RcPR5 gene for regulating and controlling gray mold resistance of Chinese rose and application of RcPR5 gene

Abstract

The invention belongs to the technical field of agricultural biology, and particularly discloses an RcPR5 gene for regulating and controlling gray mold resistance of Chinese roses and application of the RcPR5 gene, and the nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.1. The RcPR5 gene provided by the invention is remarkably associated with the gray mold resistance, plays an important role in plant immunity, regulates and controls immunity through an RNA processing complex, and has the advantages that the resistance of Chinese roses to gray mold is regulated and controlled; transcriptome difference analysis shows that the expression quantity of the RcPR5 is induced by grey mould to be remarkably up-regulated, after the RcPR5 gene is silenced, the petal scab is remarkably increased, it is proved that the RcPR5 gene really plays a role in the disease resistance process of the gray mold of the Chinese rose, breeding of the gray mold resistant Chinese rose can be conducted through the gene by adopting the modern gene engineering technology, and the breeding cost is lowered. And new germplasm is developed and introduced.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to an RcPR5 gene that regulates resistance to gray mold in roses and its application. Background Technology

[0002] The rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant belonging to the genus Rosa in the family Rosaceae. Rose cut flowers account for over 30% of the global cut flower market share and are recognized worldwide as the most economically valuable ornamental plant. Year-round rose production mainly relies on protected cultivation facilities such as greenhouses and polytunnels, where the high humidity exacerbates the spread of diseases. Among these, gray mold is one of the most significant diseases affecting the cut rose industry. Gray mold occurs throughout the rose's life cycle, infecting various tissues and organs, including flowers, sepals, leaves, and young shoots, but is most common on flowers at the third stage of opening or higher (the main commercial harvest period). After infecting petals, gray mold forms multiple lesions of varying sizes, eventually leading to discoloration, wilting, rotting, and necrosis of the petals, rendering them unusable for ornamental and economic purposes. Currently, the main disease control measure in rose cultivation is pesticide application; however, continuous and high-dose use of chemical pesticides has led to increased pesticide resistance in rose gray mold. At the same time, most modern rose varieties are not very resistant, and traditional hybridization breeding of roses has encountered a bottleneck, urgently requiring the development and introduction of new germplasm.

[0003] Disease-resistant breeding is a long-term and sustainable strategy for controlling gray mold. Its core lies in enhancing the natural disease resistance of plants by screening and introducing disease-resistant genes, thereby reducing the incidence of the disease and the damage it causes. Therefore, it is necessary to study gray mold-resistant genes in roses and combine this with molecular design and assisted selection breeding to improve the precision and efficiency of rose breeding, reduce reliance on chemical agents in crop production, thereby reducing environmental pollution and pesticide residues, and providing a basis for achieving green agriculture and sustainable production methods. Summary of the Invention

[0004] The main objective of this invention is to provide a new approach for breeding roses with an RcPR5 gene that regulates resistance to gray mold. Specifically, this invention provides the following technical solutions: This invention provides an RcPR5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] Furthermore, the present invention provides the application of products expressing the RcPR5 gene in promoting resistance to gray mold in roses, wherein the nucleotide sequence of the RcPR5 gene is shown in SEQ ID NO.1.

[0006] Furthermore, the present invention provides the use of a product expressing the RcPR5 protein in promoting resistance to gray mold in roses, wherein the amino acid sequence of the RcPR5 protein is shown in SEQ ID NO.2.

[0007] As one embodiment, the present invention provides a kit for promoting resistance to gray mold in roses, the kit containing a reagent expressing the RcPR5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] As one embodiment, the present invention provides the application of a carrier in promoting resistance to gray mold in roses, the carrier containing a base sequence as shown in SEQ ID NO:1.

[0009] As one embodiment, the present invention provides the application of engineered bacteria in promoting resistance to gray mold in roses, wherein the engineered bacteria contains the aforementioned carrier.

[0010] As one embodiment, the present invention provides a method for resisting gray mold infection in roses, which uses genetic engineering technology to express the RcPR5 gene or its translated protein, wherein the nucleotide sequence of the RcPR5 gene is shown in SEQ ID NO.1.

[0011] In one embodiment, the present invention provides the application of the RcPR5 gene in breeding roses resistant to gray mold, wherein the nucleotide sequence of the RcPR5 gene is shown in SEQ ID NO.1. In some embodiments, the breeding includes marker-assisted breeding, whole-genome selection breeding, or transgenic breeding.

[0012] As one embodiment, the present invention provides the application of a reagent for detecting the expression level of the RcPR5 gene in the breeding of roses resistant to gray mold, wherein the nucleotide sequence of the RcPR5 gene is shown in SEQ ID NO.1.

[0013] The technical effects achieved by this invention are as follows: This invention, after performing BLUE analysis on four years of two-site data points on rose gray mold resistance phenotypes, conducted genome-wide association analysis on lesion area and block analysis on the 17M-30M region of chromosome 3. The results revealed a significant association between the RcPR5 gene and gray mold resistance. This gene plays an important role in plant immunity and regulates immunity through the RNA processing complex. Transcriptome differential analysis showed that RcPR5 expression was significantly upregulated by gray mold. Silencing the RcPR5 gene significantly increased the size of petal lesions, proving its role in rose gray mold resistance. The rose gray mold resistance gene provided by this invention, combined with molecular design and assisted selection breeding, can improve the precision and efficiency of rose breeding, reduce dependence on chemical agents in rose cultivation, thereby reducing environmental pollution and pesticide residues, and providing a basis for achieving green agriculture and sustainable production methods. Attached Figure Description

[0014] Figure 1 Heatmap of RcPR5 expression levels at different time points after inoculation with *Botrytis cinerea*. The values ​​in the legend are Z-score normalized results of gene expression levels (FPKM). 18h, 30h, 36h, 48h, 60h, and 72h represent the expression levels of petals 18h, 30h, 36h, 48h, 60h, and 72h after *Botrytis cinerea* inoculation, respectively. Leaf48h represents the expression level of leaves 48h after *Botrytis cinerea* inoculation. Figure 2 Image of petal lesions after silencing RcPR5 in rose petal discs during VIGS validation (TRV: empty vector control, TRV-PR-5: silenced RcPR5). Figure 3 Statistical chart of lesion area after VIGS silencing RcPR5; the t-test was used to test the significance of the difference, * indicates p<0.05, the difference is significant.

[0015] Figure 4 Relative expression levels of RcPR5 in petals after VIGS silencing RcPR5; the t-test was used to test for significant differences, * indicates p<0.05, indicating a significant difference. Detailed Implementation

[0016] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0017] For products purchased for testing, if specific conditions are not specified, they should be tested under standard conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can be obtained from commercially available standard products.

[0018] The nucleotide sequence of the RcPR5 gene provided by this invention is shown in SEQ ID NO.1, and the protein amino acid sequence is shown in SEQ ID NO.2. RcPR5 is 479 bp in length, contains only one exon of 255 bp, has no introns, a 3'UTR (three prime UTR) of 2 bp, and a 5'UTR of 221 bp.

[0019] Example 1 1. Plant variety: Rose cultivar 'Carola'.

[0020] 2. Primer design Suitable primers were designed using Primer5 based on the candidate gene sequences (Table 1). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. A silencing vector was constructed using the Gateway method, and the constructed vector was sequenced by The Beijing Genomics Institute. Sequence alignment was performed using DNAMAN software, and the correctly aligned target plasmid was transformed into Agrobacterium GV3101.

[0021] Table 1. Transient gene silencing primers (constructed using the Gateway method)

[0022] 3. Procedure for Virus-Induced Gene Silencing (VIGS) in Rose Petals: (1) Activate the frozen Agrobacterium tumefaciens of TRV1, TRV2, and TRV2-RcPR5 by streaking them on plates with the corresponding resistance and incubating them in an incubator at 28°C for 2 days (inverted and protected from light); TRV is Tobacco rattle virus. (2) Pick the activated Agrobacterium and place it in a 10 mL centrifuge tube. Add 2 mL of LB liquid medium (0.1%: 50 µg / L Kan + 100 mg / L Rif), and shake at 28℃ and 200 rpm for 12-16 h. After activation, identify the target fragment. Continue the subsequent operations for the gene of the positive clone. (3) Add 100 mL of LB liquid medium to a 500 mL Erlenmeyer flask. Add 4 mL of bacterial culture to the medium at a ratio of 1:25. Incubate at 28 °C and 200 rpm for 12-16 h. LB liquid medium (1 mL 1 M MES, 40 µL acetylsuccinone, 100 µL 50 mg / L Kan, 100 µL 25 mg / L Rif); MES (2-morpholinoethanesulfonic acid; Kan (kanamycin); Rif (rifampicin).

[0023] 1 M MES: Weigh 19.52 g MES (2-(N-morpholino)ethanesulfonic acid) and dissolve it in 80 mL of deionized water. Adjust the pH to 6.3 with solid KOH, bring the volume to 100 mL, filter and sterilize using a 0.45 μm pore size filter membrane, and aliquot and store at -20 ℃.

[0024] LB liquid medium formulation (1L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride (NaCl).

[0025] (4) Dispense the shaken bacterial solution into 50mL centrifuge tubes and centrifuge at 5000rpm for 10min to collect the bacterial cells; (5) Discard the supernatant, resuspend the bacterial cells in freshly prepared infection solution, and use a 5 mL pipette to suspend the bacterial cells in the infection solution. Mix well by pipetting, and adjust the OD value of Agrobacterium using a spectrophotometer to achieve the final OD value. 600 ≈1.2; (6) Mix TRV1 with TRV2 or TRV2-RcPR5 Agrobacterium in a 1:1 ratio and treat in the dark for 3-5 hours.

[0026] (7) Use a vacuum pump to suction and infect the petal discs. After infection, rinse with tap water 5 times and distilled water once. Finally, place in distilled water at 8°C for 3 days to equilibrate in the dark. (8) After equilibration, the petal discs were equilibrated again for 3 days under culture conditions, and then placed on 0.4% water agar to inoculate with gray mold. Each culture dish contained 16 petals, and each gene contained 3 culture dishes as replicates. (9) Inoculation with infection solution: Inoculate each petal with 2 µL of a 10⁵ globular mold spore suspension and incubate in a light incubator for 60 h. The incubator conditions were: temperature 24℃, light / dark 16 h / 8 h. 60 h after inoculation with globular mold, take photos and save them. Use ImageJ software to measure the lesion area, and use GraphPadPrism v8.0.1 to perform statistical analysis and plotting of the data.

[0027] 4. Real-time quantitative PCR (RT-qPCR) procedure: The reaction was performed using a Novizumi real-time fluorescence quantitative reagent kit, and the procedure was followed according to the company's standard operating instructions.

[0028] (1) Assemble according to a 10 μL reaction system: cDNA template (1 μL) + 2×Realtime PCR Supermix (5 μL) + upstream primer (0.5 μL) + downstream primer (0.5 μL), add sterile water to bring the total volume to 10 μL; (2) The PCR reaction program was set as follows: 95℃ (10 min); 95℃ (15 s), 60℃ (15 s), 72℃ (30 s), 40 cycles; (3) The 2^-T method was used to process the data and calculate the relative gene expression levels; (4) Gene silencing efficiency detection: Take petal discs that have been in silencing equilibrium for 6 days, and take 3 biological replicates (16×3) for each gene. TRV:GFP (transient silencing) is used as a control. The quantitative primers used in the experiment are shown in Table 2.

[0029] (5) In this study, the quantitative experiments all used Rose RhUBI2 as the internal reference gene. The results were the average (±SD) of three technical replicates and three biological replicates were used for quantitative verification. GraphPadPrismv8.0.1 was used to statistically analyze and plot the data.

[0030] 5. Results like Figure 1 As shown, this invention analyzed the transcriptome of rose petals at different time points after inoculation with Botrytis cinerea and found that the expression level of RcPR5 was significantly upregulated compared with the control, indicating that RcPR5 was induced by Botrytis cinerea.

[0031] To verify the gene function of RcPR5, this invention used the modern rose cultivar 'Carola' for transient VIGS silencing, with the cloned fragment being 171 bp of the 3'UTR. After silencing the RcPR5 gene, compared with the TRV:GFP control group, the petal lesions of 'Carola' roses in the TRV:RcPR5 silencing group were significantly larger. Simultaneously, RT-qPCR results showed that the expression level of RcPR5 in the petals of the silenced roses was significantly reduced. Figure 2 , 3 4) This proves that RcPR5 does indeed play an important role in the resistance of roses to gray mold.

[0032] In summary, this invention, after performing BLUE analysis on two-site data points of rose gray mold resistance over four years, conducted genome-wide association analysis on lesion area and block analysis on the 17M-30M region of chromosome 3. The results showed that the RcPR5 gene was significantly associated with gray mold resistance, playing an important role in plant immunity and regulating immunity through the RNA processing complex. Transcriptome differential analysis indicated that RcPR5 gene expression was significantly upregulated by gray mold. Silencing the RcPR5 gene significantly increased the size of petal lesions, proving that it indeed plays a role in rose gray mold resistance.

[0033] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A RcPR5 gene, characterized in that, The nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.

1.

2. Use of the product expressed by the RcPR5 gene in promoting resistance to gray mold in Rosa chinensis, characterized in that, The nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.

1.

3. Use of a product expressing RcPR5 protein in promoting resistance to gray mold in Rosa chinensis, characterized in that, The amino acid sequence of the RcPR5 protein is shown as SEQ ID NO.

2.

4. Use of a kit in promoting resistance to gray mold in Rosa chinensis, characterized in that, The kit contains reagents for expressing the RcPR5 gene, and the nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.

1.

5. Use of a carrier in promoting resistance to gray mold in Rosa chinensis, characterized in that, The vector contains the base sequence shown as SEQ ID NO:

1.

6. The use of an engineered bacteria in promoting resistance to gray mold in Rosa chinensis, characterized in that, The engineering bacteria contain the vector described in claim 5.

7. A method of protecting rose plants from infection by Botrytis cinerea, characterized in that, Using genetic engineering technology, the RcPR5 gene or its translated protein is expressed, and the nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.

1.

8. Use of RcPR5 gene in breeding of rose resistant to downy mildew, characterized in that, The nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO.

1.

9. The use of the reagent for detecting the expression amount of RcPR5 gene in breeding of the rose plant resistant to gray mold, characterized in that, The nucleotide sequence of the RcPR5 gene is shown as SEQ ID NO. 1.

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