Gene for regulating and controlling sugar content of strawberry fruits and application thereof

Abstract

The invention belongs to the technical field of genetic engineering in molecular biology, and relates to a gene for regulating and controlling the sugar content of strawberry fruits and application of the gene. According to the invention, the FveSMXL7 gene is cloned from forest strawberries and is used for constructing a silent vector of the FveSMXL7 gene, and a stable transgenic technology proves that the sugar content of strawberry fruits can be increased by reducing the expression of the FveSMXL7 gene, which indicates that the gene has the function of regulating and controlling the sugar content of the strawberry fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering in molecular biology, and specifically relates to an FveSMXL7 gene for improving the sugar content of strawberry fruit and an application thereof. Background Art

[0002] Strawberry is a perennial herbaceous plant of the genus Fragaria (Fragaria spp.) of the family Rosaceae. The fruit is bright in color, sweet and sour, soft and juicy, and rich in various nutrients. It is known as the "Queen of Fruits" and is deeply loved by consumers. With the improvement of people's living standards, consumers' requirements for the quality of strawberry fruits are becoming increasingly stringent. The sugar content of strawberry fruit is one of the important factors that determine the flavor and quality of strawberries, which directly affects consumers' willingness to buy. Therefore, exploring the key genes that regulate the sugar content of strawberry fruit is of great guiding significance for improving the quality and economic value of strawberry fruit.

[0003] Strigolactones (SLs) are a new type of plant hormone that plays an important role in regulating plant branching and growth and development. A large number of studies have shown that SLs can regulate the aboveground plant type, root system architecture, photomorphogenesis, secondary structure, leaf senescence, etc. of plants such as Arabidopsis, rice, and pea (Jia KP, Li C, Bouwmeester HJ, et al. Strigolactone biosynthesis and signal transduction [J]. Strigolactones-biology and applications, 2019: 1-45.). In recent years, studies have found that SLs play a key 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 SL signaling. Previous studies have shown that FveSMXL7, a negative regulator of the strigolactone signaling pathway, and its homologous genes play an important role 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, there have been no reports on the role of SMXL7 in regulating fruit sugar content.

[0004] In this study, the strawberry FveSMXL7 gene sequence was cloned, the FveSMXL7 gene silencing expression vector was constructed, and the strawberry plants were stably genetically transformed to study the function of the gene. It was found that the gene can significantly regulate the sugar content of the fruit, which is conducive to the molecular analysis of the molecular mechanism of sugar accumulation in strawberry fruit. Summary of the invention

[0005] The purpose of the present invention is to provide a gene for regulating the sugar content of strawberry fruit and its application in view of the deficiencies of the prior art. The technical scheme of the present invention is to transform the constructed plant expression vector for silencing FveSMXL7 into diploid forest strawberry 'Ruegen' by Agrobacterium-mediated method to realize the functional analysis of FveSMXL7 gene; and to realize the regulation analysis of FveSMXL7 on the sugar content of strawberry fruit by detecting the soluble solid content of the fruit of transgenic plants and control plants.

[0006] The technical problem of the present 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] In the above technical solution, further, the silencing of FveSMXL7 gene is used to increase the sugar content of strawberry fruit.

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

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

[0011] The third aspect of the present invention is to provide a recombinant bacterium containing the recombinant plant expression vector.

[0012] The 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 strawberries, 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, the gene is silenced, and transgenic strawberries are obtained; the sugar content of the transgenic strawberry fruit is higher than that of untransformed wild-type strawberries.

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

[0015] The beneficial effects of the present invention are:

[0016] The present invention is the first to discover the application of the FveSMXL7 gene in regulating the sugar content of strawberry fruit. The present invention uses existing plant genetic engineering technology to obtain FveSMXL7-silenced strawberry transgenic plants, and reports for the first time through functional analysis that reducing the expression of the strawberry FveSMXL7 gene can significantly increase the sugar content of the fruit, laying the foundation for future high-sugar strawberry breeding through gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Wherein M is DL5000 Marker; Lanes 1-2 are the amplification results of FveSMXL7.

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

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

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

[0022] In order to further illustrate the present invention but not to limit the present invention, the following examples are used for illustration. The experimental methods described in the following examples are conventional methods unless otherwise specified. The biological materials of the kits can be obtained from commercial sources unless otherwise specified.

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

[0024] 1.1 Cloning of the FveSMXL7 gene from Fragaria serrata

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

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

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

[0028] Note: The first 6 lowercase bases in the primer sequences of FveSMXL7-F and FveSMXL7-R are restriction sites, which are artificially introduced for vector construction and do not belong to the FveSMXL7 gene sequence.

[0029] After the target fragment was recovered using a nucleic acid purification kit (TaKaRa), it was connected to the pMD18-T vector (TaKaRa), and then transformed into Escherichia coli competent cells DH10B (Shanghai Weidi Biotechnology Co., Ltd.), spread on LB medium plates containing kanamycin (50 mg / L), and cultured at 37°C for 12-16 hours. Positive single colonies were screened, plasmids were extracted, and the sequence obtained by sequencing was shown in SEQ ID NO.1.

[0030] After comparing the coding region sequences of FveSMXL7 in forest strawberry 'Ruegen' and 'Hawaii 4', the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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

[0032] (1) Using Primer 5.0 software, we designed a pair of forward primers FveSMXL7-RNAi-F1 and FveSMXL7-RNAi-R1 targeting the conserved region of the FveSMXL7 gene, which introduced XbaⅠ and SmaⅠ restriction endonucleases, respectively; and a pair of reverse primers FveSMXL7-RNAi-F2 and FveSMXL7-RNAi-R2, which introduced 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: The lowercase letters in the primer sequences of FveSMXL7-RNAi-F1, FveSMXL7-RNAi-R1, FveSMXL7-RNAi-F2 and FveSMXL7-RNAi-R2 are restriction sites, and the capital letters before the restriction sites are protective bases. The restriction sites and protective bases are artificially introduced to construct the vector and do not belong to the FveSMXL7 gene sequence.

[0038] (2) Using pMD-T-FveSMXL7 as template, perform PCR reaction;

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

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

[0041] (3) Use gel recovery kit (Beijing Tiangen Biotechnology Co., Ltd.) to recover specific fragments;

[0042] (4) using XbaⅠ and SmaⅠ restriction endonucleases to digest the recovered fragment and pRI101-RNAi vector respectively, and then performing gel recovery again after digestion, and connecting the recovered vector with the fragment;

[0043] The PCR reaction system was as follows: 7 μL of target fragment, 1 μL of pRI101-RNAi vector, 1 μL of T4 DNA ligase, and 1 μL of 10× T4 DNA ligase buffer; 16°C metal bath ligation for 16 h;

[0044] (5) Transform E. coli competent cells DH10B into LB medium containing kanamycin (50 mg / L) and culture at 37°C for 12-16 hours. Pick a single white clone and perform colony PCR amplification. The colony is then spread on a new LB medium plate containing kanamycin (50 mg / L) and cultured at 37°C for 12-16 hours. After the strain is cultured and verified to be correct, it is sent to Suzhou Genewise for sequencing. The sequencing is correct and 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 carried out. The recovered PCR product and RNAi-FveSMXL7-positive vector were digested with KpnI and BamHI restriction endonucleases respectively. After digestion, purification and recovery were performed again, vector connection, Escherichia coli transformation, colony PCR verification, positive clone recombinant plasmid extraction, and sequencing by Suzhou Genewise Co., Ltd. The analysis was performed using DNAMAN software. When the analysis results were correct, the RNAi-FveSMXL7 recombinant vector was obtained.

[0046] Example 2: RNAi silencing FveSMXL7 recombinant vector transformation of forest strawberry

[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 a -80°C refrigerator, melt on ice, preheat a metal bath at 37°C, pipette 2 μL of RNAi-FveSMXL7 plasmid into GV3101, and place 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) Place on ice for 5 min; add 800 μL YEP liquid culture medium to the centrifuge tube in a clean bench, seal the tube mouth with sealing film, and culture in a shaker at 180 rpm / min and 28°C for 4 h;

[0053] (5) After the incubation at 28°C, the cells were collected by centrifugation at 5000 rpm / min for 2 min.

[0054] (6) In the clean bench, keep 100 μL of supernatant to suspend the bacteria, apply it to YEP solid medium, and invert it in a 28°C constant temperature incubator for 2-3 days. When a full single colony grows, perform PCR verification on it.

[0055] 2.2 Agrobacterium-mediated genetic transformation of strawberry

[0056] (1) Preparation of infection solution

[0057] Take a sterile 10mL centrifuge tube, add 3mL YEP liquid culture medium, 1200μL rifampicin (25mg / L) and 3μL kanamycin (50mg / L), mark the name on the bottle, add the verified correct bacteria, and culture in a shaker at 180rpm / min and 28℃ for 8-12h to obtain an orange turbid and impurity-free shaken bacterial solution. Take a sterilized 100mL triangular flask, add 50mL YEP liquid culture medium, and mix 1mL of the shaken bacterial solution, and continue to culture in a 28℃ constant temperature shaker for about 5h, so that the OD600 of the bacterial solution is within the range of 0.4-0.5. At this time, the bacteria can be collected and transferred to a sterile 50mL centrifuge tube, set the centrifuge to 5000rpm / min, 5min, pour out the supernatant in the super bench, resuspend the bacteria with sterile MS suspension, and obtain a suspension with an OD600 between 0.4-0.5.

[0058] (2) Infection of explants

[0059] The leaves of the diploid 'Ruegen' strawberry seedlings in good condition were trimmed into small cubes of 3-5 mm, and 20 mL of liquid co-culture was poured into a sterilized 50 mL small triangular flask. The trimmed explants were quickly placed in the liquid co-culture to avoid wilting. After all the leaves were trimmed, the liquid co-culture in the 50 mL triangular flask was poured out, acetosyringone was added to the prepared suspension, and it was poured into the small triangular flask containing only the explants after being mixed upside down. The explants were soaked for 8 minutes, and the shaking was performed every 2 minutes to make the bacterial solution fully contact the wound of the explants. After the soaking was completed, the bacterial solution was poured out, and the leaves were transferred to sterile filter paper with tweezers, the bacterial solution was absorbed, and the leaves were placed evenly on the solid co-culture medium (pre-laid filter paper) with the back of the leaves facing up.

[0060] (3) Explant culture

[0061] Solid co-cultivation: Culture on solid co-culture medium (pre-coated filter paper) at 22-25°C in the dark for 3 days.

[0062] Delayed culture: Transfer the solid co-culture medium to the delayed culture medium, invert the leaves so that the back of the leaves face down, and culture in the dark at 22-25℃ for 4 days.

[0063] Selective culture: transfer the explants from the delayed culture medium to the selective culture medium with the back of the leaves facing downward in the dark at 22-25℃ until plump yellow-green callus is observed on the explants. Transfer the explants with callus to the light at a temperature of 22-25℃ to promote the growth of adventitious buds.

[0064] Differentiation culture: When the adventitious buds in the selected culture medium grow into plantlets, mark the strain name and transfer the plantlets to the differentiation culture 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 grew to 7-8 leaves, DNA was extracted from young leaves of transgenic strawberry plants (CTAB method). Water and 'Ruegen' strawberry plants were used as negative controls, and RNAi-FveSMXL7 plasmid was used as a positive control. The DNA was amplified by PCR and tested by agarose gel electrophoresis. The results were as follows: Figure 2 As shown, the transgenic plants showed a 1299bp (vector fragment 86bp + FveSMXL7 forward fragment 400bp + vector intron fragment 813bp) fragment, while the control plants did not, indicating that the strawberry FveSMXL7 silencing vector was successfully introduced into strawberries. Primers for DNA level detection of transgenic plants:

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

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

[0070] (2) When the resistant seedlings grew to 7-8 leaves, RNA was extracted from the young leaves of the transgenic strawberry plants and qRT-PCR was performed to detect whether the target gene was silenced. The quantitative primer was: 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 in the FveSMXL7 silenced transgenic plants was reduced, indicating that FveSMXL7 was successfully silenced in strawberry.

[0073] 3.2 Cultivation of transgenic forest strawberry plants

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

[0075] (2) Solar greenhouse cultivation of transgenic strawberry plants: After being cultured in a light incubator for about 20 days, the self-sealing bag or film can be removed and the plants can be transplanted into a nutrient pot filled with a new substrate (a mixture of perlite, vermiculite, earthworm castings and peat in a volume ratio of 1:2:2:4) and placed in a solar greenhouse at the Shenyang Agricultural University Teaching and Research Experimental Base for continued cultivation. The culture conditions are a daytime temperature of 20-25°C, a nighttime temperature of 12-15°C, and appropriate humidity.

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

[0077] 4.1 Experimental Principle

[0078] Sugar can be dehydrated in concentrated sulfuric acid to generate furfural or hydroxymethylfurfural. The generated furfural or hydroxymethylfurfural can react with anthrone to generate blue-green furfural. Within a certain range, the depth of color is proportional to the sugar content. The absorption peak of the colored substance generated by the reaction of sugar and anthrone in the visible light region is 620nm, so colorimetry is carried out 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, and measuring 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 pure anhydrous glucose, dissolve it in distilled water and make up to 10 mL, then dilute it 10 times.

[0085] Anthrone reagent: 0.1 g of anthrone is dissolved in 100 mL of 74% dilute sulfuric acid and is prepared for immediate use.

[0086] 4.3 Experimental steps:

[0087] (1) Preparation of standard curve

[0088] Take 6 test tubes and add various reagents according to the table. Shake each tube evenly, boil in a boiling water bath for 10 minutes, take out and cool, perform colorimetry at a wavelength of 620nm on a spectrophotometer, adjust the zero point with a blank, record the optical density value, and draw a standard curve.

[0089]

[0090] (2) Extraction of soluble sugars from samples

[0091] Weigh 0.2 g of fresh strawberry fruit freeze-dried powder sample and put it into a test tube, then add 10 mL of distilled water, put it into boiling water and boil it in a water bath for 20 min, take it out and cool it, filter it into a 25 mL volumetric flask, rinse the residue with hot water several times, make up to the scale, then take 1 mL of extract from each sample, add 5 mL of anthrone reagent and boil it in a boiling water bath for 10 min, perform colorimetry at a wavelength of 620 nm on a spectrophotometer, and record the optical density value.

[0092] 4.4 Calculation results

[0093] Soluble sugar content (%) = (sugar content (ug) × constant volume of extract (mL) / volume of extract reacted 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 fruit sugar content determination are shown in Table 1. The soluble sugar content of FveSMXL7-silenced forest strawberry fruits was 9.06%, 8.48% and 8.40%, respectively, while the soluble sugar content of the control group was only 6.87%. Compared with the control group, the sugar content of FveSMXL7-silenced forest strawberry fruits was significantly increased. This result indicates that FveSMXL7 is an important gene regulating 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] ACAGCAAGAAGACCAATATTTCTTCAAATGAAAAGTCATGCATCCCCACAG

[0132] ATGTGCAAGAGATATCCTCATCTCAGTCCAAGAGTGAAAGTTTCTCTTCCG

[0133] GGGTATGGGAAAAACCTACAAAAAGATGAAGATACTGAATCGGGTAGTGTC

[0134] AAGTCTTCTTGTAGTTTGTACAATTCAAGCATGGTTGAGGGTAGCCGAACA

[0135] TCTCCAACATCTTCGACTTCTGTGACAACAGATTTAGGATTAGGAATATGCT

[0136] CTTTCTCCTGCTAGTAAGTTGAACTTAAATCTGAATCAAGGTAGTCAACATGA

[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. Application of the FveSMXL7 gene in regulating the sugar content of strawberry fruit, the coding region sequence of the FveSMXL7 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the FveSMXL7 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: Application of silencing FveSMXL7 gene in improving sugar content of strawberry fruit.

3. The use according to claim 1 or 2, characterized in that: The strawberry is a forest strawberry.

4. A method for cultivating high-sugar-content transgenic strawberries, characterized in that: The gene of claim 1 is introduced into strawberries, the gene is silenced, and transgenic strawberries are obtained; the sugar content of the transgenic strawberry fruit is higher than that of untransformed wild-type strawberries.

5. A method for increasing the sugar content of strawberry fruit, characterized in that: The expression level of the gene according to claim 1 in strawberries is reduced, thereby increasing the sugar content of strawberry fruits.

6. Application of the gene according to claim 1 in strawberry breeding, characterized in that: The breeding purpose is to breed strawberries with high sugar content in the fruit.

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