvERF105, and VvNAC72 gene-specific RT-PCR detection fragments, and then the instructions of the kit were referred to.

[0112] Ⅴ. Fluorescence quantitative PCR

[0113] We use the highly specific dye quantitative PCR detection kit from Novagen Biotech. qPCR SYBR Green Master Mix (Without ROX) (Q121-02), and then refer to the kit instructions.

[0114] Actin1 was used for grape, Actin7 for tomato, and Actin97 for tobacco as internal references for normalization of expression levels. The gene expression detection involved in this article was repeated more than 6 times, and at least 3 representative replicates were selected for final result display.

[0115] VI. The method for determining the sugar content of grape, tomato tissues and tobacco leaves is as follows:

[0116] High performance liquid chromatography (HPLC) was used to detect the sugar composition and content in the sample. Using a differential refractometer detector, it can be directly measured, with simple operation and reasonable sensitivity.

[0117] (1) Sample processing: Grind the sample into fine powder in liquid nitrogen, weigh 0.2-0.3g of the sample and place it in a 2mL centrifuge tube, add 1mL of ultrapure water to the centrifuge tube, mix the sample every 30 minutes at room temperature, let it stand for 2-3 hours to fully dissolve the sugar, centrifuge at 10,000g for 5 minutes, and carefully collect the supernatant into a new 2mL centrifuge tube. (Note: If necessary, the precipitate can be extracted repeatedly, and the above steps can be repeated several times to ensure that the sugar in the tissue is completely penetrated). Then filter it with a 0.22μm filter membrane into a sample vial. Use high performance liquid chromatography (see below) to determine the sugar content in mg·g -1 FW.

[0118] (2) Use high-purity standard samples of glucose, fructose, and sucrose to prepare a series of gradient dilutions of known concentrations. Use HPLC to determine the peak times of these three sugars and draw standard curves of glucose, fructose, and sucrose based on the peak areas for quantitative analysis.

[0119] (3) Determination of sugar content by high performance liquid chromatography: The sugar content of the sample to be tested is determined by a high performance liquid chromatography cycle equipped with an automatic sample injector, a column oven, a differential detector and a chromatography workstation.

[0120] The chromatographic conditions were as follows:

[0121] Chromatographic column: Sugar-PakTMII column filled by Water Company

[0122] Detector: Differential detector

[0123] Detector temperature: 35°C

[0124] Mobile phase: ultrapure water

[0125] Column temperature: 80°C

[0126] Flow rate: 0.6 mL min -1

[0127] Time: 15 minutes

[0128] Injection volume: 10 μL

[0129] VII. Electrophoretic mobility shift assay (EMSA)

[0130] The Thermo Scientific LightShift Chemiluminescent EMSA Kit uses a non-isotopic approach to detect DNA-protein interactions. Biotin end-labeled DNA containing the target binding site is incubated with nuclear extracts or purified factors. The reaction is then subjected to gel electrophoresis on a non-denaturing polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA is detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. For specific methods, refer to the kit instructions (Pierce, 89880).

[0131] VIII. Chromatin Immunoprecipitation (ChIP)

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

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

[0134] 1. Yeast one-hybrid screening library

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

[0136] Table 1. Information description of cis-acting elements in the promoter of VvSWEET15 gene

[0137]

[0138]

[0139]

[0140] The yeast single-hybridization bait vector was constructed by artificially tandemly synthesizing these elements (218 bp, nucleotide sequence SEQ ID No. 5) as bait, and a yeast single-hybridization library containing cDNA of Chardonnay, Kyoho, and Kyokoyu fruits at different developmental stages was constructed. The proteins that interact with the bait element vector were screened.

[0141] 1) Synthesize the bait sequence of yeast one-hybrid screening library

[0142] The important elements were repeated in tandem to synthesize the VvSWEET15 yeast single hybrid bait sequence as follows:

[0143] VvSWEET15 bait sequence (218 bp):

[0144]

[0145] 2) Bait vector self-activation detection

[0146] The minimum inhibitory concentration of 3AT (Coolaber, SL0930) to inhibit the self-activation background of bait production was determined by single transformation of the bait vector into the Y187 strain and plating on yeast defective medium (Coolaber, PM2281-10 x 0.5L, referred to 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 can produce 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, which is located in the promoter region of the vector reporter gene HIS3. The pHIS2 vector (Zhuangmeng, ZK971) is an empty vector containing only the HIS3 reporter gene. The above three vectors are provided by Shanghai Haike Biotechnology Co., Ltd. together with the constructed library. Among them, pGAD53m and p53HIS were co-transformed into the Y187 strain and grown in different 3AT concentration gradients (0m SD / -Trp-Leu-His yeast defective medium (Coolaber, PM2152-10*0.5L) containing 0mM, 20mM, 30mM, 40mM, 60mM, 100mM) was used as a positive control, while Y187 strain co-transformed with pGAD53m and pHIS2 and grown in SD / -Trp-Leu-His yeast defective medium (Coolaber, PM2152-10*0.5L) with different 3AT concentration gradients (0mM, 20mM, 30mM, 40mM, 60mM, 100mM) was used as a negative control (Table 2), and cultured at 30°C for one week. By comparing the growth difference with the negative control, the 3AT concentration that best inhibits the self-activation of the bait vector was found.

[0148] Table 2. Yeast self-activation verification

[0149] reaction AD Plasmid BD Plasmid Transformation Plate Detection Plate Test 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 hybrid screening library

[0151] See the yeast one-hybrid screening library process for Figure 1, the specific steps refer 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. The cDNA library of grape berries was created using SMART cDNA synthesis technology. A high-complexity pre-transformation cDNA library was constructed by recombination between the cDNA library and the linear prey vector pGADT7-Rec2. The bait vector and the prey vector were co-transformed into the Y187 strain. True positive interactions were detected in colonies that could grow on the minimal medium of SD / -His / -Leu / -Trp / +3AT. Reliable positive clones were screened out after 3-4 rounds of repeated restreaking ( Figure 2 ), sequencing, obtaining cDNA sequences of Chardonnay, Kyoho, and Jingxiangyu fruits at different developmental stages that interact with the VvSWEET15 bait sequence, and comparing with the grape database (http: / / plants.ensembl.org / Vitis_vinifera / Info / Index) to determine the proteins in the library that interact with the bait element. The selected genes were sequenced and annotated in Table 3 below.

[0152] Table 3. Selected gene sequencing and annotation information

[0153]

[0154]

[0155]

[0156]

[0157] Yeast one-hybrid screening of candidate genes, AD-53m and pHIS2-S15 promoter co-transformation was used as a positive control, AD-53m and empty vector pHIS2, empty vector AD and pHIS2-S15 promoter co-transformation was used as a negative control. The cloning point was placed in SD (-Leu-Trp) medium to ensure the reliability of the experimental technology, and the amount of yeast growth was caused by interaction, not by human experimental error.

[0158] The results are as follows Figure 3 As shown: 5 candidate genes interact with the VvSWEET15 promoter (2024 bp). Compared with the negative control and the control transformed with the empty prey vector-promoter vector, the positive clones containing 5 candidate genes (VvTSO1, VvDPP6, VvSPFH, VvCML, VvERF105) interacted more strongly than the control group under each 3AT selection pressure.

[0159] 2. Connect VvSWEET15 Promoter (2024 bp) to pHIS2 vector and use point-to-point yeast one-hybrid technology to verify whether VvSWEET15 promoter interacts with candidate target protein. Use 3AT as a competitive inhibitor of yeast HIS3 protein synthesis to inhibit leaky expression of HIS3 gene. The specific method is as follows:

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

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

[0162] The structure of the interaction protein vector pGADT7-Rec2-VvERF105 is described as follows: the restriction endonuclease Sma I single enzyme recognition site of the starting vector pGADT7-Rec2 is connected to the cDNA sequence of VvERF105, and the other sequences of the starting vector pGADT7-Rec2 are kept unchanged to obtain a recombinant vector.

[0163] b. Self-activation detection of pHIS2-VvSWEET15Promoter vector: The method is the same as the self-activation detection of the cis-trans element bait vector in step 2) above.

[0164] c. Prepare competent yeast according to the Clontech yeast strain competent preparation method (Cat. No. 630439).

[0165] d. Transformation: The transformation method was in accordance with the instructions of Clontech (Cat. No. 630439).

[0166] e. After transformation, suspend the cells in 200 μL sterile water for each transformation, mix gently as possible, and apply gradient dilutions to the SD / -Trp-Leu defective screening medium.

[0167] f. Culture at 30℃ for 3-4 days.

[0168] g. Refer to Clontech yeast colony PCR identification kit Advantage 2 PCR Polymerase Mix Set (Cat. No 639201) and yeast plasmid identification kit Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433) to identify true positive clones.

[0169] h. Use the pipette tip to pick up a small number of large and round positive yeast monoclonal colonies in the culture medium and culture overnight until OD 600 =0.6-0.7, take appropriate amount of culture medium to adjust bacterial solution OD 600 =0.6, then take an appropriate amount of bacterial solution for gradient dilution and spot plate on SD / -Trp-Leu, SD / -Trp-Leu-His defective screening medium.

[0170] i. Observe and take photos after 2-4 days.

[0171] Yeast point-to-point verification revealed that VvERF105 interacted significantly with the full-length promoter of VvSWEET15 (S15p, 2024 bp) ( Figure 4 ).

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

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

[0174] To analyze the potential VvSWEET15 promoter binding site of VvERF105, the fusion protein VvERF105-His was purified, and the S15p fragment containing the reported VvERF105 binding element / motif was labeled with biotin as a hot probe. The corresponding S15p fragment not labeled with biotin was used as a cold probe.

[0175] The steps for preparing the target protein are detailed in Section VII, Electrophoretic Mobility Assay (EMSA).

[0176] The analysis revealed that the VvSWEET15 promoter sequence contained two GCC motif-containing elements (MYB: CCGTTG and LTR: CCGAAA) in addition to the DREcore element (-71). The MYB recognition element contained two copies (-1371, -987) in the VvSWEET15 promoter ( Figure 6 However, EMSA experiments showed that VvERF105 did not bind to the MYB recognition element ( Figure 6 The LTR element also contains two copies (-858, -355) in the VvSWEET15 promoter ( Figure 7 A), the experiment showed that VvERF105 and the S15p fragment containing the LTR binding element bind to each other and a gel retardation band appears. With the addition of increasing concentrations of the competitive probe, the retardation band becomes weaker or even disappears. However, when the mutant probe is added, a gel retardation band appears, indicating that VvERF105 binds to the LTR element in vitro ( Figure 6 Middle E, Figure 7 (B).

[0177] To further verify the results of EMSA experiments, ChIP-qPCR experiments were performed.

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

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

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

[0181] The construct was transiently transformed into Kyoho fruit, and chromatin was extracted from the transiently transformed fruit for co-immunoprecipitation experiment (ChIP). Magnetic beads coupled with HA antibodies were used to enrich the VvERF105-HA DNA-bound complex. The enriched promoter fragments were obtained by decrosslinking and purification. Probes (p1, p2, p3, p4) were designed near the VvERF105 binding element, and qPCR detection was performed. Kyoho fruit transformed with the pHB-35S-HA construct was used as a control.

[0182] qPCR detection

[0183] qPCR detection was performed by designing multiple pairs of quantitative primers near the target element. The primer sequences were 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 Figure 7 As shown in C, VvERF105-HA was found to be associated with the VvSWEET15 promoter fragment containing the LTR element (red p2 and p3). This further confirmed 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] ProVvSWEET15:LUC was constructed as a reporter by connecting the VvSWEET15 promoter to the upstream of the LUC reporter gene, and 35S:VvERF105 was constructed as an effector using the 35S promoter. The reporter and effector were co-transfected into tobacco leaves.

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

[0187] The VvSWEET15 promoter was connected to the pGreenⅡ-0800-LUC vector (Shanghai Zeye Biotechnology, ZY8124) carrying the firefly luciferase gene and the Renilla luciferase gene, namely pGreenⅡ0800-VvSWEET15promoter-LUC.

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

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

[0190] VvERF105 was connected to a pHB vector without any reporter gene, namely pHB-35S-VvERF105 vector (abbreviated as 35S:VvERF105).

[0191] The structure of 35S:VvERF105 is described as follows: a small fragment between the restriction endonuclease BamHI recognition site and the XbaI enzyme recognition site of the starting vector pHB is replaced with the cDNA sequence of VvERF105 (nucleotide sequence is SEQ ID No. 2), and the other sequences of the starting vector pHB are kept unchanged to obtain a recombinant vector.

[0192] 3. Agrobacterium GV3101 (pSoup) transformation

[0193] After the recombinant vector pGreenⅡ0800-VvSWEET15promoter-LUC was transferred into Escherichia coli, the positive clones were selected and the positive clone plasmids were transferred into Agrobacterium GV3101 (pSoup) to obtain the recombinant Agrobacterium GV3101 / VvSWEET15promoter-LUC and stored in a -40°C refrigerator for future use.

[0194] 4. Tobacco instant transformation

[0195] Select 5-week-old tobacco leaves, and the process of instantaneous infection of leaves is as follows:

[0196] a. Agrobacterium culture

[0197] (1) Streak the Agrobacterium GV3101 / VvSWEET15 promoter-LUC containing the target transformant onto LB solid medium (50 mg / L Kana, 50 mg / L Rfp) and culture inverted at 28°C for 2 days.

[0198] (2) The positive clones of Agrobacterium tumefaciens GV3101 / VvSWEET15 promoter-LUC with activated target gene were selected and cultured in 1 mL LB liquid medium (50 mg / L Kana, 50 mg / L Rfp) at 28°C and 200 rpm overnight.

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

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

[0201] b. Vacuum injection of Agrobacterium GV3101 / VvSWEET15 promoter-LUC into tobacco leaves

[0202] (1) Mix the suspension and use a 1 mL syringe to draw up the infection solution. Inject from the back of the leaf and avoid the main vein until the leaf is clearly soaked. Incubate at room temperature in the dark for 1 day and under normal light for 2 days.

[0203] (2) 3 days after injection, the leaves were cut directly, the injection site was sprayed with the dual-luciferase substrate, and the fluorescence was observed using a chemiluminescence instrument.

[0204] 5. Luciferase Assay

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

[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 the LUC activity using a 20 / 20 luminometer (Promega). Take 10 μL of the dilution and add 50 μL of LARⅡ, pipette 2-3 times, and immediately put it into the luminometer to record the firefly luciferase LUC activity.

[0208] (4) After adding 60 μL Stop&Glo reagent, pipette and pump 2-3 times to mix well, and immediately return the sample to the instrument to record the sea cucumber luciferase REN activity.

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

[0210] The results show that ( Figure 5 Center AC): VvERF105 acts as a transcriptional repressor to downregulate LUC expression.

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

[0212] Using grape fruit cDNA as a template, the high-fidelity enzyme KOD-Plus-Neo was used to amplify the CDS sequence of the target gene. After purification and recovery by agarose gel electrophoresis, the target fragment was seamlessly connected to the target expression vector pHB and pRI101 (TaKaRa, Code No. 3262) using a homologous recombination kit. The constructed vector was verified by sequencing, and the construct bacterial solution was stored in a -80℃ refrigerator for a long time.

[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), and keeping the other sequences of the starting vector pHB unchanged.

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

[0216] The interference fragment of VvERF105 is 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 enzyme recognition site of the starting vector pRI101 is replaced with a fragment of SEQ ID No. 6, and the interference fragment of VvERF105 is reversely linked to the starting vector pRI101 to ensure that other sequences remain unchanged to obtain a recombinant vector.

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

[0221] In order to study the functions of VvSWEET15 and VvERF105 during grape berry ripening, transient transgenic experiments were carried out on grape variety "Kyoho" fruits for three consecutive years to overexpress or inhibit VvSWEET15 and VvERF105 in grape berries.

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

[0223] This experiment was carried out two weeks before the Kyoho grape fruits began 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 test process is as follows:

[0225] 1. Every year, we conduct flower and fruit thinning management on Kyoho grape varieties to ensure that the growth of the test fruits is basically the same;

[0226] 2. Pick out the activated and transformed Agrobacterium GV3101 containing EV-OE, VvERF105-OE, EV-RNAi, and VvERF105 RNAi vectors and culture them in 1 mL LB liquid medium (50 mg / L Kana, 50 mg / L Rfp) at 28 degrees and 200 rpm overnight;

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

[0228] 4. Add the above 5 mL bacterial solution to 100 mL LB liquid medium (50 mg / L Kana, 50 mg / L Rfp) at 28 degrees and 200 rpm until the OD 600 0.8-1;

[0229] 5. Centrifuge at 5000 rpm for 10 min, discard the supernatant, suspend the Agrobacterium cells with 80 mL suspension (10 mM MES-KOH, PH5.2; 10 mM MgCl2; 100 μM acetosyringone) and adjust the OD of the suspension. 600 =1, let stand at room temperature for 3h;

[0230] 6. Before using the bacterial solution, vortex or use a pipette to aspirate the suspended bacteria. Use a 1mL sterile syringe to take 1mL of the suspension and continuously inject the bacterial solution from the stalk end or the pedicel end of the grape. Insert the needle about 0.5cm and slowly inject the recombinant Agrobacterium suspension GV3101 into the pulp to ensure that the solution slowly penetrates into the pulp. Usually, a lot of suspension will leak out, so the injection process can be repeated 2-3 times to ensure that at least 0.1mL of suspension penetrates into the flesh (the fruit with successful injection is obviously harder than before injection); 3-4 bunches of grapes (about 80-100 fruits) were injected with each suspension containing EV-OE, VvERF105-OE, EV-RNAi, and VvERF105 RNAi vectors, and each fruit was used as an independent biological repeat; in order to explore the transformation efficiency of different transient transgenic experiments after Agrobacterium GV3101 infection for a few days, dynamic sampling was performed (i.e. OE / RNAi: fruits were taken 3, 5, 7, and 9 days after infection), and each sampling ensured that 8-15 fruits were repeated; in this experiment, 38-42 fruits (repeated) were finally sampled for overexpression, and 31-61 fruits were sampled in the RNAi experiment for the next step of 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 fruits, VvSWEET15 transcripts were downregulated 0.36-fold in VvERF105-OE, and the glucose and fructose contents decreased by 33% and 28.7%, respectively, but were upregulated 1.65-fold in VvERF-RNAi, and the glucose and fructose contents increased significantly by 19.1% and 18.8%, respectively.

[0232] Example 6: Extraction of total protein from grape berries and detection of VvERF105 protein content in overexpressed and RNAi berries

[0233] Total protein was extracted from the grape berries of Chardonnay at 32, 42, 52, 63, 72, 92, 106, 119, and 134 days after flowering, and the fruits of VvSWEET15, VvERF105, transient overexpression of Kyoho, and RNAi obtained in Example 2. The total protein extraction test method was completely carried out in accordance with the Plant Total Protein Extraction Kit (PE0230-1KT, Sigma-Aldrich). Proteins were detected by SDS-PAGE gel electrophoresis and immunoblotting.

[0234] Western blot

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

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

[0237] 3) Incubation 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 sealing incubation solution and rinse with TTBS solution 4 times for 5 min.

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

[0240] 6) Discard the sealing and incubation solution and rinse with TTBS solution 4 times for 5 min.

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

[0242] Western blotting experiments showed that ( Figure 8Middle D): Before maturity (72 days after flowering or earlier) the content of VvERF105 protein changed in the opposite direction to that after maturity (72 days after flowering). That is, when the content of VvERF105 protein was high during the developmental period, the content of VvSWEET15 protein was low. This also indicates that VvERF105 inhibits the expression of VvSWEET15, resulting in an overall low level of sugar accumulation in the fruit before maturity.

[0243] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating sugar accumulation in plant fruits; 2) Application in the preparation of products for regulating sugar accumulation in plant fruits; 3) Application in breeding plants with altered fruit sugar accumulation; 4) Use in the preparation of products for cultivating plants with altered fruit sugar accumulation; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No. 1; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 after one or more amino acid residues are replaced and / or deleted and / or added; a3) a protein having an amino acid sequence identity of more than 80% with that of a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from grapes.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); 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); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); 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); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); 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); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); 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).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown in any of the following: d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No.3; d2) the coding sequence is the DNA molecule shown in SEQ ID No.2; d3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2) and encoding the protein described in claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

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

6. A method for cultivating a plant with increased fruit sugar accumulation, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein of claim 1 or 2 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.

7. The method according to claim 6, characterized in that The method of inhibiting, reducing or silencing the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing into the target plant the nucleic acid molecule described in c1) of claim 4, the expression cassette described in c2) or the recombinant vector described in c3) to obtain a plant with high glucose and fructose content.

8. A method for cultivating a plant with reduced fruit sugar accumulation, characterized in that: The method comprises enhancing, increasing or up-regulating the expression level of the gene encoding the protein of claim 1 or 2 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 of the plant with reduced fruit sugar accumulation is lower than that of the target plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) monocotyledonous or dicotyledonous plants; N2) plants of the order Vitales; N3) Plants of the grape family; N4) Grapes; N5) Grapes.

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