Gene for regulating sugar content of strawberry fruit and application thereof

Abstract

The present application belongs to the field of genetic engineering technology in molecular biology, and relates to a gene for regulating sugar content of strawberry fruits and application thereof. The present application clones FveSMXL7 gene from forest strawberry, uses the FveSMXL7 gene to construct a silencing vector of the FveSMXL7 gene, and proves that reducing expression of the FveSMXL7 gene can increase the sugar content of strawberry fruits by stable transgenic technology, and indicates that the gene has the function of regulating the sugar content of strawberry fruits.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology in molecular biology, specifically relating to the FveSMXL7 gene for increasing the sugar content of strawberry fruits and its application. Background Technology

[0002] Strawberry is a perennial herbaceous plant belonging to the genus *Fragaria* of the family Rosaceae. Its brightly colored fruit is sweet and tart, with soft, juicy flesh and rich in various nutrients, earning it the title of "Queen of Fruits" and making it a favorite among consumers. With the improvement of people's living standards, consumers have increasingly stringent requirements for the quality of strawberries. The sugar content of strawberries is one of the important factors determining their flavor and quality, directly affecting consumers' purchasing intentions. Therefore, identifying the key genes that regulate the sugar content of strawberries is of significant guiding importance for improving the quality and economic value of strawberries.

[0003] Strigolactones (SLs) are novel plant hormones that play an important role in regulating plant branching and growth. Numerous studies have shown that SLs can regulate aboveground plant architecture, root structure, photomorphogenesis, secondary structure, and leaf senescence in plants such as Arabidopsis, rice, and peas (Jia KP, Li C, Bouwmeester HJ, et al. Strigolactone biosynthesis and signal transduction[J]. Strigolactones-biology and applications, 2019:1-45.). In recent years, research has found that SLs play a crucial role in plant responses to abiotic stresses such as drought, salt, and nutrient deficiency (Zhuang L, Wang J, Huang B. Drought inhibition of tillering in Festuca arundinacea associated with axillary bud development and strigolactone signaling[J]. Environmental and Experimental Botany, 2017, 142:15-23.). In model plants, DWARF14 (D14), MORE AXILLARY GROWTH 2 (MAX2), SUPPRESSOR OF MAX2-LIKE 6, 7, and 8 (SMXL6, SMXL7, and SMXL8) and their orthologs form a complex in the perception of strigolactones and play a central role in the SL signaling pathway. Previous studies have shown that the negative regulator of the strigolactone signaling pathway, FveSMXL7, and its homologs play important roles in regulating branching, anthocyanin synthesis, and leaf elongation (Wang L, Wang B, Yu H, et al. Transcriptional regulation of strigolactone signalling in Arabidopsis[J]. Nature, 2020, 583(7815):277-281.). However, no studies have been reported on the role of SMXL7 in regulating fruit sugar content.

[0004] This study investigated the function of the strawberry FveSMXL7 gene by cloning the FveSMXL7 gene sequence, constructing a FveSMXL7 gene silencing expression vector, and performing stable genetic transformation on strawberry plants. The results showed that the gene can significantly regulate the sugar content of the fruit, which is helpful for elucidating the molecular mechanism of sugar accumulation in strawberry fruit. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a gene that regulates the sugar content of strawberry fruits and its application. The technical solution of this invention involves transforming a pre-constructed plant expression vector silencing FveSMXL7 into diploid forest strawberry 'Ruegen' using Agrobacterium-mediated transformation to achieve functional analysis of the FveSMXL7 gene. By detecting the soluble solids content of fruits from transgenic and control plants, the regulatory effect of FveSMXL7 on the sugar content of strawberry fruits is analyzed.

[0006] The technical problem of this invention can be solved by the following technical solution:

[0007] The first aspect of the present invention is to provide the application of the FveSMXL7 gene in regulating the sugar content of strawberry fruit, the coding region sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0008] Furthermore, the above technical solution includes the application of silencing the FveSMXL7 gene in increasing the sugar content of strawberry fruits.

[0009] In the above technical solution, the strawberry is further described as a forest strawberry.

[0010] A second aspect of the present invention is to provide a recombinant plant expression vector for the silenced FveSMXL7 gene.

[0011] A third aspect of the present invention is to provide recombinant bacteria containing the recombinant plant expression vector.

[0012] A fourth aspect of the present invention is to provide a method for increasing the sugar content of strawberry fruit, comprising: reducing the expression level of the FveSMXL7 gene in strawberry, thereby increasing the sugar content of strawberry fruit.

[0013] The fifth aspect of the present invention is to provide a method for cultivating transgenic strawberries with high sugar content, wherein the FveSMXL7 gene is introduced into strawberries and the gene is silenced to obtain transgenic strawberries; the transgenic strawberry fruit has a higher sugar content than untransformed wild-type strawberries.

[0014] The fifth aspect of this invention is to provide the application of the FveSMXL7 gene in strawberry breeding, wherein the breeding objective is to select strawberries with high sugar content in the fruit.

[0015] The beneficial effects of this invention are:

[0016] This invention is the first to discover the application of the FveSMXL7 gene in regulating the sugar content of strawberry fruits. Utilizing existing plant genetic engineering techniques, this invention obtained FveSMXL7-silenced transgenic strawberry plants. Through functional analysis, it is the first to report that reducing the expression of the FveSMXL7 gene in strawberries can significantly increase the sugar content of the fruit, laying the foundation for future breeding of high-sugar strawberries through gene editing. Attached Figure Description

[0017] Figure 1 Amplification of the FveSMXL7 gene cDNA sequence.

[0018] M represents the DL5000 Marker; lanes 1-2 represent the amplification results of FveSMXL7.

[0019] Figure 2 Electrophoresis diagram for DNA level identification of transgenic plants.

[0020] Wherein, W: water; P: RNAi-FveSMXL7 recombinant plasmid; M: DL2000 Marker; CK: wild-type RG DNA; #1-#3: RNAi-FveSMXL7 transgenic line DNA. Figure 3 Relative expression level of the FveSMXL7 gene.

[0021] Among them, RG: untransformed wild-type strawberry; #1-#3: three lines of RNAi-FveSMXL7 transgenic strawberry. Detailed Implementation

[0022] To further clarify the invention and not limit it, the following embodiments are provided. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods. Unless otherwise specified, the biological materials used in the reagent kits are commercially available.

[0023] Example 1: Cloning of the FveSMXL7 gene from forest strawberry and construction of its RNAi interference vector

[0024] 1.1 Cloning of the FveSMXL7 gene in forest strawberry

[0025] The diploid forest strawberry 'Ruegen' was used as the test material.

[0026] RNA extraction: Total RNA was extracted from the material using a modified CTAB method, and then the RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa).

[0027] Gene cloning: Using reverse-transcribed strawberry cDNA as a template, PCR amplification was performed using primers FveSMXL7-F and FveSMXL7-R (which introduced NdeI and BamHI restriction sites, respectively). The PCR product was recovered, yielding a 3171 bp target fragment. Figure 1 shown. FveSMXL7-F:5'-catatgATGCCTACGCCAGTTTCTGTAGCCAG-3'FveSMXL7-R:5'-ggatccTCACTTTAGTATGATTTCGGGTGGG-3'

[0028] Note: The first six lowercase bases in the primer sequences FveSMXL7-F and FveSMXL7-R are restriction enzyme sites, which were artificially introduced for vector construction and do not belong to the FveSMXL7 gene sequence.

[0029] After recovering the target fragment using a nucleic acid purification kit (TaKaRa), it was ligated into the pMD18-T vector (TaKaRa), then transformed into E. coli competent cells DH10B (Shanghai Weidi Biotechnology Co., Ltd.), plated on LB agar plates containing kanamycin (50 mg / L), and incubated at 37°C for 12-16 h. Positive single colonies were screened, plasmids were extracted, and sequencing was successful, yielding the sequence shown in SEQ ID NO.1.

[0030] The coding sequence of FveSMXL7 in forest strawberry 'Ruegen' and 'Hawaii 4' was found to be identical, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0031] 1.2 Construction of the plant interference expression vector RNAi-FveSMXL7

[0032] (1) Using Primer 5.0 software, a pair of forward fragment primers FveSMXL7-RNAi-F1 and FveSMXL7-RNAi-R1 were designed for the conserved region of the FveSMXL7 gene, which introduced XbaⅠ and SmaⅠ restriction endonucleases, respectively; and a pair of reverse fragment primers FveSMXL7-RNAi-F2 and FveSMXL7-RNAi-R2 were designed to introduce KpnⅠ and BamHⅠ restriction endonucleases, respectively.

[0033] FveSMXL7-RNAi-F1:5'-GCtctagaACGCCAGTTTCTGTAGCC-3'

[0034] FveSMXL7-RNAi-R1:5'-TCCcccgggCGCAGGACATGGA-3'

[0035] FveSMXL7-RNAi-F2:5'-GGggtaccCGCAGGACATGGA-3'

[0036] FveSMXL7-RNAi-R2:5'-CggatccACGCCAGTTTCTGTAGCC-3'

[0037] Note: In the primer sequences FveSMXL7-RNAi-F1, FveSMXL7-RNAi-R1, FveSMXL7-RNAi-F2, and FveSMXL7-RNAi-R2, the lowercase letters are restriction enzyme sites, and the uppercase letters before the restriction enzyme sites are protective bases. The restriction enzyme sites and protective bases are artificially introduced during vector construction and do not belong to the FveSMXL7 gene sequence.

[0038] (2) PCR reaction was performed using pMD-T-FveSMXL7 as a template;

[0039] The PCR reaction system was as follows: 0.5 μL of pMD-T-FveSMXL7 plasmid was added to 25 μL of Ex Taq enzyme (TaKaRa), 0.5 μL each of FveSMXL7-RNAi-F1 and FveSMXL7-RNAi-R1 primers, and finally water was added to make up to 50 μL.

[0040] PCR reaction program: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, 32 cycles; extension at 72℃ for 5 min; storage at 4℃; products were subjected to agarose gel electrophoresis.

[0041] (3) The specific fragments were recovered using a gel recovery kit (Beijing Tiangen Biotech Co., Ltd.);

[0042] (4) The recovered fragments and pRI101-RNAi vector were digested with XbaⅠ and SmaⅠ restriction endonucleases respectively. After digestion, the fragments were recovered again and the recovered vectors were ligated to the fragments.

[0043] The PCR reaction system consisted of: 7 μL of the target fragment, 1 μL of pRI101-RNAi vector, 1 μL of T4 DNA ligase, and 1 μL of 10×T4 DNA ligase buffer; ligation was performed in a 16℃ metal bath for 16 h.

[0044] (5) Transform E. coli competent cells DH10B into LB medium containing kanamycin (50 mg / L) and incubate at 37°C for 12-16 h. Pick a single white clone and perform colony PCR amplification. Simultaneously, plate the colonies onto fresh LB medium plates containing kanamycin (50 mg / L) and incubate at 37°C for 12-16 h. After verifying the correct strain, send it to Suzhou Genewiz for sequencing. The sequencing was correct, and the strain was named RNAi-FveSMXL7-positive.

[0045] (6) Using FveSMXL7-RNAi-F2 and FveSMXL7-RNAi-R2 as primers and pMD-T-FveSMXL7 as template, PCR reaction was performed. The recovered PCR product was digested with KpnI and BamHI restriction endonucleases and the RNAi-FveSMXL7-positive vector, respectively. After digestion, the product was purified and recovered again. The vector was ligated, transformed with E. coli, verified by colony PCR, and the recombinant plasmid of positive clone was extracted. The product was then sent to Suzhou Genewiz for sequencing and analyzed using DNAMAN software. Once the analysis results were correct, the RNAi-FveSMXL7 recombinant vector was obtained.

[0046] Example 2: Transformation of forest strawberry with RNAi-silencing FveSMXL7 recombinant vector

[0047] 2.1 Transformation of Agrobacterium

[0048] The RNAi-FveSMXL7 plasmid was introduced into Agrobacterium GV3101 as follows:

[0049] (1) Take 50 μL of GV3101 Agrobacterium competent cells from the -80℃ freezer, thaw them on ice, preheat them in a metal bath at 37℃, add 2 μL of RNAi-FveSMXL7 plasmid to GV3101, and incubate on ice for 5 min.

[0050] (2) Rapid freezing with liquid nitrogen for 1 min;

[0051] (3) Heat shock in metal bath at 37℃ for 2 minutes;

[0052] (4) Let stand on ice for 5 min; in a clean bench, add 800 μL of LYEP liquid culture medium to the centrifuge tube, seal the centrifuge tube opening with sealing film, and incubate in a shaker at 180 rpm / min and 28℃ for 4 h.

[0053] (5) After incubation at 28℃, collect the bacterial cells by centrifugation at 5000 rpm / min for 2 min;

[0054] (6) In a clean bench, retain 100 μL of supernatant to suspend the bacterial cells, spread them onto YEP solid medium, and incubate upside down in a 28°C incubator for 2-3 days. Once plump single colonies have grown, perform PCR verification.

[0055] 2.2 Agrobacterium-mediated genetic transformation of strawberries

[0056] (1) Preparation of infecting bacterial solution

[0057] Take a sterile 10mL centrifuge tube, add 3mL of YEP liquid medium, 1200μL of rifampicin (25mg / L), and 3μL of kanamycin (50mg / L), label the tube, add the verified bacterial cells, and incubate at 180rpm / min in a shaker at 28℃ for 8-12 hours to obtain an orange, turbid, and impurity-free first-shake bacterial suspension. Take a sterile 100mL Erlenmeyer flask, add 50mL of YEP liquid medium, and mix in 1mL of the first-shake bacterial suspension. Continue to incubate at 28℃ in a shaker for about 5 hours, until the OD600 of the bacterial suspension is in the range of 0.4-0.5. At this point, the bacterial cells can be collected and transferred to a sterile 50mL centrifuge tube. Set the centrifuge to 5000rpm / min for 5 minutes. Discard the supernatant in a super benchtop, and resuspend the bacterial cells with sterile MS suspension to obtain a suspension with an OD600 between 0.4 and 0.5.

[0058] (2) Infection of explants

[0059] Trim the leaves of healthy diploid 'Ruegen' tissue culture strawberry seedlings into 3-5mm cubes. Pour 20mL of liquid into a sterile 50mL Erlenmeyer flask for co-culture. Quickly place the trimmed explants into the liquid co-culture to prevent wilting. After all leaves have been trimmed, discard the liquid co-culture in the 50mL Erlenmeyer flask. Add acetylsyl syringone to the prepared suspension, mix thoroughly by inverting, and pour into the Erlenmeyer flask containing only the explants. Soak the explants for 8 minutes, shaking every 2 minutes to ensure the bacterial solution fully contacts the explant wounds. After soaking, discard the bacterial solution, use tweezers to transfer the leaves to sterile filter paper, blot dry, and place the leaves, underside up, evenly on a solid co-culture medium (pre-laid with filter paper).

[0060] (3) Culture of explants

[0061] Solid co-culture: Incubate in the dark at 22-25℃ for 3 days on a solid co-culture medium (pre-laid with filter paper).

[0062] Delayed culture: Transfer the solid co-culture medium to delayed culture medium, turn the leaves upside down so that the leaf back is facing down, and culture in the dark at 22-25℃ for 4 days.

[0063] Selective culture: Transfer the culture from the delayed culture medium to the selective culture medium, with the leaf underside down, and culture in the dark at 22-25℃ until plump yellow-green callus is observed on the explants. Then, transfer the explants with callus to the light and maintain the temperature at 22-25℃ to promote the emergence of adventitious buds.

[0064] Differentiation culture: After the adventitious buds in the selective medium grow into small plants, the strain name is labeled, and the small plants are transferred to the differentiation medium for differentiation and proliferation.

[0065] Example 3: Molecular identification and cultivation of transgenic forest strawberry plants

[0066] 3.1 Molecular identification of transgenic plants

[0067] (1) When the resistant seedlings have grown to 7-8 leaves, DNA is extracted from the tender leaves of the strawberry transgenic plants (CTAB method). Water and 'Ruegen' strawberry plants are used as negative controls, and the RNAi-FveSMXL7 plasmid is used as a positive control. The results are examined by PCR amplification and agarose gel electrophoresis. Figure 2 As shown, the transgenic plants exhibited a 1299bp fragment (vector fragment 86bp + FveSMXL7 forward fragment 400bp + vector intron fragment 813bp), while the control plants did not, indicating that the strawberry FveSMXL7 silencing vector was successfully introduced into strawberries. Primers for DNA level detection in transgenic plants:

[0068] CaMV35S-F:5'-GACGCACAATCCCACTATCC-3'

[0069] Intro-R:5'-GGTACCATCGATTTCGAACCCAGCTT-3'

[0070] (2) When the resistant seedlings have grown to 7-8 leaves, RNA is extracted from the tender leaves of the strawberry transgenic plants and qRT-PCR is performed to detect whether the target gene is silenced. The quantitative primer is: FveSMXL7-DL-F:5'-GTTTCCGTCGGTAGAGAAAGAC-3'

[0071] FveSMXL7-DL-R:5'-CAACATAACTCACCTGGACCTC-3'

[0072] The results are as follows Figure 3 As shown, compared with the control plants, the expression level of FveSMXL7 was reduced in the FveSMXL7-silenced transgenic plants, indicating that FveSMXL7 silencing was successful in strawberries.

[0073] 3.2 Cultivation of transgenic forest strawberry plants

[0074] (1) Cultivation of strawberry transgenic plants in the ground: The identified positive transgenic plants were transferred into rooting medium and cultured for 40 days. When the roots of the plantlets basically covered the bottom of the bottle, the bottle mouth was opened and the plants were hardened off under natural light indoors for 4-7 days. After that, the plantlets were taken out of the bottle, the base of the medium was washed off, and the plants were soaked in 0.2% calcium chloride solution and 1 mg / L IBA solution to promote rooting. Then the plants were transplanted into seedling trays filled with sterilized substrate (vermiculite, earthworm castings and peat moss mixed in a volume ratio of 2:3:6). Moisturizing was done and detailed information was marked.

[0075] (2) Cultivation of transgenic strawberry plants in a greenhouse: After about 20 days of cultivation in a light incubator, the self-sealing bag or film can be removed and the plants can be transplanted again into nutrient pots filled with new substrate (a mixture of perlite, vermiculite, earthworm castings and peat moss in a volume ratio of 1:2:2:4). The plants can then be placed in the greenhouse of the Teaching and Research Experimental Base of Shenyang Agricultural University for further cultivation. The cultivation conditions are a daytime temperature of 20-25℃, a nighttime temperature of 12-15℃, and suitable humidity.

[0076] Example 4: Determination of soluble sugar content in strawberry fruit using the anthrone method

[0077] 4.1 Experimental Principle

[0078] Sugars can undergo a dehydration reaction under concentrated sulfuric acid to produce furfural or hydroxymethylfurfural. The resulting furfural or hydroxymethylfurfural can react with anthrone to produce blue-green furfural, which is biologically active. Within a certain range, the intensity of the color is directly proportional to the sugar content. The colored substance produced by the reaction of sugars with anthrone has an absorption peak of 620 nm in the visible light region, so colorimetry is performed at this wavelength.

[0079] 4.2 Experimental Supplies:

[0080] (1) Experimental materials: freeze-dried powder of fresh strawberry fruit.

[0081] (2) Instruments and equipment: spectrophotometer, analytical balance, water bath, test tubes, volumetric flasks, graduated cylinders.

[0082] (3) Experimental reagents:

[0083] 74% dilute sulfuric acid: Add 74 mL of concentrated sulfuric acid to 26 mL of distilled water and mix well.

[0084] Glucose standard solution (100 μg / mL): Accurately weigh 0.01 g of analytical grade anhydrous glucose, dissolve it in distilled water and bring the volume to 10 mL, then dilute it 10 times.

[0085] Anthrone reagent: Dissolve 0.1g of anthrone in 100mL of 74% dilute sulfuric acid, and prepare immediately before use.

[0086] 4.3 Experimental Procedure:

[0087] (1) Standard curve preparation

[0088] Take 6 test tubes, add the various reagents according to the table, shake each tube well, boil in a boiling water bath for 10 minutes, remove and cool, and perform colorimetric analysis at a wavelength of 620 nm using a spectrophotometer. Use a blank to adjust the zero point, record the optical density value, and plot the standard curve.

[0089]

[0090] (2) Extraction of soluble sugars from the sample

[0091] Weigh 0.2g of freeze-dried fresh strawberry fruit powder into a test tube, add 10mL of distilled water, boil in a water bath for 20min, remove and cool, filter into a 25mL volumetric flask, rinse the residue several times with hot water, and dilute to the mark. Then, take 1mL of the extract from each sample, add 5mL of anthrone reagent, boil in a water bath for 10min, and measure the colorimetric value at 620nm wavelength using a spectrophotometer and record the optical density value.

[0092] 4.4 Calculation Results

[0093] Soluble sugar content (%) = (Sugar content (ug) × Volume of extract (mL) / Volume of extract reacting with anthrone (mL) × Sample mass (g) x 10 6 )×100

[0094] Table 1 Comparison of sugar content in transgenic and control strawberry fruits

[0095]

[0096] Note: Different capital letters indicate significant differences at the P<0.01 level.

[0097] The results of the sugar content determination of the fruit are shown in Table 1. The soluble sugar contents of the FveSMXL7 Silent Forest strawberry fruit were 9.06%, 8.48% and 8.40%, respectively, while the soluble sugar content of the control group fruit was only 6.87%. Compared with the control group, the sugar content of the FveSMXL7 Silent Forest strawberry fruit was significantly increased. This result indicates that FveSMXL7 is an important gene that regulates the sugar content of strawberry fruit.

[0098] FveSMXL7 gene

[0099] DNA

[0100] 3171bp

[0101] ATGCCTACGCCAGTTTCTGTAGCCAGGCAATGCTTAACACCGGAGGCAACC

[0102] CACGCGCTCGACGAGGCGGTGTCCGTCGCGCGCCGCAGAAGCCACGCGCA

[0103] GACCACATCCCTCCACGCAGTCTCTGCTCTACTCTCCCTCCCCAACTCCGCC

[0104] CTACGCGAGGCCTGCGCGCGTGCTCGCAACGGGGCCTACTCGCCGCGGCT

[0105] CCAGTTCAAGGCGCTGGAGCTCTGCCTCAGCGTATCCCTGGACCGGGTCTC

[0106] GTCGTCGACCCGGCAGCTCAGCGACGACGACCCGCCCGTTTCGAACTCCC

[0107] TCATGGCCGCCATCAAGCGCTCTCAGGCCAACCAGCGACGGCAGCCGGAG

[0108] AATTATCACCTCTACCATCAGCTCCAGCAACAACAGTCGTCCATGTCCTGCG

[0109] TTAAGGTCGAGCTCCAGCATCTTATTCTCTCCATTCTCGACGACCCGGTTGT

[0110] CAGTCGGGTCTTCGCGGAAGCGGGTTTCCGGAGCTCCGAAATCAAGATGG

[0111] CCATCCTCCGCCCTTTTCCTCCTCTTCCTCTGTTTCTCCATAATCCGGGTCCG

[0112] GGTCCGGGTCCCGGTCCGGGTCGTCGTCGCCGGCCCGTTTTCCCGTTCTCG

[0113] GGTTTCGCCAACGGAGACGAGAATTGTCGGAGAATTGGAGAAGTTTTAGG

[0114] AAGAAACAGGAATCCTCTGCTCCTGGGTGTATGTGCTTACGAAGCTCTTCA

[0115] CATGTTCATGGCATCGTTAACTAAAGAAGGTATCTTACCGGTGGAGCTGTCC

[0116] GGTGTGAGTTCAGTTTCGATCGAAAAAGAGTTATCTCAGTTTACATTAACTG

[0117] ATTCCGACAAAGGGTGCTTGAGTTCGAGGCTGGCCGAGGTGGGTGAGTTA

[0118] GTGGACAAGTGTTTGGGAGGTGGAGTTGTGGTGAATATTGGAGACTTGAA

[0119] GATGCTAGTTGGGGAGGAGTGTTTGGGTGAGTCAGTGAGATATGTGGTGGC

[0120] CCAGTTGACTAGGCTGGTGGAGGTTTACCGGGGAAGTGTTTGGTTCGTTGG

[0121] AGCTACGGCGAGCTATGGGAGCTACTTGAAGTTTGTGAGTATGTTTCCGTC

[0122] GGTAGAGAAAGACTGGGACTTGCAGCTTCTGCCGATCACTTCGGTTGGAG

[0123] CTGAATCATACCCGAGGTCCAGCTTGATGGAGTCATTTGTTCCACTTGGTGG

[0124] GTTCTTTTCTGCACCTTCTGACCTAAAGCTTCCGTTAAGTTGTTCAAATGGG

[0125] TTATTTCCCCTCGATCATCAGTGCAGTGAAAAAGATGATCAAGAAGCATATG

[0126] CTTTTCCGAAGGGAGGCTTTGCTACTTCAGTGGCAGGTCAGCATGCTAGCT

[0127] TGCCTTCTTGGATGTGGATGGCTCCACTTGGCACAAACAAGGGGTTGGATA

[0128] TGAAGACCAAAGACGATGGAGTGCTATTGAGTTCCAAAGTTACAGGACTG

[0129] CAAAAGAAATGGGATAATACACATGAATCTCATCCCTTGCCTTTAGCAAACT

[0130] TGTTTCCAACTATTGTGGGCTTTGAGTCTGGTGAAGACAAGAAGCACATTC

[0131] ACAGCAAGAAGACCAATATTTCTTCAAATGAAAAGTCATCATCCCCACAG

[0132] ATGTGCAAGAGATATCCTCATCTCAGTCCAAGAGTGAAAGTTTCTCTTCCG

[0133] GGGTATGGGAAAAACCTACAAAAGATGAAGATACTGAATCGGTAGTGTC

[0134] AAGTCTTCTTGTAGTTTGTACAATTCAAGCATGGTTGAGGGTAGCCGAACA

[0135] TCTCCAACATCTTCGACTTCTGTGACAACAGATTTAGGATTAGGAATATGCT

[0136] CTTCTCCTGCTAGTAAGTTGAACTTAAATCTGAATCAAGGTAGTCAACATGA

[0137] CATGTCAGTTTTCTCTTCTGGAAATTCCTCTATTTACACTGCTCAGTCATCAT

[0138] TCTGCTCAAGGGCTGACAAACATGGGCAGTTTGATCCAAGTGATGTCAAGA

[0139] TGCTTTTGAGAGCTCTCTTTGAGAGAGTGAGCTGGCAAACTGAGGCTATAA

[0140] GTGCTATTAGCCAACGAATAGCGCATTGCCGATCAAGAAGTGAACACCGGA

[0141] CTGGATGTAGGCACAGAAGGGATATATGGTTCAACTTTGTTGGACCAGATA

[0142] GGTATGGTAAAAAGAAAATTGCTAGTGTGCTCGCTGAGTTATTATACGGGA

[0143] GCCAGGAACAACTGATCTGTGTGGATCTGAATTCTCAGGATGGATTGCTCC

[0144] ATTCAGACACAATGTTCGGTGGTCAAGTGTTGAATGGTTATGATGCAAAGT

[0145] ATAGAGGTAAGACAGTTGTTGATTATGTGGCTGGAGAACTCTGCAGGAAAC

[0146] CATTGTCAATTGTGTTCCTGGAAAATGTAGACAAGGCAGATGTCGTTGCTC

[0147] AGCATAGTTTGTCCCAGGCGGTTTTGAGCGGTAAGTTTTCAGATTCCCATG

[0148] GAAGACAAGTCAGCACAAGTAATGCAGTGTTTATTACAACTACAGAAAAG

[0149] GGCTGCAGCACTCTCACTTCCAAAAGGGTTCCTTCCAAATATTCCGAGGAA

[0150] AAAATCTCACAAGCAAAAGGGTGGCCCGTTCAGATTACAGTTGAATGCGC

[0151] ACTTGATGGCAGGGCTGTAAGCCAAAACTGGACAACATCCTCCAATACAAC

[0152] TAAAGAAAGCATCCCTCATTTTCTGAACAAAAGGAAGCTGTCTGGTGTGGG

[0153] CAAACCTCTAGAGCAGCATTCAGTCTCAGAGATGAGTAAACGGCCTAACA

[0154] AAACGTCGACTAGGTTTCTGGATCTCAACCTTCCAGCTGAAGAGAATGCAG

[0155] TGCAACATCTAGACGCTGATGACTGCTTGTCAGAAAACTCCAGTCCCTGGT

[0156] TGCAAGAATTCACTGATCAGTTTGATGAAACAGTGGCATTCAAGCCGGTTG

[0157] ATTTCGATGCACTTGCTGAGAATATATCAAAGCAGATCAAAAACAGTTTCC

[0158] ACCAGCTCATTGGTTCAGAGTGCTTGCTAGAAATCGACACACAAGTCATGG

[0159] AACGACTACTTGCAGCCGCATATCTTTCCAACAAGTACAACGTGGTGGAAA

[0160] ACTGGGTAGACCAAGTCTTGAGCAGAAGATTCGCAGAGGTCCAGAAGCGA

[0161] CACAACCTGAATGCTCATACTATCGTAACACTCAAAGCTTGCGACGGCTTTT

[0162] GCTTGGAGGAGGAGTCATCTCCGGAAGACTACCTCCCACCCGAAATCATAC

[0163] TAAAGTGA

[0164] FveSMXL7 protein

[0165] PRT

[0166] 1056aa

[0167] MPTPVSVARQCLTPEATHALDEAVSVARRRSHAQTTSLHAVSALLSLPNSALR

[0168] EACARARNGAYSPRLQFKALELCLSVSLDRVSSSTRQLSDDDPPVSNSLMAAI

[0169] KRSQANQRRQPENYHLYHQLQQQQSSMSCVKVELQHLILSILDDPVVSRVFA

[0170] EAGFRSSEIKMAILRPFPPLPLFLHNPGPGPGPGPGRRRRPVFPFSGFANGDENC

[0171] RRIGEVLGRNRNPLLLGVCAYEALHMFMASLTKEGILPVELSGVSSVSIEKELS

[0172] QFTLTDSDKGCLSSRLAEVGELVDKCLGGGVVVNIGDLKMLVGEECLGESVR

[0173] YVVAQLTRLVEVYRGSVWFVGATASYGSYLKFVSMFPSVEKDWDLQLLPITS

[0174] VGAESYPRSSLMESFVPLGGFFSAPSDLKLPLSCSNGLFPLDHQCSEKDDQEAY

[0175] AFPKGGFATSVAGQHASLPSWMWMAPLGTNKGLDMKTKDDGVLLSSKVTG

[0176] LQKKWDNTHESHPLPLANLFPTIVGFESGEDKKHIHSKKTNISSNEKSCIPTDV

[0177] QEISSSQSKSESFSSGVWEKPTKDEDTESGSVKSSCSLYNSSMVEGSRTSPTSST

[0178] SVTTDLGLGICSSPASKLNLNLNQGSQHDMSVFSSGNSSIYTAQSSFCSRADKH

[0179] GQFDPSDVKMLLRALFERVSWQTEAISAISQRIAHCRSRSEHRTGCRHRRDIW

[0180] FNFVGPDRYGKKKIASVLAELLYGSQEQLICVDLNSQDGLLHSDTMFGGQVL

[0181] NGYDAKYRGKTVVDYVAGELCRKPLSIVFLENVDKADVVAQHSLSQAVLSG

[0182] KFSDSHGRQVSTSNAVFITTTEKGCSTLTSKRVPSKYSEEKISQAKGWPVQITV

[0183] ECALDGRAVSQNWTTSSNTTKESIPHFLNKRKLSGVGKPLEQHSVSEMSKRPN

[0184] KTSTRFLDLNLPAEENAVQHLDADDCLSENSSPWLQEFTDQFDETVAFKPVDF

[0185] DALAENISKQIKNSFHQLIGSECLLEIDTQVMERLLAAAYLSNKYNVVENWV

[0186] DQVLSRRFAEVQKRHNLNAHTIVTLKACDGFCLEEESSPEDYLPPEIILK。

Claims

1. Silence FveSMXL7 The application of genes in increasing the sugar content of strawberry fruits, the FveSMXL7 The gene coding region sequence is shown in SEQ ID NO.1; FveSMXL7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The strawberries mentioned are forest strawberries.

3. A method for cultivating transgenic strawberries with high sugar content, characterized in that, Construct the structure described in claim 1 FveSMXL7 A gene silencing vector was introduced into strawberries to obtain transgenic strawberries; the transgenic strawberries have a higher sugar content than untransformed wild-type strawberries.

4. A method for increasing the sugar content of strawberry fruit, characterized in that, The expression level of the gene described in claim 1 in strawberries is reduced, thereby increasing the sugar content of strawberry fruit.

5. The application of the gene described in claim 1 in strawberry breeding, characterized in that: The breeding objective is to select strawberries with high sugar content in their fruit.

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