New use of rcRBL1 gene and method for preventing or treating black spot and botrytis blight of Chinese rose

Abstract

The present application relates to the technical field of plant variety breeding, and particularly relates to a new use of RcRBL1 gene and a method for preventing or treating Chinese rose black spot and botrytis blight. The present application provides application of RcRBL1 gene in prevention or treatment of Chinese rose black spot and botrytis blight, and the ability of resisting infection of Chinese rose black spot and botrytis blight can be significantly improved by reducing the expression of RcRBL1 gene in Chinese rose plant, which provides an effective method for prevention and treatment of Chinese rose black spot and botrytis blight, reduces the breeding cost of Chinese rose, and improves market economic benefits.

Description

Technical Field

[0001] This invention relates to novel uses of the RcRBL1 gene and methods for preventing or treating black spot and gray mold in roses, belonging to the field of plant variety breeding technology. Background Technology

[0002] Roses belong to the Rosaceae family. Haploid roses contain seven chromosomes, bloom year-round, and boast beautiful flowers with diverse colors and rich fragrances. Currently, there are over 30,000 varieties of roses. Roses are one of the world's most popular ornamental plants, widely cultivated as a horticultural crop for thousands of years and used as cut flowers and perfume ingredients for centuries, becoming an economic crop in many countries. Besides their ornamental uses, roses also have edible and medicinal value. Due to their significant economic value, the planting area of ​​roses has surged in recent years. However, improper cultivation conditions and management methods often lead to pathogen invasion and disease outbreaks, directly affecting the growth and flowering of roses, as well as their aesthetic appeal and ornamental value, and even causing death. With economic development and rising living standards, people are paying more attention to their living environment and quality of life. The flower market is expanding rapidly, and roses occupy an important position in this market. Market demand for roses is increasing. In the process of rose cultivation, diseases have become a pressing problem that needs to be solved, mainly including rose black spot and gray mold.

[0003] Rose black spot disease, commonly known as rose black spot caused by *Diplocarpon rosae*, is a serious disease widely distributed worldwide outside of arid regions. It manifests as round spots on roses, about one centimeter in diameter, with a lobed edge. Infected plants develop yellow leaves and premature leaf drop. Affected plants may experience two leaf drop cycles during the growing season, become severely weakened, produce fewer and poorer flowers, are susceptible to canker, and are prone to death due to winter damage. Numerous spores are formed in the conidiophores and spread through splashing raindrops, dew, watering, and when gardeners work on damp plants. Spore germination and invasion of rose tissue takes approximately 9–18 hours or longer; new leaf spots appear within 3–16 days, and spores form within 10–18 days. Leaves, petioles, young shoots, pedicels, and young fruits can all be affected, but the leaves are primarily affected. There are two types of symptoms: One type initially presents as small reddish-brown to purplish-brown spots on the leaf surface, which gradually enlarge into round or irregular dark black lesions. These lesions are often surrounded by a yellow halo, with radiating edges, and are approximately 3-15 mm in diameter. Later, small black dots, the conidiophores of the pathogen, appear scattered on the lesions. In severe cases, the lower leaves of the plant turn yellow and wither, leading to premature leaf drop and the death of some branches. This cycle can repeat during the growing season.

[0004] Gray mold in roses is a disease caused by a fungus (Botrytis cinerea Pers), belonging to the class Hyphomycetes and order Hyphomyales. When this disease occurs on the leaf margins and tips of roses, it initially appears as water-soaked, light brown spots with a smooth, slightly sunken surface, which then enlarge and rot. When flower buds are infected, the lesions turn grayish-black, preventing the flowers from opening, causing the buds to turn brown and wither. When flowers are affected, some petals turn brown, wrinkle, and rot. Gray mold can also infect the tips of branches after flowering, with black lesions extending several centimeters below the point of infection. In warm, humid environments, a gray mold layer can completely cover the infected area.

[0005] Currently, the control of black spot and gray mold in roses mainly relies on chemical control. While chemical control can effectively cure these diseases, it increases rose production costs, pollutes the environment, and does not provide long-term cure. Plants are frequently exposed to various pathogens and possess a certain ability to develop resistance to pathogenic microorganisms through pre-formed effective structures, chemical barriers, and plant-induced defense mechanisms. Therefore, those skilled in the art are dedicated to developing a gene that enhances the resistance of roses to black spot and gray mold pathogens for the control of these diseases. Summary of the Invention

[0006] This invention provides the application and method of the RcRBL1 gene in the prevention or treatment of black spot and gray mold in roses.

[0007] The first aspect of this invention provides a novel use of the RcRBL1 gene, namely, its application in the prevention or treatment of black spot and gray mold in roses, wherein the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:2.

[0008] The amino acid sequence of the RcRBL1 gene is shown in SEQ ID NO:3.

[0009] The nucleotide sequence of the RcRBL1 gene is shown in SEQ ID NO:1.

[0010] Furthermore, the rose is either the 'Angela' rose or the 'Lavender Garland' rose.

[0011] A second aspect of the present invention provides a method for preventing or treating black spot disease and gray mold in roses by downregulating the expression level of the RcRBL1 gene in roses, wherein the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:2.

[0012] The third aspect of the present invention provides a method for preventing or treating gray mold in roses by downregulating the expression level of the RcRBL1 gene in roses, wherein the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:2.

[0013] The amino acid sequence of the RcRBL1 gene is shown in SEQ ID NO:3, as described in the second and third aspects above.

[0014] The nucleotide sequence of the RcRBL1 gene is shown in SEQ ID NO:1.

[0015] Furthermore, methods for downregulating the expression level of the RcRBL1 gene in the rose include gene silencing or gene knockout.

[0016] Furthermore, the gene silencing method includes the following steps:

[0017] The RcRBL1 gene was inserted into the TRV2 vector to construct a recombinant vector;

[0018] The recombinant vector was transformed into Agrobacterium to obtain recombinant transformants;

[0019] Roses were infected with the recombinant transformants to obtain silent rose lines.

[0020] Furthermore, the gene knockout method includes the following steps:

[0021] A tRNA-sgRNA that knocks out the RcRBL1 gene was constructed, and the tRNA-sgRNA was ligated into the pYLCRISPR / CAS9 vector to obtain a recombinant vector.

[0022] The recombinant vector was transformed into Agrobacterium to obtain recombinant transformants;

[0023] The recombinant transformant was used to transform rose plants to obtain RcRBL1 knockout lines.

[0024] Furthermore, the roses mentioned are 'Angela' and 'Lavender Garland' roses.

[0025] Furthermore, compared to wild-type roses, the expression level of the RcRBL1 gene decreased by at least 60% in transgenic roses after the expression level of the RcRBL1 gene was downregulated.

[0026] The fourth aspect of the present invention provides the application of the RcRBL1 gene in the preparation of rose varieties resistant to black spot or gray mold pathogens, wherein the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:2.

[0027] Furthermore, the amino acid sequence of the RcRBL1 gene is shown in SEQ ID NO:3.

[0028] Furthermore, the nucleotide sequence of the RcRBL1 gene is shown in SEQ ID NO:1.

[0029] In a fifth aspect, the present invention provides a method for breeding rose varieties resistant to black spot or gray mold by downregulating the expression level of the RcRBL1 gene in roses.

[0030] Furthermore, methods for downregulating the expression level of the RcRBL1 gene in the rose include gene silencing or gene knockout.

[0031] By implementing the above technical solution, this invention can significantly improve the resistance of rose plants to black spot and gray mold by reducing the expression of the RcRBL1 gene, providing an effective method for the prevention and treatment of rose black spot and gray mold, reducing the cost of rose cultivation and improving market economic benefits. Attached Figure Description

[0032] Figure 1 This is a graph showing the expression analysis of the RcRBL1 gene in the leaves of the 'Angela' rose at different time points after infection with the black spot pathogen;

[0033] Figure 2 This is a graph showing the expression analysis of the RcRBL1 gene in the leaves of the 'Angela' rose at different time points after infection with gray mold.

[0034] Figure 3 Figure showing the results of identifying the expression levels of the RcRBL1 gene in the silent form of the rose and the wild-type 'Lavender Garland' rose;

[0035] Figure 4 A comparison of leaf phenotypes between the silent variant of the RcRBL1 gene in rose and the wild-type 'Lavender Garland' rose;

[0036] Figure 5 Comparison of disease severity between the silent variant of the RcRBL1 gene in rose and the wild-type 'Lavender Garland' rose after inoculation with black spot and gray mold pathogens;

[0037] Figure 6 The graph shows the statistical results of lesion infection rates of the silent variant of the RcRBL1 gene in roses and the wild-type 'Lavender Garland' rose after inoculation with black spot and gray mold pathogens. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used were commercially available or publicly available.

[0040] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.

[0041] In this invention, the term "gene silencing" generally refers to post-transcriptional gene silencing (PTGS), which means affecting gene expression at the post-transcriptional level through specific intervention on target RNA. Typically, when a gene is silenced, the expression of the corresponding gene is downregulated / reduced. The term "gene knockout" generally refers to manipulation of genomic DNA. When a gene is knocked out, it usually manifests as the genomic DNA being damaged and unable to be expressed. For example, in a cell, knocking out all alleles of a specific gene results in the disappearance of that gene's expression.

[0042] In this invention, the rose variety 'Lavender Flower Circus' is a climbing rose that originated in Japan in 2014. It has slender branches and almost no thorns. It produces multiple blooms, prefers full sun, and slightly acidic soil. The flowers are blue-purple, about 6cm in diameter, double-petaled, and unscented. 'Lavender Flower Circus' grows rapidly but branches less than other varieties, making it a less prolific bloomer.

[0043] In this invention, the rose variety 'Angela' is divided into two types: climbing and shrub. The climbing type is vine-like, while the shrub type grows independently. The flowers are typically pink with light pink highlights and a paler center. They have approximately 35 petals and an average diameter of about 4 cm. The flowers are fully double-petaled, containing 26 to 40 petals, and open in clusters, forming small groups with a cup-shaped bloom.

[0044] Example 1: Cloning of the Rose RcRBL1 Gene

[0045] 1. Select the rose varieties 'Angela' and 'Lavender Garland' planted in the Rose Garden of Taoyuan Building at Shanghai Normal University. The growing conditions are outdoors. Water once a week, and fertilize and maintain the plant regularly with pesticides for disease prevention and control.

[0046] 2. RNA extraction. Take 500 mg of fresh rose plant tissue material, add liquid nitrogen and grind it into powder, then extract plant RNA (Accurate Biology SteadyPure Plant RNA Extraction Kit AG21019).

[0047] 3. Gene Cloning. Using extracted rose RNA as a template, total RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme HiScriptIII All-in-one RT SuperMix Perfect for qPCR R333-01) to obtain cDNA containing the 2158 bp RcRBL1 gene. The full-length nucleotide sequence of RcRBL1 is shown in SEQ ID NO:1. PCR amplification of the rose sample cDNA using primer pairs (forward primer (SEQ ID NO:4), reverse primer (SEQ ID NO:5)) yielded a 1257 bp coding region gene fragment. The coding region sequence is shown in SEQ ID NO:2. The protein encoded by RcRBL1 has 424 amino acid residues, and its amino acid sequence is shown in SEQ ID NO:3.

[0048] Example 2: Analysis of the expression pattern of the rose RcRBL1 gene under different pathogen infection levels

[0049] 'Angela' roses grown outdoors were inoculated with black spot and gray mold pathogens. Leaves with different disease severity were sampled at 0, 6h, 12h, 24h, 48h, and 4 days. RNA was extracted from the rose leaves using the Accurate Biology SteadyPure Plant RNA Extraction Kit AG21019. The RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme HiScript III All-in-one RT SuperMix Perfect for qPCR R333-01). Real-time PCR was then performed using primers (forward primer (SEQ ID NO:6) and reverse primer (SEQ ID NO:7)).

[0050] The results are as follows Figure 1 As shown, the expression level of gene RcRBL1 initially decreased and then increased during black spot disease infection. The results are as follows... Figure 2 As shown, the expression level of gene RcRBL1 decreases during gray mold infection.

[0051] Example 3: Identification of the silencing efficiency of the RcRBL1 silencing agent

[0052] The RcRBL1 gene was inserted into the TRV2 vector (purchased from the Plasmid Vector Culture Collection Center) to construct the TRV2-RcRBL1 vector. This vector was then transformed with Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The vector was propagated using a medium containing rifampicin and kanamycin. The bacterial cells were then collected and resuspended in magnesium chloride solution. 'Lavender Wreath' rose branches, sterilized with potassium permanganate for 15 minutes, were then infected using a vacuum method. Rose branches treated with TRV2-GFP Agrobacterium served as the control line, while those treated with TRV2-RcRBL1 Agrobacterium served as the silent line. New leaves appeared one week later. Meanwhile, TRV2-GFP with a GFP tag served as the control group. The RcRBL1 gene silencer was detected by extracting total RNA from leaves of both the silent and wild-type control plants. Primers (forward primer (SEQ ID NO: 6) and reverse primer (SEQ ID NO: 6) were used to detect the silenced RcRBL1 gene. NO:7)) Real-time PCR was performed to detect whether the RcRBL1 gene was silenced, and the silenced lines were selected.

[0053] Test results as follows Figure 3 As shown, the expression level of RcRBL1 in the silenced form was reduced by at least 60% compared to the wild type.

[0054] Example 4: Phenotypic analysis of the RcRBL1 silent strain

[0055] Leaves of both the silent plant and wild-type plants were sampled from the same batch as those used in Example 3 for the determination of the silencing efficiency of the silent plant. The results are as follows: Figure 4 As shown, there is no difference in leaf shape and leaf area between the silent plant and the wild-type plant.

[0056] Example 5: Resistance analysis of RcRBL1 silent rose 'Lavender Garland' to black spot and gray mold on leaves.

[0057] Silent plants and leaves of wild-type plants were inoculated with mycelial blocks of black spot and gray mold pathogens (belonging to the physiological race of *Podosphaera pannosa*) and cultured in the dark at 28°C for 7 days; the specific steps included the following:

[0058] (1) RcRBL1 silent plants and wild-type plants were cultured in a constant temperature incubator at 28℃ and watered regularly.

[0059] (2) Once the young leaves unfold, spray live rose leaves with black spot and gray mold pathogens respectively and culture for 4 days. Then, culture them in the dark at 28℃ for 4 days to subject them to biological stress, and photograph and record the leaf morphology. The results are as follows: Figure 5As shown, the leaves of the control group showed obvious lesions and browning after being infected by black spot fungus and gray mold fungus, but the leaves of the silent group still showed a green and healthy state.

[0060] (3) Determining the size of lesions in silent and wild-type plants. Leaves from the TRV2-GFP control group and the TRV2-RBL1 treatment group were inoculated with black spot and gray mold fungi for 10 days. The infection rate was statistically analyzed (based on pinhole enlargement). The infection rate was calculated as the ratio of the number of leaves with pinholes invaded by rose pathogens in each group (i.e., the number of leaves with enlarged pinholes) to the total number of leaves in that group. Results are as follows: Figure 6 As shown, the disease severity of leaves in the silent plant was much lower than that in the wild-type plant, indicating that downregulating the expression of the RcRBL1 gene can reduce infection during the infection process of rose black spot and gray mold pathogens. In other words, reducing the expression of the RcRBL1 gene can improve the resistance of rose to black spot and gray mold.

[0061] In summary, this invention obtained RcRBL1-silenced rose lines by silencing the RcRBL1 gene induced by a virus. Analysis of the resistance of RcRBL1-silenced lines and wild-type lines to pathogens clarified that reducing the expression of the RcRBL1 gene in rose plants can significantly improve their resistance to black spot and gray mold infections. This provides an effective method for the prevention and treatment of black spot and gray mold in roses, reduces rose cultivation costs, and improves market economic benefits.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. RcRBL1 The application of genes in the prevention of black spot and gray mold in roses is characterized by, Lowering the price of roses RcRBL1 Gene expression levels RcRBL1 The CDS sequence of the gene is shown in SEQ ID NO:2, and the rose is either the 'Angela' rose or the 'Lavender Garland' rose.

2. The application according to claim 1, characterized in that, RcRBL1 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. A method for preventing black spot and gray mold in roses, characterized in that, Lowering the price of roses RcRBL1 The expression level of the gene, the RcRBL1 The CDS sequence of the gene is shown in SEQ ID NO:2, and the rose is either the 'Angela' rose or the 'Lavender Garland' rose.

4. The method according to claim 3, characterized in that, RcRBL1 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that, Lower the mentioned rose RcRBL1 Methods for measuring gene expression include gene silencing or gene knockout.

6. Lower the price of roses RcRBL1 The application of genes in the preparation of rose varieties resistant to black spot and gray mold pathogens, the aforementioned RcRBL1 The CDS sequence of the gene is shown in SEQ ID NO:2, and the rose is either the 'Angela' rose or the 'Lavender Garland' rose.

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