Strawberry sweet transporter FvSWEET10 gene and application thereof

Abstract

The application provides a strawberry SWEET transporter protein FvSWEET10 gene and a protein encoded by the gene, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The application also provides a recombinant expression vector containing the gene, a host cell and application of the gene in regulating plant soluble sugar accumulation and fruit ripening process. The application explicitly defines FvSWEET10 the molecular mechanism of the gene in regulating plant sugar distribution and fruit ripening, fills the blank of the gene function research, can be used for high-sugar and early-ripening molecular breeding of horticultural crops such as strawberries and tomatoes, provides a new gene resource and a molecular target for quality improvement of fruit trees, and has a good breeding application prospect.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and fruit tree molecular breeding technology, and in particular to a strawberry SWEET transporter protein. FvSWEET10 Genes and their applications. Background Technology

[0002] strawberry( Fragaria × ananassa Strawberry (Fragariae Duch.) is a perennial herbaceous plant belonging to the genus Fragrance in the family Rosaceae. It is a small berry with high economic value and is widely cultivated globally. Strawberry fruits are rich in nutrients, including vitamin B1, vitamin C, pectin, protein, and organic acids. As a typical non-climacteric fruit, the quality of strawberry fruits is largely determined at harvest, and it is difficult to improve their flavor and nutritional value through post-harvest ripening. Therefore, elucidating the molecular mechanisms underlying the formation of fruit flavor, texture, and nutritional characteristics has always been a key research focus in horticulture and food science. Among these, the transport and regulation mechanisms of sugar metabolism in fruits are currently a core research hotspot for strawberry quality improvement.

[0003] Carbohydrates are the most widely distributed carbohydrates in nature and the most basic and economical energy source for life activities. In the growth and development of strawberries, soluble sugars play multiple key physiological roles: on the one hand, as energy substances and metabolic precursors, they provide material and energy support for fruit cell division, enlargement, and the synthesis of secondary metabolites such as anthocyanins, directly affecting fruit size, color, and nutrient accumulation levels; on the other hand, as signaling molecules, they can regulate the expression of genes encoding key enzymes in sugar metabolism such as sucrose phosphate synthase (SPS) and acid invertase (AI), thereby regulating the synthesis, transport, and accumulation of sugars. Simultaneously, they participate in regulating the plant's growth rhythm and physiological state through sugar signal transduction pathways. The main components of soluble sugars in strawberry fruits are glucose and fructose, while sucrose accumulates rapidly in the later stages of fruit ripening and plays a key signaling regulatory role. The synergistic effect of these three components runs through the entire process of fruit cell division, enlargement, color change, and ripening.

[0004] The SWEET family of sugar transporters are core functional proteins that mediate transmembrane sugar transport in plants and regulate carbon allocation between source and sink organs. This gene family has been extensively studied in various model plants and crops, and many sugar transporters have been identified and their functions validated. However, in strawberries, specific functional studies of SWEET family genes are still rare. Most family members remain at the bioinformatics prediction stage, and the molecular mechanisms by which they mediate sugar unloading into the fruit and regulate sugar accumulation in the fruit are unclear. Therefore, the resources of functional SWEET genes that can be directly applied to improve the high-sugar quality of strawberries are very limited, hindering the advancement of molecular breeding and quality improvement efforts in strawberries. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a strawberry sweet transporter protein. FvSWEET10 Genes and their applications.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A strawberry sweet transporter protein FvSWEET10 Genes, the ones mentioned FvSWEET10 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] A kind of the above FvSWEET10 The gene-encoded strawberry SWEET transporter protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A recombinant expression vector comprising the above FvSWEET10 Gene.

[0009] A host cell comprising the above FvSWEET10 Gene or recombinant expression vector.

[0010] One of the above FvSWEET10 Application of genes in regulating the accumulation of soluble sugars in plants and the fruit ripening process, wherein the soluble sugars include sucrose, glucose and / or fructose.

[0011] As one of the preferred embodiments of the present invention, the plant is a diploid strawberry 'Ruegen', which expresses transiently through the fruit. FvSWEET10 Genes promote the accumulation of soluble sugars in strawberry fruits and accelerate fruit ripening.

[0012] As one of the preferred embodiments of the present invention, the plant is Arabidopsis thaliana, which is obtained through stable genetic transformation. FvSWEET10 Genes enhance Arabidopsis' tolerance to high concentrations of sucrose and regulate the plant's sugar response.

[0013] As one of the preferred embodiments of the present invention, the plant is a tomato, which is subjected to stable genetic transformation. FvSWEET10 The gene promotes the transport of sucrose out of the leaves, increases the sucrose, glucose, and fructose content of tomato fruits, and advances fruit ripening.

[0014] As one of the preferred embodiments of the present invention, the FvSWEET10 The gene-encoded transporter FvSWEET10 forms a dimer with the FvSWEET1 transporter through heterologous interaction or homologous interaction, and synergistically regulates the transport and accumulation of soluble sugars in plants.

[0015] The advantages of this invention compared to the prior art are: This invention clones the strawberry sweet transporter protein. FvSWEET10This gene fills a gap in research on the function of members of the SWEET family in strawberries. The study confirmed this through transient infection experiments on strawberry fruits. FvSWEET10 Genes showed a significant positive correlation with sucrose accumulation in strawberry fruits: silencing and inhibiting this gene in green strawberry fruits. FvSWEET10 After gene expression, the sucrose content of the fruit decreases significantly; transient overexpression of the gene can significantly promote sucrose accumulation in strawberry fruit and accelerate the fruit ripening process.

[0016] This invention also utilizes a stable Arabidopsis thaliana transformation system to verify heterologous function. Compared to wild-type Arabidopsis thaliana, overexpression... FvSWEET10 The transgenic lines of the gene exhibit greater tolerance to high-concentration sucrose environments. Furthermore, through sucrose and hexose (glucose, fructose) deficient yeast complementation experiments, dual-luciferase complementation experiments, and two-hybrid multiplex systems using ubiquitin-based yeast, it was confirmed that the FvSWEET10 protein alone does not possess the ability to transport sugars such as sucrose, glucose, and fructose. Its function depends on protein-protein interactions: FvSWEET10 can heterologously interact with FvSWEET1, or homologously interact with itself, forming heterodimers and homodimer complexes, respectively, thereby synergistically regulating the transport and accumulation of soluble sugars in plants.

[0017] Existing studies mostly believe that the SWEET protein directly transports sugars, while this invention reveals for the first time a novel regulatory pathway in which FvSWEET10 indirectly and positively regulates sucrose accumulation in fruits through protein dimerization, with a novel mechanism. Moreover, this invention simultaneously verified the function of this gene in multiple species such as strawberry, Arabidopsis thaliana, and tomato, which can be used to improve the sugar content and ripening characteristics of strawberry fruits, and can also be extended to improve the sugar quality of other horticultural crops, with a wide range of breeding applications, providing new gene resources and theoretical basis for molecular breeding of high-sugar and early-maturing fruit trees. Attached Figure Description

[0018] Figure 1 In Example 1 FvSWEET10 Figure 1 shows the gene expression patterns in strawberry (Figure a shows different organs: root, petiole, functional leaf, flower; Figure b shows floral organs: sepals, petals, pistil, stamen; Figure c shows the expression patterns of different organs in strawberry). FvSWEET10 Gene expression levels; Figure d shows the expression levels in floral organs. FvSWEET10 Gene expression levels; Figure e shows the green, white, color-changing, and mature fruits of the diploid strawberry 'Ruegen'; Figure f shows the green, white, color-changing, and mature fruits of the octoploid strawberry; Figure g shows the gene expression levels in the fruits of the diploid strawberry at different stages. FvSWEET10 Gene expression levels; Figure h shows the expression levels of octoploid strawberry fruits at different stages. FvSWEET10 Gene expression levels; where G: green fruit, W: white fruit, T: color-changing fruit, R: mature fruit). Figure 2 In Example 1 FvSWEET10Observation diagram of in situ hybridization (in the figure, a~d are sections of root, functional leaf, flower and green fruit stages respectively; scale bar = 1cm); Figure 3 In Example 2 FvSWEET10 Identification results of transgenic Arabidopsis thaliana (Figure a is...) FvSWEET10 PCR positive identification of transgenic Arabidopsis thaliana; Figure b shows the transgenic Arabidopsis thaliana... FvSWEET10 Transcription levels; where M is a 2000bp DNA marker; H2O is a negative control; plasmid is a positive control; WT is a negative control, wild type; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001). Figure 4 The different concentrations of exogenous sugar treatment in Example 2 have an effect on... FvSWEET10 The effects of heterologous overexpression on the growth of Arabidopsis seedlings; Figure 5 The effects of different sugar concentrations on treatment in Example 2 FvSWEET10 Effects of heterologous expression of Arabidopsis thaliana on root length and fresh weight of seedlings (Figure a shows root length of seedlings under different sucrose concentrations; figure b shows root length of seedlings under different glucose concentrations; figure c shows root length of seedlings under different fructose concentrations; figure d shows fresh weight of seedlings under different sucrose concentrations; figure e shows fresh weight of seedlings under different glucose concentrations; figure f shows fresh weight of seedlings under different fructose concentrations; where WT represents wild-type Arabidopsis thaliana, and OE-1, OE-2, and OE-3 represent wild-type Arabidopsis thaliana). FvSWEET10 Overexpression homozygous lines; * indicates significance testing compared to WT at the same concentration; significance p<0.05 is *, p<0.01 is **, p<0.001 is ***). Figure 6 In Example 3 FvSWEET10 Phenotypic and sugar content analysis of transgenic tomatoes with overexpression (Figure a shows...) FvSWEET10 Phenotypic diagram of genetically modified tomato fruit; Figure b is... FvSWEET10 Determination of sugar content in transgenic tomato leaves; Figure c shows... FvSWEET10 Monosaccharide content of mature transgenic tomato fruit; where * indicates significance comparison with WT: p<0.05 is *, p<0.01 is **, p<0.001 is ***). Figure 7 It is in Example 4 FvSWEET10 Effects of transient overexpression on strawberry fruit ripening, sugar content, and expression of sugar metabolism-related genes (Figure a shows transient overexpression). FvSWEET10 Phenotypic diagram of 'Ruegen' fruit; Figure b is... FvSWEET10Gene expression levels; Figure c shows the sucrose, glucose, and fructose content of the injected fruit; Figure d shows the expression levels of sugar metabolism-related genes in the injected fruit; where d represents the number of days; SUC represents sucrose; GLU represents glucose; FRU represents fructose; * represents significance tests with WT: p<0.05 is *, p<0.01 is **, p<0.001 is ***). Figure 8 It is in Example 5 FvSWEET10 Effects of transient silencing on strawberry fruit ripening, sugar content, and expression of sugar metabolism-related genes (Figure a shows VIGS silencing). FvSWEET10 Phenotypes of treated and unloaded WT 'Ruegen' fruits; Figure b shows the endogenous phenotypes of fruits in each group detected by qRT-PCR. FvSWEET10 Figure c shows the transcription level; Figure d shows the sucrose, glucose, and fructose content of the fruit; Figure 3 shows the significance of the expression levels of sugar metabolism-related genes in the fruit; where WT is the pTRV empty vector control group, and Anti-1, Anti-2, and Anti-4 are three independent groups. FvSWEET10 The silent treatment group; * represents the significance test with WT: p<0.05 is *, p<0.01 is **, p<0.001 is ***). Figure 9 This is a functional verification of the FvSWEET10 protein in the sucrose-deficient yeast SUSY7 / ura3, as described in Example 6. Figure 10 This is the functional verification of the FvSWEET10 protein in hexose-deficient yeast EBY.vw4000 in Example 6 (in the figure, maltose is Maltose; galactose is Galactose; mannitol is Mannitol; sorbitol is Sorbitol; fructose is Fructose; glucose is Glucose). Figure 11 The results of LCI verification of the interaction between FvSWEETs proteins in Example 6 are shown in Figure 6 (Figure a is...). FvSWEET10 -nLUC and respectively with FvSWEET1 -cLUC、 FvSWEET4 -cLUC、 FvSWEET10 -cLUC、 FvSWEET17 Figure b shows the four interaction combinations of cLUC and nLUC, which were co-injected into tobacco leaves to detect fluorescence signals; Figure b shows the empty control cLUC+nLUC and the single-vector negative control. FvSWEET10 -nLUC+cLUC、nLUC+ FvSWEET1 -cLUC and other blank treatment group fluorescence signals; Figure c is FvSWEET10 -nLUC and FvSWEET10 -cLUC homology interaction combined fluorescence signal; Figure d shows FvSWEET10-nLUC and FvSWEET4 -cLUC co-injection combined fluorescence signal; Figure e shows FvSWEET10 -nLUC and FvSWEET17 -cLUC co-injection of combined fluorescence signals); Figure 12 The results of FvSWEET protein interaction verification in the yeast two-hybrid system of the ubiquitin membrane in Example 6 are shown in the figure (SW10-pBT3-N+pPR3-N is the negative control, and pTSU2-APP+pNubG-Fe65 is the positive control). Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0020] In the following embodiments: The diploid strawberry 'Ruegen' is a publicly available scientific research model strawberry germplasm that can be obtained from strawberry germplasm resource banks both domestically and internationally. The octoploid strawberry variety 'Xiangye' is a common commercial strawberry variety on the market and can be purchased directly. Yeast strains SUSY7 / ura3, EBY.vw4000, and NMY51, and vectors PK7WG2D, pTRV, pBT3-N, pBT3-SUC, pBT3-STE, pBT3-C, pCAMBIA-nLUC, pCAMBIA-cLUC, PNEVE, pYES2, and Agrobacterium GV3101-p19 are all commercially available or commonly used laboratory tools and vectors in this field, and can be obtained from biological reagent companies and research platforms.

[0021] In addition, unless otherwise specified, all other reagents and materials used in this invention are commercially available conventional biochemical reagents; the methods used, such as molecular cloning, quantitative real-time PCR, yeast transformation, Agrobacterium infection, transient transformation, and transgenic cultivation, are all conventional experimental methods in the field.

[0022] Example 1 FvSWEET10 Gene expression pattern analysis: FvSWEET10 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the strawberry SWEET transporter it encodes is shown in SEQ ID NO.2. To clarify the spatiotemporal expression characteristics and tissue localization of this target gene, this embodiment conducts expression pattern analysis at the transcriptional and cellular levels, providing a basis for subsequent functional studies.

[0023] 1. FvSWEET10 Spatiotemporal expression in strawberries To investigate FvSWEET10 The expression pattern of the gene in diploid strawberry 'Ruegen' was investigated, and the transcriptional level of this gene in different tissues and organs (roots, petioles, leaves, and flowers) and at four stages of fruit development (green fruit, white fruit, color change, and ripening) was detected using qRT-PCR. The results showed that... FvSWEET10 The expression level is relatively high in floral organs, with higher levels in stamens than in pistils; followed by leaves, while expression is lower in roots and petioles. Figure 1 a~d). This organizational expression characteristic suggests FvSWEET10 Genes may be involved in the development of floral organs and the loading and transport of photosynthetic products in leaves.

[0024] During the fruit development process, FvSWEET10 The expression level was highest during the green fruit stage, then gradually decreased as the fruit matured, reaching its lowest point during the overripe stage. Fruit samples from diploid 'Ruegen' and octoploid 'Xiangye' at various developmental stages are shown below. Figure 1 e and f, corresponding to the quantitative results of fruit gene quantification, see [see details]. Figure 1 g, h. It is noteworthy that this expression trend is consistent in both the diploid 'Ruegen' and the octoploid strawberry variety 'Xiangye' ( Figure 1 g, h), indicating FvSWEET10 Its function in the early stages of fruit development is conserved, suggesting that it may be involved in the unloading and accumulation of photosynthetic products during the early stages of fruit development.

[0025] 2. FvSWEET10 Tissue in situ hybridization analysis To further determine FvSWEET10 In situ hybridization analysis was performed on roots, functional leaves, flowers, and green fruit stages of the diploid 'Ruegen' to investigate gene expression in various strawberry tissues. The results showed that the positive signal of the target gene was specifically enriched in pollen grains of the pistils and stamens of the flower, while only a very faint background was observed in vegetative tissues such as the receptacle and perianth. Figure 2 c); during the green fruit stage ( Figure 2 High expression was observed in radially distributed vascular bundles (conducting tissues) in d), followed by weak positive signals in achenes (seeds) embedded in the fruit surface. Only extremely low background levels were detected in parenchyma cells (receptacle / fruit pulp tissue), with no significant expression enrichment. In leaves and roots, expression was mainly concentrated in mesophyll cells and vascular bundles. Figure 2 a, b).

[0026] Based on these results, it is inferred that this gene is involved in two core reproductive processes in strawberries: pistil development and pollen maturation, pollination, and fertilization. It is an important functional gene involved in the regulation of strawberry reproductive development, achene development, and early fruit setting.

[0027] Example 2 FvSWEET10Heterogeneous expression function verification: 1. FvSWEET10 Obtaining and molecularly identifying transgenic Arabidopsis thaliana lines To clarify FvSWEET10 The effect of gene overexpression on sugar accumulation in plants was investigated by constructing the PK7WG2D- gene. FvSWEET10 After introducing the recombinant plasmid into Agrobacterium tumefaciens strain GV3101 using an overexpression vector, wild-type Arabidopsis thaliana plants were genetically transformed using the Agrobacterium-mediated flower immersion method, successfully obtaining three independent transgenic Arabidopsis thaliana lines. To verify that these three independent lines were all positive transgenic plants, molecular identification was performed at both the genome integration and transcriptional expression levels: (1) Genomic PCR detection: Leaf DNA was extracted from wild-type Arabidopsis and T3 generation overexpressing transgenic Arabidopsis, respectively. PCR amplification was performed on the DNA samples using specific primers designed for the vector sequence. The results showed that a specific band of approximately 800 bp was amplified in the three candidate Arabidopsis lines, consistent with the positive plasmid control. No corresponding amplification products were detected in the wild-type and blank control (H2O), preliminarily confirming that the exogenous gene had been integrated into the Arabidopsis genome. Figure 3 a).

[0028] (2) Transcriptional expression analysis: In order to further detect FvSWEET10 To determine the transcriptional expression level in heterologous Arabidopsis thaliana, RNA was extracted from leaves of wild-type and transgenic plants, and the gene expression was detected by real-time quantitative PCR after reverse transcription. FvSWEET10 The relative expression levels. The results showed ( Figure 3 b) Compared to wild-type Arabidopsis thaliana FvSWEET10 Transcriptional levels were significantly upregulated in the three overexpression lines OE-1, OE-2, and OE-3, reaching 8696-, 6648-, and 1622-fold increases compared to wild-type Arabidopsis, respectively, further confirming... FvSWEET10 It has been transferred to Arabidopsis thaliana.

[0029] The above identification results collectively confirm that the three obtained FvSWEET10 Positive transgenic materials with stable overexpression can be used for subsequent sugar content determination and functional verification experiments.

[0030] 2. FvSWEET10 Response of transgenic Arabidopsis thaliana to exogenous sugars To clarify FvSWEET10To investigate the overexpression of Arabidopsis thaliana's response to exogenous sugars, seeds of wild-type (WT) and three homozygous overexpression lines (OE-1, OE-2, and OE-3) were inoculated into MS solid medium containing different concentrations (0.1%, 0.5%, 1%, 3%, and 6%) of sucrose, glucose, and fructose. After vernalization at 4°C in the dark for 2 days, the plants were transferred to a light incubator and cultured continuously for 13 days. Leaf growth and root elongation were observed, and root length and weight were measured.

[0031] The results are as follows Figure 4 , Figure 5 As shown. The results show: With increasing glucose (GLU) concentration, root length in both the wild-type and the three overexpression lines showed a trend of first increasing and then decreasing, reaching a maximum at a 3% concentration. However, a high concentration of 6% severely inhibited the germination of all seeds. At a 0.1% glucose concentration, FvSWEET10 The root length of the overexpression lines was 30% shorter and the fresh weight was reduced by about 30% compared with the wild type. At the 0.5% and 1% concentrations, there was no significant difference in root length and fresh weight between the overexpression lines and the wild type. However, at the 3% concentration, the root length of the overexpression lines was slightly shorter than that of the wild type, and the difference was statistically significant.

[0032] Compared to the wild type, FvSWEET10 Overexpression lines showed no significant response to fructose (FRU) treatment. The effect of fructose on root length and fresh weight was concentration-dependent: at low concentrations (0.1%–1%), the growth and fresh weight of overexpression lines were slightly higher than those of the wild type, but the difference was not statistically significant; when the fructose concentration increased to 3%–6%, root length and fresh weight were inhibited in all lines; among them, a high concentration of 6% fructose completely inhibited the germination of all seeds.

[0033] FvSWEET10 The overexpression lines showed a significant concentration-dependent response to sucrose (SUC). At low concentrations (0.1%–1%), there were no significant differences in root length and fresh weight between wild-type and overexpression lines. As the sucrose concentration increased to 3%, the root length of the overexpression lines was approximately 1.2 times that of the wild-type, and the fresh weight increased slightly. However, under a high sucrose concentration of 6%, the growth of the wild-type was significantly inhibited, while the overexpression lines maintained strong growth vigor. The root length of the three overexpression lines increased by 150%, 161%, and 146% compared to the wild-type, and the fresh weight increased by 41%, 37%, and 34%, respectively. The results indicate that… FvSWEET10 Overexpression of [a substance] enhances Arabidopsis thaliana's tolerance to high concentrations of sucrose.

[0034] The above results show that FvSWEET10 Overexpression of this gene enhances Arabidopsis thaliana's adaptation to high concentrations of sucrose, suggesting that the gene may play a positive role in sugar stress response and carbon allocation regulation.

[0035] Example 3, Overexpression FvSWEET10 Effects on sugar accumulation in tomato plants: This embodiment verifies heterologous stable overexpression in tomatoes and analyzes its regulatory effect on source-sink sugar allocation and fruit ripening.

[0036] To clarify FvSWEET10 Effects of heterologous stable expression on tomato sugar metabolism and fruit ripening process, using wild-type (WT) and three independent strains. FvSWEET10 Using overexpression tomato lines as materials, the changes in sugar content in leaves and fruits were systematically analyzed, and phenotypic observations were conducted in conjunction with fruit development. The results showed that the fruits of all three lines matured earlier than the wild type. The wild type took approximately 40 days from flowering and fruit set to color change, while the overexpression lines entered the color-changing stage in about 35 days, indicating a significantly earlier maturation process. Figure 6 a).

[0037] To elucidate the molecular mechanism of its early maturation, the content of monosaccharides (sucrose, glucose, and fructose) in the leaves was measured. It was found that compared to the wild type, the content of monosaccharides in the leaves was significantly higher. FvSWEET10 In overexpression strains, the sucrose content in leaves showed a decreasing trend, while the glucose and fructose content showed an increasing trend. Figure 6 b). This indicates that, FvSWEET10 Heterologous expression of sucrose may promote the export or degradation of sucrose in leaves, thereby reducing its steady-state accumulation in the source leaves.

[0038] In addition, the contents of sucrose, glucose, and fructose in mature fruits were determined. The sucrose content in fruits from overexpressing lines at maturity was significantly higher than that of the wild type, indicating that FvSWEET10 can increase the contents of sucrose, glucose, and fructose in tomatoes. Figure 6 c). Based on the result of decreased sucrose content in leaves and increased sucrose content in fruits, it is speculated that... FvSWEET10 It can transport sugars from the leaves to the fruit to play a role.

[0039] Example 4: Transient Overexpression FvSWEET10 Promotes the accumulation of sucrose in strawberry fruits: For direct parsing FvSWEET10 To investigate the function of the target species strawberry, this embodiment uses transient overexpression technology in fruit to verify the function from multiple levels, including phenotype, sugar content, and gene expression.

[0040] First, functional analysis was performed using transient overexpression techniques. FvSWEET10The recombinant plasmid PK7WG2D-GFP and the empty vector control were transformed into Agrobacterium GV3101-p19, and injected into 'Ruegen' 'green fruit stage' fruits using a transient injection method. The empty vector PK7WG2D served as a control, and the fruit development process was observed. The results showed that, compared with the control group injected with the empty vector, transient overexpression significantly increased PK7WG2D-GFP levels. FvSWEET10 The fruit changed color 2 days earlier, showing a trend of accelerated ripening. Figure 7 a) and this phenotype remained stable and consistent across three biological replicates. Based on the expression pattern described above, FvSWEET10 Gene expression levels gradually decrease as the fruit matures, but transient overexpression promotes fruit ripening. It is speculated that this may be due to the interaction between FvSWEET10 and FvSWEET1 proteins, which jointly regulate fruit ripening.

[0041] qRT-PCR test results ( Figure 7 b) Shows that in the overexpression group FvSWEET10 The transcriptional level was significantly upregulated, and the expression of genes related to glucose metabolism and transport also changed significantly. Figure 7 d): Sucrose transporter FvSWEET1 and vacuole acid invertase gene FvNINV The relative expression level of [the gene] was also significantly upregulated; while the expression level of sucrose synthase gene [was also] significantly upregulated. FvSUS1 and sucrose phosphate synthase gene FvSPS2 The expression of was significantly suppressed.

[0042] The sugar content of mature strawberry fruits was determined, and the results were as follows ( Figure 7 As shown in c), FvSWEET10 Transient overexpression significantly affected sugar accumulation in the fruit. Compared with the control group, the sucrose content in the overexpressing fruit increased sharply, reaching 3 to 4 times that of the control group; the fructose content decreased by 40 to 50%; while the glucose content did not change significantly.

[0043] In conclusion, FvSWEET10 Transient overexpression is achieved by upregulating FvSWEET1 and FvNINV The expression of this promotes the accumulation of sucrose in the fruit. And... FvSUS1 and FvSPS2 The downregulation of [a certain substance] may be a feedback regulation mechanism to maintain sugar homeostasis within the fruit. FvSWEET10 Overexpression also showed an effect of accelerating fruit color change, suggesting that it may participate in the initiation regulation of strawberry fruit ripening by affecting sugar signaling. Combined with the detection results of Example 1, during natural fruit development... FvSWEET10 Expression levels gradually decrease as the fruit matures; however, transient overexpression of exogenous substances can significantly increase the expression levels within the fruit. FvSWEET10 Transcriptional abundance alters the soluble sugar content of fruits and accelerates ripening.

[0044] Example 5: Momentary Silence FvSWEET10 Inhibits the accumulation of sucrose in strawberry fruits: For further analysis FvSWEET10 This embodiment investigates the molecular mechanisms regulating sugar accumulation in fruit. Using a virus-mediated gene silencing system, it examines the changes in sugar accumulation and ripening phenotype in strawberry fruit after the suppression of endogenous gene expression, which corroborates the results of overexpression.

[0045] pTRV1 and FvSWEET10 The recombinant plasmid pTRV2 was transformed into Agrobacterium GV3101-p19, with pTRV1+pTRV2 empty vector as a control. Endogenous endogenous silencing was detected by injection into strawberry fruits using a transient silencing system. FvSWEET10 The effect of silencing on sugar accumulation was investigated. Fruit development after injection was observed, and it was found that fruit color change was delayed compared to the wild type. Figure 8 a). Meanwhile, the qRT-PCR test results showed ( Figure 8 b) The Silent Group FvSWEET10 The transcriptional level was significantly downregulated, indicating that the silencing system was effectively established. Glycogram analysis showed that... FvSWEET10 Transient silencing led to a significant decrease in sucrose content in the fruit, decreasing by approximately 30%, 42%, and 46%, respectively; a significant increase in fructose content; and no significant change in glucose content. Figure 8 c).

[0046] Further examination of changes in related sugar metabolism genes showed that ( Figure 8 d), FvSWEET1 The relative expression level also decreased significantly; while the sucrose synthase gene FvSUS1 and sucrose phosphate synthase gene FvSPS2 The expression of [something] increased, a result consistent with transient overexpression injection. FvSWEET10 The result was the opposite.

[0047] The above results indicate that FvSWEET10 It may participate in maintaining the sugar balance of strawberry fruit by regulating sucrose accumulation or transport. The results obtained from the silencing treatment in this example are opposite to the experimental phenotype of transient overexpression in Example 4; the two sets of experiments corroborate each other. FvSWEET10 The regulatory role of genes in the accumulation of soluble sugars and the ripening process of strawberry fruits.

[0048] Example 6 FvSWEET10 Validation of yeast functional complementarity and analysis of sugar transport and protein interaction To reveal FvSWEET10 To investigate the molecular mechanisms regulating sugar accumulation, this embodiment verifies the protein's own transport activity through a yeast defect complementation experiment and explores its protein interaction mode through two sets of interaction experimental systems.

[0049] 1. Validation of FvSWEET10 sucrose transport function To verify the sugar transport function of the FvSWEET10 protein, the sucrose-deficient yeast strain SUSY7 / ura3 was used to verify whether the protein transports sucrose. This yeast strain lacks a sucrose uptake mechanism and can only grow on a medium with glucose as the carbon source. FvSWEET10 Construct the PNEVE vector, and FvSWEET10 - The PNEVE plasmid was transformed into SUSY7 / ura3. Our lab had previously verified that the FvSWEET1 protein has the ability to transport sucrose, therefore... FvSWEET1 -PNEVE was used as a positive control, and the empty vector plasmid PNEVE was used as a negative control. Results are as follows: Figure 9 Compared with the empty vector PNEVE, the positive control FvSWEET1 -PNEVE can grow normally on a medium where sucrose is the sole carbon source, while FvSWEET10 -PNEVE did not grow, indicating that the FvSWEET10 protein alone does not have the ability to directly transport sucrose in defective yeast, and further verification is needed to determine whether it transports hexose.

[0050] 2. Verification of FvSWEET10 hexose transport function Because FvSWEET10 could not grow in the sucrose-deficient yeast strain SUSY7 / ura3, the hexose-deficient strain EBY.vw4000 was used to verify the hexose transport ability of FvSWEET10, and a model was constructed. FvSWEET10 -PNEVE and FvSWEET10 The PYES2 plasmid was transformed into EBY.vw4000. This strain could not grow normally on SC medium (uracil-deficient) with hexose as the sole carbon source, but could only grow on medium with maltose as the carbon source. Results are as follows... Figure 10 This indicates that FvSWEET10 cannot transport hexoses such as glucose, fructose, and sorbitol. 3. FvSWEET10 Glycotransporter Interaction Since the FvSWEET10 protein alone does not function to transport sucrose and hexose, it is speculated that this protein may interact with other SWEET transporters to form a dimer transport sugar. To clarify the interaction relationships between the FvSWEET10 protein and its family members, this study selected the target gene of this invention. FvSWEET10 And the genes of three known families FvSWEET1 , FvSWEET4 , FvSWEET17 (The sequences of the three individuals can be obtained from the NCBI Public Genome Database and the Strawberry Germplasm Resource Bank), totaling four. FvSWEETThe potential interactions among the four FvSWEET proteins were verified using the luciferase complementation assay (LCI) and yeast double hybridization technique with a split ubiquitin membrane system.

[0051] (1) Luciferase complementation assay (LCI) to verify interactions between SWEET proteins The pCAMBIA-nLUC and pCAMBIA-cLUC vectors were used to construct [the following structures] using restriction endonucleases Kpn I and Sal I, respectively. FvSWEET1 / 4 / 10 / 17 -pCAMBIA-nLUC fusion expression vector and corresponding FvSWEET1 / 4 / 10 / 17 -pCAMBIA-cLUC fusion expression vector.

[0052] The recombinant plasmid empty vector was transformed into Agrobacterium GV3101-p19 via electroporation. After overnight culture at 28°C, the bacterial cells were collected by centrifugation and resuspended in infection solution to an OD600 of 1.0–1.2. The bacterial solution was then co-injected into tobacco leaves in the following ratio: cLUC + nLUC. FvSWEET10 -nLUC+cLUC、 FvSWEET10 -nLUC+ FvSWEET1 -cLUC、 FvSWEET10 -nLUC+ FvSWEET4 -cLUC、 FvSWEET10 -nLUC+ FvSWEET10 -cLUC、 FvSWEET10 -nLUC+ FvSWEET17 -cLUC、 nLUC+ FvSWEET1 -cLUC、nLUC+ FvSWEET4 -cLUC、nLUC+ FvSWEET10 -cLUC、nLUC+ FvSWEET17 -cLUC, etc., were co-injected into tobacco leaves, which were then cultured in darkness for 1 day, followed by 2 days of light culture. The luminescence signal of firefly luciferase was detected using a plant in vivo imaging system.

[0053] The results are as follows Figure 11 As shown, no significant signal response was observed in the co-injection region containing the empty vector; however, in the combination of FvSWEET10 co-injected with each candidate protein, only... FvSWEET10 -nLUC+ FvSWEET1 -cLUC and FvSWEET10- nLUC+ FvSWEET10 The two combinations of -cLUC detected fluorescence signals, among which FvSWEET10 and FvSWEET1 The combined fluorescence signal is particularly strong, while FvSWEET10 and FvSWEET4 , FvSWEET17No fluorescent signal was observed in the co-injection region, indicating that FvSWEET10 protein can form a dimer with FvSWEET1 protein, or it can form a homodimer on its own. This suggests that FvSWEET10 protein may exert its function by forming a homodimer or a heterodimer with FvSWEET1 protein.

[0054] (2) Yeast two-hybrid verification of SWEET protein-protein interactions in the ubiquitin membrane system. The split-ubiquitin system was used to detect interactions between membrane proteins. This system employed four bait plasmids: pBT3-N, pBT3-SUC, pBT3-STE, and pBT3-C, selected using LEU as the selection marker; and two prey plasmids, pPR3-N and pPR3-C, selected using TRP as the selection marker. Because the membrane localization signals of different Bait plasmids differ, the appropriate vector needed to be selected based on the specific membrane localization of the bait protein. This selection was confirmed through bioinformatics analysis and preliminary experiments. FvSWEET10 Genes were adapted to the pBT3-STE vector, and construction was carried out accordingly. FvSWEET10 -pBT3-STE recombinant decoy plasmid; simultaneously, FvSWEET1 , FvSWEET4 , FvSWEET10 , FvSWEET17 Four genes were cloned into the pPR3-NPrey vector to construct the corresponding recombinant prey plasmids. After transformation into E. coli, the plasmids were sent to the company for sequencing. After sequencing confirmed the correct construction of the recombinant plasmids, high-purity plasmids were extracted for subsequent yeast two-hybrid experiments.

[0055] Co-transformation with plasmids pTSU2-APP and pNubG-Fe65 was used as a positive control. FvSWEET10 -pBT3-STE and pPR3-N were co-transformed with empty vectors as a negative control. The experimental group was... FvSWEET10 -pBT3-STE and respectively with FvSWEET1- pPR3-N, FvSWEET4 -pPR3-N、 FvSWEET10 -pPR3-N、 FvSWEET17 -pPR3-N combination. The above-mentioned positive and negative control and experimental group recombinant plasmids were co-transformed into competent cells of yeast strain NMY51. If all combined strains could grow normally on SD / -Trp / -Leu medium, it indicated that the co-transformation was successful. Colony PCR was performed on the co-transformed yeast colonies using gene-specific primers. The strains that amplified the target band were identified as positive co-transformed yeast. The positive co-transformed yeast was cultured overnight in YPDA liquid medium. After centrifugation to enrich the colonies, they were resuspended in 0.9% NaCl solution and the OD was adjusted to 0.2. Each yeast cell was then subjected to 10... -1 10-2 10 -3 Serial dilutions were performed, and 1.5 μL of each was spotted onto SD / -Trp / -Leu, SD / -Trp / -Leu / -His / -Ade, and SD / -Trp / -Leu / -His / -Ade solid medium supplemented with 4 mg / mL X-gal. After incubation at 30 ℃ for 2-3 days, colony growth and color development were observed.

[0056] The results are as follows Figure 12 As shown, all co-transformed yeasts grew normally on SD / -Trp / -Leu plates. On SD / -Trp / -Leu / -His / -Ade plates, the co-transformed yeast containing the pPR3-N empty vector, serving as a negative control, could not grow, while the positive control pTSU2-APP+pNubG-Fe65 grew normally and appeared blue on plates containing X-gal. FvSWEET10 -pBT3-STE with FvSWEET1 -pPR3-N、 FvSWEET10 The -pPR3-N combination showed growth and a blue color, while the other two groups had the same phenotype as the negative control, with no obvious growth or blue reaction.

[0057] The above results indicate that the FvSWEET10 protein can interact with both FvSWEET1 and FvSWEET10 proteins in this system, which is consistent with the results of the luciferase complementation (LCI) assay. Therefore, it is inferred that FvSWEET10 can form a heterodimer with FvSWEET1 and can also form a homodimer through self-interaction, thus exerting its function in plants.

[0058] In summary, this invention confirms FvSWEET10 The gene can regulate the accumulation of soluble sugars (sucrose SUC, glucose GLU, and fructose FRU) in various plants such as Arabidopsis thaliana, tomato, and strawberry, and accelerate fruit ripening. The protein monomer encoded by this gene does not have independent sugar transport activity and needs to form a homo / heterodimer with FvSWEET1 to complete the sugar transport function. It can be applied to improve sugar content in fruit trees and breed early-maturing varieties.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strawberry sweet transporter protein FvSWEET10 Genes, characterized by, The FvSWEET10 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A device as described in claim 1 FvSWEET10 The strawberry sweet transporter protein encoded by the gene is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, Includes the claims 1 FvSWEET10 Gene.

4. A host cell, characterized in that, Includes the claims 1 FvSWEET10 The gene or the recombinant expression vector as described in claim 3.

5. A device as described in claim 1 FvSWEET10 The application of genes in regulating the accumulation of soluble sugars and the fruit ripening process in plants is characterized by, The soluble sugars include sucrose, glucose, and / or fructose.

6. The application according to claim 5, characterized in that, The plant in question is a diploid strawberry 'Ruegen', which is expressed transiently through fruit. FvSWEET10 Genes promote the accumulation of soluble sugars in strawberry fruits and accelerate fruit ripening.

7. The application according to claim 5, characterized in that, The plant in question is Arabidopsis thaliana, and it was obtained through stable genetic transformation. FvSWEET10 Genes enhance Arabidopsis' tolerance to high concentrations of sucrose and regulate the plant's sugar response.

8. The application according to claim 5, characterized in that, The plant in question is a tomato, obtained through stable genetic transformation. FvSWEET10 The gene promotes the transport of sucrose out of the leaves, increases the sucrose, glucose, and fructose content of tomato fruits, and advances fruit ripening.

9. The application according to any one of claims 5 to 8, characterized in that, The FvSWEET10 The gene-encoded transporter FvSWEET10 forms a dimer with the FvSWEET1 transporter through heterologous interaction or homologous interaction, and synergistically regulates the transport and accumulation of soluble sugars in plants.

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