CN118703557B

CN118703557B - New application of China rose auxin oxidase gene RhDAO1

Info

Publication number: CN118703557B
Application number: CN202411061928.0A
Authority: CN
Inventors: 陈雯; 崔永一; 沈元吉; 罗平
Original assignee: Jiyang College of Zhejiang A&F University
Current assignee: Jiyang College of Zhejiang A&F University
Priority date: 2024-08-05
Filing date: 2024-08-05
Publication date: 2024-12-03
Anticipated expiration: 2044-08-05
Also published as: CN118703557A

Abstract

The invention belongs to the field of plant genetic engineering, relates to application of a China rose auxin oxidase gene RhDAO, is used for regulating and controlling the number of petals of China rose, provides a new means for regulating and controlling the number of petals of half-and-full-petal China rose, and provides a theoretical basis for cultivating new varieties of China rose.

Description

New application of China rose auxin oxidase gene RhDAO1

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a new application of a China rose auxin oxidase gene RhDAO.

Background

China rose belongs to Rosaceae, is praised as queen in flowers, has a cultivation history of thousands of years, and creates about forty thousands of gardening varieties. These varieties are classified into hybrid tea rose, rose of strong flowers, rose of micro-rose and rose of vine.

The China rose has strong adaptability, does not need special treatment, and can bloom for many times in one year. Therefore, the China rose is widely applied, can be planted in flower beds, flower scenes, lawn corners and the like, and can also be arranged into China rose gardens. The vine rose is used in flower shelf, flower wall, flower fence, flower door, etc. China rose can be cultivated in a pot for ornamental, and is also an important cut flower material. The roots, leaves and flowers of China rose can be used for medicine, and have the effects of activating blood, removing toxicity and relieving swelling, and the fragrant varieties can also be used for extracting essence and eating.

The China rose has indispensable value in landscaping, and is the flower with the largest use times in the south-north gardens and viaduct isolation zones. China rose is a main ornamental flower in spring, and has long flowering period, high ornamental value and low price, and is favored by gardens in various places. Therefore, the development of new rose varieties is always the direction of research in the fields of landscape architecture and ornamental gardening.

The China rose flower can be divided into single petals, half-heavy petals and heavy petals according to the number of the petals of the China rose, and the single petals generally comprise only 5 or 6 petals. The half-and double-petal China rose refers to China rose with multiple layers (more than two layers) of petals. Wherein, the half-double petals generally have about 12 petals, and the petals of the double petals can be up to 20 petals and more. Flowers of the rose with heavy petals have better layering property and higher ornamental value, so the rose with heavy petals is always favored.

However, the current China rose varieties mainly depend on wild resource domestication breeding and conventional hybridization breeding, the consumed period is long, and how to improve ornamental properties by means of modern molecular biotechnology and efficiently cultivate new varieties becomes a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide application of a China rose auxin oxidase gene RhDAO in China rose petal number regulation, and provides a new means for regulating the petal number of a breeding target by using half-and-double-petal China rose and a theoretical basis for cultivating new varieties of China rose.

The invention provides the following technical scheme:

In a first aspect, the present invention provides a novel use of the rose auxin oxidase gene RhDAO1 for regulating the number of petals of rose.

In a second aspect, the present invention provides a novel use of the rose auxin oxidase gene RhDAO1 for controlling the number of stamens of rose.

In a third aspect, the present invention provides a novel use of rose auxin oxidase gene RhDAO1 for improving rose flower type traits.

Preferably, the improvement of the flower shape is an improvement in which the number of petals is increased and the number of layers of petals is increased.

In a fourth aspect, the present invention provides a novel use of a rose auxin oxidase gene RhDAO1 for cultivation of a new rose variety, which is a new variety with an increased number of petals or a decreased number of petals.

Preferably, the amino acid sequence of the protein encoded by the China rose auxin oxidase gene RhDAO is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the China rose auxin oxidase gene RhDAO is shown as SEQ ID NO. 2.

Compared with the prior art, the invention has the following beneficial effects:

The invention separates the rose auxin oxidase gene RhDAO from the rose, researches the improvement of the rose flower heavy petal character, provides a theoretical basis for the cultivation of new rose varieties and opens up a new direction.

Drawings

FIG. 1 is a conserved domain of RhDAO;

FIG. 2 shows RhDAO protein properties and secondary structure;

FIG. 3 is a three-dimensional structural model of RhDAO protein;

FIG. 4 shows a sequence alignment analysis of China rose RhDAO and its homologous proteins (note: blue line represents the 2OG-FeII_oxy feature region; open circle represents Fe binding residue; filled circle represents 2OG binding residue; long circle labeled with alpha letter represents alpha helix; short circle labeled with mu letter is 3 ₁₀ helix; arrow labeled with beta letter represents beta turn; TT letter represents strict beta turn; feature motifs 5 and 10 are represented by black boxes.);

FIG. 5 is a phylogenetic tree analysis of China rose RhDAO and its homologous proteins;

FIG. 6 shows the results of promoter homeotropic elements analysis of RhDAO and homologous genes thereof;

FIG. 7 shows quantitative qRT-PCR analysis results of RhDAO genes, wherein (A) shows RhDAO expression analysis results in flower buds of China rose variety 'Airiley' and 'Jie Lvzhen' in different development periods (note: grades 2 to 4 respectively show flower buds of petal primordium differentiation period, flower buds of stamen primordium differentiation period and flower buds of pistil primordium differentiation period), (B) shows RhDAO expression analysis results in different organs of China rose variety 'Airiley', and (C) shows auxin response gene RhGH3.3 (left graph) and RhDAO gene (right graph) expression analysis results (note: biological repetition (n=3), asterisks represent different significance levels (independent sample T test, P <0.05, P < 0.01)) after exogenous IAA treatment of China rose variety 'Airiley' flower discs;

FIG. 8 shows results of a RhDAO-1 subcellular localization analysis, wherein (A) is a plot of the co-localization results of RhDAO-1 with nuclear, cytoplasmic, and endoplasmic reticulum marker proteins in epidermal cells of tobacco leaves, and (B) is a Pelson correlation coefficient analysis of 2 fluorescence intensities in (A) (note: scale represents 20. Mu.M; "a.u." -arbitrary units; R-Pelson correlation coefficient);

FIG. 9 shows the results of an analysis of the dimer formation of RhDAO proteins by interaction, wherein (A) is the result of transformation verification of RhDAO and RhDAO1 in yeast Y2HGold, (B) is a fluorescence image of Agrobacterium GV 3101-mediated bimolecular fluorescence complementation assay (bimolecular fluorescence complementation, biFC) for detection of RhDAO1-nYFP and RhDAO1-cYFP interactions in tobacco leaf epidermal cells (note: scale represents 20. Mu.M);

FIG. 10 is a RhDAO1 expression analysis in RhDAO1 silenced plant petals;

FIG. 11 is a comparison of TRV and TRV-RhDAO1 petals, stamens and total;

FIG. 12 is a comparison of TRV and TRV-RhDAO1 normal petals, petal stamens;

FIG. 13 is a flower overall and anatomic map of TRV and TRV-RhDAO1 (note (A) biological repeat (n.gtoreq.5); (B) and (C) biological repeat (n.gtoreq.11); asterisks represent significant levels of difference (independent sample T test, <0.05, <0.01, <0.001,; scale bar represents 1 cm);

Fig. 14 is an analysis of expression of genes associated with the bivalve or auxin pathway in petals of RhDAO silencing plants (note: biological replicates (n=5); asterisks represent significant levels of difference (independent sample T-test, <0.05, < 0.01).

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1RhDAO Gene cloning and analysis of physiological and Biochemical Properties

1. Materials and methods

1. Plant material

1.1, A green miniature China rose variety 'Aiklier' and a heavy flap bud-changing variety 'Jieqingzhen' cutting seedling

The China rose variety 'JieLzhen' is a double-petal bud-changing variety of China rose variety 'Aiklier', and the main difference between the two flowers is that the number of petals of the 'JieLzhen' is obviously more than that of the 'Aiklier'. Cutting seedlings of 'Aiklier' and 'JieLvzhen' are planted in an 8cm black square containing peat, vermiculite and perlite (1:1:1). Incubator temperature 21+ -1deg.C, humidity 56%, illumination 40000Lux, photoperiod 16/8 (light/dark, h).

1.2 Wild Nicotiana benthamiana

Wild type nicotiana benthamiana (Nicotiana benthamiana) seeds were supplied by the laboratory. After mixing seeds and vermiculite, spreading the mixture on the surface of a wet clear clay carbon substrate, covering the surface with a preservative film, and placing the mixture in an incubator with the temperature of 21+/-1 ℃ and the humidity of 56 percent, the illumination of 40000Lux and the photoperiod of 16/8 (light/dark, h) for cultivation. When 3-4 true leaves grow out of the tobacco, the preservative film is uncovered, the young seedlings are transplanted into an 8cm black square containing peat, vermiculite and perlite (1:1:1) moist matrixes for growth, and the young seedlings are used for gene transient expression before the buds of the tobacco appear.

2. Extraction of China rose genome DNA

The method for extracting the China rose genome DNA refers to a plant genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., ltd.) for use.

1) And (3) taking down the rose sample less than 200mg, loading the rose sample into a 2mL grinding tube, adding grinding beads, putting the grinding beads into a grinding machine, grinding at 60Hz for 1min, repeating the grinding operation once, and temporarily storing the grinding operation in an ultralow temperature refrigerator.

2) In a fume hood, 700. Mu.L of buffer GP1,0.1% beta-mercaptoethanol was prepared for each sample and then pre-heated at 65 ℃.

3) Adding the preheated mixed solution into a pipe, mixing by vortex for 1min, putting into a 65 ℃ water bath for 20min, and mixing for 4 times in a midway way to help cracking.

4) 700. Mu.L of chloroform was added to the fume hood, and the mixture was thoroughly mixed and centrifuged at 12000rpm for 5 minutes.

5) After transferring the upper aqueous phase to a 2mL centrifuge tube, 700. Mu.L of buffer GP2 was added, and after thoroughly mixing, the mixture was added to an adsorption column CB3, centrifuged at 12000rpm for 30sec, and the filtrate was discarded.

6) Mu.L of buffer GD was added, centrifuged at 12000rpm for 30sec, and the filtrate was discarded and repeated once.

7) Placing the adsorption column CB3 back into a collecting pipe, centrifuging at 12000rpm for 2min, discarding the filtrate, and standing for 5min after uncapping.

8) Placing the adsorption column CB3 into a 1.5mL centrifuge tube, adding 50 mu L of elution buffer TE, standing at room temperature for 5min, centrifuging at 12000rpm for 2min, repeating the steps once, combining the two filtrates, and storing in an ultralow temperature refrigerator.

3. Total RNA extraction from China rose

The extraction method refers to the use instruction of a polysaccharide polyphenol/complex plant RNA rapid extraction kit (Nanjing Borez biotechnology Co., ltd.).

1) Taking down the rose sample less than 200mg, loading into a 2mL grinding tube without RNase, adding grinding beads, putting into a grinder, grinding at 60Hz for 1min, repeating the grinding process once, and temporarily storing in an ultralow temperature refrigerator.

2) The cracking liquid CLB is placed in a 65 ℃ water bath kettle for preheating, and then 1mL of cracking liquid CLB,2%PVP40000,5% beta-mercaptoethanol of each sample is used for preparing mixed liquid, and the mixed liquid is inverted, uniformly mixed and preheated. While pre-cooling the low temperature centrifuge to 4 ℃ and then placing RNASE FREE WATER in a 90 ℃ water bath for preheating.

3) Adding the cracking mixed solution into a sample tube, immediately vortex-mixing for 1min, then placing into a 65 ℃ water bath kettle for 5-10min, and reversely mixing for 2 times midway to assist in cracking. Centrifuge at 13000rpm for 10min.

4) Carefully transfer the supernatant to a 1.5mL RNase-free centrifuge tube, add half of the volume of absolute ethanol, immediately pipette and mix, transfer to a genomic DNA removal column, centrifuge at 13000rpm for 2min, discard the filtrate.

5) The clearing column was placed in a 2mL RNase-free centrifuge tube, 500. Mu.L of lysate RLT Plus was added and centrifuged at 13000rpm for 30sec.

6) The filtrate was collected, 245. Mu.L of absolute ethanol was added, immediately sucked and stirred evenly, transferred to an RNA adsorption column, centrifuged at 13000rpm for 2min, and the filtrate was discarded.

7) Mu.L of deproteinized solution RW1 was put into an RNA adsorption column, left standing at room temperature for 1min, centrifuged at 13000rpm for 30sec, and the filtrate was discarded.

8) Mu.L of rinse RW was added to the RNA adsorption column, centrifuged at 13000rpm for 30sec, the filtrate was discarded and repeated.

9) The adsorption column was put back into the collection tube, centrifuged at 13000rpm for 2min, and uncapped for 5min.

10 Placing the RNA adsorption column into a 1.5mL RNase-free centrifuge tube, adding 30 mu L RNASE FREE WATER, standing at room temperature for 5min, and centrifuging at 12000rpm for 1min.

11 Collecting filtrate, adding into RNA adsorption column again, standing at room temperature for 5min, centrifuging at 12000rpm for 1min, and storing in ultra-low temperature refrigerator.

4. Synthesis of China rose cDNA

The cDNA synthesis method was described with reference to Evo M-MLV reverse transcription premix kit (Hunan Ai Kerui Bio-engineering Co., ltd.).

1) RNA concentration (ng/. Mu.L) was measured using a Simer-flying Nanodrop 2000 ultra-micro spectrophotometer and the volume of RNA solution required was calculated to be 1. Mu.g total RNA concentration per reverse transcription system.

2) On the ice box, the genomic DNA removal system was configured to 200. Mu.L of enzyme-free tube (Table 1).

TABLE 1 genomic DNA clearance System

Component name	System of
		GDNA scavenging reaction mixture	2μl
Total RNA	1000ng
		RNASE FREE Water	up to 10μl

3) The system was mixed and placed in a constant temperature metal bath heater at 42 ℃ for 2min.

4) On the ice box, a reverse transcription system was configured to the sample tube (table 2).

TABLE 2 reverse transcription reaction system

Component name	System of
		Step 2) reaction solution	10μl
5 XEvo M-MLVRT reaction mixture	4μL
		RNASE FREE Water	up to 20μl

5) The system was mixed and then reacted in a PCR instrument (Table 3).

Table 3 PCR reaction procedure

Temperature (temperature)	Time of	Cycle number
			37°C	15min	1
85°C	5sec	1
			4°C	hold

6) -20 ℃ Refrigerator preservation.

5. Fragment amplification and product recovery of interest

The method for amplifying the target fragment was described with reference to the 2X PHANTA FLASH MASTER Mix kit (Nanjinouzan Biotechnology Co., ltd.). The method for recovering and purifying the amplified product is referred to the instruction book of gel DNA recovery kit (Hangzhou New scenic reagent development Co., ltd.).

1) The fragment amplification system was placed on a 200. Mu.L centrifuge tube on an ice box (Table 4).

TABLE 4 target fragment amplification System

Component name	System of
		2×Phanta Flash Master Mix	12.5μl
cDNA	20ng
		Forward primer RhDAO-all-F (10 μm)	1μl
Reverse primer RhDAO-all-R (10 μm)	1μl
		SDW	up to 10μl

The RhDAO gene promoter fragment uses DNA as an amplification template, the RhDAO gene ORF fragment uses cDNA as an amplification template, and the primer sequences are shown in Table 5.

TABLE 5 amplification primers for RhDAO1 Gene promoter and RhDAO Gene ORF fragment

Primer name	Primer sequences
		Forward primer RhDAO-promter-F	5’-AAAAGTAGACGAGGAAGCCA-3’
Reverse primer RhDAO-promoter-R	5’-ATTGGACCAAGATAGAGAGCA-3’
		Forward primer RhDAO1-ORF-F	5’-ATGGCAAATCCAATTCCAGTGA-3’
Reverse primer RhDAO-ORF-F	5’-TTAGGACTTGATGCGCAGAAGTT-3’

2) The system was mixed and then reacted in a PCR instrument (Table 6).

Table 6 PCR reaction procedure

3) And (3) performing gel electrophoresis analysis on the PCR amplification product, and recovering the target band.

6. Analysis of the biological messages of RhDAO1 Gene

1) The RhDAO gene promoter sequence was amplified and homologous gene promoter sequences were obtained by NCBI database.

2) The cis-acting elements of the promoter sequence were analyzed by PlanCARE.

3) The cis-acting element was visualized by TB Tools-II.

4) The protein conserved domain was analyzed by NCBI Conserved Domain Search (nih.gov).

5) Protein physicochemical properties were analyzed by Expasy-protParam tool.

6) Protein type and transmembrane structure were analyzed by DTU/DEEPTMHMM-BioLib.

7) The protein secondary structure was analyzed by SOPMA secondary structure prediction (ibcp. Fr).

8) The protein tertiary structure was analyzed by alpha Fold2.Ipynb-Colaboratory (google. Com).

9) Protein motifs (Motif) were analyzed by MEME-examination form (MEME-suite. Org).

7. RhDAO1 phylogenetic tree and sequence alignment

1) Alignment sequences were screened by Protein BLAST search Protein databases using a Protein query (nih. Gov).

2) Sequence alignment was performed by MAFFT 7.52.

3) The predicted optimal model parameters, LG+G+I, are adopted by the MEGA11.0, and the maximum likelihood model (Maximum Likelihood model) is adopted, and the parameters are stepped 1000 times.

4) And (3) visualizing the protein Motif comparison result through TB Tools-II, and constructing a phylogenetic tree.

5) Sequence alignment was completed by visualizing Espript bound protein secondary structure.

8. 'Aicleer' flower disc auxin treatment

1) And selecting 'Aicleer' plants with similar growth vigor in the incubator. Taking middle petals of the flowers growing consistently, taking out the flower discs by using a puncher, and soaking the flower discs in IAA solution (1mM IAA,0.1%Tween-20) in a dark place.

2) And (3) after 0h, 1h, 6h, 12h and 24h of treatment, taking flower discs, respectively mixing the flower discs into 2mL grinding pipes, quick-freezing the flower discs by liquid nitrogen, and storing the flower discs in an ultralow temperature refrigerator.

3) Extracting RNA of the flower disc, and reversely transcribing the RNA into cDNA. Relevant gene expression was analyzed by qRT-PCR.

9. Real-time fluorescence quantitative PCR (qRT-PCR)

The qRT-PCR method is described with reference to BrightCycle Universal SYBR GREEN QPCR Mix with UDG kit (Botai Ke Biotechnology Co., ltd.) in use. The quantitative primer sequences were generated by primer design website PRIMERDESIGNING TOOL (nih. Gov). The rose RhUBI2 is used as an internal reference gene to calculate and analyze the relative expression quantity of the detection gene.

1) On the ice box, cDNA of the test sample is added to a 96 Kong Yingguang quantitative test plate in advance.

2) In the dark, the qRT-PCR reaction system was configured on an ice box, and the mixture was then added to a fluorescent quantitative assay plate (Table 7).

TABLE 7 qRT-PCR reaction System

Component name	System of
		BrightCycle Universal SYBR Green qPCR Mix with UDG	5 μl
cDNA	1 μl
		Forward primer (10 μm)	0.2 μl
Reverse primer (10 μm)	0.2 μl
		SDW	up to 10 μl

The primer sequences involved in qRT-PCR are shown in Table 8.

TABLE 8 qRT primer sequences involved in PCR

Primer name	Primer sequences
		RhUBI2-RT-qPCR-F	5’-GCCCTGGTGCGTTCCCAACTG-3’
RhUBI2-RT-qPCR-R	5’-CCTGCGTGTCTGTCCGCATTG-3’
		RhDAO1-RT-qPCR-F	5’-CCTGCTTCTGGTGAGTACGA-3’
RhDAO1-RT-qPCR-R	5’-TTGTGGTTGAGAAGCCGGAA-3’
		RhGH3.1-RT-qPCR-F	5’-GCCCTTCTTTACCGCATTGG-3’
RhGH3.1-RT-qPCR-R	5’-GACCACAAAGCCGTTGACTTTTC-3’
		RhARF18-RT-qPCR-F	5’-ACAAGACTGGTCCTTTGAGCA-3’
RhARF18-RT-qPCR-R	5’-AGTTCGGTATCCCATTTCTCCT-3’
		RhPILS1-RT-qPCR-F	5’-TTGCTTCGCTATCGCTTACG-3’
RhPILS1-RT-qPCR-R	5’-GTTGCTGTTTCGGTACTCAGG-3’
		RhIAA16-RT-qPCR-F	5’-CTGCTATGGGTGGTGGTGTTA-3’
RhIAA16-RT-qPCR-R	5’-CCTTGGTCGCCGTCTTTCA-3’
		RhAP2-RT-qPCR-F	5’-CCCAAGCCTAGAGAGGATAATG-3’
RhAP2-RT-qPCR-R	5’-GGGATGGAGATTGGAGGTTT-3’
		RhPID-RT-qPCR-F	5’-TGAGAAGGCAGAACACGACG-3’
RhPID-RT-qPCR-R	5’-CACCCGCCAGTGACAAAATC-3’

3) The qRT-PCR reaction system was placed in the Siemens StepOnePlus real-time fluorescence quantification system and the reaction procedure was set (Table 9).

TABLE 9 qRT-PCR reaction procedure

10. Vector construction

10.1 Sequencing vector construction

Vector construction method was described with reference to pClone007 Blunt Simple Vector kit (Beijing Optimu Corp.).

1) On the ice box, the vector construction system was configured to 200. Mu.L of enzyme-free tubes (Table 10).

TABLE 10 blunt end vector ligation system

Component name	System of
		pClone007 Blunt Simple Vector	1μl
10×Topo Mix	1μl
		Recovery of the product from the gum	100ng
SDW	up to 10μl

2) The system was mixed and placed in a constant temperature metal bath heater at 25℃for 5min.

3) The ligation product was transferred into E.coli DH 5. Alpha. Competence.

10.2 Construction of expression vectors

Constructing an expression vector by adopting a homologous recombination method. Homologous recombination methods refer to TreliefSoSoo Cloning Kit recombinant cloning kit (Beijing Optimu Biotech Co., ltd.) product instructions. The vector double cleavage method refers to the instructions for use of the restriction enzyme QuickCut kit (Bao Ri doctor materials technology (Beijing) Co., ltd.).

1) On an ice box, a carrier double enzyme digestion system was placed into a 200. Mu.L centrifuge tube (Table 11).

Table 11 double enzyme digestion system

Component name	System of
		10 Xfast-cutting enzyme buffer	5μl
DNA	1μg
		Fast cutting enzyme A	1μl
Fast cutting enzyme B	1μl
		SDW	up to 50μl

2) The system was mixed and placed in a constant temperature metal bath heater at 37 ℃ for 30min.

3) Comparing the sizes of the fragments after agarose gel electrophoresis, cutting correct bands, and recovering the gel.

4) On ice box, the homologous recombination system was placed into a 200. Mu.L centrifuge tube (Table 12).

TABLE 12 homologous recombination System

Component name	System of
		2×SoSoo Mix	5μl
Linearization carrier	XμL
		Insertion fragment	YμL
SDW	up to 10μl

Note that the carrier is used in an amount of 10-100ng. The molar ratio of carrier X to insert Y solution is 1:2 to 1:10.

5) The system was mixed and placed in a constant temperature metal bath heater at 50 ℃ for 15min.

6) The ligation product was transferred into E.coli DH 5. Alpha. Competence.

11. Transformation of E.coli competence by heat shock method

Coli transformation methods were described in the specification of DH 5. Alpha. CHEMICALLY COMPETENT CELL (Shanghai Biotechnology Co., ltd.). Plasmid extraction methods refer to the rapid plasmid DNA miniprep kit (Hangzhou New Biochemical reagent development Co., ltd.). Transferring the vector into escherichia coli DH5 alpha, determining positive cloning, transferring to Zhejiang Shangya biotechnology Co., ltd, sequencing to determine insert, preserving bacterial liquid, extracting plasmid and preserving.

12. Transformation of Agrobacterium by Heat shock

Agrobacterium transformation methods refer to GV3101 CHEMICALLY COMPETENT CELL (Shanghai Biotechnology Co., ltd.).

13. Subcellular localization of leaf epidermis of Nicotiana benthamiana

In order to observe RhDAO < 1 > localization conditions in the tobacco epidermal cells, a GFP-carrying overexpression vector is constructed by adopting a homologous recombination method. After the modified vector pCAMBIA2300-GFP (P2300-GFP) is digested by the restriction enzymes BamH I and Sal I, primers are designed according to the full-length sequence of RhDAO gene, and the ORF fragment of China rose RhDAO1 gene with the homologous arm of the vector is inserted into the linearized P2300-GFP vector. Transferring into colibacillus DH5 alpha, sequencing to obtain positive clone, transferring into agrobacterium GV3101 after extracting plasmid to obtain agrobacterium GV3101 containing P2300-GFP-RhDAO1 plasmid.

The construction process of the pCAMBIA2300-GFP and P2300-GFP vectors described above uses the conventional method, and the amplification primer sequences are shown in Table 13.

TABLE 13 primers for construction of subcellular localization vectors

Primer name	Primer sequences
		P2300-GFP-F	5’- AGCTTTCGCGAGCTCGGTACCATGGTGAGCAAGGGCGAGG-3’
P2300-GFP -R	5’-TCTAGAGGATCCCCGGGTACCCTTGTACAGCTCGTCCATGCC-3’
		RhDAO1-GFP-F	5’-AGGCCGAATTCCCGGGGATCCATGGCAAATCCAATTCCAGTGA-3’
RhDAO1-GFP-R	5’-ATGCGGCCGCTGCAGGTCGACTTAGGACTTGATGCGCAGAAGTT-3’

Transferring the constructed vector into agrobacterium for subcellular localization experiment, specifically comprising the following steps:

1) Agrobacterium containing the P2300-GFP plasmid and the P2300-GFP-RhDAO1 plasmid, as well as the nuclear marker NF-YA4-mCherry plasmid, the cell membrane marker PM-mCherry plasmid, the endoplasmic reticulum marker ER-mCherry plasmid and the pSoup-P19 plasmid were streaked on LB plates (50 mg/L Kan,25mg/L Rif). The monoclonal was picked and re-activated on LB (Luria-Bertan) plates (50 mg/L Kan,25mg/L Rif). The monoclonal flora was picked up and inoculated into conical flasks containing 10mLLB liquid medium (50 mg/L Kan,25mg/L Rif) and placed in a 28℃shaking incubator at 200rpm for 12h.

2) The bacterial liquid after the suction culture was diluted to a fresh liquid LB medium (containing 50mg/L Kan,25mg/L Rif) in a conical flask at a ratio of 1:50 (v: v), and placed in a shaking incubator at 28 ℃.

3) And (3) centrifuging at 5000rpm for 5min at the temperature of bacterial liquid OD ₆₀₀ = 0.8-1.0,28 ℃, collecting bacterial cells, and discarding supernatant.

4) The cells were resuspended with the challenge fluid (10mM MgCl2,10mM MES,200mM AS,pH =5.6) to an OD ₆₀₀ =1.0.

5) The suspended bacterial solutions were mixed at a ratio of 1:1:1 (v: v) and allowed to stand in the dark for 4 hours (Table 14).

Table 14 conversion combinations

Experiment	Plasmid 1	Plasmid 2	Plasmid 3
				Experiment group 1	P2300-GFP-RhDAO1	NF-YA4-mCherry	pSoup-p19
Experiment group 2	P2300-GFP-RhDAO1	PM-mCherry	pSoup-p19
				Experiment group 3	P2300-GFP-RhDAO1	ER-mCherry	pSoup-p19

6) Selecting good-growth Nicotiana benthamiana, and injecting the dyeing liquor from the subleaf epidermis by using a needleless injector.

7) And placing the tobacco plants after injection treatment in a wet foam box, and culturing for about 48 hours under dark conditions.

8) And (3) taking the injected tobacco leaves to manufacture a slide, wherein the lower surface of the slide faces upwards, inverting the slide, and acquiring fluorescent signals by using an Olympus laser scanning confocal microscope and photographing.

14. Yeast vector construction

The ORF region of RhDAO gene was constructed into pGBKT7 by homologous recombination. The vectors pGBKT7 and pGADT7 were double digested by restriction enzymes EcoRI and BamHI, respectively. The ORF fragment of RhDAO gene with the homologous arm of the vector, amplified in China rose cDNA, is inserted into linearized pGBKT7 and pGADT7 vectors respectively.

The construction process of the pGBKT7-RhDAO1 and pGADT7-RhDAO1 vectors adopts the prior method, and the amplification primer sequences are shown in Table 15.

TABLE 15 primers for Yeast vector construction

Primer name	Primer sequences
		pGBKT7-RhDAO1-F	5’-ATGGCCATGGAGGCCGAATTCATGGCAAATCCAATTCCAGTGA-3’
pGBKT7-RhDAO1-R	5’-CCGCTGCAGGTCGACGGATCCTTAGGACTTGATGCGCAGAAGTT-3’
		pGADT7-RhDAO1-F	5’-GCCATGGAGGCCAGTGAATTCATGGCAAATCCAATTCCAGTGA-3’
pGADT7-RhDAO1-R	5’-CAGCTCGAGCTCGATGGATCCTTAGGACTTGATGCGCAGAAGTT-3’

After transformation of E.coli DH 5. Alpha. The positive clones were sequenced and the plasmid was transferred into yeast Y2HGold to obtain yeast Y2HGold containing pGBKT7-RhDAO1 and pGADT7-RhDAO1 vectors.

15. Preparation of competent Y2HGold Yeast

The Y2HGold Yeast competent transformation method was referred to the classical Yeast transformation kit (Beijing cool Lai Bo technology Co., ltd.) product instructions.

1) And (3) taking out the preserved Y2HGold bacterial liquid from the ultra-low temperature refrigerator, activating the bacterial liquid on a2 XYPDA solid culture medium, picking single bacterial colonies, drawing a 5mm short line on the new solid culture medium, inverting the solid culture medium, and culturing the solid culture medium in a 30 ℃ incubator for 72 hours.

2) Y2HGold was scraped off, inoculated into a conical flask containing 5mL of 2 XYPDA liquid medium, and placed in a shaking incubator at 30℃and cultured at 250rpm for 12 hours.

3) Mu.L of the bacterial liquid was aspirated, diluted into a conical flask containing 50mL of YPDA liquid medium, and centrifuged at 3000rpm for 5min until the bacterial liquid OD600 = 0.5, and the supernatant was discarded.

4) The cells were suspended in 100mL of fresh YPDA (if BD-containing plasmid, SD/-Trp) broth. After OD600 = 0.6-0.8, centrifugation at 3000rpm for 5min, the supernatant was discarded.

5) The cells were suspended with 50mL of SDW, centrifuged at 3000rpm for 5min, and the supernatant was discarded.

6) The cells were resuspended in 1.5mL of 1 XLIAc solution and centrifuged at 3000rpm for 5min, and the supernatant was discarded.

7) The cells were resuspended in 1.0mL of 1 XLiAc solution and the single pellet was transferred to 100. Mu.L without the library transfer. Centrifugation at 3000rpm for 5min, discarding supernatant and competent preparation.

16. Y2HGold Yeast plasmid extraction

The Y2HGold yeast plasmid extraction method is referred to the product specification of the yeast plasmid extraction kit (Beijing Tiangen Biochemical technology Co., ltd.).

1) 500. Mu.L of the balancing solution BL was added to the adsorption column CP2, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

2) 5ML of the bacterial liquid was added to a 2 mL centrifuge tube, centrifuged at 12000rpm for 1min, and the supernatant was discarded.

3) Mu.L of YP1 resuspended cells were added, 0.1g of 0.45 mm acid-washed glass beads were added, and vortexed for 10min.

4) 250 Μl of YP2 was added, and the mixture was gently mixed until the bacterial liquid was clear and viscous, and allowed to stand at room temperature for 10min.

5) 350. Mu.L of YP3 was added, gently mixed and centrifuged at 12000rpm for 20min.

6) The supernatant was added to the column CP2, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

7) Add 500. Mu.L deproteinized solution PD, centrifuge at 12000rpm for 1min, discard the filtrate.

8) 600 Μl of rinse PW was added and centrifuged at 12000rpm for 1min, the filtrate was discarded and repeated once.

9) The column CP2 was returned to the 2mL collection tube, centrifuged at 12000rpm for 2min, and left open for 5min.

10 Placing the adsorption column CP2 into a 1.5mL centrifuge tube, adding 50 mu L of elution buffer EB, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, and preserving at-20 ℃ in a refrigerator.

11 Transferring the plasmid into escherichia coli DH5 alpha, and carrying out plasmid separation by utilizing the resistance of the vector to obtain the yeast plasmid.

17. Bimolecular fluorescence complementation detection (BiFC) in tobacco

A p35SYC vector carrying YFP is constructed by adopting a homologous recombination method. After P35SYC-nYFP was double digested with restriction enzymes BamHI and SalI, the ORF fragment of the China rose RhDAO1 gene with the homology arm of the vector was inserted into the linearized P35SYC-cYFP vector. After double cleavage of P35SYC-cYFP by the restriction enzymes BamHI and SalI, the ORF fragment (with the terminator removed) of the China rose RhDAO1 gene with the homology arm of the vector was inserted into the linearized P35SYC-cYFP vector. After transferring into colibacillus DH5 alpha, positive clone is sequenced, and after plasmid is extracted, agrobacterium GV3101 is transferred.

The amplification primer sequences used in the construction of the P35SYC-RhDAO1-cYFP and P35SYC-nYFP-RhDAO vectors described above are shown in Table 16.

Table 16 primers for construction of fluorescence complementation experiment vector

Primer name	Primer sequences
		RhDAO1-nYFP-F	5’-ATCGAGGACGCCGGCGGATCCATGGCAAATCCAATTCCAGTGA-3’
RhDAO1-nYFP-R	5’-ACGAAAGCTCTGCAGGTCGACTTAGGACTTGATGCGCAGAAGTT-3’
		RhDAO1-cYFP-F	5’-ATTACAGGTACCCGGGGATCCATGGCAAATCCAATTCCAGTGA-3’
RhDAO1-cYFP-R	5’-CACGCTGCCACCGCCGTCGACGGACTTGATGCGCAGAAGTTCA-3’

1) The Agrobacterium GV3101 monoclonal flora containing the P35SYC-cYFP, P35SYC-RhDAO1-cYFP, P35SYC-nYFP, and P35SYC-nYFP-RhDAO1 plasmids was inoculated into a conical flask containing 10mL of LB liquid (50 mg/L Kan,25mg/L Rif), placed in a 28℃shaking incubator, and cultured at 200rpm for 12 hours.

2) The bacterial liquid was diluted 1:50 (v: v) into a conical flask of fresh liquid LB medium (containing 100mg/L Spec,25mg/L Rif), placed in a 28℃shaking incubator and cultured at 200rpm for 12-16 h.

3) And (3) centrifuging for 5min at a speed of 5000rpm until the bacterial liquid OD ₆₀₀ =0.6-0.8, collecting bacterial cells, and discarding supernatant.

4) The cells were resuspended with an infection solution (10 mM MgCl ₂, 10mM MES,200mM AS,pH =5.6) and OD ₆₀₀ =0.8.

5) The suspended bacterial liquid is mixed according to the ratio of 1:1 (v: v) and then is kept stand in the dark for 4 hours.

7) The tobacco plants after injection were placed in a humidified foam box and incubated for about 48 hours in the dark.

2. Results and analysis

1. Cloning and sequence analysis of the China rose auxin oxidase Gene RhDAO1

The RhDAO gene reference sequence is obtained through NCBI database, and the full length of RhDAO gene is obtained by using China rose Aicler' leaf cDNA amplification. Through sequence analysis, the RhDAO gene ORF region is 900 bp, the sequence is shown as SEQ ID NO.2, 299 amino acids can be encoded, and the sequence is shown as SEQ ID NO. 1.

Protein physicochemical property prediction analysis RhDAO 1.2 kDa protein molecular mass, isoelectric point pI of 5.57, instability index of 34.35, lipid solubility index of 93.91, hydrophilic index of-0.159, is hydrophilic protein and is relatively stable. The result of the conserved domain analysis of the amino acid sequence showed that RhDAO belongs to the group of 2-ketoglutarate and ferrous-dependent dioxygenase (FIG. 1). Transmembrane structure prediction showed that the protein was a globular protein, with no transmembrane domain present (fig. 2). Finally, the three-dimensional structure of RhDAO1 protein is predicted by AlphaFold (FIG. 3).

Analysis of the homologous protein sequence of RhDAO1 relative to the binding-related literature revealed that the domain of DAO was more conserved and contained Fe and 2OG binding sites (fig. 4). Three auxin oxidases RhDAO, rcDAO2 and RcDAO3 exist in China rose, and the amino acid sequences of the three are obviously different at the amino terminal. The phylogenetic tree analysis of RhDAO1 showed that RhDAO has high homology with apple MdARRO, tobacco NtDAO1, arabidopsis AtDAO, atDAO2 and rice OsDAO (fig. 5). In addition, the analysis of the evolutionary tree shows that the auxin oxidase, the glutamate biosynthetic enzyme and the gibberellin oxidase all belong to the families of 2-ketoglutarate and ferrous-dependent dioxygenase. MEME analysis showed that Motif5 and Motif10 are characteristic of auxin oxidase (Table 17).

TABLE 17 motif matching sequences

Motif sequence	Length of	Optimal matching sequences
			Motif 1	36	PCPGTLLVNLGDLAQAWSNGRLCSVKHRVQCNEATI
Motif 2	32	CQFRINKYNFTPEPVGSLGVQIHTDSGFLTIL
			Motif 3	41	VRSLLDLPMEIKKRNTDVIAGSGYMAPSKVNPLYEALGLYD
Motif 4	21	KKJREASEEWGCFRVVNHGIP
			Motif 5	41	SSQAVDTFCSQLDASPHQREVIEKYAZAIHELIVDIGRKLA
Motif 6	29	AVEAPPELVDSEHPRLYVPFTYEEYRKLR
			Motif 7	15	DDENVGGLEVMDKSG
Motif 8	11	RVSIATFLLPP
			Motif 9	11	IPVIDLSDFPD
Motif 10	15	LSTKLQAGEALELVR

To predict the expression characteristics of RhDAO gene, a 2000bp promoter sequence before the RhDAO gene transcription initiation site is cloned and analyzed, and the sequence is shown as SEQ ID NO. 4. The result shows that RhDAO gene promoter contains auxin response element, meristem regulating element and stress response element. Meanwhile, it was found that the promoter of RhDAO homologous gene contains a plant hormone responsive element and a stress responsive element. For example RcDAO, fvDAO, paDAO, ntDAO, atDAO1, osDAO-like gene promoters each contain an auxin response element (AuxRR-core), and NtDAO, atDAO1, atDAO2, osDAO gene promoters each contain a meristem regulatory element. Promoter cis-acting element analysis showed that RhDAO gene and its homologous gene might be widely involved in the growth and development process regulated by auxin (FIG. 6).

2. Analysis of expression Properties of China rose RhDAO Gene

In order to analyze whether the gene participates in the formation of the rose flower double-petal character, the expression level of RhDAO gene in the flower development process is detected by qRT-PCR technology in China rose Aicleer ' and the double-petal bud-variant variety ' JieLvzhen '. The results showed that the expression level of RhDAO gene in 'Airiley' was significantly higher than in 'JieLvzhen' (in the figure, ecl represents 'Airiley', kon represents 'JieLvzhen') in the petal primordium differentiation stage (grade 2) and in the stamen primordium differentiation stage flower bud (grade 3), whereas the difference between the two was not significant. Since the re-petal of China rose mainly originates from the petal formation of stamens, it is presumed that RhDAO gene expression levels may be inversely related to the re-petal formation degree of the micro China rose (A in FIG. 7).

Meanwhile, the expression condition of RhDAO gene in different tissues of China rose Aiklier' is detected. The results show that RhDAO gene has the highest expression level in pistil, then stamen and stems, and lower expression level in root, leaf, sepal and petal. Wherein, the RhDAO 1.2 times of the stamen is expressed in the pistil, and the 3.9 times of the petals is expressed in the stamen. The results show that RhDAO gene may be involved in the developmental processes of stamen and pistils (B in FIG. 7).

To investigate whether RhDAO1 genes responded to auxin, discs 1cm in diameter were obtained by punching petals in China rose flowers, and the flower discs were subjected to 1mM IAA soaking treatment, and the related gene expression levels were detected by qRT-PCR. The results show that the expression level of the auxin-induced gene RcGH3.3 is obviously improved after IAA treatment for 1h, and the expression level is still maintained at a higher level for 24h, which proves that IAA treatment of China rose petal discs is effective. RhDAO1 gene expression levels also increased significantly after IAA treatment for 6h and 12h, 1.1 and 2.4 fold, respectively, compared to the control (C in fig. 7).

3. Subcellular localization analysis of RhDAO protein

To further understand the localization of RhDAO.sup.1 in plant cells, agrobacterium GV3101 invader solutions containing the P2300-GFP and P2300-GFP-RhDAO plasmids, as well as the nuclear marker NF-YA4-mCherry plasmid, the plasma membrane marker PM-mCherry plasmid, and the endoplasmic reticulum marker ER-mCherry plasmid, were mixed separately. Transferring into the lower epidermic cell of Nicotiana benthamiana for transient expression, and then observing the distribution of fluorescent signals by using a laser scanning confocal microscope. The results showed that the green fluorescence in the tobacco leaf blade expressing the P2300-GFP-RhDAO1 plasmid was simultaneously distributed in the nucleus, plasma membrane and endoplasmic reticulum, fused with the red fluorescence of the nucleus, plasma membrane and endoplasmic reticulum marker plasmids. And pearson correlation coefficient analysis showed that the two fluorescence can overlap highly, suggesting RhDAO that in rose might be localized on the nucleus, plasma membrane and endoplasmic reticulum (fig. 8).

4. RhDAO1 proteins form protein complexes between them

Yeast growth test results show that the positive control, the negative control and the experimental group yeast Y2HGold can normally grow in the double-deficiency culture medium (SD/-Leu/-Trp), which indicates that each group of plasmids can successfully transform the yeast. However, only the positive control and the experimental group yeasts containing pGBKT7-RhDAO1 and pGADT7-RhDAO were able to grow and develop blue in the tetra-deficiency medium (SD/-Leu/-Trp/-Ade/-His/X-alpha-gal) containing X-alpha-gal. The RhDAO protein was shown to be capable of self-interaction in yeast cells (A in FIG. 9).

To further verify that RhDAO proteins have an interactive relationship in plants, a bimolecular fluorescence complementation (BiFC) assay was performed in nicotiana benthamiana. Agrobacterium GV3101 invader solutions containing the p35SYC-cYFP, p35SYC-RhDAO1-cYFP, p35SYC-nYFP, p35SYC-nYFP-RhDAO1 plasmids were mixed separately. Transient expression in epidermal cells under Nicotiana benthamiana, and observation of fluorescence distribution by using a laser scanning confocal microscope. There was a strong fluorescent signal in tobacco epidermal cells containing the p35SYC-RhDAO1-cYFP and p35SYC-nYFP-RhDAO plasmids after transformation, indicating that the RhDAO1 protein was able to form a dimeric complex in plant cells (B in FIG. 9).

Example 2 functional verification of RhDAO Gene in China rose Aicleer flower development

1. Materials and methods

1. Plant material

Selecting a China rose variety 'Aiklier' semi-lignified branch which grows well in a farm garden and is free of plant diseases and insect pests, and cutting off the branch which is about 8cm long, contains 1 compound leaf and has 1 to 2 bud points by using a sterilized branch scissors. And (3) after soaking rooting water, pruning the compound leaves to 4 small leaves, and inserting the plug tray filled with red jade soil upwards from the bud point. After 80% of cuttings root, carrying out VIGS experiment by using cutting seedlings with similar growth vigor, and cutting off overlong root systems and redundant old leaves before the experiment.

2. Virus induced gene silencing GENE SILENCING (VIGS)

And obtaining RhDAO gene-silenced China rose cutting seedlings by using a VIGS technology, and observing the phenotype of the silenced plants. Constructing pTRV2 silencing vector by homologous recombination, designing silencing fragment through SGN-VIGS of website, selecting non-conservative sequence, silencing fragment 275bp (sequence shown as SEQ ID NO. 3) located in RhDAO gene 5' -UTR. After the pTRV2 vector is digested by the fast cutting enzymes BamHI and EcoRI, rhDAO gene silencing fragment with a vector homology arm is inserted into the linearized pTRV2 silencing vector.

The amplification primer sequences used in the construction of the above-described TRV2-RhDAO1 vector are shown in Table 18.

Table 18 amplification primers used in construction of TRV2-RhDAO1 vector

Primer name	Primer sequences
		TRV2-RhDAO1-F	5’-GTGAGTAAGGTTACCGAATTCTGAAGGGTTCACGATGATTCCG-3’
TRV2-RhDAO1-R	5’-CGTGAGCTCGGTACCGGATCCTCTCTGGCTATGGATACTAAGTTTGG-3’

After transferring into colibacillus DH5 alpha, positive clone is determined, and the extracting plasmid is transferred into agrobacterium GV3101.

1) Agrobacterium monoclonal containing pTRV1, pTRV2 and pTRV2-RhDAO1 plasmids was added to a conical flask containing 10mL of liquid LB medium (50 mg/L Kan,25 mg/LRif), the incubator was shaken at 28℃and incubated at 200rpm for 12h.

2) The bacterial liquid is diluted to fresh LB liquid medium (50 mg/L Kan,25mg/L Rif,10mM MES and 20mM AS) according to the ratio of 1:50 (v: v), and the bacterial liquid is placed into a 28 ℃ shaking incubator for 16-20h.

3) And (3) centrifuging at 5000rpm for 10min at room temperature until the bacterial liquid OD ₆₀₀ is more than 1.5, collecting bacterial cells, and discarding supernatant.

4) The cells were resuspended in an invader solution (10 mM MgCl ₂, 10mM MES,200mM AS,0.01%SilwetL-77, pH=5.6) to give OD ₆₀₀ =1.0.

5) The suspended bacterial liquids were mixed at a ratio of 1:1 (v: v), and then left to stand in the dark for 4 hours (Table 19).

Table 19 conversion combinations

Experiment	Plasmid 1	Plasmid 2
			Blank control (TRV)	pTRV1	pTRV2
Experiment set (TRV-RhDAO 1)	pTRV1	pTRV2-RhDAO1

6) Taking out the cutting seedlings with uniform growth from the plug tray, cleaning root systems with clear water, wrapping plants with gauze, fixing with rubber bands, then immersing the whole plant in the dye liquor in an inverted manner, immersing the root parts of the plant in the dye liquor, placing the plant in a vacuum dryer, and standing for 1min after the pressure in the dryer reaches-0.1 MPa. The pressure was slowly applied again for about 30min and repeated once.

7) After the infection is finished, the plants are washed by deionized water for 6 to 8 times. After rinsing, planting the plant in a sterilized 8cm small black square containing peat, vermiculite and perlite (1:1:1) moist matrix, and watering the matrix with clear water. The incubator has the temperature of 21+/-1 ℃ and the humidity of 60 percent, the illumination of 40000Lux and the photoperiod of 16h/8h (light/dark), and is beneficial to the survival and propagation of viruses. The plant position is changed periodically and nutrients are applied.

3. Phenotype observation and flower organ number statistics of silent plants

1) And extracting young leaf RNA after 4 weeks of infection, reversely transcribing the young leaf RNA into cDNA, detecting the expression quantity of the corresponding genes of the control plant and RhDAO gene silencing plant by adopting qRT-PCR, and primarily screening the silencing plant. After the flowers are opened, observing the phenotype, recording the diameters of the flowers by using an electronic vernier caliper, photographing and counting the number of flower organs, collecting the flower organs to a 2mL grinding tube, quick-freezing by using liquid nitrogen, and storing in an ultralow temperature refrigerator.

2) Inner petal RNA was extracted and reverse transcribed into cDNA. And carrying out related gene expression analysis on RhDAO gene silencing plants by adopting qRT-PCR.

2. Results and analysis

1. Phenotype observation of RhDAO Gene-silenced China rose flowers

In order to explore the function of RhDAO gene in the process of re-flap of 'Airil', rhDAO gene of 'Airil' cutting seedling plant was silenced by using VIGS technique.

Sampling and qRT-PCR detection are carried out on petals of TRV blank control and TRV-RhDAO treatment groups, and 5 plants with higher silencing efficiency are obtained through screening. The expression level of RhDAO1 gene in TRV-RhDAO 1-silenced plants was significantly reduced compared to the control, approximately 17% of the control (FIG. 10).

Phenotypic observations of 11 flowers in the silenced plants revealed a significant increase in the number of petals of TRV-RhDAO 1. Compared with TRV control, the normal petals of the flower TRV-RhDAO1 are increased by 7.7, the malformed petioled stamens is increased by about 9.2, and the number of stamens is obviously reduced by about 19.0. While the total number of petals and stamens did not change significantly compared to the control group (fig. 11, fig. 12). This result indicates that the decrease in RhDAO gene expression results in a shift of the 'Aicleer' stamen to petals, an increase in the degree of stamen petal formation, and a phenotype of increased number of petals. Thus, rhDAO gene may play an important role in the process of China rose bivalve.

2. Detection of gene related to double valve of China rose RhDAO gene silencing plant and gene related to auxin

In order to further explore a gene regulation network related to RhDAO gene negative regulation of the number of Chinese rose petals, qRT-PCR technology is utilized to detect genes related to heavy petals and auxin in RhDAO gene silencing plant petals. In RhDAO1 gene-silenced plants, the A-class functional gene RhAP2 expression level is significantly increased by 2.2 times compared with the control, the auxin polarity transport related gene RhPID expression level is significantly increased by 2.6 times compared with the control, the auxin response gene RhIAA16 expression level is significantly increased by 2.1 times compared with the control, the auxin-induced gene RhGH3.1 expression level is significantly increased by 2.3 times compared with the control, the rose flower heavy valve positive regulatory factor RhARF expression level is significantly increased by 47.3 times compared with the control, and the auxin transport carrier gene RhPILS1 expression level is not different (FIG. 14).

From the above experimental results, it can be seen that:

1) The use of VIGS technology in 'aclei' silences RhDAO the gene, rhDAO the gene silences flowers in a total number of flower organs similar to the TRV control plants, but with an increased degree of stamen petalonization, resulting in a phenotype of reduced number of stamen and increased number of petals.

2) The expression levels of the gene related to the double valve and the gene related to the auxin are analyzed by adopting qRT-PCR technology. In the RhDAO gene silencing plant of 'Aicleer', the expression level of the gene RcAP2 and RcARF positively correlated with the heavy valve character is obviously increased, and the expression level of the gene RcPID, rcIAA16 and RcGH3.1 correlated with the auxin pathway is obviously increased, which indicates that the negative regulation of RhDAO1 genes on the petal number can be related to the change of the expression of the genes.

Claims

1. A new use of the rose auxin oxidase gene RhDAO1 , characterized in that it is used to regulate the number of rose petals, the amino acid sequence of the protein encoded by the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:1, and the nucleotide sequence of the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:2.

2. A new use of the rose auxin oxidase gene RhDAO1 , characterized in that it is used to regulate the number of stamens of roses, the amino acid sequence of the protein encoded by the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:1, and the nucleotide sequence of the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:2.

3. A new use of the rose auxin oxidase gene RhDAO1 , characterized in that it is used to perform homologous conversion between the stamens of a rose and the petals of a rose, the amino acid sequence of the protein encoded by the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:1, and the nucleotide sequence of the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:2.

4. A new use of the rose auxin oxidase gene RhDAO1 , characterized in that it is used for improving the double-petal traits of rose flowers, the amino acid sequence of the protein encoded by the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:1, and the nucleotide sequence of the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:2.

5. The new use of the rose auxin oxidase gene RhDAO1 according to claim 4, characterized in that the improvement of the double-petal trait of the flower shape is to increase the number of petals and the number of petal layers.

6. A new use of the rose auxin oxidase gene RhDAO1 , characterized in that it is used for the cultivation of new rose varieties, wherein the new rose varieties are new varieties with increased or reduced number of petals, the amino acid sequence of the protein encoded by the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:1, and the nucleotide sequence of the rose auxin oxidase gene RhDAO1 is shown in SEQ ID NO:2.

7. The new use of the rose auxin oxidase gene RhDAO1 according to claim 6, characterized in that the number of petals is increased by inhibiting the expression of the rose auxin oxidase gene RhDAO1 in the rose by gene silencing.