Gene for enhancing gray mold resistance of strawberry fruits and application thereof

Abstract

The invention discloses a gene for enhancing gray mold resistance of strawberry fruits and application of the gene, and belongs to the technical field of gene engineering. The nucleotide sequence of the gene disclosed by the invention is as shown in SEQ ID NO.7. The invention provides the gene for enhancing the gray mold resistance of the strawberry fruit, the gene is introduced into the strawberry body to realize stable overexpression, the gray mold resistance of the strawberry fruit is obviously enhanced, the gene has no influence on the quality of the strawberry fruit, and a new gene resource and a theoretical support are provided for breeding of strawberry varieties with high gray mold resistance.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a gene for enhancing the gray mold resistance of strawberry fruit and an application thereof. Background Art

[0002] China is a major strawberry producer and consumer in the world, with a planting area of ​​173,000 hm2. 2 , the annual yield increase ranks first among crops, and the production accounts for more than 50% of the world's total production. However, strawberries are very susceptible to pathogens during cultivation, transportation and storage, resulting in reduced yield, poor quality, and serious damage to economic benefits. Gray mold is one of the fungal diseases that seriously affects the fruit yield of strawberries. It is caused by Botrytis cinerea and is a plant disease with a wide host and fast reproduction rate.

[0003] Current prevention and control measures for strawberry gray mold include agricultural prevention, chemical prevention, biological prevention and physical prevention. Agricultural prevention mainly improves plant disease resistance through reasonable close planting, keeping the field well ventilated, increasing the application of organic fertilizer, and regularly cleaning diseased leaves and fruits. Chemical prevention uses pesticide spraying for prevention and control, but long-term use of a single agent easily leads to drug resistance in pathogens. Biological prevention uses antagonistic microorganisms to prevent and control Botrytis cinerea, but the prevention and control effect is greatly affected by environmental conditions. Physical prevention uses measures such as mulching and hanging silver-gray reflective film in the greenhouse to repel Botrytis cinerea. In recent years, with the rapid development of molecular biology and genetics technology, technicians have begun to explore the resistance mechanism of plants to gray mold from the gene level, in order to screen out genes with gray mold resistance, thereby cultivating gray mold high-resistant varieties. Based on this, the present invention studies the resistance to gray mold of strawberry, and successfully screens out a gene that can enhance the resistance to gray mold of strawberry fruit. Summary of the invention

[0004] The purpose of the present invention is to provide a gene for enhancing the gray mold resistance of strawberry fruit and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a gene for enhancing the gray mold resistance of strawberry fruit, and the gene is introduced into the strawberry body to make it stably overexpressed, which significantly enhances the resistance of strawberry fruit to gray mold. In addition, the gene has no effect on the quality of strawberry fruit, and provides new gene resources and theoretical support for the breeding of strawberry varieties with high resistance to gray mold.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a gene for enhancing the gray mold resistance of strawberry fruit, and the nucleotide sequence of the gene is shown in SEQ ID NO.7.

[0007] The present invention also provides a protein encoded by the gene for enhancing the gray mold resistance of strawberry fruit, and the amino acid sequence thereof is shown in SEQ ID NO.8.

[0008] The present invention also provides a recombinant vector, which contains the gene for enhancing the gray mold resistance of strawberry fruit.

[0009] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the gene for enhancing the gray mold resistance of strawberry fruit or the recombinant vector.

[0010] The present invention also provides the application of the gene for enhancing the gray mold resistance of strawberry fruit, the protein, the recombinant vector or the recombinant bacteria in enhancing the gray mold resistance of strawberry fruit or cultivating strawberry varieties with high gray mold resistance.

[0011] The present invention also provides a method for enhancing the gray mold resistance of strawberry fruit, comprising the steps of using genetic transformation technology to transfer a gene for enhancing the gray mold resistance of strawberry fruit into a strawberry plant to stably overexpress it;

[0012] The nucleotide sequence of the gene for enhancing the gray mold resistance of strawberry fruit is shown in SEQ ID NO.7.

[0013] The present invention also provides a method for cultivating a strawberry variety with high resistance to gray mold, comprising the steps of using genetic transformation technology to transfer a gene that enhances the gray mold resistance of strawberry fruit into a strawberry plant to construct a strawberry variety with high resistance to gray mold;

[0014] The nucleotide sequence of the gene for enhancing the gray mold resistance of strawberry fruit is shown in SEQ ID NO.7.

[0015] The present invention also provides a primer pair for amplifying the gene for enhancing the gray mold resistance of strawberry fruit, the primer pair comprising an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

[0016] The present invention also provides a primer pair for quantitatively detecting the expression amount of the gene enhancing the gray mold resistance of strawberry fruit, wherein the primer pair comprises an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6.

[0017] The present invention also provides the use of the primer pair in enhancing the gray mold resistance of strawberry fruit or cultivating a strawberry variety with high gray mold resistance.

[0018] The present invention discloses the following technical effects:

[0019] The invention provides a gene for enhancing the gray mold resistance of strawberry fruit, and the gene has the function of regulating the gray mold resistance of strawberry fruit. By constructing an overexpression vector, the gene is introduced into a strawberry body, and the gene is stably overexpressed, which significantly enhances the resistance of strawberry fruit to gray mold and reduces the incidence rate by 79%. In addition, the gene has no significant effect on the contents of components such as SOD, MDA, hydrogen peroxide, total phenol, flavonoids, and ascorbic acid in strawberry fruit, and does not affect the quality of strawberry fruit, thus providing new gene resources and theoretical support for the breeding of strawberry varieties with high resistance to gray mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The gene expression in plant tissues of different parts of strawberry and fruits at different developmental stages;

[0022] Figure 2 The results of the transient gene injection experiment are verified; A is the phenotype of overexpressed strawberry fruit (OE) and normal strawberry fruit (35S); B is the gene expression in A overexpressed strawberry fruit (OE) and normal strawberry fruit (35S);

[0023] Figure 3 is the effect of overexpressed gene on the resistance of strawberry fruit to gray mold; A is the fluorescence detection result of overexpressed strawberry fruit (OE) and normal strawberry fruit (35S) after treatment; B is the disease incidence of overexpressed strawberry fruit (OE) and normal strawberry fruit (35S); C is the incidence of gray mold in overexpressed strawberry fruit (OE) and normal strawberry fruit (35S);

[0024] Figure 4 This is the effect of gene overexpression on related indicators in strawberry fruit. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The primer sequences involved in the embodiments of the present invention are shown in Table 1:

[0031] Table 1 Primer sequences

[0032]

[0033] Unless otherwise specified, the biological materials involved in the embodiments of the present invention can be purchased through conventional channels; the method steps involved, unless otherwise specified, are all carried out using conventional methods in the art.

[0034] Example 1

[0035] 1. Materials and methods

[0036] 1.1 Plant materials

[0037] Plant tissues from different parts of octoploid cultivated strawberry 'Hongyan' (Fragaria×ananassa'Benihoppe') and fruits at different developmental stages were collected from a strawberry picking base near Sichuan Agricultural University. After sampling, they were quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0038] 1.2 Test methods

[0039] 1.2.1 Gene cloning

[0040] The total RNA of the collected plant materials was extracted, and the total RNA with qualified quality after electrophoresis was reverse transcribed and synthesized into cDNA using the TaKaRa reverse transcription kit. The cf and cR primer pairs in Table 1 were used for PCR amplification using the high-fidelity enzyme I-5TM 2×High-Fidelity Master Mix (Beijing Qingke Xinye Biotechnology Co., Ltd.). A 50μL reaction system was used, including 25μL of high-fidelity enzyme, 1μL of upper and lower primers, 1μL of cDNA, and 50μL was supplemented with sterile water. After mixing, it was placed on a PCR instrument for amplification. The program was set as follows: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 20s, 35 cycles, and final extension for 5min. After the PCR product was detected by electrophoresis, the target band was recovered and connected to the pEASY-Blunt cloning vector, transformed into Escherichia coli Trans1-T1, and the positive clones were screened and sent to Shanghai Shenggong Biological Co., Ltd. for sequencing.

[0041] The gene was sequenced, and its CDS sequence is shown in SEQ ID NO.7:

[0042]

[0043] The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.8:

[0044] MAATWLEALPAQHKRLIMNELVHGRNRARELQSLLNNRGHGSTSSRSEEELVMEIVKSFSESLSVLSESSAKFGGDDQQYSGTTGCGGGETVKAEQSHVEHSHCGDRSFEDSGESKKRPGVKDRRGCYKRRKNSESWATVSSTVEDGQAWRKYGQKEILNAPYPRAYFRC TRKYDQGCQATKQVQQTQDTPKLYKTTYIGNHTCRMIRAPQMIMGSSHPPALDSHAPPRATVSSESGSTPVCNKKEHHGDHGGHLSRSSSILPVKKEESKEGTTTSSGLTDNLNDTSDMWPGFDLGFPEAETTVLSDENVVSNMPFLDMHLVKSIDDFESEFDFDQVY*.

[0045] 1.2.2 Spatiotemporal expression analysis

[0046] The SYBR Green I real-time fluorescence quantitative PCR (qPCR) system was used to detect the expression level of the gene in different tissues and fruit development stages. The qF and qR primer pairs in Table 1 were used, and a 10 μL reaction system was used: 2 μL cDNA, 5 μL SYBRgreen mix, 0.4 μL upper and lower primers, 2.2 μL ddH2O, mixed and centrifuged, and then placed in the CFX96 qPCR system for reaction. The reaction program was: 95°C for 30s; (95°C for 5s, 55°C for 30s, 72°C for 30s, collect fluorescence signals, a total of 40 cycles); insert the melting curve, 95°C for 15s, cool to 60°C and maintain for 30s, start from 65°C and increase the temperature by 0.5°C each step, collect fluorescence signals to 95°C to end the reaction. The strawberry Actin (accession number: LC017712) gene was used as the internal reference, each sample had three biological replicates, each replicate had two technical replicates, and the quantitative results were taken 2 -△△CT SPSS 25.0 was used for variance analysis, and the data were presented as mean ± standard deviation.

[0047] 1.2.3 Overexpression vector construction

[0048] Use the OE-TF and OE-TR primer pairs in Table 1, and use the plasmid extracted from the positive colonies screened in 1.2.1 as a template to perform PCR amplification using a high-fidelity enzyme. The amplified product was mixed with the linearized pCAMBIA1302 vector for homologous recombination, and then transformed into E. coli competent cells using the heat shock method. After expansion and cultivation, single colonies were picked for PCR identification, and positive clones were sent to a biological company for sequencing. The returned positive bacterial solution was extracted with the alkaline lysis method to extract the overexpression plasmid and stored at -20°C for later use.

[0049] 1.2.4 Agrobacterium transformation

[0050] The overexpression plasmid and empty vector (pCAMBIA1302 vector) verified by sequencing were transferred into Agrobacterium GV3101 (laboratory storage) competent cells by freeze-thaw method. The specific operation is as follows: 100 ng of plasmid was added to 100 μL competent cells and mixed, placed on ice for 30 minutes, then placed in liquid nitrogen for rapid freezing for 5 minutes, and then placed in a 28°C water bath for 5 minutes. Add 800 μL YEB liquid culture medium, culture at 28°C, 150-200 r / min with shaking for about 5 h, centrifuge at 10,000×g for 30 s, discard the supernatant, and leave about 100 μL YEB liquid culture medium. Use a pipette tip to blow and mix, then spread on YEB solid culture medium containing rifampicin (Rif; 100 mg / L), gentamycin sulfate (Gen; 40 mg / L) and kanamycin (Kan; 100 mg / L), and culture at 28°C in the dark for 48-36 h.

[0051] 1.2.5 Strawberry fruit instant injection

[0052] (1) Preparation of infection solution: Add 100 mL and 50 mL of LB liquid medium to Agrobacterium transformed with overexpression plasmid and Agrobacterium transformed with empty vector, respectively, and culture overnight at 28°C. OD of bacterial solution was measured. 600 When the value is about 0.8-1.0, collect the cells by centrifugation (5000r / min, 5min, 20℃), pour off the supernatant, add 30mL of infiltration buffer (10mmol / LMES, 10mmol / LMgCl2, 20μmol / L acetosyringone), incubate in the dark at 28℃ and 50r / min for 4h, take out and collect the bacteria by centrifugation at room temperature, resuspend them with buffer to obtain overexpression infection fluid and empty load infection fluid.

[0053] (2) Infection of fruits: The isolated strawberry fruits were rinsed and disinfected with distilled water, 70% ethanol, and sterile water in turn. The moisture on the surface of the fruit was gently wiped dry. The stem end of the strawberry was wrapped with sterile wet cotton. Then, the overexpression infection solution or the empty infection solution was slowly injected from the fruit pedicle with a 1 mL syringe. The surface of the fruit was then wiped clean and placed in a box. The fruit was cultured in a constant temperature culture room for six days, during which time the cotton on the stem end was kept moist.

[0054] (3) Verification of infection effect: Referring to the method in 1.2.2, use the qF and qR primer pairs in Table 1 and a 10 μL reaction system to detect gene expression in overexpressed strawberry fruits (OE) and empty strawberry fruits (35S).

[0055] 1.2.6 Inoculation of Botrytis cinerea

[0056] Take a plate of Botrytis cinerea that has been cultured for 45 days, add about 5 mL of sterile water, use a sterile scraper to gently scrape the mycelium on the surface of the culture medium into the sterile water, and place it on a vortex oscillator to oscillate to fully suspend the spores. Filter the suspension with a sterilized 4-layer medical gauze into another 50 mL sterilized centrifuge tube, and rinse the filter residue with a small amount of sterile water 2 to 3 times. Adjust the concentration of the Botrytis cinerea suspension to 1×10 5 On the 5th day after the injection of the overexpression vector or the empty vector, a small hole of about 0.5 cm was poked in the middle of the strawberry fruit with a sterilized inoculation needle, and 5 μL of spore suspension was dripped in. 24 fruits were treated each time and the disease was observed. The formula for calculating the incidence rate is: incidence rate = number of cases / total number of fruits × 100%.

[0057] 1.2.7 Detection of resistance-related indicators

[0058] 1.2.7.1 Determination of superoxide dismutase

[0059] Freeze-dry the strawberry fruit, take 0.5g of strawberry sample powder, add 1mL of phosphate buffer, centrifuge at 1000r / min at 4℃ for 20min, add 1.5mL of 0.05mol / L phosphate buffer, 0.3mL of 130mmol / L Met solution, 0.3mL of 750μmol / L NBT solution, 0.3mL of 100μmol / L EDTA-Na2 solution, 0.3mL of 20μmol / L riboflavin solution, 0.25μL of distilled water, and 0.05mL of enzyme solution in a finger-shaped tube. After mixing, place one tube as a control tube in a dark place. The rest are test tubes, which are placed in 4000Lx sunlight for reaction for 20min, and then the control tube is used as a blank, and the absorbance of each tube is measured at a wavelength of 560nm to calculate the superoxide dismutase activity.

[0060] 1.2.8 Determination of malondialdehyde content

[0061] Freeze-dry the strawberry fruit, take 0.5g of strawberry sample powder, add 5mL 10% TCA, centrifuge at 3000xg for 10min, take the supernatant. Add 3mL of supernatant to the measuring tube, add 3mL of water to the blank, add 3mL of 0.5% TBA solution to each, shake well and boil in a boiling water bath for 10min, immediately cool in cold water, use the blank as a reference, measure the sample absorbance at 450nm, 532nm, and 600nm, and calculate the malondialdehyde content.

[0062] 1.2.7.2 Determination of hydrogen peroxide content

[0063] The strawberry fruit was freeze-dried, 0.3 g of strawberry sample powder was weighed, 1.5 mL of acetone was added for ice bath homogenization, centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was collected. The hydrogen peroxide content kit of Gres Biotech was used to measure the absorbance according to the instructions and calculate the hydrogen peroxide content.

[0064] 1.2.7.3 Determination of ascorbic acid content

[0065] Freeze-dry the strawberry fruit, weigh 0.2g of strawberry sample powder and add 1mL of 5% TCA solution, let stand at 4℃ for 10min, and then centrifuge at 12000r / min for 5min. Pipette 25μL of supernatant and add 375μL of 5% TCA solution, then add 200μL of anhydrous ethanol and mix well, then add 100μL of 0.4% phosphoric acid-ethanol solution, 200μL of 0.5% BP-ethanol solution, 100μL of 0.03% FeCl3-ethanol solution, mix well, react at 30℃ for 60min, and then calculate the ascorbic acid content based on the standard curve.

[0066] 1.2.7.4 Determination of total phenols

[0067] The strawberry fruit was freeze-dried, 0.5 g of strawberry sample powder was weighed, 1 mL of 80% acetone solution was added and allowed to stand for 1 h, and then centrifuged at 4500 r / min for 10 min at 4 ° C. 12.5 μL of the supernatant diluted four times was taken, 250 μL of 2% Na2CO3 solution was added, and the mixture was shaken and placed for 5 min, 12.5 μL of 50% Folin phenol reagent was added, and the mixture was shaken and placed in the dark for 30 min, and then 80% acetone solution was used as a blank reference, and the absorbance was measured at a wavelength of 655 nm, and the total phenol content was calculated based on the standard curve.

[0068] 1.2.7.5 Flavonoids determination

[0069] Freeze-dry the strawberry fruit, weigh 0.5g of strawberry fruit powder, add 1mL of 80% acetone solution and let stand for 1h, then centrifuge at 4500r / min for 10min at 4℃. Take 30μL of supernatant, add 90μL of 95% ethanol solution, 6μL of 2% AlCl3 solution, 6μL of 1mol / LKAc solution, and then add 168μL of deionized water and shake well. After standing at room temperature and away from light for 40min, use 80% acetone solution as blank reference, measure the absorbance at a wavelength of 415nm, and calculate the flavonoid content based on the standard curve.

[0070] 1.3 Data analysis and processing

[0071] The experimental data were analyzed by variance analysis using IBM SPSS 26.0 software, with P < 0.05 as the significance level. Excel 2016 was used for data processing and drawing.

[0072] 2. Test results

[0073] 2.1 Spatiotemporal expression analysis

[0074] The gene expression patterns in plant tissues of different parts of strawberry and fruits at different developmental stages are shown in Figure 2. Figure 1 As shown in the figure, it can be seen that the gene is expressed in different tissue samples of strawberry, with the highest expression level in stems, followed by leaves, flowers and roots. In the fruit, except for the high expression level in the green period, the expression level in the white fruit, red slices and full red periods is low, only about 0.01 times the expression level in the roots. It is speculated that under normal circumstances, the gene mainly plays its physiological function in stems, leaves and flowers.

[0075] 2.2 Validation of transient gene injection experiments

[0076] The constructed overexpression vector and empty vector were injected into the 'Hongyan' strawberry fruit in the green stage for transient overexpression, and the phenotypic changes were observed 6 days after injection. Figure 2 As shown in A, it can be seen that overexpression has no significant effect on the phenotype of strawberry fruit. qPCR detection of gene expression levels in injected fruits was further performed, and the results are shown in Figure 2 As shown in B, it can be seen that the gene expression level in the fruit injected with the expression vector (OE) increased by 150 times compared with the expression level in the fruit injected with the empty vector (35S), indicating that the gene was successfully overexpressed in strawberry fruit.

[0077] 2.3 Effect of overexpressed genes on fruit resistance to gray mold

[0078] In order to detect whether transient overexpression is successful, the GFP fluorescent gene carried by the vector was used to detect the fluorescence intensity of the fruit after injection of the vector. The results showed that the fruit sample (OE) injected with the overexpression vector began to show strong fluorescence on the third day after injection ( Figure 3 A), while the fruit sample (35S) injected with the empty vector showed fluorescence on the 4th day after injection, indicating that the injected exogenous gene began to express at this time. And as the number of days after injection increased, the fluorescence intensity gradually increased. However, the strawberry fruit on the 4th day after injection was still not fully mature, which was not conducive to inoculation with Botrytis cinerea. Therefore, the fruit on the 5th day after injection was selected for inoculation with Botrytis cinerea. Figure 3 As shown in Figure B, 6 days after inoculation with Botrytis cinerea, all 24 fruits injected with the empty vector showed gray mold, with an incidence rate of 100%; while only 5 fruits of the overexpressed fruit showed disease, with an incidence rate of only 21%, which was 79% lower than that of the control ( Figure 3 C). This indicates that overexpression of this gene significantly enhances the resistance of strawberry fruit to gray mold, and this gene has the effect of enhancing the resistance of strawberry fruit to gray mold.

[0079] 2.4 Effects of overexpressed genes on resistance-related indicators

[0080] In order to preliminarily explore the mechanism of the effect of this gene on the resistance of strawberry fruit to gray mold, the resistance-related physiological indicators in the overexpression samples were measured. Figure 4 As shown in the figure, it can be seen that overexpression has no significant effect on the antioxidant enzyme activity (SOD), MDA, hydrogen peroxide and non-enzymatic antioxidants (including total phenols, flavonoids and ascorbic acid) in the fruit. This shows that the gene may not affect the gray mold resistance of strawberry fruit by regulating the strawberry antioxidant system and active oxygen content, and the overexpression of this gene has no significant effect on the quality of strawberry fruit and does not affect the market value of strawberry.

[0081] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene for enhancing the resistance of strawberry fruit to gray mold, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

7.

2. A protein encoded by the gene for enhancing the gray mold resistance of strawberry fruit according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

8.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene for enhancing the gray mold resistance of strawberry fruit as claimed in claim 1.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the gene for enhancing the gray mold resistance of strawberry fruit as described in claim 1 or the recombinant vector as described in claim 2.

5. Use of the gene for enhancing the gray mold resistance of strawberry fruit according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in enhancing the gray mold resistance of strawberry fruit or cultivating strawberry varieties with high resistance to gray mold.

6. A method for enhancing the resistance of strawberry fruit to gray mold, characterized in that: The method comprises the steps of using genetic transformation technology to transfer a gene that enhances the resistance of strawberry fruit to gray mold into strawberry plants so that the gene is stably overexpressed; The nucleotide sequence of the gene for enhancing the gray mold resistance of strawberry fruit is shown in SEQ ID NO.

7.

7. A method for cultivating a strawberry variety highly resistant to gray mold, characterized in that: The method comprises the steps of using genetic transformation technology to transfer genes that enhance the resistance of strawberry fruit to gray mold into strawberry plants to construct a strawberry variety with high resistance to gray mold; The nucleotide sequence of the gene for enhancing the gray mold resistance of strawberry fruit is shown in SEQ ID NO.

7.

8. A primer pair for amplifying the gene for enhancing the gray mold resistance of strawberry fruit according to claim 1, characterized in that: The primer pair includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.

2.

9. A primer pair for quantitatively detecting the expression amount of the gene for enhancing the gray mold resistance of strawberry fruit according to claim 1, characterized in that: The primer pair includes an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.

6.

10. Use of the primer pair according to claim 8 or the primer pair according to claim 9 in enhancing the resistance of strawberry fruit to gray mold or in breeding strawberry varieties with high resistance to gray mold.

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