RcWRKY29 gene and application of RcWRKY29 gene in enhancing resistance of Chinese rose to gray mold

Abstract

The invention discloses an RcWRKY29 gene and application of the RcWRKY29 gene in enhancing the resistance of Chinese roses to gray mold. The nucleotide sequence of the RcWRKY29 gene is shown as SEQ ID NO: 1. Researches find that the gene is specifically and highly expressed in Chinese rose petals, and the expression quantity is remarkably increased within 24-48 hours after botrytis cinerea infection. After RcWRKY29 expression is instantaneously inhibited through a virus-induced gene silencing technology, the scab area of Chinese rose petals is increased, and the disease resistance is remarkably reduced; otherwise, overexpression of the gene can significantly enhance disease resistance and reduce scab area and cell damage. The invention provides a recombinant vector containing RcWRKY29, an engineering bacterium and a detection kit, and develops a method for enhancing the disease resistance of Chinese rose based on an agrobacterium-mediated transformation technology. The invention provides a new target for Chinese rose gray mold resistance molecular breeding, can effectively reduce postharvest disease loss, and has important economic value and popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding, and particularly relates to an RcWRKY29 gene and its application in enhancing the resistance of Rosa hybrida to Botrytis cinerea Background Art

[0002] Rosa hybrida is a perennial evergreen or semi-evergreen woody ornamental plant of the genus Rosa in the family Rosaceae. Rosa hybrida has the characteristics of elegant flower shape, bright flower color, and rich fragrance, and has high ornamental, economic and cultural values. It ranks first among the world's four major cut flowers and is widely used in landscaping, courtyard decoration, potted flowers, perfume and cosmetics industries, etc.

[0003] Cut rose is one of the four major cut flowers. Botrytis cinerea is the most harmful fungal disease faced by cut roses during postharvest transportation. It seriously damages the ornamental value and economic benefits of cut roses. Cut roses are extremely vulnerable to Botrytis cinerea during long-distance logistics transportation. Approximately 15-40% of roses completely lose their economic value due to postharvest spoilage. Chemical control, physical control, biological control, and agricultural control all have certain drawbacks. Therefore, the fundamental control of Botrytis cinerea in roses should start from the genetic breeding of roses resistant to Botrytis cinerea. By studying the interaction between roses and Botrytis cinerea, new rose varieties with high resistance are excavated and cultivated to reduce the economic losses of the cut rose industry due to Botrytis cinerea. At present, the specific mechanism of action of the RcWRKY29 gene in the resistance of roses to Botrytis cinerea is not clear. Therefore, it is necessary to study its function through gene editing or transgenic technology. Summary of the Invention

[0004] In order to solve the problems of the prior art, the purpose of the present invention is to provide an RcWRKY29 gene and its application in enhancing the resistance of Rosa hybrida to Botrytis cinerea. The resistance of roses to Botrytis cinerea is enhanced by overexpressing this gene, and new rose varieties with high resistance are cultivated.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions: The first aspect of the present application provides an application of an RcWRKY29 gene or a product expressed by its transcription and translation protein in enhancing the resistance of roses to Botrytis cinerea. The nucleotide sequence of the RcWRKY29 gene is shown in SEQ ID NO.1.

[0006] The second aspect of the present application provides a method for enhancing the resistance of roses to Botrytis cinerea, which is characterized by including the following steps: (1) constructing a recombinant vector containing the gene described in claim 1 RcWRKY29 gene; (2) introducing the recombinant vector into rose cells to achieve RcWRKY29Overexpression of the gene.

[0007] Further, in step (1), vector primer design and vector construction: Using EcoRI and KpnI as restriction enzyme sites, insert the RcWRKY29 silencing fragment sequence into the empty TRV2 vector, design primers by the method of homologous recombination, and construct the TRV- RcWRKY29 vector. The nucleotide sequence of the silencing fragment of the RcWRKY29 gene is shown in SEQ ID NO: 3.

[0008] Further, in step (2), select SmaI and KpnI as restriction enzyme sites, insert the RcWRKY29 gene sequence into the pSuper-1300 vector, design primers by the method of homologous recombination to construct the RcWRKY29 -GFP vector, and introduce the recombinant vector into rose cells by the Agrobacterium-mediated transformation method.

[0009] Beneficial effects: In the present invention, a disease resistance regulatory gene RcWRKY29 is identified from the genome of the rose cultivar 'Yu Yue Fen'. It is found that this gene is highly expressed in rose petal tissues and has low expression levels in tissues such as roots and leaves. In the analysis of the expression of RcWRKY29 at different times after Botrytis cinerea infection of rose flowers, it is found that the expression of RcWRKY29 increases significantly during a certain period after Botrytis cinerea infection. Therefore, it is speculated in this application that RcWRKY29 may be involved in regulating the resistance of roses to Botrytis cinerea.

[0010] Compared with the prior art, the present invention has the following advantages: (1) According to the expression results of RcWRKY29 in rose petals in the present invention, transiently silence and overexpress RcWRKY29 in petals, and it is found that the abnormal expression of RcWRKY29 will affect the resistance of rose petals to Botrytis cinerea. Overexpression of RcWRKY29 can promote the resistance of petals to Botrytis cinerea, and conversely, silencing will inhibit the resistance of petals to Botrytis cinerea.

[0011] (2) In the present invention, silence and overexpress the RcWRKY29 gene in rose petals with good growth status and no Botrytis cinerea infection. The research results show that the lesion area of petals treated with RcWRKY29 silencing is larger and the resistance to Botrytis cinerea is reduced; the resistance of petals treated with RcWRKY29 overexpression to Botrytis cinerea is enhanced. In general, RcWRKY29 is of great significance for regulating the resistance of roses to Botrytis cinerea. Description of the Drawings

[0012] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0013] Figure 1 Expression analysis diagram of RcWRKY29 provided by the present invention; Rt: root; St: stem segment; Lf: leaf; Fl: petal.

[0014] Figure 2 Subcellular localization diagram of RcWRKY29 provided by the present invention; Note: Subcellular localization in Nicotiana benthamiana leaves. RcWRKY29-GFP and DAPI (nuclear marker) were co-transfected into tobacco leaves, and the fluorescence signal was observed through a confocal microscope.

[0015] Figure 3 Graph showing the change in the expression level of RcWRKY29 over time after infection with Botrytis cinerea provided by the present invention.

[0016] Figure 4 Gene expression levels of TRV and RcWRKY29 silenced on the 3rd day after transient silencing of RcWRKY29 provided by the present invention ( Figure 4 B) and petal phenotype diagram (diameter of each petal disc is 1.5 cm) ( Figure 4 A); Petal lesion area of TRV and RcWRKY29 silenced on the 3rd day after transient silencing of RcWRKY29 ( Figure 4 C) and statistical chart of particle leakage rate ( Figure 4 D).

[0017] Figure 5 Gene expression levels of pSuper1300 and overexpressed RcWRKY29 on the 3rd day after transient overexpression of RcWRKY29 provided by the present invention ( Figure 5 B) and petal phenotype diagram ( Figure 5 A); Petal lesion area of pSuper1300 and overexpressed RcWRKY29 on the 3rd day after transient overexpression of RcWRKY29 ( Figure 5 C) and statistical chart of ion leakage rate ( Figure 5 D). Detailed implementation manners

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, or B exists alone. Here, A and B may be singular or plural. The character " / " generally represents an "or" relationship between the preceding and following associated objects.

[0020] In this application, "at least one less" means one or more, and "a plurality" means two or more. "At least one (item) less" or its similar expression refers to any combination of these items, including any combination of single item (s) or plural item (s). For example, "at least one (item) of a, b, or c", or "at least one (item) of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or plural respectively.

[0021] It should be understood that in various embodiments of this application, the magnitudes of the serial numbers of the above processes do not mean the order of execution is prior or subsequent. Some or all of the steps can be executed in parallel or successively. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0022] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "the", and "said" 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.

[0023] The weights of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific contents of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the contents of the relevant components in the specification of the embodiments of this application are scaled up or down proportionally, they are within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass described in the specification of the embodiments of this application can be mass units well - known in the chemical industry such as µg, mg, g, kg, etc.

[0024] The terms "first" and "second" are for descriptive purposes only, used to distinguish objects such as substances from each other, and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features.

[0025] The embodiments of the present application provide a RcWRKY29 Use of a gene or a product expressed by its transcription and translation proteins in enhancing the resistance of roses to Botrytis cinerea. RcWRKY29 The nucleotide sequence of the gene is shown in SEQ ID NO.1. RcWRKY29 The coding protein of the gene, and the amino acid sequence of the coding protein is shown in SEQ ID NO.2.

[0026] In the second aspect of the embodiments of the present application, a method for enhancing the resistance of roses to Botrytis cinerea is provided, including the following steps: (1) constructing a recombinant vector containing the RcWRKY29 gene; (2) introducing the recombinant vector into rose cells to achieve RcWRKY29 overexpression of the gene.

[0027] In some embodiments, in step (1), vector primer design and vector construction: using EcoRI and KpnI as restriction enzyme sites, inserting the RcWRKY29 silencing fragment sequence into the empty TRV2 vector, designing primers by the method of homologous recombination to construct the TRV- RcWRKY29 vector, and the nucleotide sequence of the silencing fragment of the RcWRKY29 gene is shown in SEQ ID NO: 3.

[0028] In some embodiments, in step (2), selecting SmaI and KpnI as restriction enzyme sites, inserting the RcWRKY29 gene sequence into the pSuper-1300 vector, designing primers by the method of homologous recombination to construct the RcWRKY29 -GFP vector, and introducing the recombinant vector into rose cells by the Agrobacterium-mediated transformation method.

[0029] Example 1 Use of an RcWRKY29 gene of the present invention or a product expressed by its transcription and translation proteins in enhancing the resistance of roses to Botrytis cinerea. The nucleotide sequence of the RcWRKY29 gene is shown in SEQ ID NO.1. The coding protein of the RcWRKY29 gene, and the amino acid sequence of the coding protein is shown in SEQ ID NO.2.

[0030] A method for enhancing the resistance of roses to Botrytis cinerea 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 enzyme sites, insert the RcWRKY29 silencing fragment sequence into the TRV2 empty vector, design primers using the homologous recombination method, and construct the TRV- RcWRKY29 vector. The nucleotide sequence of the silencing fragment of the RcWRKY29 gene is shown in SEQ ID NO: 3.

[0031] (2) Introduce the recombinant vector into rose cells to achieve RcWRKY29 overexpression of the gene. Select SmaI and KpnI as restriction enzyme sites, insert the RcWRKY29 gene sequence into the pSuper-1300 vector, design primers using the homologous recombination method to construct the RcWRKY29 -GFP vector, and introduce the recombinant vector into rose cells through the Agrobacterium-mediated transformation method.

[0032] Example 2 Based on the genomic sequence of the rose cultivar 'Yu Yue Fen', potential disease-resistant regulatory gene RcWRKY29 was mined through the identification and analysis of WRKY family members in the early stage, and it was found that the expression of the RcWRKY29 gene was significantly increased in a certain period after Botrytis cinerea infection. After transient silencing or overexpression of the RcWRKY29 gene, it was found that the resistance of rose petals to Botrytis cinerea changed significantly. Therefore, this application reveals the biological function of the RcWRKY29 gene in the disease resistance of roses to Botrytis cinerea, thus providing technical support for enhancing the resistance of roses to Botrytis cinerea, promoting the research of rose functional genomics, gene editing breeding and transgenic breeding, mining and cultivating new rose varieties with high resistance, and providing a certain theoretical basis for the integrated control of Botrytis cinerea and the cultivation of new rose varieties.

[0033] The abbreviations and their corresponding names that appear in this application are shown in Table 1.

[0034] Table 1

[0035] 1. Plant materials 1.1 'Yu Yue Fen' 'Yu Yue Fen' was obtained from the Jinning Baofeng Base of the Institute of Flowers, Yunnan Academy of Agricultural Sciences. Different tissues were taken for tissue-specific analysis. 'Carola' is a modern cut flower rose cultivar, obtained from the Baofeng Base of the Institute of Flowers, Yunnan Academy of Agricultural Sciences. Petals at the S3 stage were taken for silencing and expression verification experiments.

[0036] 1.2 Tobacco The experimental materials for subcellular localization and transcriptional activation were Nicotiana benthamiana. The seeds were sown on moist nutrient substrate, covered with a film, and cultured in a cultivation chamber.

[0037] Cultivation conditions: temperature 24 ± 1 °C, relative humidity 60 - 65%, photoperiod 16 h / 8 h 1.3 Strains and vectors Escherichia coli: DH5α, Agrobacterium tumefaciens strain EHA105, pSuper-1300 (Kan resistance) were all purchased from Beijing Tsingke Biotechnology Co., Ltd. The VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0038] 1.4 Culture medium formulations involved in the case (1) LB medium and YEB medium (Table 2) Table 2

[0039] (2) Experiment-related culture media (Table 3) Table 3

[0040] 2. Research methods 2.1 Extraction of total RNA The RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (centrifugal column type) was used to extract total RNA from the roots, stem segments, leaves, and petals of 'Yueyuefen'.

[0041] 2.2 cDNA synthesis (1) The reaction system for genomic DNA removal was shown in Table 4: Table 4

[0042] Mix well, incubate at 42 °C for 2 min.

[0043] (2) Prepare the reverse transcription reaction system (Table 5) Table 5

[0044] Pipette and mix well, incubate at 37 °C for 15 min; 85 °C for 5 s. The product was stored at -20 °C.

[0045] 2.3 Real-time fluorescence quantitative PCR Design specific primers for the gene using Primer premier5 (Table 6). Dilute the cDNA four-fold with ddH2O and perform Real-Time PCR amplification using the reverse-transcribed cDNA as a template. Use UBI2 as an internal reference and set up 3 biological replicates.

[0046] Table 6

[0047] The RT-qPCR reaction system is shown in Table 7: Table 7

[0048] The RT-qPCR reaction program is shown in Table 8: Table 8

[0049] 2.4 Vector construction 2.4.1 PCR amplification of the target gene fragment Use a high-fidelity enzyme (PhusionTM Plus PCR Master Mix) for PCR amplification of the target gene. The PCR amplification reaction system is shown in Table 9: Table 9

[0050] The reaction program is shown in Table 10: Table 10

[0051] After the amplification is completed, perform 1% gel electrophoresis and select the target band for subsequent gel extraction.

[0052] 2.4.3 Double digestion of the vector Use a DNA gel extraction kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0) to recover and purify the PCR product.

[0053] 2.4.3 Double digestion of the vector According to the restriction enzyme sites for the insertion of the target fragment sequence, use the corresponding enzymes to perform double digestion of the vector. The double digestion system is shown in Table 11: Table 11

[0054] After adding the system reagents on ice, perform double digestion of the vector according to the heat denaturation temperature of the specific enzyme.

[0055] 2.4.4 Homologous recombination The double-digested vector and the cloned target gene fragment were subjected to homologous recombination to construct the vector. The homologous recombination system is shown in Table 12: Table 12

[0056] Procedure: Run at 50 °C for 15 min in a PCR instrument.

[0057] 2.4.5 Transformation of Escherichia coli (1) Take out the competent cells from -80 °C and melt them on ice; (2) Take a 1.5 mL centrifuge tube, add 10 μL of the recombinant product and 50 μL of DH5α competent cells, pipette and mix well, and let it stand on ice for 30 min; (3) Heat shock in a 42 °C water bath for 90 s, and quickly transfer it to ice and let it stand for 2 min; (4) Add 500 μL of LB to the centrifuge tube, incubate at 37 °C and 200 rpm for 1 h; (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min; (6) Discard 400 μL of the supernatant in a laminar flow hood, pipette and mix well, then spread it on an LB solid medium containing antibiotics, and incubate it inverted overnight at 37 °C.

[0058] 2.4.6 Bacterial inspection and sequencing (1) Shaking culture: Pick a single colony grown overnight and shake it in 500 μL of LB medium containing antibiotics for 3 - 4 h (37 °C, 200 rpm).

[0059] (2) Bacterial liquid PCR: The cultured bacterial liquid was used for PCR amplification to detect whether the target band of the constructed vector was as expected. The bacterial liquid PCR amplification system is shown in Table 13: Table 13

[0060] The bacterial liquid PCR amplification reaction procedure is shown in Table 14: Table 14

[0061] The amplification product was used for 1% gel electrophoresis imaging, and the samples with the expected band size were sent to the company for sequencing.

[0062] 2.5 Plasmid extraction After the sequencing results were returned, sequence alignment was performed, and the samples that met the expectations were selected for shaking culture, and plasmids were extracted using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0063] 2.6 Agrobacterium transformation (1)Take out the Agrobacterium competent cells from the -80 °C refrigerator, insert them into ice when they are melted to the state of ice-water mixture; (2)Add 0.01 - 1 μg plasmid DNA to every 100 μL of competent cells, gently mix by pipetting, then let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and on ice for 5 min in sequence; (3)Add 500 μL of antibiotic-free YEB liquid medium, and culture it with shaking at 28 °C for 5 h; (4)Centrifuge at 5000 rpm for 2 min, discard 400 μL of the supernatant in a laminar flow hood, spread it on the YEB solid medium containing antibiotics, and culture it upside down at 28 °C for 5 d.

[0064] 2.7 Transient infection (1)Vector primer design and vector construction Using EcoRI and KpnI as restriction enzyme sites, insert the silencing fragment (SEQ ID NO: 3) sequence of RcWRKY29 into the empty TRV2 vector, design primers by the method of homologous recombination, and construct the TRV-RcWRKY29 vector.

[0065] Select SmaI and KpnI as restriction enzyme sites, insert the CDS sequence of RcWRKY29 into the pSuper-1300 vector, design primers by the method of homologous recombination to construct the RcWRKY29-GFP vector, and perform the following experiments after obtaining the Agrobacterium positive strains.

[0066] (2)Bacterial liquid culture Streak the bacterial liquid on a plate (containing 50 mg / L Kan / Rif), and culture it upside down at 28 °C for 5 days. Pick a single colony and culture it with gentle shaking in 500 μL of YEB containing antibiotics for bacterial inspection; for those with correct bands, perform medium-scale shaking and large-scale shaking (28 °C, 200 rpm).

[0067] (3)Collecting bacteria and resuspending Centrifuge to collect bacteria at 5000 rpm for 8 min, pour out the supernatant, resuspend the bacterial cells with the infection solution, gently mix by pipetting with a pipette gun, and adjust to OD600 = 1.0. When performing the transient silencing experiment, mix the TRV1 and TRV2, TRV2-RcWRKY29 bacterial solutions in a volume ratio of 1:1, and let it stand in the dark for 4 - 6 h, while for transient overexpression, just collect and let stand the Super-1300 and Super-RcWRKY29 bacteria separately.

[0068] (4)Vacuum suction The outermost petals of the 'Corolla' S3 - level flowers were made into petal discs with a diameter of 1.5 cm and were subjected to suction infiltration with a vacuum pump at 0.082 MPa for 10 min, holding pressure for 10 min, and deflating for 10 min. The whole petal disc was fully soaked in the bacterial solution and the treatment was repeated three times. After infiltration, it was rinsed with sterile water and placed in a 1% agar Petri dish. After dark cultivation at 8 °C for 3 d, it was transferred to a tissue culture room at a temperature of (22 ± 1) °C with a light cycle of 16 h light / 8 h dark. Observation was carried out until all the petal discs had completely changed color, the petal phenotypes were photographed and recorded, and the software ImageJ 15.1 was used to measure and statistically analyze the petal color changes.

[0069] 2.8 Data statistical analysis The data for statistical analysis were taken from 3 biological replicates and 3 technical replicates. The data were statistically analyzed by GraphPad Prism 9.5 software, and the comparison of two groups of data was carried out by the student - t test method (*P < 0.05, **P < 0.01, ***P < 0.001).

[0070] 2.10 Primers used in the RcWRKY29 sequence experiment. The RcWRKY29 sequence and primer list are shown in Table 15: Table 15

[0071] Example 3 Verification and subcellular localization of RcWRKY29 in different tissue parts The total RNA of rose petals, leaves, roots and stems was extracted and its quality was detected in Example 7. The genes with the most cis - acting elements related to meristems and plant growth and development were selected for fluorescence quantitative expression analysis, and it was found that the expression of different genes differed in different tissues ( Figure 1 ). Among them, RcWRKY29 was specifically expressed and had a relatively high expression level in petals, indicating that RcWRKY29 may play a certain role in the disease resistance of rose petals.

[0072] At the same time, the CDS sequence of RcWRKY29 was cloned and the RcWRKY29 - GFP vector was constructed. Using the GFP empty vector as a control, it was transferred into Agrobacterium tumefaciens EHA105 and then injected into the leaves of Nicotiana benthamiana. After 3 d, observation was carried out under a laser confocal microscope, and the results showed that RcWRKY29 - GFP was localized in the nucleus ( Figure 2 ).

[0073] Example 4 The resistance of rose petals to Botrytis cinerea weakened after silencing RcWRKY29 First, the change in the expression level of RcWRKY29 over time after the infection of Botrytis cinerea was detected ( Figure 3 ). It was found that the expression level of RcWRKY29 gradually increased 24 - 48 hours after the infection of Botrytis cinerea, indicating that RcWRKY29 is related to the resistance of rose petals to Botrytis cinerea.

[0074] To confirm the function of RcWRKY29 in the resistance of rose petals to Botrytis cinerea, RcWRKY29 in 'Carola' rose plants was silenced using the VIGS technique and Botrytis cinerea was injected into the petals. Observation and statistics were carried out 3 days after the infection. It was found that the lesion areas of the petals with gene silencing (TRV2 - RcWRKY29) were larger than those of the control (TRV) ( Figure 4 A, Figure 4 C). The relative electrolyte leakage rate was detected to prove the degree of damage to the petals with gene silencing (TRV2 - RcWRKY29) and the control (TRV). The relative electrolyte leakage rate of the petals increased after gene silencing compared with the control ( Figure 4 D). At the same time, the expression level of RcWRKY29 in the petals of the silenced gene (TRV2 - RcWRKY29) and the control gene (TRV) was detected ( Figure 4 B). The results showed that the expression level in the RcWRKY29 - silenced plants decreased significantly compared with the control (TRV). This result further indicates that RcWRKY29 is related to the resistance of rose petals to Botrytis cinerea.

[0075] Example 5 The resistance of rose petals to Botrytis cinerea is enhanced after overexpressing RcWRKY29 To confirm the function of RcWRKY29 in the resistance of rose petals to Botrytis cinerea, RcWRKY29 in 'Carola' rose plants was transiently overexpressed and Botrytis cinerea was injected into the petals. Observation and statistics were carried out 3 days after the infection. It was found that the lesion areas of the petals with gene overexpression (pSuper1300 - RcWRKY29) were smaller than those of the control (pSuper1300) ( Figure 5 A, Figure 5 C). The relative electrolyte leakage rate was detected to prove the degree of damage to the petals with gene overexpression (pSuper1300 - RcWRKY29) and the control (pSuper1300). The relative electrolyte leakage rate of the petals decreased after gene overexpression compared with the control ( Figure 5 D). At the same time, the expression level of RcWRKY29 in the petals of the overexpressed gene (pSuper1300 - RcWRKY29) and the control gene (pSuper1300) was detected ( Figure 5 B). The results showed that the expression level in the RcWRKY29 - overexpressed plants increased significantly compared with the control (TRV). This result further indicates that RcWRKY29 is related to the resistance of rose petals to Botrytis cinerea.

[0076] 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 by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection claimed by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A RcWRKY29 product of gene or its transcription and translation protein expression for enhancing the resistance of roses to Botrytis cinerea, characterized in that: RcWRKY29 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for enhancing the resistance of roses to Botrytis cinerea, characterized in that Comprising the following steps: (1) constructing a recombinant vector containing the gene described in claim 1 RcWRKY29 ; (2) Introduce the recombinant vector into rose cells to achieve RcWRKY29 overexpression of the gene.

3. The method according to claim 2, wherein: In step (1), vector primer design and vector construction: Using EcoRI and KpnI as restriction enzyme sites, insert the RcWRKY29 silencing fragment sequence into the empty TRV2 vector. Design primers using the method of homologous recombination to construct the TRV- RcWRKY29 vector. The nucleotide sequence of the silencing fragment of the RcWRKY29 gene is shown in SEQ ID NO:

3.

4. The method according to claim 2, wherein: In step (2), SmaI and KpnI were selected as restriction sites, and RcWRKY29 the gene sequence was inserted into the pSuper-1300 vector. Primers were designed using the method of homologous recombination to construct RcWRKY29 -GFP vector. The recombinant vector was introduced into rose cells by the Agrobacterium-mediated transformation method.

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