Application of RhRAP2.4 gene in regulation of axillary bud germination of Chinese rose

Abstract

The invention discloses an application of an RhRAP2.4 gene in regulation of axillary bud germination of Chinese rose. Relates to the technical field of biology, and provides application of a product for inhibiting expression of an RhRAP2.4 gene or transcriptional translation protein of the RhRAP2.4 gene in promoting germination of rosa chinensis axillary buds, and the nucleotide sequence of the RhRAP2.4 gene is shown as SEQ ID NO.1. According to expression results of the RhRAP2.4 in different site buds of rosa chinensis branches, the RhRAP2.4 in the axillary buds is instantaneously silenced, and the rosa chinensis axillary bud germination is promoted. It is found that abnormal expression of the RhRAP2.4 can influence germination and growth of the axillary buds, silent RhRAP2.4 can inhibit growth of the axillary buds, and overexpressed RhRAP2.4 can promote growth of the axillary buds.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a RhRAP2.4 The application of genes in regulating the germination of axillary buds in rose. Background Art

[0002] Rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant of the genus Rosa in the family Rosaceae. It blooms all year round and has a variety of colors and species. It can be used as an ornamental and medicinal plant and is also widely used as a cut flower. As one of the four major cut flowers in the world, rose has a high economic value because of its large and beautiful flowers and a variety of colors. It is one of the most consumed flowers in the cut flower market.

[0003] Germination is an important biological characteristic in the growth and development of plants. Axillary bud germination determines the formation of plant branches. The number of branches determines the number of nutritional organs and plays a decisive role in crop yield. The differentiation of meristems can form axillary buds. The process is mainly that the meristems differentiate into lateral bud primordia, which further differentiate into axillary buds and then develop into lateral branches. The number of lateral branches formed in the plant is positively correlated with the germination of axillary buds.

[0004] Rose axillary buds mainly develop in the leaf axils and grow into flowering branches. They are crucial to the growth and morphological formation of roses and are one of the most important proliferation materials for roses. In the production of cut roses, the speed of axillary bud germination directly determines its survival rate; during the growth and development process, the characteristics of axillary buds determine the growth cycle, yield and quality of cut roses. Therefore, studying the factors that regulate the germination of rose axillary buds is of great significance for the breeding of cut roses and the improvement of quality and efficiency of the industry. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides an application of RhRAP2.4 gene in regulating the germination of axillary buds of rose.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: The first aspect of the present application provides a rose gene RhRAP2.4, and the rose gene RhRAP2.4 has the following sequence: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) The gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.2.

[0007] The nucleotide sequence of the silent rose gene RhRAP2.4 provided in the second aspect of the present application is shown in SEQ ID NO.2.

[0008] The third aspect of the present application provides a protein encoded by the rose gene RhRAP2.4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0009] The fourth aspect of the present application provides the use of the rose gene RhRAP2.4 or the silenced rose gene RhRAP2.4 in inhibiting the germination of rose axillary buds.

[0010] The fifth aspect of the present application provides a kit comprising a reagent for inhibiting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0011] The sixth aspect of the present application provides a kit comprising reagents for detecting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0012] Furthermore, the kit contains primers for amplifying the base sequence shown in SEQ ID NO:1.

[0013] The seventh aspect of the present application provides a vector, which contains a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] The eighth aspect of the present application provides an engineered bacterium, which contains a vector.

[0015] The ninth aspect of the present application provides a kit, a vector, and an engineered bacteria for use in regulating the germination of axillary buds of roses.

[0016] A method for promoting the germination of axillary buds of roses, promoting the expression of RhRAP2.4 gene or its transcription and translation protein in roses, wherein the nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1.

[0017] The tenth aspect of the present application provides a method for breeding improved new rose varieties, which utilizes the rose gene RhRAP2.4 or the encoded protein to promote or inhibit the germination of rose axillary buds.

[0018] Beneficial effects: The present invention identified a candidate gene for axillary bud germination, RhRAP2.4, from the genome of the rose variety 'Yueyuefen', and found that the gene was highly expressed in the stems, leaves and axillary buds of rose tissues, and the expression signal was concentrated in the axillary buds, with almost no expression in the petals. In the analysis of the expression of RhRAP2.4 in buds at different locations of rose branches, it was found that the expression of RhRAP2.4 was high in the axillary buds at the base, and low in the axillary buds at the middle and above. Therefore, the present application speculates that RhRAP2.4 may be involved in the axillary bud germination of roses.

[0019] In the present invention, based on the expression results of RhRAP2.4 in buds at different locations of rose branches, RhRAP2.4 in axillary buds was transiently silenced, and it was found that abnormal expression of RhRAP2.4 would affect the germination and growth of axillary buds, silencing RhRAP2.4 could inhibit the growth of axillary buds, and overexpressing RhRAP2.4 could promote the growth of axillary buds. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 It is the expression analysis diagram of RhRAP2.4 provided by the present invention; Note: they are root, stem, leaf, petal, pistil, stamen, axillary bud and sepal respectively.

[0022] Figure 2 It is a subcellular localization diagram of RhRAP2.4 provided by the present invention; Note: (A) RhRAP2.4 subcellular localization prediction result; (B) Subcellular localization in Nicotiana benthamiana leaves, RhRAP2.4-GFP and NF-YA4-mCherry (nuclear marker) were co-infiltrated into tobacco leaves, and the fluorescence signal was observed by confocal microscopy.

[0023] Figure 3 The expression level of RhRAP2.4 in axillary buds at different locations; data are shown as mean ± SD (n = 3) (**** indicates P ≤ 0.0001), which are the second, fourth, sixth, and eighth axillary buds from top to bottom.

[0024] Figure 4 The present invention provides a diagram of the phenotypic observation results of axillary bud germination and growth at different time periods after transient silencing of RhRAP2.4; Note: 0d, 6d, 10d, 12d represent the growth of silenced axillary buds of TRV2 and TRV2-RhRAP2.4 on days 0, 6, 10, and 12 after infection.

[0025] Figure 5 It is an anatomical diagram of the growth status of axillary buds in different time periods after transient silencing of RhRAP2.4 provided by the present invention; Note: 0d, 6d, 10d, and 12d represent the anatomical observation of the growth points of the axillary buds silenced by TRV2 and TRV2-RhRAP2.4 on days 0, 6, 10, and 12 after infection.

[0026] Figure 6It is a statistical graph of gene expression and axillary bud length measurement after transient silencing of RhRAP2.4 provided by the present invention; Note: (A) RT-qPCR analysis of RhRAP2.4 relative expression after silencing; (B) Bud length of the control group TRV2 and the treatment group TRV2-RhRAP2.4 silenced at 0d, 4d, 4d, 8d and 12d after silencing (* indicates P≤0.05, ** indicates P≤0.01).

[0027] Figure 7 The present invention provides a phenotypic observation result diagram of axillary bud germination and growth at different time periods after transient overexpression of RhRAP2.4; Note: 0d, 4d, 8d, 10d represent the growth of silenced axillary buds of Super1300 and Super1300-RhRAP2.4 on days 0, 4, 8, and 10 after infection.

[0028] Figure 8 It is an anatomical diagram of the growth status of axillary buds in different time periods after transient overexpression of RhRAP2.4 provided by the present invention; Note: 0d, 4d, 8d, and 10d represent the anatomical observation of the growth points of the axillary buds overexpressing Super1300 and Super1300-RhRAP2.4 on days 0, 4, 8, and 10 after infection.

[0029] Fig. 9 It is a statistical graph of gene expression and axillary bud length measurement after transient overexpression of RhRAP2.4 provided by the present invention; Note: (A) RT-qPCR analysis of RhRAP2.4 relative expression after overexpression; (B) Bud length of the control group Super1300 and the treatment group Super1300-RhRAP2.4 overexpression at 0d, 4d, 8d, and 10d after overexpression (* indicates P≤0.05, ** indicates P≤0.01). DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] The first aspect of the embodiment of the present application provides a rose gene RhRAP2.4, characterized in that: the rose gene RhRAP2.4 has the following sequence: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) The gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.2.

[0032] The nucleotide sequence of the silent rose gene RhRAP2.4 provided in the second aspect of the embodiment of the present application is shown as SEQ ID NO.2.

[0033] The third aspect of the embodiment of the present application provides a protein encoded by the rose gene RhRAP2.4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0034] The fourth aspect of the embodiments of the present application provides the use of the rose gene RhRAP2.4 or the silenced rose gene RhRAP2.4 in promoting the germination of rose axillary buds.

[0035] A fifth aspect of the embodiments of the present application provides a kit comprising a reagent for inhibiting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0036] A sixth aspect of the present application provides a kit comprising reagents for detecting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0037] In some embodiments, the kit contains primers for amplifying the base sequence shown in SEQ ID NO:1.

[0038] A seventh aspect of the present application provides a vector, which contains a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0039] An eighth aspect of the present application embodiment provides an engineered bacterium, wherein the engineered bacterium contains a vector.

[0040] The ninth aspect of the present application provides a kit, a vector, and an engineered bacteria for use in regulating the germination of axillary buds of roses.

[0041] A method for promoting the germination of axillary buds of roses, promoting the expression of RhRAP2.4 gene or its transcription and translation protein in roses, wherein the nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1.

[0042] The tenth aspect of the embodiment of the present application provides a method for breeding improved new rose varieties, which utilizes the rose gene RhRAP2.4 or the encoded protein to promote or inhibit the germination of rose axillary buds. Example 1

[0043] A rose gene RhRAP2.4 of the present invention has the following sequence: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) The gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.2. Example 2

[0044] The nucleotide sequence of the silent rose gene RhRAP2.4 of the present invention is shown in SEQ ID NO.2.

[0045] The encoded protein of the rose gene RhRAP2.4 of the present invention has an amino acid sequence as shown in SEQ ID NO.3. Example 3

[0046] The invention discloses an application of the rose gene RhRAP2.4 or the silent rose gene RhRAP2.4 in promoting the germination of axillary buds of roses. Example 4

[0047] A kit of the present invention comprises a reagent for inhibiting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0048] A kit of the present invention comprises a reagent for detecting the expression of a nucleotide sequence as shown in SEQ ID NO:1.

[0049] The kit contains primers for amplifying the base sequence shown in SEQ ID NO:1.

[0050] A vector of the present invention comprises a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0051] The invention discloses an engineered bacterium, wherein the engineered bacterium contains a vector.

[0052] Application of the kit, the carrier and the engineering bacteria of the invention in regulating the germination of axillary buds of roses. Example 5

[0053] The invention discloses a method for cultivating improved new rose varieties, which utilizes rose gene RhRAP2.4 or the encoded protein to promote or inhibit the germination of rose axillary buds.

[0054] Test Example 1

[0055] This application is based on the genome sequence of the rose variety 'Yueyuefen'. In the early stage, transcriptome sequencing was performed on axillary buds at different time points after pruning, and the significantly differentially expressed gene RhRAP2.4 related to the germination of rose axillary buds was screened out. In the axillary buds, the expression level of RhRAP2.4 is negatively correlated with the activity of axillary buds, with high expression in the axillary buds at the base and low expression in the active buds in the middle and upper parts. After transient silencing of RhRAP2.4, it was found that the germination and growth rate of axillary buds were affected. Therefore, this application reveals the biological function of RhRAP2.4 in the germination of rose axillary buds, which is of great significance to the breeding of cut roses and the improvement of quality and efficiency of the industry.

[0056] This test example provides the nucleotide sequence of the RhRAP2.4 gene as shown in SEQ ID NO: 1. The abbreviations and their corresponding names appearing in this application are shown in Table 1.

[0057] Table 1

[0058] 1. Plant materials 1.1 'Pink Snow Mountain' 'Pink Snow Mountain' is a modern cut rose variety, taken from the Baofeng Base of the Institute of Flowers and Plants of Yunnan Academy of Agricultural Sciences. The stem segments with single buds were used as materials for the rose axillary bud germination experiment.

[0059] 1.2 Tobacco The experimental material for subcellular localization and transcriptional activation was Nicotiana benthamiana. The seeds were sown in a moist nutrient matrix, covered with a film, and cultured in a culture room.

[0060] Culture conditions: temperature 24±1℃, relative humidity 60-65%, photoperiod 16h / 8h.

[0061] 1.3 Strains and vectors Escherichia coli: DH5α and Agrobacterium tumefaciens strains: EHA105, GV3101 (pSoup), pSuper-1300 (Kan resistance) were purchased from Beijing Qingke Biotechnology Co., Ltd. VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0062] 1.4 Culture medium formula involved in the case (1) LB medium and YEB medium (Table 2) Table 2

[0063] 2. Research Methods 2.1 Total RNA extraction Total RNA was extracted from the petals, leaves, receptacles, sepals and axillary buds of 'Pink Snow Mountain' using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (spin column type).

[0064] 2.2 cDNA synthesis (1) Genomic DNA removal reaction system (Table 3) Table 3

[0065] Mix well and incubate at 42°C for 2 min.

[0066] (2) Prepare the reverse transcription reaction system (Table 4) Table 4

[0067] Mix by pipetting, incubate at 37°C for 15 min, and 85°C for 5 s. Store the product at -20°C.

[0068] 2.3 Real-time fluorescence quantitative PCR Primer premier5 was used to design RT-qPCR specific primers for the gene (Table 5). The cDNA was diluted four times with ddH2O, and the cDNA obtained by reverse transcription was used as a template for Real-Time PCR amplification. GAPDH was used as an internal reference, and three biological replicates were set.

[0069] Table 5

[0070] The RT-qPCR reaction system is shown in Table 6: Table 6

[0071] The RT-qPCR reaction procedure is shown in Table 7: Table 7

[0072] 2.4 Vector construction 2.4.1 PCR amplification of target gene fragments A high-fidelity enzyme (PhusionTM Plus PCR Master Mix) was used for PCR target gene amplification, and the PCR amplification reaction system is shown in Table 8: Table 8

[0073] The PCR amplification reaction program is shown in Table 9: Table 9

[0074] After amplification, 1% gel electrophoresis was performed and the target band was selected for subsequent gel recovery.

[0075] 2.4.2 Rubber Recycling The PCR product was recovered and purified using a DNA gel recovery kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0).

[0076] 2.4.3 Double restriction digestion of vector According to the restriction site where the target fragment sequence is inserted, the vector is double-digested with the corresponding enzyme. The double-digestion system is shown in Table 10: Table 10

[0077] After adding the system reagents on ice, perform double enzyme digestion of the vector according to the thermal denaturation temperature of the specific enzyme.

[0078] 2.4.4 Homologous recombination The double-digested vector and the cloned target gene fragment are homologously recombined to construct the vector. The homologous recombination system is shown in Table 11: Table 11

[0079] Procedure: Run in PCR instrument at 50°C for 15 min.

[0080] 2.4.5 E. coli transformation (1) Take out the competent cells from -80 °C and thaw them in ice; (2) Take a 1.5 mL centrifuge tube, add 10 μL of the recombinant product and 50 μL of DHα competent cell, mix well by pipetting, and place on ice for 30 min; (3) Heat shock in a 42°C water bath for 90 s, then quickly transfer to ice and let stand for 2 min; (4) Add 500 μL LB to the centrifuge tube and incubate at 37 °C, 200 rpm, for 1 h. (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min; (6) Discard 400 μL of supernatant in a clean bench, mix thoroughly by pipetting, and then spread on LB solid medium containing antibiotics, and culture inverted at 37°C overnight.

[0081] 2.4.6 Bacterial testing and sequencing (1) Shaking: Pick a single colony grown from the overnight culture and culture it in 500 μL of LB medium containing antibiotics with shaking for 3-4 h (37 ℃, 200 rpm).

[0082] (2) Bacterial liquid PCR: The cultured bacterial liquid is used for PCR amplification to detect whether the target band of the constructed vector meets the expectations. The bacterial liquid PCR amplification system is shown in Table 12: Table 12

[0083] The bacterial solution PCR amplification reaction program is shown in Table 13: Table 13

[0084] The amplified products were used for 1% gel electrophoresis imaging, and those with expected band sizes were sent to the company for sequencing.

[0085] 2.5 Plasmid extraction After the test results are returned, sequence comparison is performed, and the samples that meet the expectations are selected for bacterial culture, and the plasmid is extracted using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0086] 2.6 Agrobacterium transformation (1) Take out the competent stem from the -80 ℃ freezer and put it into ice when it melts to an ice-water mixture; (2) Add 0.01-1 μg of plasmid DNA per 100 μL competent medium, mix thoroughly, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. (3) Add 500 μL of YEB liquid medium without antibiotics and culture at 28 °C with shaking for 2-3 h; (4) Centrifuge at 5000 rpm for 2 min. Discard 400 μL of supernatant on a clean bench and spread onto YEB solid medium containing antibiotics. Incubate inverted at 28 °C for 2-3 d.

[0087] 2.7 Subcellular localization (1) Vector construction Select SmaI and KpnI as restriction sites. RhRAP2.4 The complete coding region (SEQ ID NO: 1) was inserted into the pSuper-1300 vector, and primers were designed by homologous recombination to construct the RhRAP2.4-GFP vector. After obtaining Agrobacterium-positive strains, the following experiments were performed.

[0088] (2) Shaking bacteria 1) Streak culture of bacterial liquid: Take out the stored bacterial liquid and streak it on YEB medium containing corresponding antibiotics, and culture it upside down at 28℃ for 2-3 days; 2) Shaking: Pick a single colony into a 1.5 mL centrifuge tube, add 500 μL of YEB liquid culture medium containing antibiotics and shake overnight (28 ℃, 200 rpm), perform PCR bacterial test, and continue to shake the bacterial solution with the correct band at medium and high speed.

[0089] (3) Tobacco injection 1) Collection of bacteria: Culture to OD 600 =0.4-0.6, centrifuge (5000 rpm, 8 min), discard the supernatant, and collect the bacteria. Resuspend the bacteria in the infection solution and adjust the OD 600Adjust to 1.0, mix at a ratio of RhRAP2.4-GFP / GFP: NF-YA4-mCherry: P19 = 1:1:0.5, and incubate in the dark for 3 h.

[0090] 2) Injection: After the rest period, use a needle to gently cut a hole on the back of the tobacco (when it grows to 4-6 leaves), and use a 1 mL syringe to inject the mixed bacterial solution into the tobacco leaves in the right proportion, and mark them; 3) Cultivation: Place the injected tobacco in a 25 ℃ incubator, incubate in the dark for one day, and then transfer to light for 2 days; pay attention to watering during this period to maintain normal growth of the tobacco.

[0091] (4) Observation and photography Select the tobacco leaves near the injection port, cut them into squares with a blade and tweezers, place them on a glass slide, drip distilled water on them, and cover them with a coverslip (be careful to avoid bubbles). Use a laser confocal microscope from the State Key Laboratory of the Institute of Biology, Yunnan Academy of Agricultural Sciences to observe and take pictures.

[0092] 2.8 Momentary Silence (1) Vector primer design and vector construction The silencing fragment of RhRAP2.4 (SEQ ID NO: 2) was inserted into the pTRV2 empty vector using EcoRI and Kpn1 as restriction sites, and primers were designed using homologous recombination to construct the TRV2-RhRAP2.4 vector.

[0093] (2) Bacterial culture Streak the bacterial solution plate (containing Kan / Rif 50 mg / L) and invert and culture at 28 ℃ for 2-3 days. Pick a single colony and shake it in 500 μL YEB containing antibiotics for bacterial inspection; shake it medium and high (28 ℃, 200 rpm) if the strip is correct.

[0094] (3) Collection and resuspension Centrifuge at 5000 rpm for 8 min to collect the bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix with a pipette, and adjust to OD600 = 1.0. When performing transient silencing experiments, mix TRV1 and TRV2, and TRV2-RhRAP2.4 bacterial solutions in a volume ratio of 1:1 and let stand in the dark for 4-6 h.

[0095] (4) Vacuum suction Use a vacuum pump to infect the stem segments with single buds, 0.082 MPa, suction for 10 min, hold pressure for 10 min, release air for 10 min, and immerse the entire stem segment in the bacterial solution as much as possible. Repeat the process three times. After infection, rinse with sterile water, place in a culture room at 8℃ for 3 days, and then cut the cuttings. Observe every two days, take samples, and take photos for record.

[0096] 2.9 Transient overexpression (1) Vector primer design and vector construction Using Sam1 and Kpn1 as restriction sites, the overexpression fragment of RhRAP2.4 was inserted into the pSuper1300 empty vector. Primers were designed using homologous recombination to construct the Super1300-RhRAP2.4 vector.

[0097] (2) Preparation of bacterial solution Prepare pSuper1300 and pSuper1300-RhRAP2.4 Agrobacterium. The bacterial collection method is the same as the transient silencing method in 2.8 of this chapter. After the bacterial solution is prepared, place it at 28℃ and shake at 200rpm for 45 minutes.

[0098] (2) Vacuum suction The method is the same as the instantaneous silencing method in 2.8 of this chapter. After infection, gently rinse the bacterial solution on the surface of the stem segment with RO H2O, and then cut the stems in the culture room. Take photos, take samples, and observe the body every other day.

[0099] 2.10 Primers used in the RhRAP2.4 sequence experiment Table 14

[0100] 3 Test results 3.1 Verification of RhRAP2.4 expression in different tissues In this application, total RNA from roots, stems, leaves, petals, pistils, stamens, axillary buds, and sepals of Rose 'Pink Snow Mountain' was extracted and quality tested. Genes with the most cis-acting elements related to meristem and plant growth and development were selected for fluorescence quantitative expression analysis. It was found that different genes were expressed differently in different tissues ( Figure 1 ). Among them, RhRAP2.4 was expressed more specifically and at higher levels in stems, leaves and axillary buds, indicating that RhRAP2.4 may be essential for the growth and development of rose axillary buds.

[0101] 3.2 Analysis of transcriptional activation and subcellular localization of RhRAP2.4 In order to explore the subcellular location of RhRAP2.4 function, this application first used Cell-PLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2) for online prediction, which showed that it was localized in the nucleus ( Figure 2A). To further verify the accuracy of localization, the CDS sequence of RhRAP2.4 was cloned and the RhRAP2.4-GFP vector was constructed. Using GFP empty vector as control, it was transferred into Agrogan EH105 and injected into Nicotiana benthamiana leaves. After 3 days, the leaves were observed under a laser confocal microscope. The results showed that both GFP empty vector and RhRAP2.4-GFP were localized in the cell nucleus ( Figure 2 B).

[0102] 3.3 Analysis of RhRAP2.4 expression in different active buds In this application, the expression level of RhRAP2.4 in axillary buds at different nodes of the cut rose 'Pink Snow Mountain' was analyzed by real-time fluorescence quantitative PCR. It was found that the expression of RhRAP2.4 was high in the axillary buds at the base ( Figure 3 ). Therefore, the present application speculates that RhRAP2.4 is closely related to the germination of axillary buds.

[0103] 3.5 RhRAP2.4 promotes rose axillary bud germination Based on the expression analysis results of RhRAP2.4 in buds at different sites, the specific segment of the RhRAP2.4 sequence (SEQ ID NO: 2) was selected, and the TRV2-RhRAP2.4 transient silencing vector was constructed by homologous recombination. TRV1, TRV2 and TRV2-RhRAP2.4 were transferred into Agrogan EHA105 and used to infect the middle axillary bud stem segments to silence RhRAP2.4. Phenotypic observations and records were performed at 0 d, 6 d, 10 d and 12 d after infection ( Figure 4 ). From the bud phenotype, compared with the TRV2 control, the germination speed of TRV2-RhRAP2.4 buds is slower. The dissection results of the buds also show that the development of the buds slows down after RhRAP2.4 silencing ( Figure 5 ). In order to confirm the silencing effect, total RNA of buds before and after RhRAP2.4 silencing was extracted, and its expression level was analyzed by RT-qPCR. It was found that the expression of RhRAP2.4 was significantly reduced after silencing, indicating that the silencing was effective ( Figure 6 A). In addition, the bud lengths of the control group TRV2 and the experimental group TRV2-RhRAP2.4 at different time points were measured, and it was found that the difference was extremely significant on the 12th day after silencing ( Figure 6 B).

[0104] Based on the expression analysis results of RhRAP2.4 in buds at different sites, the CDS sequence of RhRAP2.4 was selected, and the Super1300-RhRAP2.4 transient overexpression vector was constructed by homologous recombination. Super1300 and Super1300-RhRAP2.4 were transferred into Agrogan EHA105 and used to infect the middle axillary bud stem segments to overexpress RhRAP2.4. Phenotypic observations and records were performed at 0 d, 4 d, 8 d, and 10 d after infection ( Figure 7 ). From the phenotype of the buds, compared with the Super1300 control, the buds of Super1300-RhRAP2.4 germinated faster. The dissection results of the buds also showed that the development of the buds was accelerated after RhRAP2.4 was silenced ( Figure 8 ). In order to confirm the overexpression effect, total RNA of buds before and after RhRAP2.4 overexpression was extracted, and its expression level was analyzed by RT-qPCR. It was found that the expression level of RhRAP2.4 increased significantly after overexpression, indicating that the overexpression was effective ( Fig. 9 A). In addition, the bud lengths of the control group Super1300 and the experimental group Super1300-RhRAP2.4 at different time points were measured, and it was found that the difference was extremely significant on the 10th day after overexpression ( Fig. 9 B).

[0105] It should be noted that the above specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention description and its drawings are illustrative and do not constitute a limitation of the claims. The protection scope of the present invention is defined by the claims and their equivalents.

Claims

1. A rose gene RhRAP2.4 , characterized in that: The rose gene RhRAP2.4 has the following sequence: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) The gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.

2.

2. The nucleotide sequence of a silent rose gene RhRAP2.4 is shown in SEQ ID NO.

2.

3. The protein encoded by the rose gene RhRAP2.4 according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

3.

4. Use of the rose gene RhRAP2.4 according to claim 1 or the silent rose gene RhRAP2.4 according to claim 2 in inhibiting the germination of rose axillary buds.

5. A kit, characterized in that: Contains reagents for inhibiting or detecting the expression of the nucleotide sequence shown in SEQ ID NO:

1.

6. The kit according to claim 5, characterized in that: The kit contains primers for amplifying the base sequence shown in SEQ ID NO:

1.

7. A carrier, characterized in that: The vector contains the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

8. An engineered bacterium, characterized in that: The engineered bacteria contains the vector according to claim 7.

9. Use of the kit according to claim 5 or 6, or the vector according to claim 7, or the engineered bacteria according to claim 8 in regulating the germination of axillary buds of roses.

10. A method for cultivating an improved new variety of rose, characterized in that: The rose gene RhRAP2.4 described in claim 1 or the encoded protein described in claim 2 is used to promote or inhibit the germination of rose axillary buds.

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