An RcWRKY29 gene and its application in enhancing rose resistance to gray mold.

Abstract

This invention discloses a RcWRKY29 Genes and their application in enhancing rose resistance to gray mold RcWRKY29 The nucleotide sequence of the gene is shown in SEQ ID NO:1. Studies have found that this gene is specifically highly expressed in rose petals, and its expression level significantly increases within 24-48 hours after Botrytis cinerea infection. Transient inhibition was achieved using virus-induced gene silencing technology. RcWRKY29 After expression, the area of ​​lesions on rose petals increases, and disease resistance is significantly reduced; conversely, overexpression of this gene can significantly enhance disease resistance and reduce lesion area and cell damage. This invention provides a [container name missing] gene. RcWRKY29 This invention developed recombinant vectors, engineered bacteria, and detection kits, and employed Agrobacterium-mediated transformation technology to enhance the disease resistance of roses. This invention provides a new target for molecular breeding of roses to resist gray mold, effectively reducing post-harvest disease losses, and has significant economic value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to an RcWRKY29 gene and its application in enhancing the resistance of rose (Rosa hybrida) to gray mold. Background Technology

[0002] The rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant belonging to the genus Rosa in the family Rosaceae. Roses are characterized by their elegant flower shape, vibrant colors, and rich fragrance, possessing high ornamental, economic, and cultural value. They rank first among the world's four major cut flowers and are widely used in landscaping, garden decoration, potted plants, and the fragrance and cosmetics industries.

[0003] Cut roses are one of the four major cut flowers. Gray mold is the most damaging fungal disease affecting cut roses during post-harvest transportation, severely damaging their ornamental and economic value. Cut roses are highly susceptible to gray mold during long-distance transport, with approximately 15-40% losing their economic value due to post-harvest spoilage. Chemical, physical, biological, and agricultural control methods all have drawbacks. Therefore, fundamentally controlling gray mold in roses should begin with genetic breeding to combat it. By studying the interaction between roses and gray mold, we can discover and cultivate highly resistant rose varieties, reducing economic losses to the cut rose industry caused by gray mold. Currently, the specific mechanism of action of the RcWRKY29 gene in rose resistance to gray mold is unclear; therefore, it is necessary to study its function through gene editing or transgenic technology. Summary of the Invention

[0004] To address the problems of existing technologies, the purpose of this invention is to provide an RcWRKY29 gene and its application in enhancing the resistance of Rosa hybrida to gray mold. Overexpression of this gene enhances the resistance of roses to gray mold, leading to the cultivation of highly resistant new rose varieties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this application provides the application of an expression product of the RcWRKY29 gene or its transcribed and translated protein in enhancing the resistance of roses to gray mold, wherein the nucleotide sequence of the RcWRKY29 gene is shown in SEQ ID NO.1.

[0007] The second aspect of this application provides a method for enhancing the resistance of roses to gray mold, characterized by the following steps: (1) constructing a recombinant vector containing the RcWRKY29 gene;

[0008] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcWRKY29 gene.

[0009] Further, in step (1), the vector primer design and vector construction are as follows: using EcoRI and KpnI as restriction sites, the silenced fragment sequence of RcWRKY29 is inserted into the empty TRV2 vector. Primers are designed using homologous recombination to construct the TRV-RcWRKY29 vector. The nucleotide sequence of the silenced fragment of the RcWRKY29 gene is shown in SEQ ID NO:3.

[0010] Further, in step (2), SmaI and KpnI were selected as restriction enzyme sites, and the RcWRKY29 gene sequence was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the vector.

[0011] The RcWRKY29-GFP vector, a recombinant vector, was introduced into rose cells via Agrobacterium-mediated transformation.

[0012] Beneficial Effects: This invention identified a disease resistance regulatory gene, RcWRKY29, from the genome of the rose variety 'Yueyuefen'. The gene was found to be highly expressed in rose petal tissues but low in root and leaf tissues. Analysis of RcWRKY29 expression at different time points after gray mold infection of rose flowers revealed a significant increase in RcWRKY29 expression for a certain period following infection. Therefore, this application hypothesizes that RcWRKY29 may be involved in regulating the resistance of roses to gray mold.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) Based on the expression results of RcWRKY29 in rose petals, the present invention transiently silences and overexpresses RcWRKY29 in petals and finds that abnormal expression of RcWRKY29 affects the resistance of rose petals to gray mold. Overexpression of RcWRKY29 can promote the resistance of petals to gray mold, while silencing it will inhibit the resistance of petals to gray mold.

[0015] (2) In this invention, the RcWRKY29 gene was silenced and overexpressed in rose petals that were in good growth condition and free from gray mold infection. The results showed that the petals treated with silenced RcWRKY29 had larger lesion areas and reduced resistance to gray mold; the petals treated with overexpressed RcWRKY29 had enhanced resistance to gray mold. In conclusion, RcWRKY29 is of great significance in regulating the resistance of roses to gray mold. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The expression analysis diagram of RcWRKY29 provided for this invention; Rt: root; St: stem segment; Lf: leaf; Fl: petal.

[0018] Figure 2 This is a subcellular localization map of RcWRKY29 provided by the present invention; Note: Subcellular localization in tobacco leaves was achieved by co-transfecting RcWRKY29-GFP with DAPI (nuclear marker) into tobacco leaves and observing the fluorescence signal using a confocal microscope.

[0019] Figure 3 The graph shows the change in the expression level of RcWRKY29 over time after infection with Botrytis cinerea, as provided in this invention.

[0020] Figure 4 The gene expression levels of TRV and RcWRKY29 silencing on day 3 after transient silencing of RcWRKY29 provided by this invention ( Figure 4 B) and petal phenotypic diagram (each petal disc is 1.5cm in diameter) Figure 4 A); The area of ​​petal lesions silencing TRV and RcWRKY29 on the 3rd day after transient silencing of RcWRKY29 (A); Figure 4 C) and particle leakage rate statistics ( Figure 4 D).

[0021] Figure 5 The present invention provides pSuper1300 and pSuper1300 on day 3 after transient overexpression of RcWRKY29.

[0022] RcWRKY29 overexpression level ( Figure 5 B) and petal phenotypic diagram ( Figure 5 A); The area of ​​petal lesions overexpressing pSuper1300 and RcWRKY29 on day 3 after transient overexpression of RcWRKY29 ( Figure 5 C) and ion leakage rate statistics ( Figure 5 D). Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In this application, "-one less" means one or more, and "more than" means two or more. "-one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "-one less item (item) in a, b, or c", or "-one less item (item) in a, b, and c", can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0026] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0027] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0028] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0029] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0030] This application provides an application of the RcWRKY29 gene or a product expressing its transcribed and translated protein in enhancing the resistance of roses to gray mold. The nucleotide sequence of the RcWRKY29 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the RcWRKY29 gene is shown in SEQ ID NO.2.

[0031] The second aspect of this application provides a method for enhancing the resistance of roses to gray mold, comprising the following steps: (1) constructing a recombinant vector containing the RcWRKY29 gene;

[0032] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcWRKY29 gene.

[0033] In some embodiments, in step (1), the vector primer design and vector construction are as follows: using EcoRI and KpnI as restriction sites, the silenced fragment sequence of RcWRKY29 is inserted into the empty TRV2 vector, and primers are designed using homologous recombination to construct the TRV-RcWRKY29 vector. The nucleotide sequence of the silenced fragment of the RcWRKY29 gene is shown in SEQ ID NO:3.

[0034] In some embodiments, in step (2), SmaI and KpnI are selected as restriction enzyme sites, and the enzyme is digested by...

[0035] The RcWRKY29 gene sequence was inserted into the pSuper-1300 vector, and primers were designed using homologous recombination to construct the RcWRKY29-GFP vector. The recombinant vector was introduced into rose cells via Agrobacterium-mediated transformation.

[0036] Example 1

[0037] This invention relates to the application of an expression product of the RcWRKY29 gene or its transcribed and translated protein in enhancing the resistance of roses to gray mold. The nucleotide sequence of the RcWRKY29 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the RcWRKY29 gene is shown in SEQ ID NO.2.

[0038] A method for enhancing the resistance of roses to gray mold according to the present invention includes the following steps: (1) constructing a recombinant vector containing the RcWRKY29 gene; vector primer design and vector construction: using EcoRI and KpnI as restriction sites, the silent fragment sequence of RcWRKY29 is inserted into the empty vector of TRV2, primers are designed using homologous recombination, and the TRV-RcWRKY29 vector is constructed, wherein the nucleotide sequence of the silent fragment of the RcWRKY29 gene is shown in SEQ ID NO:3.

[0039] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcWRKY29 gene. SmaI and KpnI were selected as restriction sites, and the RcWRKY29 gene sequence was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RcWRKY29-GFP vector. The recombinant vector was introduced into rose cells by Agrobacterium-mediated transformation.

[0040] Example 2

[0041] This application is based on the genome sequence of the rose variety 'Yueyuefen'. Previous analysis of the WRKY family members identified the potential disease resistance regulatory gene RcWRKY29, and further discovered the gene...

[0042] The expression of the RcWRKY29 gene significantly increases within a certain period after gray mold infection. Transient silencing or overexpression of the RcWRKY29 gene revealed significant changes in the resistance of rose petals to gray mold. Therefore, this application reveals the biological function of the RcWRKY29 gene in rose resistance to gray mold, providing technical support for enhancing rose resistance to gray mold, promoting research in rose functional genomics, gene editing breeding, and transgenic breeding, discovering and cultivating highly resistant new rose varieties, and providing a theoretical basis for the integrated control of gray mold and the cultivation of new rose varieties.

[0043] The abbreviations and their corresponding names that appear in this application are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] 1. Plant materials

[0048] 1.1'Monthly Powder'

[0049] 'Yueyuefen' was taken from the Jinning Baofeng Base of the Flower Research Institute of Yunnan Academy of Agricultural Sciences. Different tissue sections were used for tissue-specific analysis. 'Kaluola' is a modern cut flower rose variety. It was taken from the Baofeng Base of the Flower Research Institute of Yunnan Academy of Agricultural Sciences. Petals from the S3 stage were used for silencing and expression verification experiments.

[0050] 1.2 Tobacco

[0051] The subcellular localization and transcriptional activation experiments used Nicotiana benthamiana as the experimental material. The seeds were sown in a moist nutrient substrate, covered with a film, and cultured in a culture room.

[0052] Cultivation conditions: Temperature 24±1℃, relative humidity 60-65%, photoperiod 16h / 8h

[0053] 1.3 Strains and Vectors

[0054] Escherichia coli DH5α, Agrobacterium tumefaciens strain EHA105, and pSuper-1300 (Kan resistant) were all purchased from Beijing Qingke Biotechnology Co., Ltd. The VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0055] 1.4 Culture medium formulations involved in the case study

[0056] (1) LB medium and YEB medium (Table 2)

[0057] Table 2

[0058]

[0059]

[0060] (2) Experimental culture media (Table 3)

[0061] Table 3

[0062]

[0063] 2. Research Methods

[0064] 2.1 Extraction of total RNA

[0065] Total RNA was extracted from the roots, stems, leaves, and petals of 'Yueyuefen' using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (centrifuge column type).

[0066] 2.2cDNA synthesis

[0067] (1) The genomic DNA removal reaction system is shown in Table 4:

[0068] Table 4

[0069]

[0070] Mix well, incubate at 42°C for 2 minutes.

[0071] (2) Preparation of the reverse transcription reaction system (Table 5)

[0072] Table 5

[0073]

[0074] Mix thoroughly by blowing and heating at 37°C for 15 min; then at 85°C for 5 s. Store the product at -20°C.

[0075] 2.3 Real-time quantitative PCR

[0076] Gene-specific primers were designed using Primerpremier5 (Table 6). The cDNA was diluted four-fold with ddH2O, and Real-Time PCR amplification was performed using the cDNA obtained from reverse transcription as a template. Three biological replicates were set up using UBI2 as an internal control.

[0077] Table 6

[0078]

[0079] The RT-qPCR reaction system is shown in Table 7:

[0080] Table 7

[0081]

[0082] The RT-qPCR reaction procedure is shown in Table 8:

[0083] Table 8

[0084]

[0085] 2.4 Vector Construction

[0086] 2.4.1 PCR amplification of the target gene fragment

[0087] The high-fidelity enzyme (Phusion™ Plus PCR Master Mix) was used for PCR amplification of the target gene. The PCR amplification reaction system is shown in Table 9.

[0088] Table 9

[0089]

[0090] The reaction procedure is shown in Table 10:

[0091] Table 10

[0092]

[0093] After amplification, 1% gel electrophoresis was performed, and the target band was selected for subsequent gel recovery.

[0094] 2.4.2 Glue Recycling

[0095] The PCR products were recovered and purified using a DNA gel extraction kit (TaKaRa MiniBESTAgarose Gel DNAExtraction Kit Ver. 4.0).

[0096] 2.4.3 Vector double enzyme digestion

[0097] Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-digested with the corresponding enzyme.

[0098] The enzyme digestion system is shown in Table 11:

[0099] Table 11

[0100]

[0101] After adding the system reagents on ice, perform double digestion of the vector according to the specific enzyme denaturation temperature.

[0102] 2.4.4 Homologous recombination

[0103] The double-digested vector was then subjected to homologous recombination with the cloned target gene fragment to construct the vector. The homologous recombination system is shown in Table 12:

[0104] Table 12

[0105]

[0106] Procedure: Run the PCR machine at 50℃ for 15 minutes.

[0107] 2.4.5 Escherichia coli transformation

[0108] (1) Remove the competent states from -80℃ and melt them in ice;

[0109] (2) Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of LDH5α competent cells, mix by pipetting, and let stand on ice for 30 min.

[0110] (3) Heat shock in a 42℃ water bath for 90 seconds, then quickly transfer to ice and let stand for 2 minutes;

[0111] (4) Add 500 μL LB to the centrifuge tube, incubate at 37°C and 200 rpm for 1 hour;

[0112] (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min;

[0113] (6) Discard 400 μL of supernatant in a clean bench, mix thoroughly by pipetting, spread on LB solid medium containing antibiotics, and incubate overnight at 37°C with the medium inverted.

[0114] 2.4.6 Microbial Detection and Sequencing

[0115] (1) Shaking culture: Pick a single colony that has grown overnight and shake it in 500 μL of LB medium containing antibiotics for 3-4 h (37℃, 200 rpm).

[0116] (2) Bacterial culture PCR: The cultured bacterial culture was used for PCR amplification to detect whether the target band of the constructed vector met expectations. The bacterial culture PCR amplification system is shown in Table 13:

[0117] Table 13

[0118]

[0119]

[0120] The bacterial culture PCR amplification reaction procedure is shown in Table 14:

[0121] Table 14

[0122]

[0123] The amplified products were used for 1% gel electrophoresis imaging, and the band sizes, which met expectations, were sent to the company for sequencing.

[0124] 2.5 Plasmid Extraction

[0125] After the test results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation and plasmid extraction is carried out using a plasmid extraction kit (TaKaRaMiniBEST PlasmidPurificationKitVer.4.0).

[0126] 2.6 Agrobacterium-mediated transformation

[0127] (1) When the competent state of the straw is taken out of the -80℃ freezer and melted into an ice-water mixture, it is inserted into ice;

[0128] (2) Add 0.01-1 μg plasmid DNA to each 100 μL competent cells, mix well and then incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min.

[0129] (3) Add 500 μL of antibiotic-free YEB liquid culture medium and incubate at 28°C with shaking for 5 h;

[0130] (4) Centrifuge at 5000 rpm for 2 min, discard 400 μL of supernatant in a clean bench, spread it onto YEB solid medium containing antibiotics, and incubate upside down at 28℃ for 5 days.

[0131] 2.7 Instantaneous infection

[0132] (1) Vector and primer design and vector construction

[0133] Using EcoRI and KpnI as restriction enzyme sites, the silent fragment (SEQ ID NO:3) sequence of RcWRKY29 was inserted into the empty TRV2 vector. Primers were designed using homologous recombination to construct the TRV-RcWRKY29 vector.

[0134] SmaI and KpnI were selected as restriction enzyme sites. The CDS sequence of RcWRKY29 was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RcWRKY29-GFP vector. After obtaining Agrobacterium-positive strains, the following experiments were performed.

[0135] (2) Bacterial culture

[0136] Streak the bacterial culture on agar plates (containing 50 mg / L Kan / Rif) and incubate upside down at 28°C for 5 days. Pick a single colony and gently shake it into 500 μL of YEB containing antibiotics for bacterial testing; if the bands are correct, perform medium and large shaking (28°C, 200 rpm).

[0137] (3) Collection and resuspension of bacteria

[0138] Centrifuge at 5000 rpm for 8 minutes to collect bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix thoroughly by pipetting, and adjust to OD600 = 1.0. For transient silencing experiments, mix TRV1, TRV2, and TRV2-RcWRKY29 bacterial cultures at a 1:1 volume ratio and incubate in the dark for 4-6 hours. For transient overexpression, [the following steps are not specified in the original text].

[0139] Both Super-1300 and Super-RcWRKY29 can be left to stand for bacterial collection.

[0140] (4) Vacuum suction

[0141] Vacuum pumps were used to inoculate 1.5 cm diameter petal discs made from the outermost petals of 'Carola' S3 grade flowers. The pressure was 0.082 MPa, aspirated for 10 min, held for 10 min, and then degassed for 10 min, ensuring the entire petal disc was fully immersed in the bacterial solution. This process was repeated three times. After inoculation, the discs were rinsed with sterile water and placed in 1% agar plates. They were incubated in the dark at 8°C for 3 days, then transferred to a tissue culture room at (22±1)°C with a light / dark cycle of 16 h and 8 h respectively. Observation continued until all petal discs showed complete color change. Petal phenotypes were photographed, and the color changes were measured and statistically analyzed using ImageJ 15.1 software.

[0142] 2.8 Data Statistical Analysis

[0143] Data used for statistical analysis were obtained from three biological replicates and three technical replicates. Data were statistically analyzed using GraphPad Prism 9.5 software. The student-t test was used to compare the two groups (*P<0.05, **P<0.01, ***P<0.001).

[0144] 2.10 Primers used in the RcWRKY29 sequence experiment. The RcWRKY29 sequence and primer list are shown in Table 15:

[0145] Table 15

[0146]

[0147]

[0148] Example 3

[0149] Validation and subcellular localization of RcWRKY29 in different tissue sites

[0150] Total RNA was extracted from rose petals, leaves, roots, and stems and its quality was assessed. Genes with the most cis-regulatory elements related to meristem and plant growth and development were selected, and quantitative fluorescence expression analysis revealed differences in gene expression across different tissues. Figure 1 Among them, RcWRKY29 was expressed specifically and at a high level in the petals, indicating that RcWRKY29 may play a certain role in the disease resistance of rose petals.

[0151] Simultaneously, the CDS sequence of RcWRKY29 was cloned, and the RcWRKY29-GFP vector was constructed. Using the empty GFP vector as a control, the vector was transformed into Agrobacterium EHA105 and injected into Tobacco Benzoenta leaves. After 3 days, observation under a laser confocal microscope showed that RcWRKY29-GFP was localized in the cell nucleus (…). Figure 2 ).

[0152] Example 4

[0153] Silent RcWRKY29 reduces resistance to gray mold on flower petals.

[0154] First, the expression level of RcWRKY29 after infection with Botrytis cinerea was detected over time. Figure 3 The study found that the expression level of RcWRKY29 gradually increased 24-48 hours after infection with gray mold, indicating that RcWRKY29 is related to the resistance of rose petals to gray mold.

[0155] To confirm the function of RcWRKY29 in the resistance of rose petals to gray mold, RcWRKY29 in 'Carola' rose plants was silenced using VIGS technology, and gray mold fungus was injected into the petals. Observations were conducted 3 days after infection, and it was found that the area of ​​lesions on petals with the gene silenced (TRV2-RcWRKY29) was larger than that of the control (TRV). Figure 4 A, Figure 4 C) The relative particle leakage rate was measured to demonstrate the extent of damage to petals in gene-silenced (TRV2-RcWRKY29) and control (TRV) petals. Compared with the control, the relative particle leakage rate of petals increased after gene silencing. Figure 4 D). The expression levels of RcWRKY29 in petals of both the silenced gene (TRV2-RcWRKY29) and the control gene (TRV) were simultaneously detected. Figure 4 B) The results showed that the expression level of RcWRKY29 was significantly reduced in silent plants compared with the control (TRV), which further illustrates that RcWRKY29 is associated with resistance to gray mold in rose petals.

[0156] Example 5

[0157] Overexpression of RcWRKY29 enhances resistance to gray mold on flower petals.

[0158] To confirm the function of RcWRKY29 in the resistance of rose petals to gray mold, RcWRKY29 overexpressing in 'Carola' rose plants was transiently infected, and gray mold fungus was injected into the petals. Observations were performed 3 days post-infection, and it was found that the petal lesion area of ​​the overexpressed gene (pSuper1300-RcWRKY29) was smaller than that of the control (pSuper1300). Figure 5 A, Figure 5 C) The relative particle leakage rate was measured to demonstrate the degree of damage to petals in gene overexpression (pSuper1300-RcWRKY29) and the control (pSuper1300). Compared with the control, the relative particle leakage rate of petals decreased after gene overexpression. Figure 5D). The expression levels of RcWRKY29 in the petals of both the overexpressed gene (pSuper1300-RcWRKY29) and the control gene (pSuper1300) were simultaneously detected. Figure 5 B) The results showed that the expression level of RcWRKY29 was significantly increased in silent plants compared with the control (TRV), which further illustrates that RcWRKY29 is associated with the resistance of rose petals to gray mold.

[0159] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. An overexpression RcWRKY29 The application of genes in enhancing the resistance of roses to gray mold is characterized by: RcWRKY29 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for enhancing the resistance of roses to gray mold, characterized in that... The steps include: (1) constructing a system comprising the contents of claim 1 RcWRKY29 (2) Introducing the recombinant vector into rose cells to achieve... RcWRKY29 Gene overexpression.

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