Application of RhRAP2.4 Gene in Regulating Axillary Bud Germination of Rosa chinensis

Abstract

The present invention discloses the application of the RhRAP2.4 gene in regulating the germination of axillary buds in roses. Relating to the field of biotechnology, the present invention provides the application of a product that inhibits the expression of the RhRAP2.4 gene or its transcription and translation protein in promoting the germination of axillary buds in roses. The nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1. In the present invention, according to the expression results of RhRAP2.4 in buds at different sites of rose flower branches, RhRAP2.4 in axillary buds was transiently silenced, and it was found that the 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.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of a RhRAP2.4 gene in regulating the axillary bud germination of roses. 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 throughout the year with rich colors and varieties, and can be used as an ornamental and medicinal plant, and is also widely used in cut flowers. As one of the world's four major cut flowers, roses have high economic value due to their large and beautiful flowers and diverse colors, and are one of the most consumed flowers in the cut flower market.

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

[0004] Rose axillary buds mainly develop in leaf axils and grow into flowering branches, which are crucial for the growth and morphogenesis of roses and are also one of the most important proliferation materials of 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 regulating rose axillary bud germination is of great significance for cut rose breeding and improving the quality and efficiency of the industry. Summary of the Invention

[0005] To solve the problems of the prior art, the present invention provides the application of a RhRAP2.4 gene in regulating the axillary bud germination of roses.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A rose gene RhRAP2.4 provided in the first aspect of the present application, the rose gene RhRAP2.4 has the following sequences:

[0008] (1) The nucleotide sequence shown by SEQ ID NO.1;

[0009] (2) A gene obtained by silencing or knocking out the nucleotide sequence shown by SEQ ID NO.2.

[0010] The nucleotide sequence of the RhRAP2.4 gene for silencing roses provided in the second aspect of the present application is shown in SEQ ID NO.2.

[0011] The encoded protein of the RhRAP2.4 gene of roses provided in the third aspect of the present application, the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0012] The application of the RhRAP2.4 gene of roses or the RhRAP2.4 gene for silencing roses in inhibiting the germination of axillary buds of roses.

[0013] A kit provided in the fifth aspect of the present application, containing a reagent for inhibiting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0014] A kit provided in the sixth aspect of the present application, containing a reagent for detecting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0015] Furthermore, the kit contains primers for amplifying the base sequence shown in SEQ ID NO:1.

[0016] A vector provided in the seventh aspect of the present application, the vector contains the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0017] An engineered bacterium provided in the eighth aspect of the present application, the engineered bacterium contains the vector.

[0018] The application of the kit, vector, and engineered bacterium provided in the ninth aspect of the present application in regulating the germination of axillary buds of roses.

[0019] A method for promoting the germination of axillary buds of roses, promoting the expression of the RhRAP2.4 gene or its transcription and translation protein in roses, and the nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1.

[0020] A method for cultivating an improved new rose variety provided in the tenth aspect of the present application, using the RhRAP2.4 gene of roses or the encoded protein to promote or inhibit the germination of axillary buds of roses.

[0021] Beneficial effects: The present invention identified a candidate gene RhRAP2.4 for axillary bud germination from the genome of the rose variety 'Monthly Pink'. It was found that this gene was highly expressed in the stems, leaves, and axillary buds of rose tissues, and the expression signal was concentrated in the axillary buds and hardly expressed in the petals. In the analysis of the expression of RhRAP2.4 in axillary buds at different positions of rose flower branches, it was found that the expression of RhRAP2.4 was high in the basal axillary buds and low in the axillary buds in the middle and upper parts. Therefore, the present application speculates that RhRAP2.4 may be involved in the germination of axillary buds of roses.

[0022] In the present invention, based on the expression results of RhRAP2.4 in buds at different positions of rose branches, RhRAP2.4 in axillary buds was transiently silenced, and it was found that the 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 overexpression of RhRAP2.4 could promote the growth of axillary buds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] 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, sepal respectively.

[0025] Figure 2 It is the subcellular localization diagram of RhRAP2.4 provided by the present invention; Note: (A) Prediction result of subcellular localization of RhRAP2.4; (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 through a confocal microscope.

[0026] Figure 3 It is the diagram of the expression levels of axillary buds at different parts of RhRAP2.4; The data are shown as mean ± SD (n = 3) (**** indicates P ≤ 0.0001), and they are axillary buds at the second, fourth, sixth, and eighth nodes from top to bottom respectively.

[0027] Figure 4 It is the diagram of the phenotypic observation results of the germination and growth of axillary buds at different time periods after transient silencing of RhRAP2.4 provided by the present invention; Note: 0d, 6d, 10d, 12d represent the growth conditions of the silenced axillary buds of TRV2 and TRV2-RhRAP2.4 at the 0th, 6th, 10th, and 12th days after infection.

[0028] Figure 5 It is the anatomical diagram of the growth status of axillary buds at different time periods after transient silencing of RhRAP2.4 provided by the present invention; Note: 0d, 6d, 10d, 12d represent the stereoscopic anatomical observation conditions of the growth points of the silenced axillary buds of TRV2 and TRV2-RhRAP2.4 at the 0th, 6th, 10th, and 12th days after infection.

[0029] Figure 6It is a statistical chart of gene expression levels and axillary bud lengths after transient silencing of RhRAP2.4 provided by the present invention; Note: (A) RT-qPCR analysis of the relative expression level of RhRAP2.4 after silencing; (B) Bud lengths of the control group TRV2 and the treatment group TRV2-RhRAP2.4 silenced at 0 d, 4 d, 8 d, and 12 d after silencing (* indicates P≤0.05, ** indicates P≤0.01).

[0030] Figure 7 It is a figure showing the phenotypic observation results of axillary bud germination and growth at different time periods after transient overexpression of RhRAP2.4 provided by the present invention; Note: 0 d, 4 d, 8 d, and 10 d represent the growth conditions of the silenced axillary buds of Super1300 and Super1300-RhRAP2.4 on the 0th, 4th, 8th, and 10th days after infection.

[0031] Figure 8 It is a sectional view of the growth state of axillary buds at different time periods after transient overexpression of RhRAP2.4 provided by the present invention; Note: 0 d, 4 d, 8 d, and 10 d represent the stereoscopic anatomical observation of the growth points of the overexpressed axillary buds of Super1300 and Super1300-RhRAP2.4 on the 0th, 4th, 8th, and 10th days after infection.

[0032] Figure 9 It is a statistical chart of gene expression levels and axillary bud lengths after transient overexpression of RhRAP2.4 provided by the present invention; Note: (A) RT-qPCR analysis of the relative expression level of RhRAP2.4 after overexpression; (B) Bud lengths of the control group Super1300 and the treatment group Super1300-RhRAP2.4 overexpressed at 0 d, 4 d, 8 d, and 10 d after overexpression (* indicates P≤0.05, ** indicates P≤0.01). Detailed implementation manners

[0033] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the accompanying drawings required for use in the embodiments or the description of the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0034] A rose gene RhRAP2.4 provided in the first aspect of the embodiments of the present application, characterized in that: the rose gene RhRAP2.4 has the following sequences:

[0035] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0036] (2) A gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.2.

[0037] The nucleotide sequence of the RhRAP2.4 gene of Rosa chinensis provided in the second aspect of the embodiments of the present application is shown in SEQ ID NO.2.

[0038] The encoded protein of the RhRAP2.4 gene of Rosa chinensis provided in the third aspect of the embodiments of the present application, the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0039] The application of the RhRAP2.4 gene of Rosa chinensis or the silenced RhRAP2.4 gene of Rosa chinensis provided in the fourth aspect of the embodiments of the present application in promoting the germination of axillary buds of Rosa chinensis.

[0040] A kit provided in the fifth aspect of the embodiments of the present application, containing a reagent for inhibiting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0041] A kit provided in the sixth aspect of the embodiments of the present application, containing a reagent for detecting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0042] In some embodiments, the kit contains primers for amplifying the nucleotide sequence shown in SEQ ID NO:1.

[0043] A vector provided in the seventh aspect of the embodiments of the present application, the vector contains the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0044] An engineered bacterium provided in the eighth aspect of the embodiments of the present application, the engineered bacterium contains a vector.

[0045] The application of the kit, vector, and engineered bacterium provided in the ninth aspect of the embodiments of the present application in regulating the germination of axillary buds of Rosa chinensis.

[0046] A method for promoting the germination of axillary buds of Rosa chinensis, promoting the expression of the RhRAP2.4 gene or its transcription and translation protein in Rosa chinensis, and the nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1.

[0047] A method for cultivating a new improved variety of Rosa chinensis provided in the tenth aspect of the embodiments of the present application, using the RhRAP2.4 gene of Rosa chinensis or the encoded protein to promote or inhibit the germination of axillary buds of Rosa chinensis. Example 1

[0048] A RhRAP2.4 gene of Rosa chinensis of the present invention, the RhRAP2.4 gene of Rosa chinensis has the following sequence:

[0049] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0050] (2)The gene obtained by silencing or knocking out the nucleotide sequence shown in SEQ ID NO.2. Example 2

[0051] The nucleotide sequence for silencing the rose gene RhRAP2.4 of the present invention is shown in SEQ ID NO.2.

[0052] The encoded protein of the rose gene RhRAP2.4 of the present invention, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3. Example 3

[0053] The application of the rose gene RhRAP2.4 or the silenced rose gene RhRAP2.4 of the present invention in promoting the germination of rose axillary buds. Example 4

[0054] A kit of the present invention, containing a reagent for inhibiting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0055] A kit of the present invention, containing a reagent for detecting the expression of the nucleotide sequence shown in SEQ ID NO:1.

[0056] The kit contains primers for amplifying the base sequence shown in SEQ ID NO:1.

[0057] A vector of the present invention, the vector contains the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0058] An engineered bacterium of the present invention, the engineered bacterium contains a vector.

[0059] The application of the kit, vector, and engineered bacterium of the present invention in regulating the germination of rose axillary buds. Example 5

[0060] A method for cultivating an improved new rose variety of the present invention, using the rose gene RhRAP2.4 or the encoded protein to promote or inhibit the germination of rose axillary buds.

[0061] Test Example 1

[0062] This application is based on the genomic sequence of the rose cultivar 'Yu Yue Fen'. Previously, transcriptome sequencing was performed on axillary buds at different time points after pruning, and a significantly differentially expressed gene, RhRAP2.4, related to rose axillary bud germination was screened out. In axillary buds, the expression level of RhRAP2.4 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 RhRAP2.4, it was found that axillary bud germination and growth rate were affected. Therefore, this application reveals the biological function of RhRAP2.4 in rose axillary bud germination, which is of great significance for cut rose breeding and improving the quality and efficiency of the industry.

[0063] This test example provides that the nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO: 1.

[0064] The abbreviations and their corresponding names that appear in this application are shown in Table 1.

[0065] Table 1

[0066] 1. Plant materials

[0067] 1.1 'Pink Avalanche'

[0068] 'Pink Avalanche' is a modern cut rose cultivar, obtained from the Baofeng Base of the Institute of Floriculture, Yunnan Academy of Agricultural Sciences. Single-bud stem segments were used as materials for the rose axillary bud germination experiment.

[0069] 1.2 Tobacco

[0070] The materials for subcellular localization and transcriptional activation experiments were Nicotiana benthamiana. Seeds were sown on moist nutrient substrate and covered with a film, and cultured in a culture room.

[0071] Culture conditions: temperature 24 ± 1 °C, relative humidity 60 - 65%, photoperiod 16 h / 8 h.

[0072] 1.3 Strains and vectors

[0073] Escherichia coli: DH5α and Agrobacterium tumefaciens strains: EHA105, GV3101 (pSoup), pSuper-1300 (Kan resistance) were all purchased from Beijing Tsingke Biotechnology Co., Ltd. The VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0074] 1.4 Culture medium formulations involved in the case

[0075] (1) LB medium and YEB medium (Table 2)

[0076] Table 2

[0077] 2. Research Methods

[0078] 2.1 Total RNA Extraction

[0079] The total RNA was extracted from the petals, leaves, receptacles, sepals, and axillary buds of 'Pink Avalanche' using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (column type).

[0080] 2.2 cDNA Synthesis

[0081] (1)Genomic DNA Removal Reaction System (Table 3)

[0082] Table 3

[0083] Mix well and incubate at 42 °C for 2 min.

[0084] (2)Prepare the Reverse Transcription Reaction System (Table 4)

[0085] Table 4

[0086] Mix well by pipetting, incubate at 37 °C for 15 min; 85 °C for 5 s. Store the product at -20 °C.

[0087] 2.3 Real-Time Fluorescent Quantitative PCR

[0088] Specific primers for RT-qPCR of the gene were designed using Primer premier5 (Table 5). The cDNA was diluted four-fold with ddH 2 O, and Real-Time PCR amplification was performed using the reverse-transcribed cDNA as a template. GAPDH was used as an internal reference, and 3 biological replicates were set up.

[0089] Table 5

[0090] The RT-qPCR reaction system is shown in Table 6:[[]]END]]

[0091] Table 6

[0092] The RT-qPCR reaction program is shown in Table 7:[[]]END]]

[0093] Table 7

[0094] 2.4 Vector Construction

[0095] 2.4.1 PCR Amplification of the Target Gene Fragment

[0096] Use the high-fidelity enzyme for PCR amplification of the target gene (PhusionTM Plus PCR Master Mix). The PCR amplification reaction system is shown in Table 8:

[0097] Table 8

[0098] The PCR amplification reaction program is shown in Table 9:

[0099] Table 9

[0100] After the amplification is completed, perform 1% gel electrophoresis and select the target band for subsequent gel extraction.

[0101] 2.4.2 Gel Extraction

[0102] Use a DNA gel extraction kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0) to recover and purify the PCR product.

[0103] 2.4.3 Double Digestion of the Vector

[0104] According to the restriction enzyme sites inserted in the target fragment sequence, use the corresponding enzymes to perform double digestion on the vector. The double digestion system is shown in Table 10:

[0105] Table 10

[0106] After adding the system reagents on ice, perform double digestion of the vector according to the heat denaturation temperature of the specific enzyme.

[0107] 2.4.4 Homologous Recombination

[0108] Perform homologous recombination on the vector after double digestion and the cloned target gene fragment to construct the vector. The homologous recombination system is shown in Table 11:

[0109] Table 11

[0110] Procedure: Run at 50 °C for 15 min in a PCR instrument.

[0111] 2.4.5 Transformation of Escherichia coli

[0112] (1) Take out the competent cells from -80 °C and thaw them on ice;

[0113] (2) Take a 1.5 mL centrifuge tube, add 10 μL of the recombinant product and 50 μL of DHα competent cells, pipette and mix well, and let it stand on ice for 30 min;

[0114] (3) Heat shock in a 42 °C water bath for 90 s, and quickly transfer it to ice and let it stand for 2 min;

[0115] (4) Add 500 μL of LB to the centrifuge tube, incubate at 37 °C and 200 rpm for 1 h;

[0116] (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min;

[0117] (6) Discard 400 μL of the supernatant in the laminar flow hood, pipette and mix well, and then spread it on the LB solid medium containing antibiotics, and incubate it inverted overnight at 37 °C.

[0118] 2.4.6 Bacterial Inspection and Sequencing

[0119] (1) Shake the bacteria: Pick a single colony grown overnight and incubate it in 500 μL of LB medium containing antibiotics for 3 - 4 h (37 °C, 200 rpm).

[0120] (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 expectation. The bacterial liquid PCR amplification system is shown in Table 12:

[0121] Table 12

[0122] The bacterial liquid PCR amplification reaction procedure is shown in Table 13:

[0123] Table 13

[0124] The amplification product is used for 1% gel electrophoresis imaging, and the samples with the expected band size are sent to the company for sequencing.

[0125] 2.5 Plasmid Extraction

[0126] After the sequencing results are returned, perform sequence alignment, select the samples that meet the expectation to shake the bacteria, and extract the plasmid using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0127] 2.6 Agrobacterium Transformation

[0128] (1) Take out the Agrobacterium competent cells from the -80 °C refrigerator, insert them into ice when they are melted to the state of ice - water mixture;

[0129] (2) Add 0.01 - 1 μg of plasmid DNA to every 100 μL of competent cells. After gently pipetting to mix, incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and then on ice for 5 min in sequence.

[0130] (3) Add 500 μL of antibiotic-free YEB liquid medium and culture with shaking at 28 °C for 2 - 3 h.

[0131] (4) Centrifuge at 5000 rpm for 2 min. In a laminar flow hood, discard 400 μL of the supernatant and spread the remaining liquid onto YEB solid medium containing antibiotics. Incubate in an inverted position at 28 °C for 2 - 3 d.

[0132] 2.7 Subcellular localization

[0133] (1) Vector construction

[0134] Select SmaI and KpnI as restriction enzyme sites and insert the complete coding region (SEQ ID NO: 1) sequence of RhRAP2.4 into the pSuper - 1300 vector. Design primers using the method of homologous recombination to construct the RhRAP2.4 - GFP vector. After obtaining the positive Agrobacterium strain, perform the following experiments.

[0135] (2) Bacterial shaking culture

[0136] 1) Streak culture of bacterial liquid: Take out the preserved bacterial liquid and streak it on YEB medium containing the corresponding antibiotics. Incubate in an inverted position at 28 °C for 2 - 3 days.

[0137] 2) Shaking culture: Pick a single colony into a 1.5 mL centrifuge tube, add 500 μL of YEB liquid medium containing antibiotics, and perform a small overnight shake (28 °C, 200 rpm). Check the bacteria by PCR. For the bacterial liquid with the correct band, continue with medium - scale and large - scale shaking.

[0138] (3) Tobacco injection

[0139] 1) Bacterial collection: Centrifuge the bacterial liquid cultured to OD 600 = 0.4 - 0.6 (5000 rpm, 8 min), discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells with the infection solution and adjust the OD 600 to 1.0. Mix in the ratio of RhRAP2.4 - GFP / GFP:NF - YA4 - mCherry:P19 = 1:1:0.5 and let it stand in the dark for 3 h.

[0140] 2) Injection: After standing, gently make a small cut on the back of the tobacco leaves (grown to 4 - 6 leaves) with a needle, and use a 1 mL syringe to inject the proportionally mixed bacterial liquid into the tobacco leaves and make marks.

[0141] 3) Cultivation: Place the injected tobacco in an incubator at 25 °C, first incubate it in the dark for one day, and then transfer it to the light for 2 days; pay attention to watering during this period to keep the tobacco growing normally.

[0142] (4)Observation and photography

[0143] Select the leaves near the injection site of the tobacco, gently cut them into squares with a blade and forceps, place them on a glass slide, drop distilled water on them, and cover them with a cover slip (pay attention to avoiding the generation of bubbles). Observe and photograph them with a laser confocal microscope in the State Key Laboratory of the Institute of Biology, Yunnan Academy of Agricultural Sciences.

[0144] 2.8 Transient silencing

[0145] (1)Vector primer design and vector construction

[0146] Using EcoRI and Kpn1 as restriction enzyme sites, insert the silencing fragment (SEQ ID NO: 2) sequence of RhRAP2.4 into the empty pTRV2 vector, design primers using the method of homologous recombination, and construct the TRV2-RhRAP2.4 vector.

[0147] (2)Bacterial liquid culture

[0148] Streak the bacterial liquid on a plate (containing 50 mg / L Kan / Rif), and incubate it upside down at 28 °C for 2 - 3 days. Pick a single colony and shake it gently in 500 μL of YEB containing antibiotics for bacterial inspection; if the band is correct, perform medium shaking and large shaking (28 °C, 200 rpm).

[0149] (3)Bacterial collection and resuspension

[0150] Centrifuge to collect bacteria at 5000 rpm for 8 min, pour off the supernatant, resuspend the bacterial cells with the infection solution, pipette and mix well, and adjust to OD600 = 1.0. When performing the transient silencing experiment, mix the TRV1 and TRV2, and TRV2-RhRAP2.4 bacterial solutions in a volume ratio of 1:1, and let it stand in the dark for 4 - 6 h.

[0151] (4)Vacuum aspiration

[0152] Use a vacuum pump to aspirate and infect the stem segments with single buds, at 0.082 MPa, aspirate for 10 min, hold the pressure for 10 min, release the air for 10 min, and try to immerse the entire stem segment in the bacterial solution as much as possible, and repeat the treatment three times. After infection, rinse with sterile water, place it in an 8 °C incubator for 3 d and then cuttage, observe every two days, and take samples and photograph for recording.

[0153] 2.9 Transient overexpression

[0154] (1)Vector primer design and vector construction

[0155] Using Sam1 and Kpn1 as restriction sites, the overexpression fragment of RhRAP2.4 was inserted into the empty pSuper1300 vector. Primers were designed using homologous recombination to construct the Super1300-RhRAP2.4 vector.

[0156] (2)Bacterial liquid preparation

[0157] Prepare Agrobacterium tumefaciens of pSuper1300 and pSuper1300-RhRAP2.4. The method of collecting bacteria is the same as that of the transient silencing method in Section 2.8 of this chapter. After the bacterial liquid preparation is completed, it is placed in a shaker at 28 °C and 200 rpm for 45 min.

[0158] (2)Vacuum aspiration

[0159] The method is the same as that of the transient silencing method in Section 2.8 of this chapter. After infection, gently rinse the bacterial liquid on the surface of the stem segments with sterile water, and then cutt them in the culture room. Take pictures, sample, and observe with a stereoscope every other day. 2 Gently rinse the bacterial liquid on the surface of the stem segments with sterile water, and then cutt them in the culture room. Take pictures, sample, and observe with a stereoscope every other day.

[0160] 2.10 Primers used in the RhRAP2.4 sequence experiment

[0161] Table 14

[0162] 3 Experimental results

[0163] 3.1 Expression verification of RhRAP2.4 in different tissue parts

[0164] In this application, total RNA was extracted from the roots, stems, leaves, petals, pistils, stamens, axillary buds, and sepals of Rosa hybrida 'Pink Avalanche' and its quality was detected. The genes with the most cis-acting elements related to meristems and plant growth and development were selected for fluorescence quantitative expression analysis. It was found that the expression of different genes varied in different tissues ( Figure 1 ). Among them, RhRAP2.4 was specifically expressed and had a relatively high expression level in stems, leaves, and axillary buds, indicating that RhRAP2.4 may be crucial for the growth and development of axillary buds in roses.

[0165] 3.2 Transcriptional activation and subcellular localization analysis of RhRAP2.4

[0166] To explore the subcellular location where RhRAP2.4 functions, this application first performed an online prediction using Cell-PLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2), which showed localization 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 the GFP empty vector as a control, it was transferred into Agrobacterium EH105 and then injected into the leaves of Nicotiana benthamiana. After 3 days, observation was carried out under a laser confocal microscope. The results showed that both the GFP empty vector and RhRAP2.4-GFP were localized in the nucleus ( Figure 2 B).

[0167] 3.3 Analysis of the expression level of RhRAP2.4 in different active buds

[0168] In this application, real-time fluorescence quantitative PCR analysis was performed on the expression level of RhRAP2.4 in the axillary buds at different node positions of the cut rose 'Pink Avalanche' flower branches (buds not showing white tips), and it was found that the expression of RhRAP2.4 was high in the basal axillary buds ( Figure 3 ). Therefore, this application speculates that RhRAP2.4 is closely related to the germination of axillary buds.

[0169] 3.5 RhRAP2.4 promotes the germination of rose axillary buds

[0170] Based on the analysis results of the expression level of RhRAP2.4 in buds at different positions, a specific segment (SEQ ID NO: 2) of the RhRAP2.4 sequence was selected in this application, and a TRV2-RhRAP2.4 transient silencing vector was constructed by homologous recombination. After transferring TRV1, TRV2, and TRV2-RhRAP2.4 into Agrobacterium EHA105, they were used to infect the stem segments of the middle axillary buds to silence RhRAP2.4, and phenotypic observations and records were carried out at 0 d, 6 d, 10 d, and 12 d after infection ( Figure 4 ). From the phenotypic perspective of the buds, compared with the TRV2 control, the germination rate of the TRV2-RhRAP2.4 buds was slower, and the anatomical results of the buds also showed that the development of the buds slowed down after RhRAP2.4 was silenced ( Figure 5 ). To confirm the silencing effect, total RNA of the buds before and after RhRAP2.4 silencing was extracted, and RT-qPCR analysis was performed on its expression level. It was found that the expression decreased extremely significantly after RhRAP2.4 was silenced, 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 also measured, and it was found that the difference was extremely significant at the 12th day after silencing ( Figure 6 B).

[0171] Based on the analysis results of the expression levels of RhRAP2.4 in buds at different sites, the CDS sequence of the RhRAP2.4 sequence was selected, and a transient overexpression vector of Super1300-RhRAP2.4 was constructed by homologous recombination. After transferring Super1300 and Super1300-RhRAP2.4 into Agrobacterium EHA105, they were used for the infection of the middle axillary bud stem segments to overexpress RhRAP2.4. Phenotype observations and records were carried out at 0 d, 4 d, 8 d, and 10 d after infection ( Figure 7 ). From the perspective of the bud phenotype, compared with the Super1300 control, the germination rate of the buds of Super1300-RhRAP2.4 was faster. The anatomical results of the buds also showed that the development of the buds was accelerated after RhRAP2.4 silencing ( Figure 8 ). To confirm the overexpression effect, the total RNA of the buds before and after the overexpression of RhRAP2.4 was extracted, and RT-qPCR analysis was performed on its expression level. It was found that the expression level increased extremely significantly after the overexpression of RhRAP2.4, indicating that the overexpression was effective ( Figure 9 A). In addition, the bud lengths of the control group Super1300 and the experimental group Super1300-RhRAP2.4 at different time points were also measured, and it was found that the difference was extremely significant on the 10th day after the overexpression ( Figure 9 B).

[0172] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosed content of the present invention, and these solutions also belong to the scope of the disclosure of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation on the claims. The protection scope of the present invention is defined by the claims and their equivalents.

Claims

1. An application for inhibiting the germination of axillary buds of roses by silencing the RhRAP2.4 gene or promoting the germination of axillary buds of roses by overexpressing the RhRAP2.4 gene, characterized in that: The nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.

1.

2. A method for cultivating an improved new variety of rose, characterized in that: The germination of axillary buds of roses is inhibited by silencing the RhRAP2.4 gene or promoted by overexpressing the RhRAP2.4 gene. The nucleotide sequence of the RhRAP2.4 gene is shown in SEQ ID NO.1.

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