Application of a rose gene RhMYB1 in regulating rose axillary bud germination

Abstract

This invention discloses the application of the rose gene RhMYB1 in regulating axillary bud germination in roses. Relating to the field of molecular biology, this invention provides the application of products that inhibit the expression of the RhMYB1 gene or its transcribed and translated protein in regulating axillary bud germination in roses. The nucleotide sequence of the RhMYB1 gene is shown in SEQ ID NO.1. Based on the expression results of the RhMYB1 gene at different sites in rose buds, this invention found that its expression level is higher in upper buds (i.e., active buds). Transient silencing of the RhMYB1 gene in axillary buds showed that silencing the RhMYB1 gene significantly inhibited the germination and growth of axillary buds, while overexpression of RhMYB1 promoted the growth of axillary buds.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of a rose gene RhMYB1 in regulating the germination of rose axillary buds. Background Technology

[0002] The rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant belonging to the genus Rosa in the family Rosaceae. It blooms year-round, with a rich variety of colors and types, and is used for ornamental and medicinal purposes. It is also widely used as a cut flower. As one of the world's four major cut flowers, the rose has high economic value due to its large, beautiful flowers and numerous colors, and is one of the most consumed flowers in the cut flower market.

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

[0004] Rose axillary buds primarily develop in the leaf axils and grow into flowering branches. They are crucial for the growth and morphological formation of roses and are one of the most important propagation materials. In cut rose production, the speed of axillary bud germination directly determines its survival rate; during growth and development, 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 paramount importance for cut rose breeding and for improving the quality and efficiency of the industry. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides an application of the rose gene RhMYB1 in inhibiting the germination of rose axillary buds.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: Firstly, this application provides the application of the rose gene RhMYB1 in regulating the germination of rose axillary buds. Secondly, this application provides the application of the protein encoded by the rose gene RhMYB1, consisting of the amino acid sequence shown in SEQ ID NO:3, in regulating the germination of rose axillary buds. Thirdly, this application provides a kit for inhibiting or promoting the expression of the rose gene RhMYB1. Fourthly, this application provides the application of a nucleic acid construct in regulating the germination of rose axillary buds. Fifthly, this application provides the application of an engineered bacterium in regulating the germination of rose axillary buds. Sixthly, this application provides a method for cultivating improved new rose varieties.

[0007] The first aspect of this application provides an application of the rose gene RhMYB1 in regulating the germination of rose axillary buds, wherein the rose gene RhMYB1 comprises:

[0008] (a) The nucleotide sequence shown in SEQ ID NO:1; or

[0009] (b) A sequence having ≥90% identity and function with SEQ ID NO:1; the regulation is achieved by inhibiting or promoting the expression of the rose gene RhMYB1.

[0010] Furthermore, the germination of axillary buds of roses was promoted by overexpressing the rose gene RhMYB1, and the overexpression vector was the Super1300-RhMYB1 transient expression vector.

[0011] Furthermore, axillary bud germination was inhibited by silencing RhMYB1, wherein the silencing target fragment is the sequence shown in SEQ ID NO:2, and the fragment is complementary to RhMYB1 mRNA.

[0012] The second aspect of this application provides the application of the protein encoded by the rose gene RhMYB1, which consists of the amino acid sequence shown in SEQ ID NO:3, in regulating the germination of rose axillary buds.

[0013] A third aspect of this application provides a kit for inhibiting or promoting the expression of the rose gene RhMYB1, the kit comprising at least:

[0014] (a) RT-qPCR primer pair capable of amplifying the SEQ ID NO:1 fragment;

[0015] (b) Negative control primers;

[0016] (c) Reverse transcription and quantitative PCR reaction solution.

[0017] A fourth aspect of this application provides an application of a nucleic acid construct in regulating axillary bud germination in roses, the nucleic acid construct comprising:

[0018] (a) Silencing construct: Insertion of the target fragment SEQ ID NO:2 into the multiple cloning site of the pTRV2 viral vector; or

[0019] (b) Overexpression construct: The complete ORF of SEQ ID NO:1 was inserted downstream of the 35S promoter of the Super1300 plant expression vector; and the construct could reduce or increase RhMYB1 expression after transformation with Agrobacterium.

[0020] The fifth aspect of this application provides the application of engineered bacteria in regulating the germination of axillary buds in roses, wherein the engineered bacteria contain nucleic acid constructs.

[0021] The sixth aspect of this application provides a method for cultivating improved new rose varieties, comprising: (a) constructing a nucleic acid construct; and (b) transforming the construct into rose axillary bud explants.

[0022] Beneficial effects: This invention is the first to propose the application of the rose gene RhMYB1 in inhibiting the germination of rose axillary buds. Transient silencing of the RhMYB1 gene in axillary buds showed that silencing the RhMYB1 gene significantly inhibited the germination and growth of axillary buds, while overexpression of RhMYB1 promoted the growth of axillary buds.

[0023] Compared with the prior art, the present invention has the following advantages: (1) Based on the expression results of RhMYB1 in different buds of rose branches, the present invention transiently silences RhMYB1 in axillary buds and finds that silencing RhMYB1 can inhibit the germination and growth of axillary buds, while overexpression of RhMYB1 can promote the growth of axillary buds. (2) The present invention identifies a candidate gene for axillary bud germination, RhMYB1, from the genome of the rose variety 'Samantha'. The analysis of the expression of RhMYB1 in different buds of rose branches shows that the expression level of RhMYB1 is high in the upper axillary buds and low in the middle and basal axillary buds. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a graph showing the expression levels of RhMYB1 in axillary buds at different nodes in this invention; the data are shown as mean ± SD (n=3) (*** indicates P ≤ 0.001, **** indicates P ≤ 0.0001), which are upper, middle, and basal axillary buds, respectively.

[0026] Figure 2 The phenotypic observation results of axillary bud germination and growth after transiently silencing RhMYB1 for 10 days in this invention; Note: 10 days represents the growth of silent axillary buds of TRV2 and TRV2-RhMYB1 on the 10th day after infection.

[0027] Figure 3 These are anatomical diagrams of axillary bud growth at different time points after 10 days of instantaneous silencing of RhMYB1 according to the present invention; Note: 10 days represents the anatomical observation of the growth points of TRV2 and TRV2-RhMYB1-silenced axillary buds on the 10th day after infection.

[0028] Figure 4A is a statistical graph showing the gene expression level and axillary bud length measurement after transient silencing of RhMYB1 according to the present invention; B is a graph showing the relative expression level of RhMYB1 after silencing provided by the present invention using RT-qPCR analysis; C is a graph showing the bud length of TRV2 in the control group and TRV2-RhMYB1 in the treatment group 10 days after silencing provided by the present invention (* indicates P≤0.05, *** indicates P≤0.001).

[0029] Figure 5 The phenotypic observation results of axillary bud germination and growth after transient overexpression of RhMYB1 for 10 days in this invention; Note: 10d represents the growth of axillary buds overexpressed by Super1300 and Super1300-RhMYB1 on the 10th day after infection.

[0030] Figure 6 These are anatomical diagrams of axillary bud growth at different time points after transient overexpression of RhMYB1 for 10 days according to the present invention; Note: 10d represents the anatomical observation of the axillary bud growth points of Super1300 and Super1300-RhMYB1 overexpression on the 10th day after infection.

[0031] Figure 7 A is a statistical graph showing the gene expression level and axillary bud length measurement after transient overexpression of RhMYB1 in this invention; B is a graph showing the relative expression level of RhMYB1 after overexpression in this invention using RT-qPCR; C is the length of the Super1300 and Super1300-RhMYB1 buds in the control group 10 days after overexpression in this invention (* indicates P≤0.05, *** indicates P≤0.001). Detailed Implementation

[0032] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] First aspect of the embodiments of this application

[0034] The first aspect of this application provides an application of the rose gene RhMYB1 in regulating the germination of rose axillary buds, wherein the rose gene RhMYB1 comprises:

[0035] (a) The nucleotide sequence shown in SEQ ID NO:1; or

[0036] (b) A sequence having ≥90% identity and function with SEQ ID NO:1; the regulation is achieved by inhibiting or promoting the expression of the rose gene RhMYB1.

[0037] In some embodiments, rose axillary bud germination is promoted by overexpressing the rose gene RhMYB1, and the overexpression vector is the Super1300-RhMYB1 transient expression vector.

[0038] In some embodiments, axillary bud germination is inhibited by silencing RhMYB1, with the silencing target fragment being the sequence shown in SEQ ID NO:2, and the fragment being complementary to RhMYB1 mRNA.

[0039] The second aspect of this application provides the application of the protein encoded by the rose gene RhMYB1, which consists of the amino acid sequence shown in SEQ ID NO:3, in regulating the germination of rose axillary buds.

[0040] A third aspect of this application provides a kit for inhibiting or promoting the expression of the rose gene RhMYB1, the kit comprising at least:

[0041] (a) RT-qPCR primer pair capable of amplifying the SEQ ID NO:1 fragment;

[0042] (b) Negative control primers;

[0043] (c) Reverse transcription and quantitative PCR reaction solution.

[0044] A fourth aspect of this application provides an application of a nucleic acid construct in regulating the germination of axillary buds in roses, the nucleic acid construct comprising:

[0045] (a) Silencing construct: Insertion of the target fragment SEQ ID NO:2 into the multiple cloning site of the pTRV2 viral vector; or

[0046] (b) Overexpression construct: The complete ORF of SEQ ID NO:1 was inserted downstream of the 35S promoter of the Super1300 plant expression vector; and the construct could reduce or increase RhMYB1 expression after transformation with Agrobacterium.

[0047] The fifth aspect of this application provides the application of engineered bacteria in regulating the germination of axillary buds in roses, wherein the engineered bacteria contain nucleic acid constructs.

[0048] The sixth aspect of this application provides a method for cultivating improved new rose varieties, comprising: (a) constructing a nucleic acid construct; and (b) transforming the construct into rose axillary bud explants. Example 1

[0049] The present invention relates to the application of the rose gene RhMYB1 in regulating the germination of rose axillary buds. The rose gene RhMYB1 comprises:

[0050] (a) The nucleotide sequence shown in SEQ ID NO:1; or

[0051] (b) A sequence having ≥90% identity and function with SEQ ID NO:1; the regulation is achieved by inhibiting or promoting the expression of the rose gene RhMYB1.

[0052] The germination of axillary buds of roses was promoted by overexpressing the rose gene RhMYB1, and the overexpression vector was the Super1300-RhMYB1 transient expression vector.

[0053] Axillary bud germination is inhibited by silencing RhMYB1, wherein the silencing target fragment is the sequence shown in SEQ ID NO:2, and the fragment is complementary to RhMYB1 mRNA. Example 2

[0054] The present invention relates to the application of a protein encoded by the rose gene RhMYB1, which consists of the amino acid sequence shown in SEQ ID NO:3, in regulating the germination of rose axillary buds.

[0055] A third aspect of this application provides a kit for inhibiting or promoting the expression of the rose gene RhMYB1, the kit comprising at least:

[0056] (a) RT-qPCR primer pair capable of amplifying the SEQ ID NO:1 fragment;

[0057] (b) Negative control primers;

[0058] (c) Reverse transcription and quantitative PCR reaction solution. Example 3

[0059] The present invention discloses the application of a nucleic acid construct in regulating the germination of axillary buds of roses. The nucleic acid construct comprises: (a) a silencing construct: inserting the target fragment SEQ ID NO:2 into the multiple cloning site of the pTRV2 viral vector; or (b) an overexpression construct: inserting the complete ORF SEQ ID NO:1 downstream of the 35S promoter of the Super1300 plant expression vector; and the construct can reduce or increase RhMYB1 expression after transformation with Agrobacterium. Example 4

[0060] The present invention relates to the application of an engineered bacterium in regulating the germination of axillary buds of roses, wherein the engineered bacterium contains a nucleic acid construct. Example 5

[0061] The present invention provides a method for cultivating and improving new rose varieties, comprising: (a) constructing a nucleic acid construct; and (b) transforming the construct into rose axillary bud explants. Example 6

[0062] This application, based on the genome sequence of the rose variety 'Samantha', previously involved transcriptome sequencing of axillary buds at different time points after pruning, and screening for the significantly differentially expressed gene RhMYB1 associated with rose axillary bud germination. In axillary buds, RhMYB1 expression was high in upper axillary buds and low in basal and middle axillary buds. Transient silencing of RhMYB1 inhibited axillary bud germination, while overexpression promoted it. Therefore, this application reveals the biological function of the rose gene RhMYB1 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 experimental example provides the nucleotide sequence of the rose gene RhMYB1 as 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]

[0067] 1. Plant materials

[0068] 1.1 "Pink Snow Mountain"

[0069] "Pink Snow Mountain" is a modern cut rose variety. It was taken from the Baofeng Base of the Flower Research Institute of Yunnan Academy of Agricultural Sciences. Flower buds that had not yet shown color were selected and brought back to the laboratory. Stem segments with single buds were used as materials for rose axillary bud germination experiments.

[0070] 1.2 Strains and Vectors

[0071] Escherichia coli DH5α and Agrobacterium tumefaciens strains EHA105, GV3101, and pSuper-1300 (Kan resistant) were all purchased from Beijing Qingke Biotechnology Co., Ltd. The VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0072] 1.3 Culture medium formulations involved in the case study

[0073] (1) LB medium and YEB medium (Table 2)

[0074] Table 2

[0075]

[0076] (2) Experimental culture media (Table 3)

[0077] Table 3

[0078]

[0079] 2. Research Methods

[0080] 2.1 Extraction of total RNA

[0081] 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).

[0082] 2.2 cDNA Synthesis

[0083] (1) Genomic DNA removal reaction system (Table 4)

[0084] Table 4

[0085]

[0086] Mix well, incubate at 42°C for 2 minutes.

[0087] (2) Preparation of reverse transcription reaction system (Table 5)

[0088] Table 5

[0089]

[0090] Mix thoroughly by blowing and heating at 37°C for 15 min; then at 85°C for 5 s. Store the product at -20°C.

[0091] 2.3 Real-time quantitative PCR

[0092] RT-qPCR-specific primers for the gene were designed using Primer Premier5 (Table 6). The cDNA was diluted four-fold with ddH2O, and the cDNA obtained from reverse transcription was used as a template for RT-qPCR amplification. RhUBI2 was used as an internal control, and three biological replicates were set up.

[0093] Table 6

[0094]

[0095] The RT-qPCR reaction system is shown in Table 7:

[0096] Table 7

[0097]

[0098] The RT-qPCR reaction procedure is shown in Table 8:

[0099] Table 8

[0100]

[0101] 2.4 Carrier Construction

[0102] 2.4.1 PCR amplification of the target gene fragment

[0103] The target gene was amplified by PCR using a high-fidelity enzyme (Phusion™ Plus PCR Master Mix). The PCR amplification reaction system is shown in Table 9.

[0104] Table 9

[0105]

[0106] The PCR amplification reaction procedure is shown in Table 10:

[0107] Table 10

[0108]

[0109] After amplification, gel electrophoresis was performed to detect the target band and then the gel was recovered.

[0110] 2.4.2 Glue Recycling

[0111] The PCR products were recovered and purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0.

[0112] 2.4.3 Vector double enzyme digestion

[0113] Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-digested with the corresponding enzymes. The double-digestion system is shown in Table 11:

[0114] Table 11

[0115]

[0116] After adding the enzyme on ice, perform double digestion of the vector according to the enzyme's thermal denaturation temperature.

[0117] 2.4.4 Homologous recombination

[0118] The double-digested vector was then subjected to homologous recombination with the cloned target gene fragment to construct the vector. The homologous recombination system is shown in Table 12:

[0119] Table 12

[0120]

[0121] Incubate at 50°C for 15 min in a PCR instrument.

[0122] 2.4.5 Escherichia coli transformation

[0123] (1) Remove the competent states from -80℃ and melt them on ice;

[0124] (2) Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of DH5α competent cells, mix by pipetting, and let stand on ice for 30 min;

[0125] (3) Heat shock at 42℃ for 90 s, then quickly transfer to ice and let stand for 2 min;

[0126] (4) Add 500 μL of LB liquid culture medium to the centrifuge tube, incubate at 37°C and 200 rpm for 1 h;

[0127] (5) After the culture is completed, centrifuge at 5000 rpm for 5 min;

[0128] (6) After discarding 400 μL of supernatant in a clean bench, mix the mixture by pipetting and spread it on LB solid medium containing 50 mg / L Kan, and incubate overnight at 37°C with the medium inverted.

[0129] 2.4.6 Microbial detection and sequencing

[0130] (1) Shaking culture: Pick single colonies that have grown overnight and shake them in 500 μL of LB medium containing 50 mg / L Kan for 3-4 h (37℃, 200 rpm).

[0131] (2) Bacterial culture PCR: Using bacterial culture as a template, PCR amplification was performed to detect whether the target band of the constructed vector met expectations. The bacterial culture PCR amplification system is shown in Table 13:

[0132] Table 13

[0133]

[0134] The bacterial culture PCR amplification reaction procedure is shown in Table 14:

[0135] Table 14

[0136]

[0137] The amplified products were detected by gel electrophoresis, and those with band sizes that met expectations were sent to the company for sequencing.

[0138] 2.5 Plasmid Extraction

[0139] After the test results are returned, sequence alignment is performed, positive bacterial cultures are selected for inoculum culture, and plasmids are extracted using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0140] 2.6 Agrobacterium-mediated transformation

[0141] (1) When the competent state of the straw is taken out of the -80℃ freezer and melted into an ice-water mixture, it is inserted into ice;

[0142] (2) Add 0.01-1 μg plasmid DNA to each 100 μL competent cells, mix well and then incubate on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37℃ for 5 min, and in an ice bath for 5 min.

[0143] (3) Add 500 μL of antibiotic-free YEB liquid medium and incubate at 28°C with shaking for 2-3 h;

[0144] (4) Centrifuge at 5000 rpm for 2 min, discard 400 μL of supernatant in a clean bench, spread it onto YEB solid medium containing 50-72 mg / L Kan, and incubate upside down at 28℃ for 2-3 days.

[0145] 2.7 Momentary Silence

[0146] (1) Vector primer design and vector construction

[0147] Using EcoRI and Xho1 as restriction sites, the silent fragment of MYB1 (SEQ ID NO: 2) was inserted into the pTRV2 empty vector. Primers were designed using homologous recombination to construct the TRV2-MYB1 vector.

[0148] (2) Bacterial culture

[0149] Streaking the bacterial culture onto YEB plates containing 50 mg / L Kan and 50 mg / L Rif, and incubating upside down at 28°C for 2-3 days. Single bacteria were picked for culture PCR; positive bacteria were subjected to medium shaking, while large shaking was used for subsequent experiments.

[0150] (3) Collection and resuspension of bacteria

[0151] 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 OD600 to 1.0. For transient silencing experiments, mix TRV1 with TRV2 and TRV2-MYB1 bacterial suspensions at a volume ratio of 1:1, and incubate in the dark for 4-6 h.

[0152] (4) Vacuum suction

[0153] The stem segments with single buds were infected by suction using a vacuum pump at 0.082 MPa for 10 min, followed by holding the pressure for 10 min and releasing the pressure for 10 min, ensuring the entire stem segment was submerged in the bacterial solution. This treatment was repeated three times. After infection, the segments were rinsed with sterile water and incubated in an 8°C incubator for 3 days before being planted. Observations were conducted every two days, and samples were taken, photographed, and recorded. Axillary buds were observed under a stereomicroscope in longitudinal sections.

[0154] 2.8 Transient overexpression

[0155] (1) Vector primer design and vector construction

[0156] Using Sam1 and Kpn1 as restriction sites, the overexpression fragment of RhMYB1 was inserted into the empty pSuper1300 vector. Primers were designed using homologous recombination to construct the Super1300-RhMYB1 vector.

[0157] (2) Preparation of bacterial culture

[0158] Prepare Agrobacterium bacterial suspensions of pSuper1300 and pSuper1300-RhMYB1. The bacterial collection method is the same as the transient silencing method in section 2.7 of this invention. After the bacterial suspension is prepared, it is placed at 28°C and shaken at 200 rpm for 45 min.

[0159] (3) Vacuum suction

[0160] The method is the same as the transient silencing method in section 2.7 of this invention. After infection, the bacterial solution on the surface of the stem segments is gently rinsed with sterile water, and then the segments are propagated in a culture room. Phenotypic observations are performed every two days, and samples are taken, photographed, and the axillary buds are observed under a stereomicroscope in longitudinal sections.

[0161] 2.9 Primers used in the RhMYB1 sequence experiment (Table 15)

[0162] Table 15

[0163] Example 7

[0164] Analysis of RhMYB1 expression levels in different active buds

[0165] This application used real-time quantitative PCR to analyze the expression level of RhMYB1 in axillary buds at different nodes of the cut rose 'Pink Snow Mountain' (before the flower buds show white). The results showed that the expression level of RhMYB1 was high in the upper axillary buds. Figure 1 Therefore, this application hypothesizes that RhMYB1 is closely related to the germination of axillary buds. Example 8

[0166] RhMYB1 regulates axillary bud germination in roses.

[0167] Based on the expression analysis of RhMYB1 in buds at different sites, this application selected a specific segment of the RhMYB1 sequence (SEQ ID NO: 2) and constructed a transient silencing vector, TRV2-RhMYB1, using homologous recombination. TRV1, TRV2, and TRV2-RhMYB1 were transformed into Agrobacterium EHA105 and used to infect axillary bud stem segments to silence RhMYB1. Phenotypic observation and recording were performed 10 days after infection. Figure 2 From the bud phenotype, compared with the TRV2 control, the germination of axillary buds in TRV2-RhMYB1 was inhibited. Anatomical results of the buds also showed that bud development was hindered after RhMYB1 silencing. Figure 3 To confirm the silencing effect, total RNA was extracted from the axillary buds of TRV2 in the control group and TRV2-RhMYB1 in the experimental group, and RT-qPCR analysis was performed. The results showed that RhMYB1 expression was significantly reduced after silencing, indicating that silencing was effective. Figure 4 (A). In addition, shoot length measurements were performed on the control group TRV2 and the experimental group TRV2-RhMYB1, and it was found that the shoot length on day 10 after RhMYB1 gene silencing was significantly lower than that of the control group ( ). Figure 4 (B)

[0168] Based on the expression analysis results of RhMYB1 in buds at different sites, this application selected the CDs sequence of RhMYB1 and constructed the Super1300-RhMYB1 transient overexpression vector using homologous recombination. The Super1300 empty vector and Super1300-RhMYB1 were transformed into Agrobacterium EHA105 and used for infection of the central axillary bud stem segment to overexpress RhMYB1. Phenotypic observation and recording were performed 10 days after infection. Figure 5 From the phenotype of the buds, compared with the Super1300 control group, the Super1300-RhMYB1 buds germinated faster. Anatomical results of the buds also showed that RhMYB1 overexpression accelerated bud development. Figure 6 To confirm the overexpression effect, total RNA was extracted from the Super1300 control group and the Super1300-RhMYB1 overexpression buds, and RT-qPCR analysis was performed. The results showed a significant increase in RhMYB1 expression after overexpression, indicating that the overexpression was effective. Figure 7 (A). In addition, shoot length was measured in both the control group (Super1300) and the experimental group (Super1300-RhMYB1). It was found that shoot length on day 10 after RhMYB1 overexpression was significantly higher than that in the control group (A). Figure 7 (B)

[0169] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. The use of a Rosa chinensis gene RhMYB1 in promoting the germination of Rosa chinensis axillary buds, characterized in that: The nucleotide sequence of the rose gene RhMYB1 is shown in SEQ ID NO:1; the promotion is achieved by overexpressing the rose gene RhMYB1.

2. Use according to claim 1, characterized in that: The overexpression was achieved by constructing the rose gene RhMYB1 into an overexpression vector and then transforming it into roses.

3. The application according to claim 2, characterized in that: The overexpression vector is the Super1300 plant expression vector.

4. The application of a protein encoded by the rose gene RhMYB1, consisting of the amino acid sequence shown in SEQ ID NO:3, in promoting the germination of axillary buds in roses, characterized in that: The content or activity of the protein is increased.

5. The application of a nucleic acid construct in promoting the germination of axillary buds in roses, characterized in that: The nucleic acid construct is an overexpression construct, wherein the overexpression construct is formed by inserting the rose gene RhMYB1 shown in SEQ ID NO:1 downstream of the 35S promoter of the Super1300 plant expression vector; and the nucleic acid construct can enhance the expression of RhMYB1 after transformation with Agrobacterium.

6. The application of an engineered bacterium in promoting the germination of axillary buds in roses, characterized in that: The engineered bacteria contain the nucleic acid construct described in claim 5.

7. A method for cultivating new rose varieties with improved axillary bud germination characteristics, characterized in that: include: (a) Constructing the nucleic acid construct as described in claim 5; (b) Transform the nucleic acid construct into rose axillary bud explants; (c) Obtain rose strains with improved axillary bud germination characteristics.

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