CN114438097A - Rose skin thorn regulation gene RrCPC and application thereof - Google Patents

Abstract

The invention relates to a rose skin thorn regulation geneRrCPCThe application of the rose buds with the purple branches as the material is cloned by RACE technologyRrCPCA gene. Detection by real-time fluorescent quantitative detection techniqueRrCPCThe expression modes of the genes on different levels of lateral branches of roses are proved to be negative regulatory factors of epidermal hair generation by constructing an overexpression vector to transform arabidopsis thaliana, and the expression vectors have important application values in cultivating new species of roses with few thorns or no thorns and improving the rose management efficiency. The invention provides a rose skin thorn regulation gene RrCPC and application thereof in the aspects of few thorn and no thorn rosesHas important application value in the breeding field of the rose.

Description

Rose skin thorn regulation geneRrCPCAnd uses thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rose skin thorn regulation geneRrCPCAnd applications thereof.

Background

Roses (A), (B)Rosa rugosa Thunb.) Is Rosa multiflora of RosaceaeBelongs to deciduous shrubs, has pleasant flower fragrance and various varieties, is a traditional famous flower in China, and is also a rare natural perfume plant and an excellent landscaping material. The flower can be used for extracting rose essential oil, preparing rose tea, rose sauce and the like, is widely applied to the industries of spice, food, cosmetics and the like, and has extremely high economic and health-care values.

With the development of social economy and the improvement of living standard of people, the demand of international and domestic markets on rose products is gradually increased, the rose products have diversified characteristics, and the requirements on the fragrance, quality, appreciation, variety and the like of roses are increasingly improved. However, the rose stems have many thorns, so that inconvenience is caused in garden application, field management and flower picking processes, and if rose varieties with few thorns or no thorns can be cultivated, the work efficiency of field management and picking of the roses is greatly improved, and the economic benefit of rose planting is further improved.

Research shows that the plant skin thorn is a sharp protrusion formed by the stem epidermis, is formed by continuous growth and hardening modification of epidermal hair in the long-term evolution process for defending herbivores, and is a multicellular glandless special-form epidermal hair which is generated by the skin part, is not connected with the xylem and does not contain vascular bundles. A model plant Arabidopsis thaliana epidermal hair molecule regulation network reveals that: the initiation of plant epidermal hair is controlled by a complex (MBW) composed of products of GL1/MYB23 (MYB transcription factors), GL3/EGL3 (bHLH transcription factors) and TTG1 (WD 40 repeat protein transcription factors), and the MBW directly acts on downstream genesGL2Control the initiation of Arabidopsis epidermal hair. Meanwhile, MBW can also regulate and control negative regulatory factors such as TRY, CPC and the like to move to nearby epidermal cells, so that the development of epidermal hair is inhibited.

CPCThe gene is expressed in arabidopsis young leaf primordium and developing epidermal hair cells,CPCoverexpression results in a decrease in coat, and cpc mutant coat fasciculates.CPCEncodes a R3-MYB transcription factor which only contains 1 DNA binding domain, lacks a transcription activation domain, can move among cells, interacts with the ends of GL3 and EGL3, and competitively binds with GL1 to bHLH protein to form an inactive complex, therebyInhibiting the development of epidermal hair. The research discovers that the purple branch rose has the characteristics of more first-stage lateral branch skin thorn, sparse second-stage lateral branch skin thorn and smooth and thornless third-stage lateral branch through investigation on the variety characteristics of the rose skin thorn, and the rose is cloned by taking the purple branch rose as a test materialCPCThe gene is verified by fluorescent quantitative PCR and transformation of Arabidopsis thaliana, is helpful for clarifying the molecular mechanism of rose skin thorn regulation, provides a molecular tool for genetic engineering breeding of rose with few thorns and no thorns, and has potential economic value for reducing the labor cost of rose management and picking.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a rose skin thorn regulation geneRrCPCAnd the application thereof, and one aim is to provide a rose skin thorn regulation geneRrCPCNegative regulation of the development of the skin thorn, and the other purpose is to provide the rose skin thorn regulation geneRrCPCThe use of (1).

The invention is realized by the following technical scheme: rose skin thorn regulation geneRrCPCThe nucleotide sequence is shown in SEQ ID NO. 1. The rose fragrance regulating geneRrCPThe amino acid sequence of the expression protein of C is shown in SEQ ID NO. 2.

Contains the rose skin thorn regulatory geneRrCPCThe overexpression vector of (1).

Contains the rose skin thorn regulatory geneRrCPCThe host cell of (1).

The rose skin thorn regulation geneRrCPCApplication in reducing rose skin prick.

Has the advantages that: the invention takes the purple branch rose as the material and clones the purple branch rose by the RACE technologyRrCPCA gene. Detection by real-time fluorescent quantitative detection techniqueRrCPCThe expression modes of the genes on different levels of lateral branches of roses are proved to be negative regulatory factors of epidermal hair generation by constructing an overexpression vector to transform arabidopsis thaliana, and the expression vectors have important application values in cultivating new species of roses with few thorns or no thorns and improving the rose management efficiency.

The invention provides a rose skin thorn regulation gene RrCPC and application thereof in the aspects of few thorns and no thorns. Has important application value in the field of thornless rose breeding.

Drawings

FIG. 1 isRrCPCA map of gene spatiotemporal expression patterns;

FIG. 2 is a plasmid map of plant overexpression vector pCAMBIA 1304;

FIG. 3 is T2 generation transgenic Arabidopsis thalianaRrCPCRelative expression level of the gene;

FIG. 4 is the T3 generationRrCPCOverexpresses the phenotype of arabidopsis and wild-type arabidopsis;

FIG. 5 isRrCPCThe expression quantity of related genes in the epidermal hair molecule regulation network is changed by over-expressing arabidopsis thaliana and wild arabidopsis thaliana.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

1. Cloning by RACE techniqueRrCPCA gene;

based on the rose transcript, RACE primers are designed to take total RNA of rose organs as a template, 5 'and 3' terminal fragments are obtained and cloned into a T-vector, the inserted fragments are subjected to positive screening and then are subjected to sequencing, and sequencing result sequences are spliced through overlapping regions to obtain the full length of cDNA.

I. Designing a primer:

3' RACE specific primers:

Outer Primer：5’-GAAGAGGTAAGCAGCATT-3’；

Inner Primer：5’-CAGGTCGGAAAGCAGAAG-3’；

3' RACE adapter primer:

Outer Primer：5’-TACCGTCGTTCCACTAGTGATTT-3’；

Inner Primer：5’-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3’；

CGCAGAGT-3' was used for the universal adaptor primer.

5' RACE adapter primer:

Outer Primer：5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；

Inner Primer：5’-CTAATACGACTCACTATAGGGC-3’；

5' RACE specific primers:

Gene-Specific Primers ：5’-TTGCTATTATTAAGCTGCTGGGGTTGCTG-3’；

II.3' RACE reaction Process:

(1) reverse transcription, add the following ingredients to a small centrifuge tube of RNase-free placed on ice: mu.g of Total RNA, 2. mu.L of 5 XPrimeScript Buffer, 1. mu.L of 3' RACE Adapter (5. mu.M), 1. mu.L of dNTP mix (10mM each), 0.25. mu.L of RNase Inhibitor (40U/. mu.L), 0.25. mu.L of PrimeScript RTase (200U/. mu.L), RNase Free dH₂And O is supplemented to 10 mu L.

(2) Mixing, centrifuging for a short time, and incubating at 42 deg.C for 1 h; entering into PCR step, or storing the reactant at-20 ℃;

(3) 3' RACE nested PCR;

the reaction system is as follows: 50 μ L of Outer 3' RACE composition: 5.0 μ L10 × LA PCR Buffer II (Mg)²⁺Free)，8.0μL 1×cDNA Dilution Buffer II，2.0μL 3’ RACE gene-specific outer primer，2.0μL 3’ RACE Outer Primer (10μM)，2.0 μL RT reaction product，0.5μL TakaRa LA Taq(5U/μL)，30.5μL ddH₂O。

Reaction procedure: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 20 cycles; 10min at 72 ℃.

μ L Inner 3' RACE composition: 5.0 μ L10 × LA PCR Buffer II (Mg)²⁺ Free)，8.0μL dNTP Mixture(2.5mM each)，2.0μL gene-specific Inner primer，2.0μL 3’ RACE Inner Primer(10μM)，1.0μL Outer 3’ RACE PCR product，0.5μL TakaRa LA Taq(5U/μL)，31.5μL ddH₂O。

Reaction procedure: 3min at 94 ℃; 30cycles at 94 ℃ for 30sec, 55 ℃ for 30sec, 72 ℃ for 1 min; 10min at 72 ℃.

(4) Ligation of purified fragments to cloning vectors

The desired DNA molecule was cloned using pMD19-T simple vector from TaKaRa.

Reaction (5 μ L): 2.2. mu.L of purified recovered PCR product, 0.3. mu.L of pMD-19 Simple Vector, 2.5. mu.L of Solution I.

Reaction conditions are as follows: 30min at 16 ℃; storing at 4 ℃.

(5) E, transformation of escherichia coli: thawing freshly prepared or frozen-stored E.coli TOP10 competent cells at-70 ℃ on ice; adding 5 μ L of the ligation product of the purified fragment and the cloning vector into 100 μ L of competent cells, gently mixing, and performing ice bath for about 30 min; heating in 42 deg.C water bath for 90sec, and rapidly placing on ice for 3-5 min; adding 800 μ L LB liquid culture medium, shaking at 37 deg.C and 100rmp for 1 h; centrifuging at 4000rmp for 3min, sucking off 800 μ L of culture medium at the upper layer, and mixing the rest bacteria solution; the bacterial liquid was smeared on an LB screening plate containing km and cultured in an inverted state at 37 ℃ overnight.

(6) Positive clone screening and sequencing analysis

Selecting a single colony from a screening culture plate, inoculating the single colony in an LB liquid culture medium, and shaking the bacteria at 37 ℃ and 250rmp overnight; and directly carrying out PCR detection on the recombinant transformant by taking the overnight cultured bacterial liquid as a template.

Reaction system (20.0 μ L): 2.0 μ L10 XPCR Buffer (Mg)²⁺ free)，1.5μL MgCl₂ (25mM), 1.3. mu.L dNTP mix (each 2.5mM), 1.0. mu.L 3 'RACE gene specific Inner Primer (10. mu.M), 1.0. mu.L 3' RACE Inner Primer (10. mu.M), 0.1. mu.L bacterial fluid, 1.0. mu.L rTaq, 12.1. mu.L Milli-Q Water.

Reaction procedure: 3min at 94 ℃; 94 ℃ 30sec, 60 ℃ 30sec, 72 ℃ 1min, 28 cycles; 7min at 72 ℃.

The clone with positive PCR detection of the bacterial liquid is sent to the Biotech company (Shanghai) for sequencing.

Generation of 5' RACE-Ready cDNA

(1) Buffer Mix was prepared for cDNA synthesis reaction at the following volumes: mu.L of 5 XFirst-Strand Buffer, 0.5. mu.L of DTT (100mM), 1.0. mu.L of dNTPs (20mM), 1 additional amount of 1 reaction was prepared to ensure sufficient volume. Mixing the above reagents, rotating on a micro centrifuge for a short time, standing at room temperature, and performing subsequent experiments;

(2) the following reagents were added to a separate microcentrifuge tube: 1.0 μ g Total RNA, 1.0 μ L5' -CDS primer A, water to make up to 11.0 μ L;

(3) mixing the components and briefly rotating on a microcentrifuge;

(4) incubating at 72 deg.C for 3min, and cooling at 42 deg.C for 2 min. After cooling, centrifuge at 14,000 Xg for 10sec to collect the reagents on the bottom of the tube (while incubating the tube, the mixture from step 6 can be prepared);

(5) 1. mu.L of SMARTer II Oligonucleotide was added to the 5' RACE cDNA synthesis reaction solution;

(6) 5' RACE cDNA synthesis reaction solution was prepared in the following volumes. Mixing at room temperature in the following order: 5.5. mu.L Buffer Mix (previous step), 0.5. mu.L RNase Inhibitor (40U/. mu.L), 2.0. mu.L SMARTScripte Reverse Transcriptase (100U);

(7) adding 8 mu L of Master Mix obtained in the step 6 into the denatured RNA (5' RACE cDNA) obtained in the step 5, wherein the total volume of the cDNA synthesis reaction is 20 mu L;

(8) sucking and beating the mixture gently and evenly, and centrifuging the mixture for a short time to collect components at the bottom of the tube;

(9) incubating at 42 deg.C for 90min, and heating at 70 deg.C for 10 min;

(10) diluting the first strand cDNA synthesis reaction product with 50 μ L Tricine-EDTA Buffer, standing at normal temperature for 15min, and storing the reactant at-20 deg.C;

IV, 5' RACE nested PCR

Reaction system (50.0 μ L): 5.0. mu.L of 10 × Advantage 2 PCR Buffer, 5.0. mu.L of dNTP Mix (reach 10 mM), 2.5. mu.L of 5' -RACE Ready cDNA, 1.0. mu.L of GSP, 1.0. mu.L of UPM (10X), 1.0. mu.L of 50 × Advantage 2 Polymerase Mix, 34.5. mu.L of PCR-Grade water.

Reaction procedure: 94 ℃ for 30sec, 72 ℃ for 3min, 5 cycles; 94 ℃ for 30sec, 70 ℃ for 30sec, 72 ℃ for 3min, 5 cycles; 94 ℃ 30sec, 68 ℃ 30sec, 72 ℃ 3min, 25 cycles.

(4) The PCR product was cloned and sequenced, and the operation was identical to that of the 3' RACE clone.

Splicing 3 'RACE and 5' RACE sequences to obtain full-length cDNA, predicting the open reading frame by using NCBI-ORF finder tool, designing forward primer 5'-GAGCGGTTCAAAGATGAGCA-3' and reverse primer 5'-CCTTGTGAACACACATAGGTAGG-3', amplifyingRrCPCThe expression cassette of the gene verifies its authenticity.RrCPCThe cDNA of (1) has a total length of 797bp, the sequence of which is shown as SEQ ID NO.1, and has a complete open reading frame (339bp), a 5 'non-coding region (251bp) and a 3' non-coding regionThe coding region (195bp), the poly (A) tail (12bp), and the corresponding RrCPC protein sequence is 113 amino acid residues, and the sequence is shown as SEQID NO. 2.

Example 2

A spatio-temporal expression mode of the RrCPC gene is explored through a fluorescent quantitative PCR technology, a fluorescent quantitative PCR primer forward primer 5'-AAGCCAAGACTTGCAGCAGTTCC-3' and a fluorescent quantitative PCR primer reverse 5'-ACCTGTCTCCGACGAGCTTGTAC-3' are designed based on an exon region of the RrCPC gene, and a reference primer forward primer 5'-TGAGGCCATTTACGACAT-3' and a reference primer reverse primer 5'-AGATCACAGGAGCATAGGAG-3' are designed based on an Actin gene. Using SYBR Premix Ex Taq (Takara Inc.) and a quantitative fluorescence PCR instrument CFX96TM (Bio-RAD Inc.), the number of cycles reaching the fluorescence threshold was obtained by quantitative fluorescence PCR with reference to the PCR system and procedure described in the manufacturer's instructions, and the relative expression amounts of the RrCPC gene on the first, second and third branches of the purple rose were calculated.

As shown in FIG. 1, the RrCPC gene has the lowest expression level in the epidermal tissue of the first lateral branch (FCB) of the purple-branch rose, slightly improved expression level in the epidermal tissue of the second lateral branch (SCB) and the highest expression level in the epidermal tissue of the third lateral branch (TCB). The RrCPC gene expression mode has certain site specificity, and the expression is up-regulated along with the reduction of skin thorn in the first, second and third-level lateral branches of the rose.

Example 3 RrCPCOverexpression vector construction and Arabidopsis genetic transformation

In order to better elucidate the function of the RrCPC gene, the applicant overexpressed it in Arabidopsis plants and functionally verified according to the phenotype of the transgenic plants. The method comprises the following steps: inserting the RrCPC expression cassette into the multiple cloning site of an overexpression vector pCAMBIA1304 by using an NcoI enzyme cleavage site (FIG. 2), transforming agrobacterium tumefaciens EHA105 competence by the obtained plasmid through a liquid nitrogen freeze-thaw method, and identifying pCAMBIA 1304: an agrobacterium-positive strain of RrCPC.

The arabidopsis thaliana is infected by an inflorescence dip-dyeing method (a conventional method), and a transgenic plant is obtained by screening a transformation seedling with hygromycin resistance.

The main steps and reagents used for genetic transformation according to the invention are as follows:

I. reagent and solution abbreviations

The abbreviations for the phytohormones used in the medium of the present invention are as follows: km (Kanamycin ); hyg (Hygromycin B ); arabidopsis thaliana transformation helper reagent (Silwet L-77).

Agrobacterium-mediated genetic transformation procedure

(1) Cultivation of Agrobacterium

First, Agrobacterium EHA105 containing the vector of interest was cultured on solid LB medium (10g/L peptone +5g/L yeast extract +10g/L sodium chloride +50mg/L Km +15g/L agar) containing the selection for the corresponding resistance (Km) at 28 ℃ for 2-3 days, and the positive monoclonal was picked up and inoculated on liquid LB medium (10g/L peptone +5g/L yeast extract +10g/L sodium chloride +50mg/L Km) containing the selection for the corresponding resistance (Km) and shake-cultured overnight at 28 ℃ at 200rpm until the OD600 value of the bacterial liquid concentration became 1.5-2.0.

(2) Dipping dyeing method for arabidopsis inflorescence

The shaken bacterial solution was centrifuged at 4500rpm for 10min, and then soaked in a 5% sucrose solution (supplemented with 20. mu.L/100 mL of Arabidopsis thaliana transformation surfactant for resuspension) to soak the Arabidopsis thaliana inflorescence in the resuspension solution for about 30s, shaded and moisturized, and placed horizontally for 12-24 h.

(3) Screening of Positive seedlings

After infection placement, placing arabidopsis thaliana into an incubator for normal culture, disinfecting the harvested seeds on an ultra-clean workbench through 15s-30s of 70% alcohol and 15min of 2% sodium hypochlorite, washing with sterile water for 3-4 times, and then flatly paving the seeds on 1/2MS culture media (1/2 MS +30g/L sucrose +7g/L agar +20mg/L Hyg) containing resistance for screening; and carrying out PCR positive detection on the screened T1 generation plants, and transplanting the plants to a light incubator to wait for seed collection.

Example 4 RrCPC Gene transgenic plants qRT-PCR detection and phenotype Observation

Culturing the transgenic arabidopsis thaliana transplanted into soil in a 16h illumination/8 h dark illumination incubator, screening by using the same disinfection mode and resistance culture medium after mature seed harvest to obtain T2 generation transgenic plants, and carrying out PCR positive detection. Through observation and comparison, the rotor is foundRrCPCPart of the gene Arabidopsis thaliana had reduced epidermal hair (essentially no epidermal hair).

To verify whether the T2 generation transgenic Arabidopsis has been altered in coat with the transferred oneRrCPCThe invention adopts a common fluorescent quantitative PCR method to perform gene correlation on five T2 transgenic plants with obvious phenotypeRrCPCThe expression level of the gene was measured, and the results are shown in FIG. 3. The method comprises the following specific steps: total RNA of leaf was extracted from transgenic Arabidopsis thaliana using Takara reagent (purchased from Takara Co.) (the extraction method was performed according to the Takara reagent instructions described above), and reverse-transcribed to synthesize cDNA using reverse transcriptase (Takara Co.) (reaction conditions were 42 ℃ for 2min, 37 ℃ for 15min, and 85 ℃ for 5s (see the reverse transcription kit instructions of Takara Co.). The fluorescent quantitative PCR was performed in the same manner as in example 2.

Based on the test results shown in FIG. 3, the reference gene was expressed at a high or low levelAtActin7For comparison, the number of the first and second reference points,RrCPCthe gene is not expressed in wild arabidopsis, but the majority of the five transgenic samples are compared with the gene in the selected transgenic samplesAtActin7The expression level is similar to or even higher thanAtActin7The expression level. This means thatRrCPCThe genes have been overexpressed in different transgenic lines. FromRrCPCSelecting over-expression strain with stable quantitative resultRrCPC-2、RrCPC-3、RrCPC-4And (4) harvesting the strain single plants to T3 generation, sterilizing and sowing, and observing the phenotype after the strain grows up. The results in FIG. 4A show that, in general, the rosette leaf coat hair of three over-expression strains is obviously reduced compared with the wild type, and when fresh leaves with similar sizes are taken and placed under a stereoscope for enlarged observation (FIG. 4B), the density of the coat hair of partial over-expression strain leaves is not more than half of that of the wild type, a certain number of coat hairs exist at the edges of partial leaves, and the epidermis of partial leaves is smooth and completely has no coat hair. Similarly, the near-root part and inflorescence of the main stems of the two types of arabidopsis are taken for magnified observation, compared with the wild type,RrCPCthe over-expressed lines appeared smooth and hairless in both sites (FIG. 4C, FIG. 4D). While wild type andRrCPCdownstream genes in transgenic ArabidopsisAtGL2(NM-001198514.1), as shown in FIG. 5, the results indicated the latterAtGL2The expression level of the gene is obviously reduced, which showsRrCPCThe gene is universal in transgenic arabidopsis thalianaOver-regulation of downstream genesAtGL2To regulate epidermal hair development.

The rose skin thorn and the skin fur are congeners, the negative regulatory factor of the skin fur is presumed to exist in the rose as the negative regulatory factor of the skin thorn, the expression level of the rose is improved through transgenosis, gene editing and other modes, a new rose germplasm with few thorns and no thorns can be created, and convenience is provided for daily field management and manual picking of the rose.

SEQ ID NO.1

RrCPCNucleotide sequence of gene

ACATGGGGAGCGGTTCAAAGATGAGCAAGCTCTTCTCTCAATGAGCCATTTAAAACTGAGCCTGTTTTTTTTCCTGTAGAAAACAAATTCCTCTTGGGAAACTATTTAAAACCCAATTCTTCCCCCTTCTAATCTTATCACTCCAAAAATTCTACTTGGGTTCCTCTCCCTCCTCTCTCTTTCATCTCCCTGTGTTCATTCTCAAATTGGCCCAACTACTCCAAAAGTGCTTGTGGAAAAAGCTAGCAGCTATGGACGTCAAACGCCGCAGAAAACAAGCCAAGACTTGCAGCAGTTCCTTCTCGGAAGAGGTAAGCAGCATTGAGTGGGAGTTCATAAACATGTCGGAACAAGAAGAAGATCTCATTTGTAGAATGTACAAGCTCGTCGGAGACAGGTGGGGACTTATAGCCGGGAGGCTTCCGGGCCGGAAAGCAGAAGAAATAGAGAGGTTCTGGTTAATGAGACACGGAGAAGTATTTGCAAGTAGAAGAAGAACAGAGCAGGAGAAGTACCAGCAACCCCAGCAGCTTAATAATAGCAATAACATGAATAAGTCTACTAAGAATAGTAGGAGATACAATTCTTGATCCTGATCAATCATCATCATTGGTTTTCCTTGTCAGTAGTAGTAAACAGTAGGACTTAGAACTAGCTAAAAGAAATTTAATTTCTAATTCATGTCTCTTTTTTTCCTTTTTGACCACCTACCTATGTGTGTTCACAAGGTAAGGTAGCTGTGACTTACCTCTTTGGTTAATCTATTTAGACTTATTACCCCTCTTAAAAAAAAAAAA

SEQ ID NO.2

RrCPC protein amino acid sequence

MDVKRRRKQAKTCSSSFSEEVSSIEWEFINMSEQEEDLICRMYKLVGDRWGLIAGRLPGRKAEEIERFWLMRHGEVFASRRRTEQEKYQQPQQLNNSNNMNKSTKNSRRYNS

Sequence listing

<110> Yangzhou university

<120> rose skin thorn regulation gene RrCPC and application thereof

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aagccaagac ttgcagcagt tcc 23

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Claims (7)

1. Rose skin thorn regulation geneRrCPCThe method is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1.

2. The rose skin regulating gene of claim 1RrCPCThe expression protein of (a), characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

3. Comprising the rose skin thorn regulatory gene of claim 1RrCPCA plant overexpression vector.

4. The carrier of claim 3, wherein: the carrier is inRrCPCThe 5 'end of the gene is assembled with a strong expression promoter P35S, and the 3' end is assembled with a strong terminator NOS, so that the gene can be overexpressed in plants.

5. The carrier of claim 2, wherein: the vector is assembled with an HPT gene expression cassette and a GUS reporter gene, is used as a screening marker, and can be used for screening transgenic plant materials through hygromycin and GUS staining.

6. The rose skin thorn development key gene of claim 1RrCPCThe plant host cell of (1).

7. The rose skin thorn regulatory gene of claim 1RrCPCThe application in regulating and controlling the development of rose skin thorn.

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