Application of RhWRKY40 gene in regulating the germination of Rosa hybrida axillary buds

Abstract

This invention belongs to the field of biotechnology and discloses... RhWRKY40 Application of genes in regulating axillary bud germination in roses RhWRKY40 The gene nucleotide sequence is shown in SEQ ID NO:1. RhWRKY40 The gene is highly expressed in the basal axillary buds of roses, and its expression level is significantly negatively correlated with axillary bud activity, making it a negative regulator of rose axillary bud germination. This can be addressed by silencing the gene. RhWRKY40 The expression of this gene can significantly promote the germination and growth of rose axillary buds; overexpression of this gene significantly inhibits the germination and growth of rose axillary buds. This invention provides new gene resources and technical means for molecular breeding and plant type improvement of roses, and can effectively improve the survival rate of cut rose cuttings, shorten the seedling cycle, and increase the yield of flower branches, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to... RhWRKY40 Application of genes in regulating axillary bud germination in roses. Background Technology

[0002] Rose ( Rosa hybrida Rosa is a perennial evergreen or semi-evergreen woody plant belonging to the genus Rosa in the family Rosaceae. It is one of the most important ornamental crops, occupies an absolutely dominant position in the flower industry, and has high economic value.

[0003] In the process of plant growth and development, germination is a fundamental step in completing morphogenesis and growth. The germination of axillary buds directly determines the occurrence and formation of lateral branches. The formation of lateral branches not only affects the plant's canopy structure and photosynthetic efficiency, but is also closely related to physiological processes such as nutrient absorption, flowering, and fruiting. From the perspective of developmental biology, the formation of axillary buds originates from the differentiation activity of apical meristems or lateral meristems, undergoing stages such as primordium initiation, cell proliferation, and tissue differentiation, ultimately forming lateral buds with independent growth potential. The effective execution of this process is an important guarantee for plants to achieve morphological plasticity and adapt to the environment.

[0004] For cut roses, axillary buds are not only the foundation for new shoot development but also the main source of flowering branches. Their germination efficiency, uniformity, and robustness directly affect the survival rate of cuttings, seedling growth cycle, flower yield, and the commercial quality of cut flowers. Therefore, in-depth analysis of the molecular regulatory mechanisms of rose axillary bud germination, and the isolation and identification of key regulatory genes, are of significant theoretical and practical value for optimizing rose plant type, shortening the production cycle, and improving cut flower yield and quality through molecular breeding methods, thus promoting the rose industry. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention proposes... RhWRKY40 The application of genes in regulating the germination of rose axillary buds, through RhWRKY40 The negative regulatory role of genes in rose axillary bud germination provides new genetic resources and technical means for rose molecular breeding and plant type improvement.

[0006] To achieve the above objectives, the present invention provides a RhWRKY40 Gene, RhWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] It also provides a kind of silence RhWRKY40 Genes, silence RhWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] It also provides RhWRKY40Application of genes in regulating axillary bud germination in roses, and regulation RhWRKY40 The expression level of genes regulates the germination and growth of rose axillary buds.

[0009] Preferred, regulation RhWRKY40 Gene expression levels include silencing. RhWRKY40 Genes that promote the germination and growth of rose axillary buds.

[0010] Preferred, regulation RhWRKY40 Gene expression levels include overexpression. RhWRKY40 Genes that inhibit the germination and growth of rose axillary buds.

[0011] A kit is also provided, which contains reagents for inhibiting or detecting the expression of nucleotide sequences as shown in SEQ ID NO:1.

[0012] A vector containing a nucleotide sequence as shown in SEQ ID NO:1 is also provided.

[0013] An engineered bacterium containing the aforementioned carrier is also provided.

[0014] A method for improving axillary bud germination in roses is also provided, which includes: reducing or blocking the growth of axillary buds in roses. RhWRKY40 Gene expression; and / or reduction or blockage of rose [gene expression]. RhWRKY40 Protein expression; in, RhWRKY40 The gene sequence is shown in SEQ ID NO:1. RhWRKY40 The amino acid sequence of the protein is shown in SEQ ID NO:3.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention obtained by screening from the genome of the rose variety 'Yueyuefen' RhWRKY40 The gene was highly expressed in basal axillary buds and lowly expressed in middle and upper axillary buds. Its negative regulatory function in rose axillary bud germination was identified, and expression pattern analysis revealed that… RhWRKY40 Gene expression levels were significantly negatively correlated with rose axillary bud activity, and silencing... RhWRKY40 The gene can significantly promote the germination and growth of axillary buds in roses, and overexpression of this gene can significantly promote the germination and growth of axillary buds in roses. RhWRKY40 The gene significantly inhibits the germination and growth of rose axillary buds. Simultaneously, a recombinant expression vector, genetically engineered bacteria, and reagent kit based on this gene are provided, offering an efficient and convenient tool for regulating rose axillary bud germination using genetic engineering technology. This can effectively improve the survival rate of cut rose cuttings, shorten the seedling cycle, and increase flower branch yield. It can also be used to cultivate new rose varieties with different plant type characteristics, demonstrating broad application prospects and significant economic benefits.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 For the present invention RhWRKY40 The graph shows the expression levels of axillary buds at different locations. In the graph, 1-9 represent the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth axillary buds from the morphological top to the bottom of the flower branch (before the flower buds show white). In the graph, ns represents P>0.05, ** represents P≤0.01, and **** represents P≤0.0001. Figure 2 For the present invention RhWRKY40 Subcellular localization map in tobacco leaves, RhWRKY40 -GFP and NF-YA4-mCherry (nuclear marker) were co-infected into tobacco leaves, and the fluorescence signal was observed by confocal microscopy; Figure 3 For the present invention, instantaneous silence RhWRKY40 Phenotypic observation results of axillary bud germination and growth at different time points after infection. In the figure, days 0, 4, 8, and 12 represent TRV2 and TRV2- at days 0, 4, 8, and 12 post-infection. RhWRKY40 The growth status of silent axillary buds, TRV2 represents the control group (pTRV2 empty vector) plants, TRV2- RhWRKY40 Plants representing the treatment group (gene silencing), scale bar is 2cm; Figure 4 For the present invention, instantaneous silence RhWRKY40 Anatomical diagrams of axillary bud growth at different time points after infection. Days 0, 4, 8, and 12 in the diagram represent days 0, 4, 8, and 12 post-infection, representing TRV2 and TRV2-. RhWRKY40 Stereoscopic anatomical observation of silent axillary bud growth points; TRV2 represents the control group (pTRV2 empty plant); TRV2- RhWRKY40 Plants representing the treatment group (gene silencing), scale bar is 2cm; Figure 5 For the present invention, instantaneous silence RhWRKY40 Statistical graph of gene expression levels and axillary bud length measurements after silencing. Figure A shows the RT-qPCR analysis after silencing. RhWRKY40 Relative expression levels, B represents the levels at 0, 4, 8, and 12 days after silencing, and the TRV2 expression levels in the control group (empty vector) and the treatment group (TRV2-). RhWRKY40 The shoot length of gene-silenced plants, where ns represents P>0.05, ** represents P≤0.01, *** represents P≤0.001, and **** represents P≤0.0001; Figure 6 For the transient overexpression of this invention RhWRKY40Phenotypic observation results of axillary bud germination and growth at different time points after infection. In the figure, days 0, 4, 8, and 12 represent pSuper1300 (empty vector) and pSuper1300- on days 0, 4, 8, and 12 after infection. RhWRKY40 The growth of axillary buds in (overexpressing gene plants), where pSuper represents unexpressed plants. RhWRKY40 OE represents overexpressing plants; the scale bar is 2cm. Figure 7 For the transient overexpression of this invention RhWRKY40 Anatomical diagrams of axillary bud growth at different time points after infection. Days 0, 4, 8, and 12 in the diagram represent days 0, 4, 8, and 12 post-infection, respectively, for pSuper1300 (empty-plant) and pSuper1300- RhWRKY40 (Stereoscopic observation of axillary bud growth points in overexpressing plants), pSuper represents unloaded plants. RhWRKY40 OE represents overexpressing plants; the scale bar is 2cm. Figure 8 For the transient overexpression of this invention RhWRKY40 The graph shows the gene expression levels and axillary bud length measurements after overexpression. Figure A represents the RT-qPCR analysis after overexpression. RhWRKY40 Relative expression levels, B represents the levels at 0, 4, 8, and 12 days after overexpression, compared to the control group (pSuper1300 empty vector) and the treatment group. RhWRKY40 :OE overexpression of shoot length in plants, where ns represents P>0.05, ** represents P≤0.01, *** represents P≤0.001, and **** represents P≤0.0001. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0021] Unless otherwise defined or specified, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0022] The present application is based on the rose cultivar 'Old Blush' ( Rosa chinensis 'Pink China' ) plant (collected from Qingdao, Shandong Province, China on April 4, 2025) genome sequence (the genome sequence is obtained from https: / / lipm-browsers.toulouse.inra.fr / pub / RchiOBHm-V2 / ). In the early stage, transcriptome sequencing was performed on axillary buds at different time nodes after pruning, and significantly differentially expressed genes related to rose axillary bud germination were screened out RhWRKY40 . In axillary buds, RhWRKY40 expression level is negatively correlated with axillary bud activity, with high expression in basal axillary buds and low expression in active buds in the middle and upper parts. After transient silencing of RhWRKY40 , it was found that the germination and growth rate of axillary buds were affected. Therefore, the present application discloses the RhWRKY40 biological function in rose axillary bud germination, which is of great significance for cut rose breeding and industrial quality improvement and efficiency enhancement.

[0023] In the examples RhWRKY40 the nucleotide sequence of the gene is shown in SEQ ID NO: 1; the inserted TRV2 vector RhWRKY40 the nucleotide sequence of the silencing fragment is shown in SEQ ID NO: 2; RhWRKY40 the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 3.

[0024] SEQ ID NO: 1: 5'-ATGGACTACTCATCAGCTGCTGCGTATGATACTTCTCTGGATCTTAACACCAGGCCTCTCCGGCTTTTCGATGATTCTCCTCCGATCAAGAAAGAGGTGCAGAGCAAGATTTTGTCCGACTTTGGAAGGGAGCTCTCAGCAAAAGAAGAGAGTGGAGCTTTGGTAGAGGAATTGCACAGGGTGAGCACCGAAAACAAGAAGCTGACTGAAATGTTGACGGTGATGGGTGAGAGCTACAATGCTTTGAGAAGCCAATTGTTGGAGTACATGAGCAAGAACCCAGAGAAGGAGCTCAGCCCGATATCCAAGAAAAGAAAGTCTGAAAGTAGTAACAACAACAACAACAACAACAGTGCTAACAACATCCTTAATGGCATGAACAATGGAAACTCTGAGAGCAGCTCCAGTGATGGAGAATCATGCAAGAAGCCAAGGGAAGAGACCATCAAGGCCAAAATTTATAGGTCTTATGTCAGGACCGAACCATCCGATACAACAAGCTTGGTTGTCAAGGATGGGTACCAATGGAGAAAATATGGTCAAAAAGTTACAAGGGACAATCCTTGTCCTAGAGCTTACTTCAAGTGCTCTTTTGCTCCAACCTGCCCTGTCAAAAAGAAGGTTCAGAGAAGTGTTGAGGACCAATCAATTCTGGTGGCAACTTATGAAGGTGAACACAACCATCCCAATCCTTCTCAACTTGAAGCATCATCGGGCTCAAACCGCTGCATGACCTTAGGATCAGTCCCCTGCTCAACCTCTCTTGGCACATCCGGACCAACCATCACTCTTGACTTGACAAAATCCAAGTCCAATACTTCTGATACCAAGAGCCCGAAACCAAGAACTGAAACACCGGAAGTTCGAAAGCTTTTGGTGGAACAGATGGCGTCTTCCTTGACCAAAGACCCCAACTTCACAAAAGCACTTGCAGCAGCCATTTCAGGAAGATTTCTTCAGCACGATACTTACTGA-3'。

[0025] SEQ ID NO: 2: 5'-CTGATACCAAGAGCCCGAAACCAAGAACTGAAACACCGGAAGTTCGAAAGCTTTTGGTGGAACAGATGGCGTCTTCCTTGACCAAAGACCCCAACTTCACAAAAGCACTTGCAGCAGCCATTTCAGGAAGATTTCTTCAGCACGATACTTACTGATAGAAGAACTGGTGTAAAAGTCAATTAAGAAAATTGATTTTTTCCACTTGTTAGACTACTTTGAAGATTAGGTGTATTTGTAAATACAACATTTAAATGGAGGTTCGAATTCAATTTACGCAGCGGCTAAATTTTTCCCCAAACTTGAACTGTAATTTGAAGCTTAAATACAACAGTGCTTGGTTATCAATTGAGCTATAGGTGTTGATCTAGCAGAGTGAGAGAAGTATGATGAAAATGTACGTACTCATTAAACGTGTGATCTTTTCCCTTAAGAATTAAGTAATATGCTAGAAAGACCACATTTTCAGACCACAT-3'.

[0026] SEQ ID NO: 3: MDYSSAAAYDTSLDLNTRPLRLFDDSPPIKKEVQSKILSDFGRELSAKEESGALVEELHRVSTENKKLTEMLTVMGESYNALRSQLLEYMSKNPEKELSPISKKRKSESSNNNNNNNSANNILNGMNNGNSESSSSDGESCKKPREETIKAKIYRSYVRTEPSDTTSLVVKDGYQWRKYGQKVTRDNPCPRAYFKCSFAPTCPVKKKVQRSVEDQSILVATYEGEHNHPNPSQLEASSGSNRCMTLGSVPCSTSLGTSGPTITLDLTKSKSNTSDTKSPKPRTETPEVRKLLVEQMASSLTKDPNFTKALAAAISGRFLQHDTY.

[0027] The abbreviations appearing in the present application and their corresponding names are shown in Table 1.

[0028] Table 1 List of Abbreviations

[0029] Experimental materials used in the examples: 1. 'Pink Snow Mountain': The rose variety 'Pink Snow Mountain' ( Rosa 'Sweet Avalanche' The plants are modern cut rose varieties, and were taken from the germplasm resource nursery of Qingdao Agricultural University in Qingdao, Shandong Province, China on April 4, 2025. Stem segments with single buds were used as materials for the rose axillary bud germination experiment.

[0030] 2. Tobacco: The experimental material for subcellular localization and transcriptional activation was Nicotiana benthamiana. The seeds were sown in a moist nutrient substrate, covered with a film, and cultured in a culture room.

[0031] Cultivation conditions: temperature 24±1℃, relative humidity 60-65%, photoperiod 16h / 8h.

[0032] 3. Strains and vectors: Escherichia coli DH5α, Agrobacterium tumefaciens strains EHA105 and GV3101, and pSuper-1300 (Kan resistant) were all purchased from Beijing Qingke Biotechnology Co., Ltd.; VIGS vectors were pTRV1 and pTRV2 (Kan resistant) purchased from HonorGene.

[0033] 4. Culture medium formulation: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and ddH2O to a final volume of 1 L.

[0034] YEB medium: tryptone 5g / L, yeast extract 1g / L, beef extract 5g / L, MgSO4·7H2O 0.493g / L, ddH2O to a final volume of 1L.

[0035] Example 1 I. Experimental process.

[0036] 1. Extraction of total RNA.

[0037] Total RNA was extracted from the axillary buds of 'Pink Snow Mountain' using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (centrifuge column type).

[0038] 2. cDNA synthesis.

[0039] (1) Genomic DNA removal (as shown in Table 2).

[0040] Table 2 Genomic DNA Removal Reaction System

[0041] Mix well, incubate at 42°C for 2 minutes.

[0042] (2) Prepare the reverse transcription reaction system (as shown in Table 3).

[0043] Table 3 Transcription reaction system

[0044] 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.

[0045] 3. Real-time quantitative PCR.

[0046] RT-qPCR-specific primers for the gene were designed using Primer Premier5 (as shown in Table 4). The cDNA was diluted three-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.

[0047] Table 4 RT-qPCR Primer Sequences

[0048] The RT-qPCR reaction system is shown in Table 5, and the RT-qPCR reaction procedure is shown in Table 6.

[0049] Table 5 RT-qPCR reaction system

[0050] Table 6 RT-qPCR reaction procedure

[0051] 4. Carrier construction.

[0052] (1) PCR amplification of the target gene fragment: 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 7.

[0053] Table 7 PCR amplification reaction system

[0054] The PCR amplification reaction procedure is shown in Table 8.

[0055] Table 8 PCR Amplification Reaction Procedure

[0056] After amplification, 1% gel electrophoresis was performed, and the target band was selected for subsequent gel recovery.

[0057] (2) Gel recovery: The PCR products were recovered and purified using the DNA gel recovery kit (TaKaRa MiniBEST Agarose Gel DNAExtraction Kit Ver.4.0).

[0058] (3) Vector double digestion.

[0059] Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-digested with the corresponding enzyme. The double digestion system is shown in Table 9.

[0060] Table 9 Double enzyme digestion system

[0061] After adding the system reagents on ice, perform double digestion of the vector according to the specific enzyme denaturation temperature.

[0062] (4) Homologous recombination.

[0063] The double-digested vector was homologously recombinated with the cloned target gene fragment to construct the vector. The homologous recombination system is shown in Table 10.

[0064] Table 10 Homologous Recombination System

[0065] Procedure: Run the PCR machine at 50℃ for 15 minutes.

[0066] (5) Escherichia coli transformation.

[0067] S1. Remove the competent states from -80℃ and melt them in ice; S2. Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of LDHα competent cells, mix by pipetting, and let stand on ice for 30 min. S3, heat shock in a 42℃ water bath for 90 seconds, then quickly transfer to ice and let stand for 2 minutes; S4. Add 500 μL of LB medium to the centrifuge tube, incubate at 37°C and 200 rpm for 1 hour. S5. Centrifuge the cultured bacterial solution at 5000 rpm for 5 min. S6. 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.

[0068] (6) Microbial testing and sequencing.

[0069] S1. 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).

[0070] S2, Bacterial Culture PCR: The cultured bacterial culture is used for PCR amplification to detect whether the target band of the constructed vector meets expectations. The bacterial culture PCR amplification system is shown in Table 11.

[0071] Table 11 Bacterial PCR Amplification System

[0072] The bacterial culture PCR amplification reaction procedure is shown in Table 12.

[0073] Table 12 PCR amplification reaction procedure for bacterial culture

[0074] The amplified products were used for 1% gel electrophoresis imaging. The bands that met the expected size were sent to the company (Sangon Biotech (Shanghai) Co., Ltd.) for sequencing.

[0075] 5. Plasmid extraction.

[0076] 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 the plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0077] 6. Agrobacterium-mediated transformation.

[0078] (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; (2) Add 0.01-1 μg of 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 water bath at 37℃ for 5 min, and in an ice bath for 5 min. (3) Add 500 μL of antibiotic-free YEB liquid medium and incubate at 28°C with shaking for 2-3 hours; (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 2-3 days.

[0079] 7. Subcellular localization.

[0080] (1) Vector construction: SmaI and KpnI were selected as restriction sites, and the vector was constructed. RhWRKY40 The complete coding region (SEQ ID NO:1) sequence was inserted into the pSuper-1300 vector, and primers (as shown in SEQ ID NO:10 and SEQ ID NO:11) were designed using homologous recombination to construct... RhWRKY40 -GFP vector was used to obtain Agrobacterium-positive strain (GV3101), which was then preserved.

[0081] (2) Shaking.

[0082] S1. Streak culture of bacterial culture: Take out the preserved bacterial culture and streak it on YEB medium containing the corresponding antibiotics, and incubate it upside down at 28°C for 2-3 days; S2. Shaking: Pick a single colony into a 1.5mL centrifuge tube, add 500μL of YEB liquid medium containing antibiotics, and shake overnight with a light shake (28℃, 200rpm). PCR test is performed. If the bacterial solution with the correct bands is detected, continue with a medium shake (take 100μL of the culture from the light shake into a 50mL centrifuge tube, add 10mL of YEB liquid medium containing antibiotics, and shake overnight with a medium shake (28℃, 200rpm)). PCR test is performed again. If the bacterial solution with the correct bands is detected, continue with a large shake (take 1mL of the culture from the medium shake into a 250mL Erlenmeyer flask, add 100mL of YEB liquid medium containing antibiotics, and shake overnight with a large shake (28℃, 200rpm)). PCR test is performed again. If the bacterial solution with the correct bands is detected, continue with subsequent experiments.

[0083] (3) Tobacco injection.

[0084] S1. Bacterial Collection: Culture to OD... 600 Centrifuge the bacterial culture with an OD value of 0.4-0.6 (5000 rpm, 8 min), discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells in the infection solution and adjust the OD value accordingly. 600 Adjust to 1.0, press RhWRKY40 Mix -GFP / GFP:NF-YA4-mCherry in a 1:1 ratio and let stand in the dark for 45 min; S2. Injection: After the static period, gently make a small incision on the back of the tobacco (when it has grown to 4-6 leaves) with a needle, inject the mixed bacterial solution into the tobacco leaves using a 1mL syringe, and mark the incision. S3. Cultivation: Place the injected tobacco in a 25℃ incubator and cultivate it in the dark for one day, then transfer it to light for one day; during this period, pay attention to watering to ensure normal growth of the tobacco.

[0085] (4) Observation and photography: Select leaves near the tobacco injection site, gently cut them into squares with a blade and tweezers, place them on a glass slide, add distilled water, and cover with a coverslip (be careful to avoid air bubbles). Observe and photograph using a laser confocal microscope from the Analysis and Testing Center of Qingdao Agricultural University.

[0086] 8. Momentary silence.

[0087] (1) Vector primer design and vector construction: by EcoR I and Kpn I is the enzyme cleavage site, which will... RhWRKY40The silent fragment sequence (SEQ ID NO:2) was inserted into pTRV2, and primers (as shown in SEQ ID NO:12 and SEQ ID NO:13) were designed using homologous recombination to construct TRV2- RhWRKY40 Carrier (processing unit).

[0088] (2) Bacterial culture: Streaking the bacterial culture onto plates (containing 50 mg / L Kan / Rif) and incubating upside down at 28°C for 2-3 days. Pick a single colony and gently shake it in 500 μL of antibiotic-containing YEB medium (pick a single colony into a 1.5 mL centrifuge tube, add 500 μL of antibiotic-containing YEB liquid medium, and gently shake overnight (28°C, 200 rpm)). Perform PCR for bacterial testing. If the bacterial culture shows the correct bands, continue with medium shaking (take 100 μL of the culture from the medium shaking, add 10 mL of antibiotic-containing YEB liquid medium, and gently shake overnight (28°C, 200 rpm)). Perform PCR for bacterial testing. If the bacterial culture shows the correct bands, continue with large shaking (take 1 mL of the culture from the medium shaking, add 100 mL of antibiotic-containing YEB liquid medium, and gently shake overnight (28°C, 200 rpm)). Perform PCR for bacterial testing. If the bacterial culture shows the correct bands, continue with subsequent experiments.

[0089] (3) Collection and resuspension of bacteria: Centrifuge at 5000 rpm for 8 minutes to collect the bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix well by pipetting, and adjust to OD200. 600 =1.0. During the transient silence experiment, TRV1 and TRV2, TRV2- RhWRKY40 Mix the bacterial solutions at a 1:1 volume ratio and let stand in the dark for 4-6 hours.

[0090] (4) Vacuum suction: The stem segments with single buds were infected by suction using a vacuum pump at 0.082 MPa for 10 minutes, followed by holding the pressure for 10 minutes and releasing the pressure for 10 minutes, ensuring that the entire stem segment was submerged in the bacterial solution. This treatment was repeated three times. After infection, the segments were rinsed with sterile water and incubated in an 8°C incubator for 3 days before being planted. Observations were conducted every two days, and samples were taken and photographed for record-keeping.

[0091] 9. Transient overexpression.

[0092] (1) Vector primer design and vector construction: by Sam I and Kpn I is the enzyme cleavage site, which will... RhWRKY40 The overexpressed fragment was inserted into the pSuper1300 empty vector, and primers were designed using homologous recombination to construct pSuper1300- RhWRKY40 Carrier.

[0093] (2) Preparation of bacterial culture: Prepare pSuper1300 and pSuper1300- RhWRKY40 Agrobacterium. The method for collecting the bacteria is the same as the transient silencing method described above. After the bacterial culture is prepared, it is placed at 28°C and incubated with shaking at 200 rpm for 45 min.

[0094] (3) Vacuum suction: The method is the same as the transient silencing method described above. After infection, gently rinse the surface of the stem segments with RO H2O to obtain the bacterial solution, and then insert them into the culture room. Take photos, samples, and observe the microstructure every other day.

[0095] 10. RhWRKY40 Primers used in sequence experiments (as shown in Table 13).

[0096] Table 13 RhWRKY40 Sequence and primer list

[0097] II. Experimental Results.

[0098] 1. In different active buds RhWRKY40 Expression level analysis.

[0099] For cut rose 'Pink Snow Mountain', flower branches (flower buds not yet showing white) at different node axillary buds RhWRKY40 Real-time quantitative PCR analysis of the expression level revealed... RhWRKY40 The expression level is high in basal axillary buds (e.g. Figure 1 (As shown). Therefore, this application speculates that... RhWRKY40 It is closely related to the germination of axillary buds.

[0100] 2. RhWRKY40 Transcriptional activation and subcellular localization analysis.

[0101] In order to explore RhWRKY40 To determine the functional subcellular location, this application first used Cell-PLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2) for online prediction, which showed that it was located in the cell nucleus.

[0102] To further verify the accuracy of the positioning, the image was cloned. RhWRKY40 CDS sequences, construct RhWRKY40 -GFP vector. Using an empty GFP vector as a control, the GFP vector was transformed into EH105 and injected into leaves of Tobacco Benedict. Observations were made under a laser confocal microscope 2 days later, showing that the results of the empty GFP vector and... RhWRKY40 -GFP is located in the cell nucleus (e.g. Figure 2 (As shown).

[0103] 3. RhWRKY40 Inhibits the germination of axillary buds in roses.

[0104] (1) Momentary silence.

[0105] This application is based on RhWRKY40 The expression level analysis results in buds at different sites were selected. RhWRKY40 Specific regions of the sequence were used to construct TRV2- using homologous recombination. RhWRKY40 Transient silencing vectors, including TRV1, TRV2 (control group), and TRV2- RhWRKY40 (Treatment group) After being transformed with Agrobacterium EHA105, it was used to infect the basal axillary bud stem segments to silence them. RhWRKY40 Phenotypic observations and records were performed at 0, 4, 8, and 12 days post-infection (e.g., Figure 3 (As shown).

[0106] From the phenotype of the buds, compared with the TRV2 control, TRV2- RhWRKY40 The buds germinate relatively quickly, as can also be seen from the anatomical results of the buds. RhWRKY40 The development of silent buds accelerates (e.g., Figure 4 (As shown). To confirm the silence effect, extract... RhWRKY40 Total RNA from buds before and after silencing was analyzed by RT-qPCR, and its expression levels were found to be... RhWRKY40 The significant decrease in expression after silence indicates that silence is effective (e.g.) Figure 5 (As shown in Figure A). In addition, TRV2 in the control group and TRV2 in the treatment group were also compared. RhWRKY40 Bud length was measured at different time points, and the difference was found to be highly significant on day 12 after silencing (e.g., Figure 5 (As shown in B).

[0107] (2) Instantaneous overexpression.

[0108] This application is based on RhWRKY40 The expression level analysis results in buds at different sites were selected. RhWRKY40 The CDS sequence of the sequence was used to construct pSuper1300 using homologous recombination. RhWRKY40 Transient overexpression vectors, pSuper1300 and pSuper1300- RhWRKY40 After being transformed into EHA105, it was used to infect the axillary buds of the stem for overexpression. RhWRKY40 Phenotypic observations and records were performed at 0, 4, 8, and 12 days post-infection (e.g., Figure 6 (As shown).

[0109] From the phenotype of the buds, compared with the pSuper1300 control, pSuper1300- RhWRKY40 ( RhWRKY40The germination rate of buds (OE) is relatively slow, as can also be seen from the anatomical results of the buds. RhWRKY40 Overexpression slows down bud development (e.g.) Figure 7 (As shown).

[0110] To confirm the overexpression effect, extract RhWRKY40 Total RNA from both pre- and post-bud overexpression was analyzed by RT-qPCR, and the expression levels were found to be... RhWRKY40 The expression level increased significantly after overexpression, indicating that overexpression is effective (e.g. Figure 8 (As shown in Figure A). Furthermore, the control group pSuper1300 and the treatment group pSuper1300- RhWRKY40 ( RhWRKY40 :OE) bud length was measured at different time points, and it was found that the difference was highly significant on day 12 after overexpression (e.g. Figure 8 (As shown in B).

[0111] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Silence RhWRKY40 The application of genetic material in promoting the germination and growth of rose axillary buds is characterized by, RhWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO:1; silencing RhWRKY40 Genes that promote the germination and growth of rose axillary buds.

2. A method for improving the germination of axillary buds in roses, characterized in that, Methods include: reducing or blocking the rose's... RhWRKY40 Gene expression; in, RhWRKY40 The gene sequence is shown in SEQ ID NO:1.

Images