Application, reagent kit, and regulation method of RhATHB12 gene in regulating axillary bud germination in roses

Abstract

This invention relates to the field of gene regulation technology, specifically to the application of the RhATHB12 gene in regulating axillary bud germination in roses, along with its kit and regulation method. The nucleotide sequence of the RhATHB12 gene is shown in SEQ ID NO.1. Regulation involves silencing, knocking out, or inhibiting the expression of the RhATHB12 gene, which may promote axillary bud germination; overexpression of RhATHB12, which may inhibit axillary bud germination. This gene and its regulation method are of paramount importance for cut rose breeding and for improving the quality and efficiency of the industry.

Description

Technical Field

[0001] This invention relates to the field of gene regulation technology, and more specifically, to the application of the RhATHB12 gene in regulating the germination of axillary buds in roses, as well as reagent kits and regulation methods. Background Technology

[0002] The rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody plant belonging to the genus Rosa in the family Rosaceae. It is characterized by blooming in all four seasons, a rich variety of flower colors, and diverse types. It possesses both ornamental and medicinal value and is widely used in the cut flower industry. As one of the world's four major cut flowers, the rose is highly favored for its beautiful flower shape and diverse colors, possessing high economic value and being one of the main consumer varieties in the cut flower market.

[0003] Germination is a crucial biological process in plant growth and development, and the germination of axillary buds directly affects the formation of plant branches. The number of branches determines the number of vegetative organs and plays a key regulatory role in crop yield. During morphogenesis, meristematic tissue differentiates into lateral bud primordia, which gradually develop into axillary buds and further grow into lateral branches. The final number of branches in a plant is positively correlated with the degree of axillary bud germination.

[0004] Axillary buds of roses mainly develop in the leaf axils and eventually form flowering branches, playing a vital role in their overall growth and morphological development. They are also important materials for rose propagation. In cut rose production, the germination rate of axillary buds directly affects the survival rate of seedlings; and in subsequent growth and development, the germination characteristics of axillary buds are related to the growth cycle, yield, and quality of cut roses. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide the application, kit and regulation method of the RhATHB12 gene in regulating the axillary bud germination of roses.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] Application of the RhATHB12 gene in regulating the germination of axillary buds of roses, the nucleotide sequence of the RhATHB12 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the application includes any of the following methods:

[0009] The RhATHB12 gene was silenced, knocked out, or its expression was suppressed.

[0010] The RhATHB12 gene was overexpressed;

[0011] The expression of the protein encoded by the RhATHB12 gene was inhibited;

[0012] The expression of the protein encoded by the RhATHB12 gene was promoted.

[0013] Furthermore, the amino acid sequence of the protein encoded by the RhATHB12 gene is shown in SEQ ID NO.3.

[0014] The present invention also provides a kit for regulating the germination of axillary buds of roses, comprising reagents capable of regulating the expression or function of the RhATHB12 gene.

[0015] Furthermore, the reagents capable of regulating the expression or function of the RhATHB12 gene include any one of the following biological materials:

[0016] a) A recombinant vector containing the silent gene as shown in SEQ ID NO.2;

[0017] b) Engineered bacteria containing the recombinant vector in a);

[0018] c) A recombinant vector containing the overexpressed gene as shown in SEQ ID NO.4;

[0019] d) Engineered bacteria containing the recombinant vector in c).

[0020] The present invention also provides a method for regulating the germination of axillary buds of roses. The method involves treating rose plants with the kit described above and promoting or inhibiting the germination of axillary buds of roses by regulating the expression of the RhATHB12 gene.

[0021] Furthermore, silencing, knocking out, or inhibiting the expression of the RhATHB12 gene can promote the germination of axillary buds; overexpression of the RhATHB12 gene can inhibit the germination of axillary buds.

[0022] Furthermore, the treatment is performed on a stem segment with a single bud from the rose plant.

[0023] The beneficial effects of this invention are as follows:

[0024] In rose axillary buds, the expression level of the RhATHB12 gene was 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. Transient silencing of the RhATHB12 gene was found to affect axillary bud germination and growth rate.

[0025] Regulating the germination of axillary buds in roses is of great significance for improving the quality and efficiency of cut rose breeding and the industry. This invention applies the RhATHB12 gene to the regulation of axillary bud germination in roses, which can not only effectively reduce the cost of cultivation or breeding, but also improve efficiency and effectiveness, providing a new and reliable approach for the development, breeding, cultivation, planting and development of rose plants. Attached Figure Description

[0026] Figure 1 This is a comparison of RhATHB12 expression levels in axillary buds at different locations in Example 1 of the present invention.

[0027] Figure 2 This is a subcellular localization map of RhATHB12 in Embodiment 2 of the present invention;

[0028] Figure 3 This is a phenotypic observation result of axillary bud germination and growth at different time points after transient silencing of RhATHB12 in Example 3 of the present invention;

[0029] Figure 4 This is an anatomical diagram of axillary bud growth status at different time points after transient silencing of RhATHB12 in Embodiment 3 of the present invention.

[0030] Figure 5 This is a comparison diagram of the effects of momentarily silencing RhATHB12 in Embodiment 3 of the present invention. Figure 5 Figure A shows a comparison of the relative expression levels of RhATHB12 using RT-qPCR. Figure 5 Figure B shows a comparison of the shoot length of the control group TRV2 and the treatment group TRV2-RhATHB12 at each time point;

[0031] Figure 6 This is a phenotypic observation result of axillary bud germination and growth at different time points after transient overexpression of RhATHB12 in Example 4 of the present invention;

[0032] Figure 7 This is an anatomical diagram of axillary bud growth at different time points after transient overexpression of RhATHB12 in Example 4 of the present invention.

[0033] Figure 8 This is a comparison chart showing the effects of transient overexpression of RhATHB12 in Embodiment 4 of the present invention. Figure 8 Figure A shows a comparison of the relative expression levels of RhATHB12 using RT-qPCR. Figure 8 B represents the control group pSuper and the treatment group. RhATHB12 Comparison of axillary bud length in OE. Detailed Implementation

[0034] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] This invention provides the application of the RhATHB12 gene in regulating the germination of axillary buds of roses. The nucleotide sequence of the RhATHB12 gene is shown in SEQ ID NO.1.

[0036] In rose axillary buds, the expression level of the RhATHB12 gene is negatively correlated with axillary bud activity, with high expression in basal axillary buds and low expression in middle and upper active buds. Transient silencing of the RhATHB12 gene was found to affect axillary bud germination and growth rate. Therefore, this invention, based on the biological function of the RhATHB12 gene in rose axillary bud germination, aims to regulate rose axillary bud germination, which is of great significance for cut rose breeding and improving the quality and efficiency of the industry.

[0037] Specifically, the application of this invention includes any of the following methods:

[0038] Silencing, knocking out, or inhibiting the expression of the RhATHB12 gene;

[0039] Overexpression of the RhATHB12 gene;

[0040] The expression of the protein encoded by the RhATHB12 gene was inhibited;

[0041] The expression of the protein encoded by the RhATHB12 gene was promoted.

[0042] Preferably, the amino acid sequence of the protein encoded by the RhATHB12 gene is shown in SEQ ID NO.3.

[0043] The kit of the present invention for regulating the germination of axillary buds of roses contains reagents capable of regulating the expression or function of the RhATHB12 gene.

[0044] Preferably, the kit includes reagents that can silence, knock out, or inhibit the expression of the RhATHB12 gene, or includes reagents that can overexpress the RhATHB12 gene.

[0045] Preferably, the kit may also include relevant primers, reagents for detecting the expression level of the RhATHB12 gene, and other conventional biological reagents.

[0046] The nucleotide sequence of the silenced gene of the present invention is shown in SEQ ID NO.2, and the overexpressed gene is shown in SEQ ID NO.4. The nucleotide sequence shown in SEQ ID NO.2 is a specific segment of the RhATHB12 sequence, which was determined based on the expression level analysis results of RhATHB12 in buds at different sites.

[0047] The present invention relates to a method for regulating the germination of axillary buds in roses. By treating rose plants and regulating the expression of the RhATHB12 gene, the germination of axillary buds in roses can be promoted or inhibited.

[0048] Specifically, silencing, knocking out, or inhibiting the expression of the RhATHB12 gene can promote the germination of axillary buds; overexpression of the RhATHB12 gene can inhibit the germination of axillary buds.

[0049] Based on the genome sequence of the rose variety 'Yueyuefen', this invention uses 'Pink Snow Mountain' as the experimental material. Transcriptome sequencing was performed on axillary buds at different time points after pruning, and the significantly differentially expressed gene RhATHB12 associated with the germination of rose axillary buds was screened out.

[0050] 'Pink Snow Mountain' is a modern cut rose variety. It was obtained from the Baofeng Base of the Flower Research Institute of Yunnan Academy of Agricultural Sciences, and the stem segments with single buds were used as materials for the rose axillary bud germination experiment.

[0051] In the specific experiments of this invention, the subcellular localization and transcriptional activation experimental material used was Nicotiana benthamiana. The seeds were sown in a moist nutrient substrate, covered with a film, and cultured in a culture room under the following conditions: temperature 24±1℃, relative humidity 60-65%, and photoperiod 16h / 8h.

[0052] In the specific experiments of this invention, the strains and vectors used are as follows:

[0053] Escherichia coli DH5α and Agrobacterium tumefaciens strains EHA105, GV3101 (pSoup), and pSuper-1300 (Kan resistant) were all purchased from Beijing Qingke Biotechnology Co., Ltd. The VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0054] The culture medium formulations involved in this invention are shown in Table 1:

[0055] Table 1

[0056]

[0057] In this invention, the specific procedures for biological experiments such as screening, extraction, recombinant plasmid construction, homologous recombination, sequencing, and transfection of the relevant target genes are as follows:

[0058] (1) Extraction of total RNA: 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) to obtain template RNA.

[0059] (2) cDNA synthesis: The template RNA was mixed evenly with the other components in Table 2 to obtain the genomic DNA removal reaction system, and reacted at 42℃ for 2 min to obtain the reaction solution. Then, the reverse transcription reaction system shown in Table 3 was prepared using the reaction solution, mixed by pipetting, and cDNA synthesis was performed. The reaction program was: 37℃, 15 min; 85℃, 5 s. The obtained product was stored at -20℃.

[0060] Table 2

[0061]

[0062] Table 3

[0063]

[0064] (3) Real-time quantitative PCR: RT-qPCR specific primers for the gene were designed using Primer Premier5 (Table 4). The cDNA obtained from reverse transcription was diluted three times with ddH2O, and Real-Time PCR amplification was performed using the cDNA as a template. UBI2 was used as an internal control, and three biological replicates were set up.

[0065] Table 4

[0066]

[0067] The RT-qPCR reaction system is shown in Table 5:

[0068] Table 5

[0069]

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

[0071] Table 6

[0072]

[0073] (4) Carrier construction:

[0074] PCR amplification of the target gene fragment: High-fidelity enzyme (Phusion™ Plus PCRMaster Mix) was used for PCR amplification of the target gene. The PCR amplification reaction system is shown in Table 7.

[0075] Table 7

[0076]

[0077] The PCR amplification reaction procedure is shown in Table 8:

[0078] Table 8

[0079]

[0080] After amplification, 1% gel electrophoresis was performed, and the target band was selected for subsequent gel recovery.

[0081] The PCR products were recovered and purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0.

[0082] (5) Double digestion of the vector: The vector is double-digested with the corresponding enzyme according to the restriction sites inserted in the target fragment sequence. The double digestion system is shown in Table 9. After adding the system reagents on ice, the vector is double-digested according to the heat denaturation temperature of the specific enzyme.

[0083] Table 9

[0084]

[0085] (6) Homologous recombination: The double-digested vector and the cloned target gene fragment are subjected to homologous recombination to construct the vector. The homologous recombination system is shown in Table 10. The reaction program is as follows: run in a PCR instrument at 50 °C for 15 min.

[0086] Table 10

[0087]

[0088] (7) Escherichia coli transformation: Take out competent cells from -80 ℃ and thaw them on ice; take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of DHα competent cells, mix by pipetting, and let stand on ice for 30 min; heat shock in a 42 ℃ water bath for 90 s, and quickly transfer to ice and let stand for 2 min; add 500 μL of LB to the centrifuge tube, incubate at 37 ℃, 200 rpm for 1 h; centrifuge the cultured bacterial solution at 5000 rpm for 5 min; discard 400 μL of supernatant in a clean bench, mix by pipetting, spread on LB solid medium containing antibiotics, and incubate overnight at 37 ℃ inverted.

[0089] (8) Microbial detection and sequencing:

[0090] Shaking culture: Pick a single colony that has grown overnight and culture it in 500 μL of LB medium containing antibiotics with shaking for 3-4 h (37 ℃, 200 rpm).

[0091] 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:

[0092] Table 11

[0093]

[0094] The bacterial culture PCR amplification reaction procedure is shown in Table 12:

[0095] Table 12

[0096]

[0097] The amplified products were used for 1% gel electrophoresis imaging, and those with band sizes that met expectations were sent to the company for sequencing.

[0098] (9) Plasmid extraction: After the test results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation. Plasmids are extracted using the plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0099] (10) Agrobacterium transformation: When the competent Agrobacterium cells are taken out of the -80 ℃ freezer and thawed to the state of ice-water mixture, they are inserted into ice; 0.01-1 μg plasmid DNA is added to every 100 μL of competent cells, and after mixing, they are placed on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ℃ water bath for 5 min, and in an ice bath for 5 min; 500 μL of antibiotic-free YEB liquid medium is added, and the cells are cultured at 28 ℃ with shaking for 2-3 h; centrifuged at 5000 rpm for 2 min, 400 μL of supernatant is discarded in a clean bench, and the supernatant is spread onto YEB solid medium containing antibiotics and cultured upside down at 28 ℃ for 2-3 days.

[0100] Example 1: Analysis of RhATHB12 expression levels in different active buds

[0101] This embodiment uses the specific experimental methods of the present invention to perform real-time quantitative PCR (RT-qPCR) analysis on the expression level of RhATHB12 in axillary buds at different nodes of the cut rose 'Pink Snow Mountain' (flower buds before whitening). The primers used for RT-qPCR were qRT-UBI2-F, qRT-UBI2-R, qRT-RhATHB12-F, and qRT-RhATHB12-R, and their specific sequences are shown in Table 13. The RT-qPCR analysis results are as follows: Figure 1 As shown.

[0102] Figure 1 The data in the data are average ± SD (n=3) (**** indicates P ≤ 0.0001), which are the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth axillary buds from top to bottom.

[0103] according to Figure 1It can be seen that RhATHB12 expression is high in basal axillary buds. Therefore, this application hypothesizes that RhATHB12 is closely related to the germination of axillary buds.

[0104] Example 2: Transcriptional activation and subcellular localization analysis of RhATHB12

[0105] To explore the subcellular location where RhATHB12 functions, this embodiment first used Cell-PLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2) for online prediction, which showed that it is located in the cell nucleus. To further verify the accuracy of the localization, the CDS sequence of RhATHB12 was cloned using the relevant experimental methods of this invention, and the RhATHB12-GFP vector was constructed. Using the empty GFP vector as a control, the vector was transformed into EH105 and then injected into leaves of Nicotiana benthamiana. The specific experimental procedure is as follows:

[0106] (1) Vector construction: SmaI and KpnI were selected as restriction sites. The complete coding region (SEQ ID NO: 4) sequence of RhATHB12 was inserted into the pSuper-1300 vector. Primers were designed using homologous recombination to construct the RhATHB12-GFP vector. After obtaining Agrobacterium tumefaciens positive strains, the following experiments were performed.

[0107] The subcellular localization primers were RhATHB12-GFP-pSuper-F and RhATHB12-GFP-pSuper-R, and their specific sequences are shown in Table 13.

[0108] (2) Shaking:

[0109] Streak culture: Take out the preserved bacterial culture and streak it on YEB medium containing the corresponding antibiotics, and incubate it upside down at 28 ℃ for 2-3 days;

[0110] Shaking: Pick a single colony into a 1.5 mL centrifuge tube, add 500 μL of YEB liquid medium containing antibiotics and shake overnight with a small shake (28 ℃, 200 rpm). PCR bacterial test is performed. If the bacterial solution with the correct band is found, continue with a medium shake and then a large shake.

[0111] (3) Tobacco injection:

[0112] Bacterial collection: culturing 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, mix at a ratio of RhATHB12-GFP / GFP:NF-YA4-mCherry=1:1, and let stand in the dark for 45 minutes.

[0113] Injection: After the plant has settled, make a small incision on the back of the tobacco leaf (when it has grown to 4-6 leaves) with a needle, inject the bacterial solution mixed in the correct proportion into the tobacco leaf using a 1 mL syringe, and mark the incision.

[0114] 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 maintain the normal growth of the tobacco.

[0115] (4) Observe and take photos:

[0116] 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 (taking care to avoid air bubbles). Two days later, photograph and observe using a laser confocal microscope at the State Key Laboratory of Biology, Yunnan Academy of Agricultural Sciences.

[0117] according to Figure 2 It can be seen that both the empty GFP vector and RhATHB12-GFP are localized in the cell nucleus.

[0118] Example 3: Momentary Silence

[0119] Based on the expression analysis of RhATHB12 in buds at different sites, this application selected a specific segment of the RhATHB12 sequence (SEQ ID NO:2) and constructed a transient silencing vector TRV2-RhATHB12 using homologous recombination. TRV1, TRV2, and TRV2-RhATHB12 were transformed into EHA105 of agricultural stem and then used to infect the basal axillary bud stem segments to silence RhATHB12. Phenotypic observations and records were performed at 0, 4, 8, and 12 days after infection.

[0120] The specific experimental procedure is as follows:

[0121] (1) Vector primer design and vector construction: Using EcoRI and Kpn1 as restriction sites, the silent fragment (SEQ ID NO:2) sequence of RhATHB12 was inserted into the pTRV2 empty vector. Primers were designed using homologous recombination to construct the TRV2-RhATHB12 vector.

[0122] The transient silencing primers were TRV2-RhATHB12-F and TRV2-RhATHB12-R, and their specific sequences are shown in Table 13.

[0123] (2) Bacterial culture: Streak the bacterial culture on agar plates (containing 50 mg / L Kan / Rif) and incubate upside down at 28 ℃ for 2-3 days. Pick a single colony and gently shake it in 500 μL of YEB containing antibiotics for bacterial testing; if the band is correct, shake it medium and then shake it vigorously (28 ℃, 200 rpm).

[0124] (3) Collection and resuspension of bacteria: Collect bacteria by centrifugation at 5000 rpm for 8 min, discard the supernatant, resuspend the bacteria in the infection solution, mix well by pipetting, and adjust to OD. 600 =1.0. When conducting transient silencing experiments, TRV1 and TRV2, TRV2-RhATHB12 bacterial cultures were mixed at a volume ratio of 1:1 and allowed to stand in the dark for 4-6 hours.

[0125] (4) Vacuum suction: Use a vacuum pump to suction and infect stem segments with single buds at 0.082 MPa for 10 min, hold the pressure for 10 min, and release the gas for 10 min, so that the entire stem segment is submerged in the bacterial solution as much as possible. Repeat the treatment three times. After infection, rinse with sterile water, place in an 8 ℃ incubator for 3 days, and then insert cuttings. Observe and take samples every two days, and take photos for record.

[0126] Figure 3 Days 0, 4, 8, and 12 represent the growth status of silent axillary buds of TRV2 and TRV2-RhATHB12 on days 0, 4, 8, and 12 after infection. Figure 4 Note: Days 0, 4, 8, and 12 represent the post-infection anatomical observation of the silent axillary bud growth points of TRV2 and TRV2-RhATHB12 on days 0, 4, 8, and 12 after infection.

[0127] like Figure 3 and Figure 4 As shown, from the phenotype of the buds, the germination rate of TRV2-RhATHB12 buds is faster than that of the TRV2 control. The anatomical results of the buds also show that the development of buds is accelerated after RhATHB12 is silenced.

[0128] To confirm the silencing effect, total RNA was extracted from shoots before and after RhATHB12 silencing, and its expression level was analyzed by RT-qPCR. The results showed a highly significant decrease in RhATHB12 expression after silencing, indicating that silencing was effective. Figure 5 A).

[0129] In addition, such as Figure 5 As shown in Figure B, this embodiment also measured the bud length of the control group TRV2 and the experimental group TRV2-RhATHB12 at different time points. In the figure, * indicates P≤0.05, ** indicates P≤0.01, *** indicates P≤0.001, and **** indicates P≤0.0001. It can be seen that the difference reached extremely significant on day 12 after transient silencing.

[0130] Example 4: Transient overexpression

[0131] This embodiment is based on the expression level analysis results of RhATHB12 in buds at different sites. The CDS sequence of the RhATHB12 sequence was selected, and homologous recombination was used to construct... RhATHB12 OE transient overexpression vector, pSuper and RhATHB12 OE was transferred into EHA105 straw and used to infect the axillary buds of the stem to overexpress RhATHB12. Phenotypic observation and recording were performed at 0, 4, 8, and 12 days post-infection. The specific experimental procedure is as follows:

[0132] (1) Vector primer design and vector construction: Using Sam1 and Kpn1 as restriction sites, the overexpression fragment of RhATHB12 (SEQ ID NO:4) was inserted into the pSuper empty vector. Primers were designed using homologous recombination and the vector was constructed. RhATHB12 OE vector. The transient overexpression primers are RhATHB12-GFP-pSuper-F and RhATHB12-GFP-pSuper-R, and their specific sequences are shown in Table 13.

[0133] (2) Preparation of bacterial culture: Prepare pSuper and RhATHB12 Agrobacterium oxyphylla (OE). The bacterial collection method is the same as the transient silencing method. After the bacterial culture is prepared, it is placed at 28°C and incubated with shaking at 200 rpm for 45 min.

[0134] (3) Vacuum aspiration: The method is the same as the instantaneous silencing method. After infection, gently rinse the bacterial solution on the surface of the stem segment with RO H2O, and then insert the cuttings in the culture room. Take photos, samples, and observe the stereotactic properties every other day.

[0135] Figure 6 pSuper (control group) and treatment group were observed on days 0, 4, 8, and 12 post-infection. RhATHB12 The growth status of silent axillary buds in OE. Figure 7 pSuper (control group) and treatment group were observed on days 0, 4, 8, and 12 post-infection. RhATHB12 Anatomical observation of overexpression of axillary bud growth points in OE.

[0136] like Figure 6 and Figure 7 As shown, based on the bud phenotype, compared to the pSuper control, RhATHB12 OE buds germinate slowly, and the anatomical results of the buds also show that bud development is slowed down after RhATHB12 overexpression.

[0137] To confirm the overexpression effect, total RNA was extracted from shoots before and after RhATHB12 overexpression, and its expression level was analyzed by RT-qPCR. RhATHB12In OE, the expression level of RhATHB12 increased significantly relative to pSuper after overexpression, indicating that the overexpression was effective. Figure 8 A).

[0138] In addition, such as Figure 8 As shown in Figure B, this embodiment also compares the control group pSuper and the treatment group. RhATHB12 The axillary bud length at different time points after OE was measured. * indicates P≤0.05, ** indicates P≤0.01, *** indicates P≤0.001, and **** indicates P≤0.0001. It can be seen that the difference reached highly significant on day 12 after overexpression.

[0139] The primers used in various embodiments of the present invention are shown in Table 13:

[0140] Table 13

[0141]

[0142] The abbreviations and their corresponding names that appear in this invention are shown in Table 14.

[0143] Table 14

[0144]

[0145] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. RhATHB12 The application of genes in regulating axillary bud germination in roses is characterized by, The RhATHB12 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The application can be any of the following methods: Regarding the RhATHB12 Gene expression is suppressed to promote the germination of axillary buds; Regarding the RhATHB12 The gene is overexpressed to suppress the germination of axillary buds; Regarding the RhATHB12 The expression of the gene-encoded protein is suppressed to promote the germination of axillary buds; Regarding the RhATHB12 The expression of the gene-encoded protein is promoted to inhibit the germination of axillary buds.

2. A method for regulating the germination of axillary buds in roses, characterized in that, The rose plants were treated using a kit, and the treatment was carried out by... RhATHB12 Suppressing gene expression promotes the germination of rose axillary buds, or by inhibiting gene expression. RhATHB12 Gene overexpression inhibits the germination of rose axillary buds; The kit includes any one of the following biological materials: a) A recombinant vector containing the silent gene as shown in SEQ ID NO.2; b) Engineered bacteria containing the recombinant vector in a); c) A recombinant vector containing the overexpressed gene as shown in SEQ ID NO.4; d) Engineered bacteria containing the recombinant vector in c).

3. The method for regulating the germination of axillary buds of roses according to claim 2, characterized in that, The treatment is performed on a stem segment with a single bud from the rose plant.

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