A gene for enhancing the resistance of strawberry fruits to Botrytis cinerea and its application

Abstract

The present invention discloses a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea and its application, belonging to the technical field of genetic engineering. The nucleotide sequence of the gene of the present invention is as shown in SEQ ID NO.7. The present invention provides a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea. By introducing this gene into strawberry plants and enabling its stable overexpression, the resistance of strawberry fruits to Botrytis cinerea is significantly enhanced. Moreover, this gene has no impact on the quality of strawberry fruits, providing new gene resources and theoretical support for the breeding of strawberry varieties with high resistance to Botrytis cinerea.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea and its application. Background Art

[0002] China is a major country in strawberry production and consumption in the world, and the planting area has reached 173,000 hm 2 , and the annual output increase ranks among the top in crops, and the production accounts for more than 50% of the world's total output. However, strawberries are extremely vulnerable to the invasion of pathogenic bacteria during cultivation, transportation and storage, resulting in a decrease in yield, a deterioration in quality, and a serious damage to economic benefits. Botrytis cinerea is one of the fungal diseases that seriously affect the fruit yield of strawberries. It is caused by Botrytis cinerea and is a plant disease with a wide host range and a fast reproduction rate.

[0003] Current control measures for strawberry Botrytis cinerea include agricultural control, chemical control, biological control and physical control, etc. Agricultural control mainly improves the disease resistance of plants through measures such as reasonable close planting, keeping good ventilation in the field, increasing the application of organic fertilizers, and regularly cleaning diseased leaves and fruits. Chemical control uses medicine spraying for prevention and control, but long-term use of a single medicine is likely to cause the generation of drug resistance in the bacteria. Biological control uses antagonistic microorganisms to control Botrytis cinerea, but the control effect is greatly affected by environmental conditions. Physical control adopts measures such as mulching with plastic film 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 technologies, technicians have begun to explore the resistance mechanism of plants to Botrytis cinerea at the gene level, with a view to screening out genes with resistance to Botrytis cinerea, so as to cultivate high-resistant varieties to Botrytis cinerea. Based on this, the present invention has studied the resistance of strawberries to Botrytis cinerea and successfully screened out a gene that can enhance the resistance of strawberry fruits to Botrytis cinerea. Summary of the Invention

[0004] The purpose of the present invention is to provide a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea. By introducing this gene into strawberries and making it stably overexpressed, the resistance of strawberry fruits to Botrytis cinerea is significantly enhanced. Moreover, this gene has no influence on the quality of strawberry fruits, providing new gene resources and theoretical support for the breeding of strawberry varieties with high resistance to Botrytis cinerea.

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

[0006] The present invention provides a gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, and its nucleotide sequence is as shown in SEQ ID NO.7.

[0007] The present invention also provides a protein encoded by the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, and its amino acid sequence is shown as SEQ ID NO.8.

[0008] The present invention also provides a recombinant vector, which contains the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea as described above.

[0009] The present invention also provides a recombinant bacterium, which contains the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea or the recombinant vector as described above.

[0010] The present invention also provides the application of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, the protein, the recombinant vector or the recombinant bacterium as described above in enhancing the resistance of strawberry fruits to Botrytis cinerea or cultivating strawberry varieties highly resistant to Botrytis cinerea.

[0011] The present invention also provides a method for enhancing the resistance of strawberry fruits to Botrytis cinerea, including the step of using genetic transformation technology to transfer the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea into strawberry plants and enabling its stable overexpression;

[0012] The nucleotide sequence of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea is shown as SEQ ID NO.7.

[0013] The present invention also provides a method for cultivating strawberry varieties highly resistant to Botrytis cinerea, including the step of using genetic transformation technology to transfer the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea into strawberry plants to construct strawberry varieties highly resistant to Botrytis cinerea;

[0014] The nucleotide sequence of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea is shown as SEQ ID NO.7.

[0015] The present invention also provides a primer pair for amplifying the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, and the primer pair includes an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO.2.

[0016] The present invention also provides a primer pair for quantitatively detecting the expression level of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, and the primer pair includes an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO.6.

[0017] The present invention also provides the application of the primer pair in enhancing the resistance of strawberry fruits to Botrytis cinerea or cultivating strawberry varieties highly resistant to Botrytis cinerea.

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

[0019] The present invention provides a gene that enhances the resistance of strawberry fruits to Botrytis cinerea, and this gene has the function of regulating the resistance of strawberry fruits to Botrytis cinerea. By constructing an overexpression vector and introducing this gene into strawberries to achieve stable overexpression, the resistance of strawberry fruits to Botrytis cinerea is significantly enhanced, and the incidence rate can be reduced by 79%. Moreover, this gene has no significant effect on the contents of components such as SOD, MDA, hydrogen peroxide, total phenols, flavonoids, and ascorbic acid in strawberry fruits, and does not affect the quality of strawberry fruits, providing new gene resources and theoretical support for the breeding of strawberry varieties with high resistance to Botrytis cinerea. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0022] Figure 2 shows the verification results of the transient injection gene experiment; among them, A shows the phenotypes of overexpressed strawberry fruits (OE) and normal strawberry fruits (35S); B shows the gene expression in overexpressed strawberry fruits (OE) and normal strawberry fruits (35S).

[0023] Figure 3 shows the effect of overexpressing the gene on the resistance of strawberry fruits to Botrytis cinerea; among them, A shows the fluorescence detection results after treatment of overexpressed strawberry fruits (OE) and normal strawberry fruits (35S); B shows the disease incidence of overexpressed strawberry fruits (OE) and normal strawberry fruits (35S); C shows the incidence rate of Botrytis cinerea in overexpressed strawberry fruits (OE) and normal strawberry fruits (35S).

[0024] Figure 4 shows the effect of gene overexpression on related indexes in strawberry fruits. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all 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] The biological materials involved in the embodiments of the present invention, unless otherwise specified, can be purchased through conventional channels; the method steps involved, unless otherwise specified, are carried out by conventional methods in the art.

[0034] Example 1

[0035] 1. Materials and Methods

[0036] 1.1 Plant Materials

[0037] Plant tissues from different parts and fruits at different developmental stages of the octoploid cultivated strawberry 'Benihoppe' (Fragaria×ananassa 'Benihoppe') 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 method

[0039] 1.2.1 Gene cloning

[0040] Extract the total RNA of the collected plant materials. The total RNA with qualified quality detected by electrophoresis is reverse transcribed into cDNA using the reverse transcription kit of TaKaRa. Using the cf and cR primer pairs in Table 1, PCR amplification is carried out using the high-fidelity enzyme I-5TM 2×High-Fidelity Master Mix (Beijing Qingke New Industry Biotechnology Co., Ltd.). A 50 μL reaction system is adopted, including 25 μL of high-fidelity enzyme, 1 μL of each upstream and downstream primer, 1 μL of cDNA, and made up to 50 μL with sterilized water. After mixing, it is placed on a PCR instrument for amplification. The program is set as follows: pre-denaturation at 98 °C for 3 min, denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, extension at 72 °C for 20 s, 35 cycles, and final extension at 5 min. After the PCR product is detected by electrophoresis, the target band is recovered and ligated to the pEASY-Blunt cloning vector, transformed into Escherichia coli Trans1-T1, and the positive clones are screened and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0041] After sequencing, the CDS sequence of this gene 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] MAATWLEALPAQHKRLIMNELVHGRNRARELQSLLNNRGHGSTSSRSEEELVMEIVKSFSESLSVLSESSAKFGGDDQQYSGTTGCGGGETVKAEQSHVEHSHCGDRSFEDSGESKKRPGVKDRRGCYKRRKNSESWATVSSTVEDGQAWRKYGQKEILNAPYPRAYFRCTRKYDQGCQATKQVQQTQDTPKLYKTTYIGNHTCRMIRAPQMIMGSSHPPALDSHAPPRATVSSESGSTPVCNKKEHHGDHGGHLSRSSSILPVKKEESKEGTTTSSGLTDNLNDTSDMWPGFDLGFPEAETTVLSDENVVSNMPFLDMHLVKSIDDFESEFDFDQVY*。

[0045] 1.2.2 Spatial and temporal expression analysis

[0046] The SYBR Green I real-time fluorescence quantitative PCR (qPCR) system was used to detect the expression levels of this gene in different tissues and at different fruit development stages. Using the qF and qR primer pairs in Table 1, a 10 μL reaction system was adopted: 2 μL of cDNA, 5 μL of SYBRgreen mix, 0.4 μL of each upstream and downstream primer, and 2.2 μL of ddH2O. After mixing and centrifugation, the reaction was carried out on the CFX96 qPCR system. The reaction program was: 95°C for 30 s; (95°C for 5 s, 55°C for 30 s, 72°C for 30 s, fluorescence signal was collected, a total of 40 cycles); the melting curve was inserted, 95°C for 15 s, cooled to 60°C and maintained for 30 s, and the temperature was increased by 0.5°C step by step starting from 65°C, and the fluorescence signal was collected until the reaction ended at 95°C. Using the strawberry Actin (accession number: LC017712) gene as an internal reference, each sample had three biological replicates, and each replicate had two technical replicates in two wells. The quantitative results were calculated by the 2 -△△CT method, and variance analysis was performed using SPSS 25.0. The data were presented as mean ± standard deviation.

[0047] 1.2.3 Overexpression vector construction

[0048] Using the OE-TF and OE-TR primer pairs in Table 1, with the plasmid extracted from the positive colonies screened in 1.2.1 as the template, PCR amplification was carried out using a high-fidelity enzyme. The amplification product was mixed with the linearized pCAMBIA1302 vector and reacted for homologous recombination. Then, it was transformed into competent Escherichia coli cells by heat shock method. After expanded culture, single colonies were picked for PCR identification, and the positive clones were sent to a biological company for sequencing. The returned positive bacterial liquid was used to extract the overexpression plasmid by alkaline lysis method and stored at -20 °C for standby.

[0049] 1.2.4 Agrobacterium transformation

[0050] The overexpression plasmid and the empty vector (pCAMBIA1302 vector) verified by sequencing were transferred into competent cells of Agrobacterium tumefaciens GV3101 (preserved in the laboratory) by the freeze-thaw method. The specific operation is as follows: Add 100 ng of the plasmid to 100 μL of competent cells and mix well. Place it on ice for 30 min, then quickly freeze it in liquid nitrogen for 5 min, and then incubate it in a water bath at 28 °C for 5 min. Add 800 μL of YEB liquid medium, and incubate it at 28 °C with shaking at 150 - 200 r / min for about 5 h. Centrifuge at 10000×g for 30 s, discard the supernatant, and leave about 100 μL of YEB liquid medium. Pipette and mix well, then spread it on YEB solid medium containing rifampicin (Rif; 100 mg / L), gentamycin sulfate (Gen; 40 mg / L), and kanamycin (Kan; 100 mg / L), and culture it in the dark at 28 °C for 48 - 36 h.

[0051] 1.2.5 Transient injection of strawberry fruits

[0052] (1) Preparation of infection solution: The Agrobacterium tumefaciens transformed with the overexpression plasmid and the Agrobacterium tumefaciens transformed with the empty vector were respectively added to 100 mL and 50 mL of LB liquid medium and cultured overnight at 28 °C. When the OD 600 value was about 0.8 - 1.0, the cells were collected by centrifugation (5000 r / min, 5 min, 20 °C). The supernatant was poured off, and 30 mL of infiltration buffer (10 mmol / L MES, 10 mmol / L MgCl2, 20 μmol / L acetosyringone) was added. Incubate in the dark at 50 r / min at 28 °C for 4 h. After taking out, centrifuge at room temperature to collect the bacterial cells and resuspend them with the buffer to obtain the overexpression infection solution and the empty vector infection solution.

[0053] (2) Infecting the fruits: Wash the detached strawberry fruits successively with distilled water, 70% ethanol, and sterile water for disinfection, gently dry the water on the fruit surface, wrap the strawberry stem end with sterile wet cotton, then slowly inject the overexpression infection solution or the empty vector infection solution from the fruit stalk with a 1 mL syringe, then wipe the fruit surface clean and place it in a box, and culture it in a constant temperature incubator for six days, keeping the moisture of the cotton at the stem end during this period.

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

[0055] 1.2.6 Inoculation with Botrytis cinerea

[0056] Take the Botrytis cinerea plate that has been cultured for 45 days, add about 5 mL of sterile water, gently scrape the mycelium on the surface of the culture medium into the sterile water with a sterile scraper, place it on a vortex oscillator and shake it to fully suspend the spores. Filter the suspension through a sterilized four-layer medical gauze into another sterilized 50 mL centrifuge tube, and rinse the filter residue 2 - 3 times with a small amount of sterile water. Adjust the concentration of the Botrytis cinerea suspension to about 1×10 5 cells / mL. On the 5th day after injecting the overexpression vector or the empty vector, use a disinfected inoculation needle to pierce a small hole about 0.5 cm in the middle of the strawberry fruit, and drop 5 μL of the spore suspension. There are 24 fruits in each treatment, and observe the disease incidence. Incidence calculation formula: Incidence = Number of diseased fruits ÷ 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 fruits, take 0.5 g of strawberry sample powder, add 1 mL of phosphate buffer solution, centrifuge at 1000 r / min for 20 min at 4°C. In a finger tube, add 1.5 mL of 0.05 mol / L phosphate buffer solution, 0.3 mL of 130 mmol / L Met solution, 0.3 mL of 750 μmol / L NBT solution, 0.3 mL of 100 μmol / L EDTA-Na2 solution, 0.3 mL of 20 μmol / L riboflavin solution, 0.25 μL of distilled water, and 0.05 mL of enzyme solution. After mixing, use one tube as the control tube and place it in the dark. The rest are the determination tubes, place them under a 4000 Lx sunlight for reaction for 20 min, then use the control tube as the blank, measure the absorbance of each tube at a wavelength of 560 nm, and calculate the superoxide dismutase activity.

[0060] 1.2.8 Determination of malondialdehyde content

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

[0062] 1.2.7.2 Determination of hydrogen peroxide content

[0063] Freeze-dry strawberry fruits, weigh 0.3 g of strawberry sample powder, add 1.5 mL of acetone and homogenize in an ice bath, centrifuge at 4 °C and 12000 rpm for 10 min, and take the supernatant. Use the hydrogen peroxide content kit of Grees Biotechnology Co., Ltd., 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 strawberry fruits, weigh 0.2 g of strawberry sample powder, add 1 mL of 5% TCA solution, let stand at 4 °C for 10 min, and then centrifuge at 12000 r / min for 5 min. Pipette 25 μL of the supernatant, add 375 μL of 5% TCA solution, then add 200 μL of absolute ethanol and mix well. Then add 100 μL of 0.4% phosphoric acid-ethanol solution, 200 μL of 0.5% BP-ethanol solution, and 100 μL of 0.03% FeCl3-ethanol solution, mix well and react at 30 °C for 60 min, and then calculate the ascorbic acid content according to the standard curve.

[0066] 1.2.7.4 Determination of total phenols

[0067] Freeze-dry strawberry fruits, weigh 0.5 g of strawberry sample powder, add 1 mL of 80% acetone solution and let stand for 1 h, then centrifuge at 4 °C and 4500 r / min for 10 min. Take 12.5 μL of the supernatant diluted four times, add 250 μL of 2% Na2CO3 solution, shake well and let stand for 5 min, add 12.5 μL of 50% Folin-Ciocalteu reagent, shake well and keep in the dark for 30 min, then use 80% acetone solution as the blank reference, measure the absorbance at 655 nm wavelength, and calculate the total phenol content according to the standard curve.

[0068] 1.2.7.5 Determination of flavonoids

[0069] Freeze-dry strawberry fruits, weigh 0.5 g of strawberry fruit powder, add 1 mL of 80% acetone solution, let it stand for 1 h, and then centrifuge at 4500 r / min for 10 min at 4 °C. Pipette 30 μL of the supernatant, add 90 μL of 95% ethanol solution, 6 μL of 2% AlCl3 solution, 6 μL of 1 mol / L KAc solution, and then add 168 μL of deionized water and shake well. After standing at room temperature in the dark for 40 min, use 80% acetone solution as the blank reference, measure the absorbance at a wavelength of 415 nm, and calculate the flavonoid content according to the standard curve.

[0070] 1.3 Data analysis and processing

[0071] The experimental data was analyzed by IBM SPSS 26.0 software at a significant level of P < 0.05. Excel 2016 was used for data sorting and plotting.

[0072] 2. Experimental results

[0073] 2.1 Spatial and temporal expression analysis

[0074] The gene expression in different plant tissues of strawberry and fruits at different developmental stages is as Figure 1 shown. It can be seen that the gene is expressed in different tissue samples of strawberry, with the highest expression level in the stem, followed by the leaf, flower, and root. In the fruit, except for a relatively high expression level during the large green stage, the expression levels during the white fruit, partial red, and full red stages are relatively low, only about 0.01 times that in the root. It is speculated that under normal circumstances, this gene mainly plays its physiological functions in the stem, leaf, and flower.

[0075] 2.2 Verification of transient injection gene experiment

[0076] Inject the constructed overexpression vector and empty vector into the fruits of 'Hongyan' strawberries at the large green stage for transient overexpression, and observe the phenotypic changes 6 days after injection. The results are as Figure 2 shown in A. It can be seen that overexpression has no significant effect on the phenotype of strawberry fruits. Further, qPCR was used to detect the gene expression level in the injected fruits. The results are as Figure 2 shown in B. It can be seen that the gene expression level in the fruits injected with the overexpression vector (OE) increased by 150 times compared with that in the fruits injected with the empty vector (35S), indicating that the gene was successfully overexpressed in strawberry fruits.

[0077] 2.3 Effect of overexpressing the gene on fruit resistance to Botrytis cinerea

[0078] To detect whether transient overexpression was successful, the fluorescence intensity of the fruits after injecting the vector was detected using the GFP fluorescent gene carried on the vector. The results showed that strong fluorescence began to appear in the fruit samples (OE) injected with the overexpression vector on the 3rd day after injection ( Figure 3 A), while fluorescence appeared in the fruit samples (35S) injected with the empty vector on the 4th day after injection, indicating that the exogenous gene injected at this time began to express. And with the increase in the number of days after injection, the fluorescence intensity gradually increased. However, the strawberry fruits on the 4th day after injection were still not fully ripe, which was not conducive to inoculating Botrytis cinerea. Therefore, the fruits on the 5th day after injection were selected for inoculating Botrytis cinerea. As shown in Figure 3 B, 6 days after inoculating Botrytis cinerea, all 24 fruits injected with the empty vector showed gray mold, and the incidence rate was 100%; only 5 fruits of the overexpressed fruits were diseased, and the incidence rate was only 21%, which was 79% lower than that of the control ( Figure 3 C). It shows that overexpressing this gene significantly enhanced the resistance of strawberry fruits to gray mold, and this gene has the effect of enhancing the resistance of strawberry fruits to gray mold.

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

[0080] To preliminarily explore the mechanism of the effect of this gene on the resistance of strawberry fruits to gray mold, the physiological indicators related to resistance in the overexpressed samples were measured. The results are as shown in Figure 4 . It can be seen that overexpression had 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 fruits. It shows that this gene may not affect the resistance of strawberry fruits to gray mold by regulating the antioxidant system and the content of reactive oxygen species in strawberries, and the overexpression treatment of this gene had no significant effect on the quality of strawberry fruits and did not affect the market value of strawberries.

[0081] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene for enhancing the resistance of strawberry fruits to Botrytis cinerea, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

7.

2. The protein encoded by the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea as described in 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 resistance of strawberry fruits to Botrytis cinerea as claimed in claim 1.

4. A recombinant bacterium, characterized in that, The recombinant bacterium contains the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea as claimed in claim 1 or the recombinant vector as claimed in claim 3.

5. Use of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea as claimed in claim 1, the protein as claimed in claim 2, the recombinant vector as claimed in claim 3 or the recombinant bacterium as claimed in claim 4 in enhancing the resistance of strawberry fruits to Botrytis cinerea or cultivating strawberry varieties highly resistant to Botrytis cinerea.

6. A method for enhancing the resistance of strawberry fruits to Botrytis cinerea, characterized in that, It includes the step of using genetic transformation technology to transfer the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea into strawberry plants and enabling its stable overexpression; The nucleotide sequence of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea is shown in SEQ ID NO.

7.

7. A cultivation method for a strawberry variety highly resistant to Botrytis cinerea, characterized in that, It includes the step of using genetic transformation technology to transfer the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea into strawberry plants to construct strawberry varieties highly resistant to Botrytis cinerea; The nucleotide sequence of the gene for enhancing the resistance of strawberry fruits to Botrytis cinerea is shown in SEQ ID NO.

7.

8. A primer pair for amplifying the gene enhancing the resistance of strawberry fruits to Botrytis cinerea described in claim 1, characterized in that, The primer pair includes an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

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