New application of RcRBL1 gene and method for preventing or treating black spot and gray mold of Chinese rose

Abstract

The invention relates to the technical field of plant variety cultivation, in particular to new application of an RcRBL1 gene and a method for preventing or treating black spot and gray mold of Chinese roses. According to the application of the RcRBL1 gene in preventing or treating the black spot and the gray mold of the Chinese rose, the capability of resisting the black spot and the gray mold of the Chinese rose plant can be remarkably improved by reducing the expression of the RcRBL1 gene in the Chinese rose plant, an effective method is provided for preventing and treating the black spot and the gray mold of the Chinese rose, and the cultivation cost of the Chinese rose is reduced; and the market economic benefits are improved.

Description

Technical Field

[0001] The invention relates to a new use of RcRBL1 gene and a method for preventing or treating black spot disease and gray mold disease of rose, belonging to the technical field of plant variety breeding. Background Art

[0002] Rose belongs to the Rosaceae family. The haploid contains 7 chromosomes. It blooms all year round, has beautiful flowers, complex colors, and strong fragrance. There are more than 30,000 rose varieties. Rose is one of the most popular ornamental plants in the world. It has been widely planted as a horticultural crop for thousands of years. It has also been used as a cut flower and perfume raw material for hundreds of years. It has become an economic crop in many countries. In addition to its ornamental use, rose also has edible and medicinal value. Due to its outstanding economic value, the planting area of ​​rose has increased sharply in recent years. However, improper rose cultivation conditions and management methods often cause pathogens to invade and cause diseases, which not only directly affects the growth and flowering of roses, but also affects its beautification effect and ornamental value, and even causes death. With the development of the economy, people's quality of life has been continuously improved, and they are paying more and more attention to the living environment and quality. The scale of the flower market has expanded rapidly. Rose occupies an important position in the flower market, and the market demand for roses is increasing. In the maintenance process of roses, rose diseases have become a difficult problem that needs to be solved in the rose cultivation process, mainly including rose black spot and gray mold.

[0003] Rose black spot disease often refers to rose black spot disease caused by Diplocarpon rosae, which is a serious disease widely distributed throughout the world except in arid areas. It appears as round spots on roses, one centimeter in diameter, with fluffy edges. The leaves of infected plants turn yellow and fall early. Infected plants may shed their leaves twice during the growing season, become severely weak, have few and poor flowers, are prone to canker, and are prone to death due to winter damage. Countless spores are formed in the conidial disk, which are spread from splashing raindrops, dew, watering, and gardeners working in wet plants. It takes about 9 to 18 hours or longer for the spores to germinate and invade the rose tissue. New leaf spots appear within 3 to 16 days, and spores are formed within 10 to 18 days. Leaves, petioles, young branches, pedicels and young fruits can all be affected, but the leaves are mainly affected. There are two types of symptoms: one is that reddish brown to purple brown spots appear on the leaf surface at the early stage of the disease, gradually expanding into round or amorphous dark black spots, often surrounded by yellow halos, with radial edges and a diameter of about 3-15 mm. In the later stage, small black dots are scattered on the spots, which are the conidia of the pathogen. In severe cases, the lower leaves of the plant turn yellow and fall off early, causing individual branches to die. This cycle can be repeated during the growing season.

[0004] Gray mold of roses is a disease caused by a fungus (Botrytis cinerea Pers), belonging to the Hyphomycetes, Hyphomyceales. When the disease occurs on the leaf margins and tips of roses, it initially appears as water-soaked light brown spots with a smooth and slightly sunken surface, which then expand and rot. When the flower buds are infected, the spots are gray-black, which prevents the flowers from opening, causing the diseased buds to turn brown and die. When the flowers are infected, some petals will turn brown, shrink and rot. Gray mold fungi will also attack the ends of branches after the flowers are folded, and the black diseased parts can be several centimeters below the infection point. In warm and humid conditions, the gray mold layer can completely cover the infected area.

[0005] At present, the prevention and treatment of rose black spot and gray mold is mainly chemical prevention and treatment, which can effectively cure rose black spot and gray mold, but will increase the production cost of roses, pollute the environment, and cannot exert the ability of long-term cure. Plants are often exposed to various pathogens and have a certain ability to achieve resistance to pathogenic microorganisms through pre-formed effective structures and chemical barriers and plant-induced defense mechanisms. Therefore, those skilled in the art are committed to developing a gene that improves rose resistance to black spot and gray mold infection, which is applied to the prevention and treatment of rose black spot and gray mold. Summary of the invention

[0006] The present invention provides application and method of RcRBL1 gene in preventing or treating black spot disease and gray mold disease of rose.

[0007] In a first aspect, the present invention provides a new use of the RcRBL1 gene, namely, use in preventing or treating rose black spot and gray mold, 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 'Angela' rose or 'Lavender Wreath' rose.

[0011] The second aspect of the present invention provides a method for preventing or treating black spot disease and gray mold disease of rose, which down-regulates the expression level of RcRBL1 gene in rose, and the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:2.

[0012] A third aspect of the present invention provides a method for preventing or treating gray mold of roses, which downregulates 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] In the above second and third aspects, the amino acid sequence of the RcRBL1 gene is shown in SEQ ID NO:3.

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

[0015] Furthermore, the method for down-regulating the expression level of the RcRBL1 gene in the rose includes gene silencing or gene knockout.

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

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

[0018] Transforming the recombinant vector into Agrobacterium to obtain a recombinant transformant;

[0019] The recombinant transformant is used to infect rose to obtain a rose silent strain.

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

[0021] Constructing a tRNA-sgRNA for knocking out the RcRBL1 gene, and connecting the tRNA-sgRNA into a pYLCRISPR / CAS9 vector to construct a recombinant vector;

[0022] Transforming the recombinant vector into Agrobacterium to obtain a recombinant transformant;

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

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

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

[0026] A fourth aspect of the present invention provides the use of the RcRBL1 gene in preparing rose varieties resistant to black spot disease 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] A fifth aspect of the present invention provides a method for breeding rose varieties resistant to black spot disease or gray mold disease, by down-regulating the expression level of the RcRBL1 gene in the rose.

[0030] Furthermore, the method for down-regulating the expression level of the RcRBL1 gene in the rose includes gene silencing or gene knockout.

[0031] By implementing the above technical scheme, the present invention can significantly improve the ability of rose plants to resist infection by black spot and gray mold by reducing the expression of RcRBL1 gene in rose plants, providing an effective method for the prevention and treatment of black spot and gray mold of roses, reducing the cost of rose cultivation, and improving market economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the expression analysis diagram of RcRBL1 gene in the leaves of 'Angela' rose at different times after black spot disease pathogen infection;

[0033] Figure 2 This is the expression analysis diagram of RcRBL1 gene in the leaves of 'Angela' rose at different times after infection by gray mold pathogen;

[0034] Figure 3 This is the result of identifying the expression of RcRBL1 gene in the rose RcRBL1 gene silencer and the wild type 'Lavender Wreath' rose;

[0035] Figure 4 This is a comparison of the leaf phenotypes of the rose RcRBL1 gene silencer and the wild type 'Lavender Wreath' rose;

[0036] Figure 5 This is a comparison of the disease severity of the rose RcRBL1 gene silencer and the wild type 'Lavender Wreath' rose after being inoculated with black spot pathogens and gray mold pathogens;

[0037] Figure 6 This is the statistical result of the infection rate of lesions of the rose RcRBL1 gene silencer and the wild type 'Lavender Wreath' rose after inoculation with black spot and gray mold pathogens. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The reagents used, unless otherwise specified, are commercially available or publicly available.

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

[0041] In the present invention, the term "gene silencing" generally refers to post-transcriptional gene silencing (PTGS), that is, the expression of a gene is affected by specific intervention on the target RNA at the level after gene transcription. Usually, when a gene is silenced, the expression of the corresponding gene is downregulated / reduced; the term "gene knockout" generally refers to the operation of genomic DNA, and when a gene is knocked out, it is usually manifested as the genomic DNA being destroyed and unable to be expressed. For example, in a cell, when all alleles of a specific gene are knocked out, the expression of the gene disappears.

[0042] In the present invention, the rose variety 'Lavender Flower Circus' is a climbing rose born in Japan in 2014, with thin branches and almost no thorns. It blooms in multiple heads and prefers full sun and slightly acidic soil. The flower color is blue-purple, the flower diameter is 6 cm, the flower has double petals, and has no fragrance. 'Lavender Flower Circus' grows rapidly, but its branching is not as good as other varieties, and it is a lazy flower.

[0043] In the present invention, the rose variety 'Angela' is divided into two types: vines and shrubs. The vine type has climbing properties, while the shrub type grows independently, and the flowers are usually pink with light pink highlights and a light center, with about 35 petals and an average diameter of about 4 cm. The flowers are fully double, i.e., contain 26 to 40 petals, and bloom in clusters to form small clusters with a cup-shaped flowering form.

[0044] Example 1 Cloning of Rose RcRBL1 Gene

[0045] 1. Select the rose varieties 'Angela' and 'Lavender Wreath' planted in the Rose Garden of Taoyuan Building, Shanghai Normal University. The growing conditions are open air, watered once a week, and regularly fertilized and maintained with pesticides for disease 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 the extracted rose RNA as a template, the 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 a cDNA containing a 2158bp long RcRBL1 gene, and the full-length nucleotide sequence of RcRBL1 is shown in SEQ ID NO: 1. Using a primer pair (forward primer (SEQ ID NO: 4), reverse primer (SEQ ID NO: 5)) to perform PCR amplification on the rose sample cDNA, a 1257bp long coding region gene fragment can be obtained, and the coding region sequence is shown in SEQ ID NO: 2. The protein encoded by RcRBL1 has a total of 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 at different pathogen infection levels

[0049] The 'Angela' rose cultivated in the open air was inoculated with black spot and gray mold pathogens, and samples were taken at 0, 6h, 12h, 24h, 48h and 4 days to obtain leaves with different disease severity, and RNA was extracted from the rose leaves (Accurate BiologySteadyPure 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), and real-time PCR detection was performed using primers (forward primer (SEQ ID NO: 6), reverse primer (SEQ ID NO: 7)).

[0050] The results are as follows Figure 1 As shown in Figure 2, the expression level of the gene RcRBL1 first decreased and then increased when infected by black spot disease. Figure 2 As shown, the expression level of gene RcRBL1 showed a downward trend when infected by gray mold.

[0051] Example 3 Identification of Silencing Efficiency of RcRBL1 Silencers

[0052] The RcRBL1 gene was inserted into the TRV2 vector (purchased from the Plasmid Vector Bacteria Cell Gene Collection Center) to construct the TRV2-RcRBL1 vector, and transformed with Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and propagated using a culture medium containing rifampicin and kanamycin. The bacteria were then collected and resuspended in a magnesium chloride solution, and the 'Lavender Wreath' rose branches that had been disinfected with potassium permanganate for 15 minutes were infected by the immersion vacuum method. The rose 'Lavender Wreath' branches treated with TRV2-GFP Agrobacterium were used as the control strain, and the rose 'Lavender Wreath' branches treated with TRV2-RcRBL1 Agrobacterium were used as the silenced strain. New leaves grew out after one week, and TRV2-GFP with a GFP tag was used as the control group. The RcRBL1 gene silencer was detected, that is, total RNA was extracted from the leaves of the silenced plants and the wild-type plant control group, and primers (forward primer (SEQ ID NO: 6), reverse primer (SEQ ID NO: 7) were used to detect the silenced RcRBL1 gene. NO:7)) Realtime-PCR was performed to detect whether the RcRBL1 gene was silenced, and the silent strain was screened.

[0053] Test results such as Figure 3 As shown, the expression level of RcRBL1 in the silencer was at least 60% lower than that in the wild type.

[0054] Example 4 Phenotypic analysis of RcRBL1 silenced strains

[0055] The leaves of the silent body plants and wild-type plants were sampled, and the same batch of samples were taken as in Example 3 for the identification of the silencing efficiency of the silent body. The results are as follows: Figure 4 As shown, there is no difference in leaf shape and leaf area between the silent plants and the wild-type plants.

[0056] Example 5 Analysis of resistance of RcRBL1 silent rose 'Lavender Wreath' to leaf black spot and gray mold

[0057] The leaves of the silent plants and wild-type plants were inoculated with mycelial blocks of black spot and gray mold pathogens (belonging to the physiological species of Podosphaera pannosa) and cultured in the dark at 28° C. for 7 days. Specifically, the steps include:

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

[0059] (2) When young leaves unfold, live rose leaves are sprayed with black spot and gray mold pathogens and cultured for 4 days. They are then cultured in the dark at 28°C for 4 days to provide biological stress, and leaf morphology is recorded by photographing. Figure 5As shown, the leaves of the control group showed obvious lesions and browning after being infected by black spot pathogens and gray mold pathogens, but the leaves of the silent group remained green and healthy.

[0060] (3) Determine the lesion size of the silent and wild-type roses. Ten days after the leaves of the TRV2-GFP control group and the TRV2-RBL1 treatment group were inoculated with black spot and gray mold, the infection rate was statistically analyzed (based on whether the pinholes were enlarged) and the incidence of infection was calculated. The infection rate was the number of leaves with pinholes invading the rose pathogen (i.e., the number of leaves with enlarged pinholes) divided by the total number of treated leaves in the group. The results are shown in Figure 2. Figure 6 As shown, the degree of infection of the leaves of the silent plants was much lower than that of the leaves of the wild-type plants, indicating that down-regulating the expression of the RcRBL1 gene can reduce the infection during the infection of rose black spot and gray mold pathogens, that is, by reducing the expression of the RcRBL1 gene, the resistance to rose black spot and gray mold can be improved.

[0061] In summary, the present invention obtains RcRBL1-silenced rose strains through virus-induced gene silencing of RcRBL1, analyzes the resistance of RcRBL1-silenced strains and wild-type strains to pathogens, and clarifies that reducing the expression of RcRBL1 gene in rose plants can significantly improve their ability to resist black spot and gray mold infection, thereby providing an effective method for the prevention and treatment of black spot and gray mold of roses, reducing the cost of rose cultivation, and improving 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. The use of RcRBL1 gene in preventing or treating black spot disease and gray mold disease of rose, characterized in that: The CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:

2.

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

3.

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

1.

4. The use according to claim 1, characterized in that: The rose is 'Angela' rose or 'Lavender Wreath' rose.

5. A method for preventing or treating black spot disease and gray mold disease of rose, characterized in that: The expression level of the RcRBL1 gene in rose is down-regulated, and the CDS sequence of the RcRBL1 gene is shown in SEQ ID NO:

2.

6. The method according to claim 5, characterized in that The amino acid sequence of the RcRBL1 gene is shown in SEQ ID NO:

3.

7. The method according to claim 5, characterized in that The nucleotide sequence of the RcRBL1 gene is shown in SEQ ID NO:

1.

8. The method according to claim 5, characterized in that The rose is 'Angela' rose or 'Lavender Wreath' rose.

9. The method according to claim 5, characterized in that The method for down-regulating the expression level of the RcRBL1 gene in the rose includes gene silencing or gene knockout.

10. Application of RcRBL1 gene in preparing rose varieties resistant to black spot and gray mold pathogens.

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