Use of RcARF8 gene in enhancing resistance of Chinese rose to botrytis cinerea

Abstract

The application discloses application of RcARF8 genes in enhancing resistance of Chinese rose to botrytis blight, and a nucleotide sequence of the RcARF8 gene is shown as SEQ ID NO. 1. In the application, the RcARF8 gene is silenced and overexpressed in petals of Chinese rose in a good growth state and free from infection of botrytis blight, and it is found that petals treated by silencing the RcARF8 gene have a larger disease spot area and a reduced resistance to botrytis blight, and petals treated by overexpressing the RcARF8 gene have enhanced resistance to botrytis blight.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to the application of an RcARF8 gene in enhancing the resistance of roses to gray mold. Background Technology

[0002] The rose 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 are ranked 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 rot. 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 RcARF8 gene in rose resistance to gray mold is unclear; therefore, it is necessary to study its function through gene editing or transgenic technology.

[0004] Therefore, we aim to develop an application of the RcARF8 gene in enhancing the resistance of roses to gray mold. Summary of the Invention

[0005] To address the problems of existing technologies, the purpose of this invention is to provide an application of the RcARF8 gene in enhancing the resistance of roses 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.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In one aspect, this application provides the use of a product expressing the RcARF8 gene or its transcribed and translated protein in regulating gray mold disease in roses.

[0008] Secondly, this application provides a method for enhancing the resistance of roses to gray mold.

[0009] Thirdly, this application provides a recombinant vector for silencing the RcARF8 gene to study its role in resistance to gray mold in roses.

[0010] Fourthly, this application provides a method for regulating the expression of the RcARF8 gene in rose tissues.

[0011] Fifthly, this application provides a method for detecting the function of the RcARF8 gene in the resistance of rose petals to gray mold.

[0012] Sixthly, this application provides a method for verifying the function of the RcARF8 gene in the resistance of rose petals to gray mold.

[0013] Seventhly, this application provides a method for cultivating transgenic rose plants that enhance resistance to gray mold.

[0014] The first aspect of this application provides the use of a product expressing the RcARF8 gene or its transcribed and translated protein in regulating gray mold disease in roses, wherein the nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO.1.

[0015] The second aspect of this application provides a method for enhancing the resistance of roses to gray mold, comprising the following steps:

[0016] (1) Construct a recombinant vector containing the RcARF8 gene;

[0017] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene.

[0018] Further, in step (1), the vector primer design and vector construction are as follows: using EcoRI and KpnI as restriction sites, the silent fragment sequence of RcARF8 is inserted into the empty TRV2 vector. Primers are designed using homologous recombination to construct the TRV-RcARF8 vector. The nucleotide sequence of the silent fragment of RcARF8 is shown in SEQ ID NO:2.

[0019] Further, in step (2), SmaI and KpnI are selected as restriction sites, the complete coding region sequence of RcARF8 is inserted into the pSuper-1300 vector, primers are designed using homologous recombination to construct the RcARF8-GFP vector, and the recombinant vector is introduced into rose cells by Agrobacterium-mediated transformation.

[0020] A third aspect of this application provides a recombinant vector for silencing the RcARF8 gene to study its role in resistance to gray mold in roses. The recombinant vector contains the complete coding region sequence of the RcARF8 gene, and the nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO:1. The recombinant vector contains a silenced fragment sequence of RcARF8, and the nucleotide sequence of the silenced fragment of RcARF8 is shown in SEQ ID NO:2.

[0021] The fourth aspect of this application provides a method for regulating the expression of the RcARF8 gene in rose tissues. By detecting the expression level of the RcARF8 gene in different rose tissues, the expression differences in petals, leaves, roots and stems are determined. The nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO.1.

[0022] The fifth aspect of this application provides a method for detecting the function of the RcARF8 gene in the resistance of rose petals to gray mold, characterized by comprising the following steps:

[0023] (1) Silencing the RcARF8 gene in rose petals using the virus-induced gene silencing (VIGS) technology;

[0024] (2) Treat the silent petals with gray mold infection;

[0025] (3) Compare the lesion area and relative particle leakage rate of petals with silenced RcARF8 gene with those of control petals to assess the effect of RcARF8 gene on gray mold resistance.

[0026] The sixth aspect of this application provides a method for verifying the function of the RcARF8 gene in the resistance of rose petals to gray mold, comprising the following steps:

[0027] (1) Construct an overexpression vector containing the RcARF8 gene;

[0028] (2) The overexpression vector was introduced into rose petal cells;

[0029] (3) Petals overexpressing the RcARF8 gene were treated with gray mold infection;

[0030] (4) Compare the lesion area and relative particle leakage rate of petals overexpressing the RcARF8 gene with those of the control petals to assess the effect of the RcARF8 gene on gray mold resistance.

[0031] The seventh aspect of this application provides a method for cultivating transgenic rose plants to enhance resistance to gray mold, comprising the following steps:

[0032] (1) Construct a recombinant vector containing the RcARF8 gene;

[0033] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene;

[0034] (3) Breed transgenic rose plants that overexpress the RcARF8 gene and evaluate their resistance to gray mold.

[0035] Beneficial Effects: This invention identified a disease resistance regulatory gene, RcARF8, from the genome of the rose variety 'Yueyuefen'. The gene was found to be highly expressed in the petals, but expressed at low levels in the roots and leaves. Analysis of RcARF8 gene expression at different time points after gray mold infection of rose flowers revealed a significant increase in RcARF8 gene expression for a certain period following infection. Therefore, this application hypothesizes that the RcARF8 gene may be involved in regulating the resistance of roses to gray mold.

[0036] Compared with the prior art, the present invention has the following advantages: (1) Based on the expression results of RcARF8 gene in rose petals, the present invention transiently silences and overexpresses RcARF8 gene in petals and finds that abnormal expression of RcARF8 gene will affect the resistance of rose petals to gray mold. Overexpression of RcARF8 gene can promote the resistance of petals to gray mold, while silence will inhibit the resistance of petals to gray mold.

[0037] (2) In this invention, the RcARF8 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 RcARF8 gene had larger lesion areas and reduced resistance to gray mold; the petals treated with overexpressed RcARF8 gene had enhanced resistance to gray mold. In conclusion, the RcARF8 gene is of great significance in regulating the resistance of roses to gray mold. Attached Figure Description

[0038] 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.

[0039] Figure 1 This is an expression analysis diagram of RcARF8 provided by the present invention.

[0040] Figure 2 This is a subcellular localization map of RcARF8 provided by the present invention; Note: Subcellular localization in tobacco leaves was achieved by co-infiltrating RcARF8-GFP with DAPI (nuclear marker) into tobacco leaves and observing the fluorescence signal using a confocal microscope.

[0041] Figure 3 This invention provides the change in RcARF8 expression level over time after infection with Botrytis cinerea.

[0042] Figure 4 This invention provides the gene expression levels of TRV and RcARF8 silencing on day 3 after transient silencing of RcARF8. Figure 4 C) and petal phenotypic diagram (each petal disc is 1.5cm in diameter) Figure 4 A); The area of ​​petal lesions silencing TRV and RcARF8 on day 3 after transient silencing of RcARF8 (A); Figure 4 B) and particle leakage rate statistics ( Figure 4 D).

[0043] Figure 5 The expression levels of pSuper1300 and RcARF8 overexpression on day 3 after transient overexpression of RcARF8 provided by this invention are ( Figure 5 C) and petal phenotypic diagram ( Figure 5 A); The area of ​​petal lesions with pSuper1300 and RcARF8 overexpression on day 3 after transient RcARF8 overexpression ( Figure 5 B) and particle leakage rate statistics ( Figure 5 D). Detailed Implementation

[0044] 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.

[0045] 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 the following: A exists alone; A and B exist simultaneously; or B exists alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] The following detailed description is provided in conjunction with embodiments and accompanying drawings. Based on the genome sequence of the rose variety 'Yueyuefen', this application, through previous identification and analysis of WRKY family members, identified the potential disease resistance regulatory gene RcARF8, and found that RcARF8 gene expression significantly increased after a certain period following gray mold infection. Transient silencing or overexpression of the RcARF8 gene revealed significant changes in the resistance of rose petals to gray mold. Therefore, this application reveals the biological function of the RcARF8 gene in rose resistance to gray mold, thus providing technical support for enhancing rose resistance to gray mold, promoting rose functional genomics research, gene editing breeding, and transgenic breeding research, 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.

[0052] The first aspect of this application provides the application of a product expressing the RcARF8 gene or its transcribed and translated protein in regulating gray mold disease in roses, wherein the nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO.1.

[0053] A second aspect of this application provides a method for enhancing the resistance of roses to gray mold, comprising the following steps:

[0054] (1) Construct a recombinant vector containing the RcARF8 gene;

[0055] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene.

[0056] In some embodiments, in step (1), the vector primer design and vector construction are as follows: using EcoRI and KpnI as restriction sites, the silent fragment sequence of RcARF8 is inserted into the empty TRV2 vector, primers are designed using homologous recombination, and the TRV-RcARF8 vector is constructed. The nucleotide sequence of the silent fragment of RcARF8 is shown in SEQ ID NO:2.

[0057] In some embodiments, in step (2), SmaI and KpnI are selected as restriction sites, the complete coding region sequence of RcARF8 is inserted into the pSuper-1300 vector, primers are designed using homologous recombination to construct the RcARF8-GFP vector, and the recombinant vector is introduced into rose cells by Agrobacterium-mediated transformation.

[0058] A third aspect of this application provides a recombinant vector for silencing the RcARF8 gene to study its role in resistance to gray mold in roses. The recombinant vector contains the complete coding region sequence of the RcARF8 gene, and the nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO:1. The recombinant vector contains a silenced fragment sequence of RcARF8, and the nucleotide sequence of the silenced fragment of RcARF8 is shown in SEQ ID NO:2.

[0059] The fourth aspect of this application provides a method for regulating the expression of the RcARF8 gene in rose tissues. By detecting the expression level of the RcARF8 gene in different rose tissues, the expression differences in petals, leaves, roots and stems are determined. The nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO.1.

[0060] A fifth aspect of this application provides a method for detecting the function of the RcARF8 gene in the resistance of rose petals to gray mold, comprising the following steps:

[0061] (1) Silencing the RcARF8 gene in rose petals using the virus-induced gene silencing (VIGS) technology;

[0062] (2) Treat the silent petals with gray mold infection;

[0063] (3) Compare the lesion area and relative particle leakage rate of petals with silenced RcARF8 gene with those of control petals to assess the effect of RcARF8 gene on gray mold resistance.

[0064] A sixth aspect of this application provides a method for verifying the function of the RcARF8 gene in the resistance of rose petals to gray mold, comprising the following steps:

[0065] (1) Construct an overexpression vector containing the RcARF8 gene;

[0066] (2) The overexpression vector was introduced into rose petal cells;

[0067] (3) Petals overexpressing the RcARF8 gene were treated with gray mold infection;

[0068] (4) Compare the lesion area and relative particle leakage rate of petals overexpressing the RcARF8 gene with those of the control petals to assess the effect of the RcARF8 gene on gray mold resistance.

[0069] The seventh aspect of this application provides a method for cultivating transgenic rose plants to enhance resistance to gray mold, comprising the following steps:

[0070] (1) Construct a recombinant vector containing the RcARF8 gene;

[0071] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene;

[0072] (3) Breed transgenic rose plants that overexpress the RcARF8 gene and evaluate their resistance to gray mold.

[0073] Example 1

[0074] The abbreviations and their corresponding names that appear in this application are shown in Table 1.

[0075] Table 1

[0076]

[0077] This invention relates to the application of a product expressing the RcARF8 gene or its transcribed and translated protein in the regulation of gray mold disease in roses. The nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO:1. The amino acid sequence of the protein encoded by the RcARF8 gene is shown in SEQ ID NO:2.

[0078] The present invention provides a method for enhancing the resistance of roses to gray mold, comprising the following steps: (1) constructing a recombinant vector containing the RcARF8 gene; vector primer design and vector construction: using EcoRI and KpnI as restriction sites, the silent fragment sequence of RcARF8 is inserted into the empty TRV2 vector, primers are designed using homologous recombination, and the TRV-RcARF8 vector is constructed, the nucleotide sequence of the silent fragment of RcARF8 is shown in SEQ ID NO:2.

[0079] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene. SmaI and KpnI were selected as restriction sites, and the complete coding region sequence of RcARF8 was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RcARF8-GFP vector. The recombinant vector was introduced into rose cells by Agrobacterium-mediated transformation.

[0080] Example 2

[0081] The present invention provides a method for enhancing the resistance of roses to gray mold, comprising the following steps:

[0082] (1) Construct a recombinant vector containing the RcARF8 gene; Vector primer design and vector construction: Using EcoRI and KpnI as restriction sites, the silenced fragment sequence of RcARF8 was inserted into the empty TRV2 vector. Primers were designed using homologous recombination to construct the TRV-RcARF8 vector. The nucleotide sequence of the silenced fragment of RcARF8 is shown in SEQ ID NO:2.

[0083] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene. SmaI and KpnI were selected as restriction sites, and the complete coding region sequence of RcARF8 was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RcARF8-GFP vector. The recombinant vector was introduced into rose cells by Agrobacterium-mediated transformation.

[0084] Example 3

[0085] This invention discloses a recombinant vector for silencing the RcARF8 gene to study its role in resistance to gray mold in roses. The recombinant vector contains the complete coding region sequence of the RcARF8 gene, and the nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO. 1. The recombinant vector also contains a silenced fragment sequence of RcARF8, and the nucleotide sequence of the silenced fragment of RcARF8 is shown in SEQ ID NO: 2.

[0086] Example 4

[0087] The present invention discloses a method for regulating the expression of the RcARF8 gene in rose tissues. By detecting the expression level of the RcARF8 gene in different rose tissues, the expression differences of the RcARF8 gene in petals, leaves, roots and stems are determined. The nucleotide sequence of the RcARF8 gene is shown in SEQ ID NO.1.

[0088] A method for detecting the function of the RcARF8 gene in the resistance of rose petals to gray mold according to the present invention includes the following steps:

[0089] (1) Silencing the RcARF8 gene in rose petals using the virus-induced gene silencing (VIGS) technology;

[0090] (2) Treat the silent petals with gray mold infection;

[0091] (3) Compare the lesion area and relative particle leakage rate of petals with silenced RcARF8 gene with those of control petals to assess the effect of RcARF8 gene on gray mold resistance.

[0092] Example 5

[0093] A method for verifying the function of the RcARF8 gene in the resistance of rose petals to gray mold according to the present invention includes the following steps:

[0094] (1) Construct an overexpression vector containing the RcARF8 gene;

[0095] (2) The overexpression vector was introduced into rose petal cells;

[0096] (3) Petals overexpressing the RcARF8 gene were treated with gray mold infection;

[0097] (4) Compare the lesion area and relative particle leakage rate of petals overexpressing the RcARF8 gene with those of the control petals to assess the effect of the RcARF8 gene on gray mold resistance.

[0098] Example 6

[0099] The present invention discloses a method for cultivating transgenic rose plants to enhance resistance to gray mold, comprising the following steps:

[0100] (1) Construct a recombinant vector containing the RcARF8 gene;

[0101] (2) The recombinant vector was introduced into rose cells to achieve overexpression of the RcARF8 gene;

[0102] (3) Breed transgenic rose plants that overexpress the RcARF8 gene and evaluate their resistance to gray mold.

[0103] Example 7

[0104] 1. Plant materials

[0105] 1.1'Monthly Powder'

[0106] '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.

[0107] 1.2 Tobacco

[0108] 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.

[0109] Cultivation conditions: Temperature 24±1℃, relative humidity 60-65%, photoperiod 16h / 8h

[0110] 1.3 Strains and Vectors

[0111] 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.

[0112] 1.4 Culture medium formulations involved in the case study

[0113] (1) LB medium and YEB medium (Table 2)

[0114] Table 2

[0115]

[0116] (2) Experimental culture media (Table 3)

[0117] Table 3

[0118]

[0119] 2. Research Methods

[0120] 2.1 Extraction of total RNA

[0121] 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).

[0122] 2.2cDNA synthesis

[0123] (1) Genomic DNA removal reaction system (Table 4)

[0124] Table 4

[0125]

[0126] Mix well, incubate at 42°C for 2 minutes.

[0127] (2) Preparation of the reverse transcription reaction system (Table 5)

[0128] Table 5

[0129]

[0130] 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.

[0131] 2.3 Real-time quantitative PCR

[0132] RT-qPCR-specific primers for the gene were designed using Primer Premier5 (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. UBI2 was used as an internal control, and three biological replicates were set up.

[0133] Table 6

[0134]

[0135] The RT-qPCR reaction system is shown in Table 7:

[0136] Table 7

[0137]

[0138] The RT-qPCR reaction procedure is shown in Table 8:

[0139] Table 8

[0140]

[0141]

[0142] 2.4 Vector Construction

[0143] 2.4.1 PCR amplification of the target gene fragment

[0144] 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.

[0145] Table 9

[0146]

[0147] The PCR amplification reaction procedure is shown in Table 10:

[0148] Table 10

[0149]

[0150] After amplification, 1% gel electrophoresis was performed, and the target band was selected for subsequent gel recovery.

[0151] 2.4.2 Glue Recycling

[0152] The PCR products were recovered and purified using a DNA gel extraction kit (TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0).

[0153] 2.4.3 Vector double enzyme digestion

[0154] Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-cleaved with the corresponding enzyme.

[0155] Enzyme digestion. The double enzyme digestion system is shown in Table 11:

[0156] Table 11

[0157]

[0158] After adding the system reagents on ice, perform double digestion of the vector according to the specific enzyme denaturation temperature.

[0159] 2.4.4 Homologous recombination

[0160] 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:

[0161] Table 12

[0162]

[0163] Procedure: Run the PCR machine at 50℃ for 15 minutes.

[0164] 2.4.5 Escherichia coli transformation

[0165] (1) Remove the competent states from -80℃ and melt them in ice;

[0166] (2) Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of DH5α competent cells, mix by pipetting, and let stand on ice for 30 min.

[0167] (3) Heat shock in a 42℃ water bath for 90 seconds, then quickly transfer to ice and let stand for 2 minutes;

[0168] (4) Add 500 μL LB to the centrifuge tube, incubate at 37°C and 200 rpm for 1 h;

[0169] (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min;

[0170] (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.

[0171] 2.4.6 Microbial Detection and Sequencing

[0172] (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).

[0173] (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:

[0174] Table 13

[0175]

[0176] The bacterial culture PCR amplification reaction procedure is shown in Table 14:

[0177] Table 14

[0178]

[0179] The amplified products were used for 1% gel electrophoresis imaging, and the band sizes, which met expectations, were sent to the company for sequencing.

[0180] 2.5 Plasmid Extraction

[0181] 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).

[0182] 2.6 Agrobacterium-mediated transformation

[0183] (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;

[0184] (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.

[0185] (3) Add 500 μL of antibiotic-free YEB liquid culture medium and incubate at 28°C with shaking for 5 h;

[0186] (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.

[0187] 2.7 Instantaneous infection

[0188] (1) Vector and primer design and vector construction

[0189] Using EcoRI and KpnI as restriction sites, the silent fragment (SEQ ID NO:3) sequence of RcARF8 was inserted into the empty TRV2 vector. Primers were designed using homologous recombination to construct the TRV-RcARF8 vector.

[0190] SmaI and KpnI were selected as restriction sites. The CDS sequence of RcARF8 was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RcARF8-GFP vector. After obtaining Agrobacterium-positive strains, the following experiments were performed.

[0191] (2) Bacterial culture

[0192] 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).

[0193] (3) Collection and resuspension of bacteria

[0194] Centrifuge at 5000 rpm for 8 minutes to collect the 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-RcARF8 bacterial cultures at a 1:1 volume ratio and incubate in the dark for 4-6 hours. For transient overexpression, collect Super-1300 and Super-RcARF8 bacteria separately and incubate.

[0195] (4) Vacuum suction

[0196] 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% Agr culture dishes. 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 exposure of 16 h and a dark exposure of 8 h. 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.

[0197] 2.8 Data Statistical Analysis

[0198] 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).

[0199] 2.10 Primers used in the RcARF8 sequence experiment. The RcARF8 gene sequence and primer list are shown in Table 15:

[0200] Table 15

[0201]

[0202] Experimental Example 1

[0203] Validation and subcellular localization of RcARF8 in different tissue sites

[0204] Example 7: 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 for quantitative fluorescence expression analysis. The analysis revealed differences in gene expression across different tissues. The expression level of RcARF8 in different tissues, from highest to lowest, was: sepals, petals, leaves, and stem segments. Figure 1 Among them, RcARF8 was expressed specifically and at a high level in petals and sepals, indicating that the RcARF8 gene plays a certain role in the disease resistance of rose petals.

[0205] Simultaneously, the CDS sequence of the RcARF8 gene was cloned, and the RcARF8-GFP vector was constructed. Using the empty GFP vector as a control, the vector was transformed into EHA105 and injected into leaves of *Nicotiana benthamiana*. After 3 days, observation under a laser confocal microscope showed that RcARF8-GFP was localized in the cell nucleus (…). Figure 2 ).

[0206] Experimental Example 2

[0207] Silent RcARF8 reduces resistance to gray mold on flower petals.

[0208] First, the change in the expression level of the RcARF8 gene over time after Botrytis cinerea infection was detected. Figure 3 The study found that the expression level of RcARF8 gradually increased from 0 to 72 hours after infection with gray mold, indicating that RcARF8 is related to the resistance of rose petals to gray mold.

[0209] To confirm the function of the RcARF8 gene in rose petal resistance to gray mold, RcARF8 in 'Carola' rose plants was silenced using VIGS technology, and gray mold fungus was injected into the petals. Observations were performed 3 days post-infection, revealing that the lesion area on petals with the gene silenced (TRV2-RcARF8) was larger than that of the control (TRV). Figure 4 A, Figure 4 B), relative particle leakage rate was measured to demonstrate the extent of damage to petals in gene-silenced (TRV2-RcARF8) 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 the RcARF8 gene in the petals of both the silenced gene (TRV2-RcARF8) and the control gene (TRV) were simultaneously detected. Figure 4 C) The results showed that the expression level of RcARF8 was significantly reduced in plants with silenced RcARF8 compared with the control (TRV), which further illustrates that the RcARF8 gene is associated with the resistance of rose petals to gray mold.

[0210] Experimental Example 3

[0211] Overexpression of RcARF8 enhances resistance to gray mold on flower petals.

[0212] To confirm the function of the RcARF8 gene in the resistance of rose petals to gray mold, a transient infection of 'Carola' rose plants overexpressing RcARF8 was performed, followed by injection of gray mold fungus into the petals. Observations were conducted 3 days post-infection, revealing that the overexpressed gene (pSuper1300-RcARF8) resulted in smaller lesion areas on the petals compared to the control (pSuper1300). Figure 5 A, Figure 5 B) The relative particle leakage rate was measured to demonstrate the degree of petal damage in patients with gene overexpression (pSuper1300-RcARF8) and the control (pSuper1300). Compared with the control, the relative particle leakage rate of petals decreased after gene overexpression. Figure 5 D). The expression levels of RcARF8 in the petals of both the overexpressed gene (pSuper1300-RcARF8) and the control gene (pSuper1300) were simultaneously detected. Figure 5 C) The results showed that the expression level of RcARF8 was significantly increased in plants overexpressing RcARF8 compared with the control (pSuper1300), which further illustrates that RcARF8 is associated with the resistance of rose petals to gray mold.

[0213] 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 application of overexpressing RcARF8 gene in enhancing the resistance of Rosa chinensis to Botrytis cinerea, characterized in that: The nucleotide sequence of the RcARF8 gene is shown as SEQ ID NO.

1. ​ 2. A method of enhancing resistance to gray mold in Rosa hybrida, characterized by The method comprises the following steps: (1) constructing a recombinant vector comprising the RcARF8 gene according to claim 1; (2) introducing the recombinant vector into a Rosa cell to realize overexpression of the RcARF8 gene.

3. A method for breeding a transgenic Rosa hybrida plant with enhanced resistance to gray mold, characterized in that The method comprises the following steps: (1) constructing a recombinant vector comprising the RcARF8 gene according to claim 1; (2) introducing the recombinant vector into a Rosa cell to realize overexpression of the RcARF8 gene; (3) cultivating a transgenic Rosa plant overexpressing the RcARF8 gene, and evaluating the resistance of the transgenic Rosa plant to gray mold.

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