Rose organ development regulation gene and small peptide and application thereof

Abstract

The application discloses a Rosa chinensis organ development regulation gene, a small peptide thereof and application, and belongs to the technical field of plant genetic engineering.The application provides a kind of gene RwCLV3 for regulating the development of Rosa chinensis flower organ, and the nucleotide sequence is shown as SEQ ID NO.1;The application effectively regulates the development of Rosa chinensis and Arabidopsis thaliana flower organ by means of genetic engineering, can significantly change the size of apical meristem and the development and quantity of flower organ.The application provides a new gene resource for improving plant flower organ development and improving ornamental.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a gene regulating the development of rose flower organs, its small peptides, and their applications. Background Technology

[0002] Roses are the world's most traded cut flower, possessing significant horticultural and economic value, with over 37,000 cultivars currently. The number of petals in roses is subject to intense selection during breeding; wild roses predominantly have only four or five petals, exhibiting a single-petal trait, while cultivated varieties are mostly double-petaled. As the most important ornamental and economically important organ of the rose, the genetic regulation mechanism of floral organs (especially petals) has attracted considerable attention from scientists and breeders. However, research on the regulatory mechanism of rose floral organ number has largely focused on homeomorphic transformation; whether other regulatory pathways influence the number of rose floral organs remains unclear. Whether floral organ traits can be altered through the transformation of key genes or the application of conserved small peptides has significant scientific and industrial value. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a gene regulating the development of rose flower organs, its small peptide, and its applications. This invention effectively regulates the development of flower organs in roses and Arabidopsis thaliana through genetic engineering, significantly altering the size of the apical meristem and the development and number of flower organs. This invention provides a new genetic resource for improving plant flower organ development and enhancing ornamental value.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a gene for regulating the development of rose organs, wherein the gene is RwCLV3 and its nucleotide sequence is shown in SEQ ID NO. 1.

[0005] The beneficial effects of this invention are: (1) The present invention provides the rose flower organ development regulatory gene RwCLV3; its expression domain is negatively correlated with the total number of rose flower organs. Genetic transformation in rose and Arabidopsis to change the expression of CLV3 significantly altered the size of the apical meristem and the development and number of flower organs. Exogenous application of the CLAVATA3 / EMBRYO SURROUNDING REGION-related (CLE) polypeptide encoded by RwCLV3 to rose and Arabidopsis plants resulted in smaller apical meristems and shorter roots, providing new insights into the molecular mechanisms of the quantitative traits of flower organs in roses and other ornamental plants.

[0006] (2) The CLV3 expression of the rose in this invention negatively regulates the size of the apical meristem, thereby regulating the total number of floral organs in roses and Arabidopsis thaliana. In addition to the homeomorphic conversion mechanism reported in previous studies, this invention establishes for the first time a correlation between CLV3 expression and variation in the number of floral organs in roses. The analysis in this invention provides new insights into the genetic and molecular regulatory mechanisms of floral organ number diversity in roses, thus laying a theoretical foundation for the genetic improvement and germplasm innovation of various horticultural plant varieties in next-generation breeding programs.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] The second objective of this invention is to provide a small peptide for regulating the development of rose organs, wherein the small peptide is CLV3p, and its amino acid sequence is shown in SEQ ID NO. 2.

[0009] The third objective of this invention is to provide a vector for genes regulating the development of rose organs.

[0010] The fourth objective of this invention is to provide a recombinant bacterium that regulates the development of rose organs.

[0011] The fifth objective of this invention is to provide a cell containing genes that regulate the development of rose organs.

[0012] The sixth objective of this invention is to provide an application of a rose flower organ development regulatory gene, specifically the application of the rose flower organ development regulatory gene RwCLV3 in improving the traits of plant flower organs.

[0013] Furthermore, the plant floral organ trait refers to the number of floral organs.

[0014] Furthermore, the rose organ development regulatory gene RwCLV3 is also used to regulate the size of the plant apical meristem.

[0015] Furthermore, the rose organ development regulatory gene RwCLV3 is also used to regulate plant root length.

[0016] Furthermore, the plant in question is either a rose or Arabidopsis thaliana. Attached Figure Description

[0017] Figure 1Phylogenetic clustering of the rose CLV3 gene and its specific expression in floral meristems were presented. (A) shows the identification of a single copy of the RwCLV3 gene in the genome of 'Rosa spp.' using a phylogenetic tree constructed by neighbor-joining. Red, brown, and purple represent the three main branches. Bold red IDs label Arabidopsis thaliana AtCLV3 and its rose homolog (RwCLV3, Rw3G014940); numbers and asterisks on the branches indicate support rates, with red asterisks indicating support rates higher than 0.8. (B) shows the gene and protein structure of RwCLV3. (C) shows the specific expression of CLV3 in floral meristems as shown by RT-qPCR. (DF) shows the subcellular localization of 35S:GFP detected in transiently transformed tobacco epidermal cells. (GI) shows the subcellular localization of the 35S:RwCLV3-GFP fusion protein detected in transiently transformed tobacco epidermal cells; GFP is green fluorescent protein; BF is bright field; MERGE is the merge channel.

[0018] Figure 2 This is an in situ hybridization analysis diagram of Example 4 of the present invention; where (AC) represent the expression patterns of CLV3 at the apex of the reproductive stage of C195 ("Sweet Beauty") at different developmental stages (S1, S2, S3); (EG) represent the expression patterns of CLV3 at the apex of the reproductive stage of C244 ("Drunken Beauty") at different developmental stages (S1, S2, S3); and (D, H) represent negative controls obtained by detecting the expression of CLV3 at the apex of the reproductive stage of C195 and C244 using orthogonal probes.

[0019] Figure 3 The RwCLV3-encoded CLE peptide (CLV3p) of Example 5 of this invention significantly inhibited the size of the apical meristem and the length of the primary root in Arabidopsis thaliana; (A) compared with the application of sCLV3p (random sequence control peptide) and no application of the small peptide, the exogenous application of 10 μM CLV3p significantly inhibited the size of the apical meristem, scale bar = 50 μm; (B) Arabidopsis thaliana treated with 10 μM CLV3p, sCLV3p and no application of the small peptide, respectively, and the size of the apical meristem was measured 11 days after germination; (C) root growth profile after exogenous application of CLV3p compared with the control treatment; (D) the length of the primary root of two-week-old Arabidopsis thaliana after exogenous application of CLV3p, sCLV3p and no application of the small peptide was quantified.

[0020] Figure 4Example 5 of this invention describes the use of exogenous CLV3p to inhibit the size of the apical meristem and root growth of roses. (A) Compared to the application of sCLV3p and no small peptide, the size of the apical meristem of roses was significantly inhibited after two weeks of exogenous application of 60 μM CLV3p. (B) Compared to the application of sCLV3p and no small peptide, the root length of roses was inhibited after two weeks of exogenous application of 50 μM CLV3p (scale bar = 1 cm). (C) The size of the apical meristem was quantitatively analyzed after treating roses with 60 μM CLV3p, sCLV3p, and no small peptide. (D) The length of the primary root was quantitatively analyzed after treatment with 50 μM CLV3p, sCLV3p, and no small peptide. Statistical analysis was performed using one-way ANOVA, and the results are represented by lowercase letters a and b (P < 0.05).

[0021] Figure 5 This section describes the regulation of rose flower organ number by CLV3 in Example 6. (A) shows the quantitative analysis of flower organ number in transient transgenic plants of pTRV control (n=10), pTRV-CLV3 (n=7), and pTRV-OE-CLV3 (n=17); (B) shows the data distribution of the total number of flower organs in transient transgenic plants of pTRV control, pTRV-CLV3, and pTRV-OE-CLV3, with the numbers in the rectangles representing the number of flowers distributed in different intervals; (C) shows the data distribution of the number of petals in plants of pTRV control, pTRV-CLV3, and pTRV-OE-CLV3, with the numbers in the rectangles representing the number of flowers distributed in different intervals; (D) shows the anatomical phenotype of flower organs in plants of pTRV control, pTRV-CLV3, and pTRV-OE-CLV3 (scale bar = 1 cm).

[0022] Figure 6Example 7 illustrates the regulation of Arabidopsis flower organ number by CLV3. (A) shows the growth phenotype of a 2-month-old T2 generation transgenic positive plantlet. Scale bar = 1 cm; (B) shows the growth phenotype of a 2-week-old wild-type Col-0 seedling. Scale bar = 2 mm; (CD) shows the growth phenotypes of two 2-week-old T2 generation transgenic lines (C, D), with arrows indicating early cessation of shoot apical meristem development. Scale bar = 2 mm; (E) compares the siliques of Col-0 (left) with those of the two transgenic lines (middle, right). Scale bar = 2 mm. mm; (F) Shoot apical meristem (SAM) phenotype of Col-0; black dashed lines indicate meristem size, scale bar = 50 μm; (G) Shoot apical meristem (SAM) phenotype of transgenic lines; black dashed lines indicate meristem size, scale bar = 50 μm; (H) Distribution of SAM size data between Col-0 and T2 generation lines from #20, #24, and #25; (I) Floral phenotype of Col-0, scale bar = 500 μm; (J) Floral organ phenotype of Col-0, scale bar = 2 mm; (K) Floral phenotype of T2 generation transgenic line #24-9, scale bar = 500 μm; (L) Floral organ phenotype of T2 generation transgenic line #24-9, scale bar = 2 mm. (M) is the floral phenotype of the T2 generation transgenic line #25-8, scale bar = 500 μm; (N) is the floral organ phenotype of the T2 generation transgenic line #25-8, scale bar = 2 mm; (O) is the floral phenotype of the T2 generation transgenic line #24-6, scale bar = 500 μm; (P) is the floral organ phenotype of the T2 generation transgenic line #24-6, scale bar = 2 mm; (Q) is the statistical count of floral organs of Col-0 and T2 generation plants from #17, #18, #20, #24, and #25. Data are expressed as mean ± standard deviation. “n” indicates the number of flowers counted. An asterisk indicates a significant difference in the number of floral organs in the transgenic lines compared to Col-0 (Student's t-test; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). Detailed Implementation

[0023] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1: Phylogenetic Clustering and Sequence Analysis Protein sequences of 31 members of the Arabidopsis CLE gene family were identified by searching the Arabidopsis database (https: / / www.arabidopsis.org / ). BLASTp (parameter setting: e=10) was used to analyze the sequences. -5(Minimum sequence identity = 0.5, minimum length coverage = 0.6) Searched for rose homologous sequences in the genome of 'Rosa wichuraiana Basye's Thornless (BT) (Zhong MC, Jiang XD, Yang GQ, Cui WH, Suo ZQ, Wang WJ, Sun YB, Wang D, Cheng XC, Li XM, Dong X, Tang KX, Li DZ, Hu JY (2021b) Rose without prickle:genomic insights linked to moisture adaptation. Natl Sci Rev 8(12): nwab092). The retrieved rose homologous sequences were compared with the protein sequences of 31 members of the Arabidopsis CLE family using MAFFT. Following the method used in the study by Zhong MC, Jiang XD, Cui WH, Hu JY (2021a) Expansion and expression diversity of FAR1 / FRS-like genes provides insights into flowering time regulation in roses. Plant Divers 43(2): 173-179, a phylogenetic tree was constructed using FastTree 2.1 (Price MN, Dehal PS, Arkin AP (2010) FastTree 2--approximatelymaximum-likelihood trees for large alignments. PLoS ONE 5(3): e9490) through neighbor-joining, and a bootstrap replicate test was performed. Conserved protein domains were analyzed using InterProScan, and visualization was achieved using DOG 2.0 software.

[0025] The results are as follows Figure 1 (A) Figure 1 As shown in (B): BLAST alignment of 31 Arabidopsis CLE gene family members identified 15 highly homologous genes in the BT rose genome. For example... Figure 1As shown in (A), phylogenetic cluster analysis revealed that the CLE family in roses comprises 15 members, forming three main branches. Among them, Rw3G014940 is a single-copy gene with the highest similarity to the Arabidopsis CLV3 gene, and is hereby named RwCLV3, with its nucleotide sequence shown in SEQ ID NO. 1. RwCLV3 corresponds to the RcHm3g0470211 gene in the "Old Blush" (OB) rose.

[0026] like Figure 1 As shown in (B), the RwCLV3 gene is located on chromosome 3 of BT, 2806 bp upstream and 16.763 kb downstream. The full-length RwCLV3 gene sequence is 474 bp, containing two introns and three exons, encoding a protein of 95 amino acids. Domain homology analysis identified a signal peptide domain and a non-cytoplasmic domain encoding a 12-amino acid CLE peptide.

[0027] Example 2: RT-qPCR analysis of RwCLV3 expression profile In this embodiment, the rose RNA extraction kit was purchased from Kangwei Century (CW2598, CW0553S); the reverse transcription and real-time quantitative PCR kit was purchased from Nearshore Protein (E047-01B, E096-01B). To elucidate the tissue-specific expression of RwCLV3, seven tissue samples were collected from BT (Rosa buds): young shoots, leaves, flower primordia, sepals, petals, stamens, and pistils, for RNA extraction using a rose RNA extraction kit. RNA isolation and quality control followed the protocol described in Sun YB, Zhang XJ, Zhong MC, Dong X, Yu DM, Jiang XD, Wang D, Cui WH, Chen JH, Hu JY (2020) Genome-wide identification of WD40 genes reveals a functional diversification of COP1-like genes in Rosaceae. Plant Mol Biol 104(1-2): 81-95. Transcription and real-time quantitative PCR kits were used, with RwPP2A (Rw4G029830) as an internal control gene, and quantitative analysis was performed using an Applied Biosystems QuantStudio 7 Flex real-time quantitative PCR system. At least three biological replicates were set up for each type of tissue sample, each containing three technical replicates. Primer sequences are detailed in Table 1 (where qRwPP2A-F / R was used to amplify RwPP2A, and qRwCLV3-2F / R was used to amplify RwCLV3). The results are as follows: Figure 1 As shown in (C): Table 1 Depend on Figure 1 (C) We can obtain: RwCLV3 is specifically expressed in floral meristems, but its expression level is extremely low in leaves, stems, or fully developed floral organs, indicating that RwCLV3 mainly functions in meristems and affects the development process of roses.

[0028] Subsequently, its function was verified through transgenic experiments in roses and Arabidopsis thaliana.

[0029] Example 3: Detection of subcellular localization in tobacco leaves by transient transformation with RwCLV3 Primers RwCLV3-P30F / R were designed and synthesized (primer sequences are detailed in Table 2). The full-length coding sequence of RwCLV3 was amplified from a cDNA library of BT shoot apex meristem (SAM). This sequence was inserted into all available p30-3XMYC-GFP vectors in our laboratory, and the accuracy of the p30-3XMYC-RwCLV3-GFP recombinant vector was verified by Sanger sequencing. The p30-3XMYC-GFP vector carrying 35S::RwCLV3-GFP and 35S::GFP was independently transformed into Agrobacterium GV3101 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) using the heat shock method. Single clones were picked and cultured at 28°C for two days. After collecting the cells, they were resuspended in infection buffer (10 mM MES, 10 mM MgCl2, 200 μM acetylsyleugenol, pH 5.8) to prepare the infection solution. 600 The concentration was 1.0, and the mixture was left to stand in the dark for 2 hours. The infecting bacterial solution was injected onto the underside of well-grown tobacco leaves using a syringe. After culturing for 48 hours, the lower epidermal cells of the tobacco leaves were treated with 0.8 M mannitol solution to induce plasmolysis. The RwCLV3-GFP and GFP signals were then observed using a laser confocal microscope (FV10-ASW, Olympus, Japan).

[0030] Table 2 The results are as follows Figure 1 (D)- Figure 1 (I) We can obtain: In the region where plasmolysis occurs in tobacco lower epidermal cells, no signal was detected in the intercellular spaces of adjacent cells expressing 35S:GFP, indicating that the signal is located in the cell membrane; however, a signal was detected in the intercellular spaces of adjacent cells expressing 35S:RwCLV3-GFP chimeric protein, indicating that the protein encoded by RwCLV3 is similar to the protein encoded by Arabidopsis thaliana CLV3 and is mainly located in the intercellular spaces.

[0031] Example 4: In situ hybridization The sense and antisense strand sequences of RwCLV3 were cloned, and the primer sequences used are detailed in Table 3. RoCLV3-F (AS) and T7-RoCLV3-R (AS) were used to amplify the antisense strand, and RoCLV3-F (S) and T7-RoCLV3-R (S) were used to amplify the sense strand. They were then ligated into the pEASY-Blunt cloning vector CB101 (TransGen, China), and RNA probes were prepared using 10xDIG RNALabeling Mix solution (11277073910, Roche, Switzerland). Shoot apical meristems of single-petaled C195 (“Sweet Beauty”) and double-petaled C244 (“Drunken Beauty”) at different developmental stages (S1, S2, S3) were isolated and sectioned using a Leica RM 225587 microtome. In situ hybridization experiments were performed according to the method described in the study of Liu Y, Yang Y, Wang R, Liu M, Ji X, He Y, Zhao B, Li W, Mo X, Zhang X, Gu Z, Pan B, Liu Y, Tadege M, Chen J, He L (2023) Control of compound leaf patterning by MULTI-PINNATE LEAF1 (MPL1) inchickpea. Nat. Commun. 14(1): 8088. Observation was performed under an Olympus BX63 microscope.

[0032] Table 3 like Figure 2 As shown: When sepal primordia appear in the lateral regions of the floral meristem (S1 stage), CLV3 mRNA is detected in layer L3 of double-flowered C244, while in single-flowered C195, CLV3 signal is clearly visible in layers L1-L3. Figure 2 A, Figure 2 E). When the petal primordia attach (S2 stage), the expression domain of CLV3 in C195 expands ( Figure 2 B Figure 2 F). In the S3 stage of stamen primordium appearance, the CLV3 signal drops sharply in C195 and is almost undetectable in C244. Figure 2 C Figure 2 G). In summary, the CLV3 expression domain is negatively correlated with the size of floral meristems and the number of floral organs.

[0033] Example 5: Treatment of Arabidopsis thaliana and rose plants with exogenous CLV3p-related peptides (1) Exogenous application of CLV3p to Arabidopsis thaliana The CLE peptide encoded by CLV3 of the rose (CLV3p; amino acid sequence as shown in SEQ ID NO. 2: RKVPSGPDPLHH) and its random sequence control peptide (sCLV3p; sequence: PPTRGLSHHPVD) were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity >98%. 10 μM of CLV3p and sCLV3p were added to 1 / 2 MS medium, and a blank control group (1 / 2 MS medium without any form of small peptide) was set up.

[0034] After surface sterilization, wild-type Arabidopsis thaliana Col-0 seeds were randomly divided into three groups. The seeds were sown in three different media: 1 / 2 MS medium without peptides, 1 / 2 MS medium containing 10 μM CLV3p, and 1 / 2 MS medium containing 10 μM sCLV3p. The media were cultured at 22°C under long-day conditions (16 hours light / 8 hours dark). After 11 days, the shoot tips were dissected and fixed in FAA solution. The size of the shoot tip meristem (SAM) in each group was measured. On day 14 after germination, photographs were taken and root length was measured. Figure 3 As shown.

[0035] (2) Exogenous application of CLV3p to rose plants Aseptic 'Samantha' cuttings were inserted into proliferation media supplemented with 60 μM CLV3p, 60 μM sCLV3p, and peptide-free (-CLV3p) media (4.4 g / L MS; 30 g / L sucrose; 1.0 mg / L 6-benzylaminopurine (6-BA); 0.05 mg / L naphthaleneacetic acid (1-NAA); 6.8 g agar; pH 6.0). After two weeks of culture at 22°C under long-day conditions (16 hours light / 8 hours dark), shoot tips were harvested and fixed in FAA solution (1.9% formaldehyde, 5% acetic acid, and 63% ethanol) for SAM size measurement.

[0036] Aseptic 'Samantha' cuttings were inserted into rooting medium (2.2 g / L MS salt, 30 g / L sucrose, 0.1 mg / L NAA, 7.5 g / L agar, pH 6.0) and cultured for three weeks under long-day conditions (16 hours light / 8 hours dark) at 22°C. Cuttings showing signs of rooting were selected and transferred to rooting medium containing 50 μM CLV3p, 50 μM sCLV3p, and no peptide (-CLV3p) (2.2 g / L MS salt, 30 g / L sucrose, 0.1 mg / L NAA, 7.5 g / L agar, pH 6.0). They were then cultured for another two weeks under long-day conditions (16 hours light / 8 hours dark) at 22°C. Root length was photographed and measured.

[0037] Shoot tips fixed in FAA solution were used to prepare paraffin sections. After immersing the material in FAA fixative (1.9% formaldehyde, 5% glacial acetic acid, 63% ethanol) for approximately 48 hours, followed by dehydration, paraffin infiltration, embedding, sectioning, dewaxing, staining, and mounting, the size of the shoot tips (SAM) was measured using an optical microscope (Zeiss, Germany; Axio Lab.A1). Root length data were acquired using a digital camera (Canon, Japan: EOS 70D). Both root length and SAM size were measured using ImageJ software.

[0038] The results are as follows Figure 3 , 4 As shown: Figure 3 (A) Figure 3 (B) shows that exogenous application of 10 μM CLV3p significantly inhibited the size of Arabidopsis thaliana SAM compared with application of sCLV3p and no application of small peptide. Figure 3 (C) Figure 3 (D) indicates that exogenous application of 10 μM CLV3p significantly inhibited root length compared to application of sCLV3p and no application of the small peptide. In conclusion, exogenous application of CLV3p encoded by rose CLV3 in Arabidopsis plants leads to smaller apical meristems and shorter root length.

[0039] Figure 4 (A) Figure 4 (C) indicates that, compared with the application of sCLV3p and the absence of small peptides, the size of rose apical meristem was significantly inhibited after two weeks of exogenous application of 60 μM CLV3p. Figure 4 (B) Figure 4 (D) Compared with the application of sCLV3p and no small peptide, the root length of rose was inhibited after two weeks of exogenous application of 50 μM CLV3p. It can be seen that, consistent with the phenotype observed in Arabidopsis, exogenous application of CLV3p significantly inhibited the SAM size and root growth of rose.

[0040] Example 6: Instant Transformation of Roses (1) A 261bp fragment was amplified from the CLV3 coding region sequence and inserted into the pTRV2 vector (Nanjing Agricultural University) to construct the pTRV2-CLV3 recombinant vector for transient silencing experiments. The full-length CLV3 CDS was cloned into the pTRV2-OE vector (Nanjing Agricultural University) to construct the pTRV2-OE-CLV3 recombinant vector for transient overexpression experiments. Primer sequences are detailed in Table 4. Among them, CLV3-TRV2-F / R was used to amplify the 261bp fragment, and CLV3-TRV2-OE-F / R was used to amplify the full-length CDS sequence. The pTRV1 (Nanjing Agricultural University), pTRV2, pTRV2-CLV3, and pTRV2-OE-CLV3 vectors were independently transformed into Agrobacterium GV3101 strain by heat shock.

[0041] Table 4 (2) Centrifuge the Agrobacterium GV3101 strain cultured in step (1) (5000 rpm, 7 min), and resuspend the collected bacterial cells in infection buffer (10 mM MgCl2, 200 mM AS, 10 mM MES, pH 5.6) to OD. 600 =1.2. For the control group, pTRV1 and pTRV2 were mixed in equal volumes; for the experimental group, pTRV1 and pTRV2-CLV3 or pTRV2-OE:CLV3 were mixed in equal volumes. Incubate in the dark for 3 hours.

[0042] (3) Rose (R. chinensis 'Old Blush', 'Monthly Pink') seedlings were immersed in Agrobacterium suspension in the experimental and control groups and subjected to two vacuum treatments (-25 kPa, 5 minutes each time) to promote bacterial penetration. After infection, the seedlings were placed in a dark, high-humidity environment for 16 hours (to enhance Agrobacterium adhesion). The seedlings were rinsed with running water, transplanted into culture pots, and cultured in an artificial climate chamber (22℃, 65% humidity, photoperiod of 16 hours light / 8 hours dark). (4) After 30 days of Agrobacterium infiltration treatment, positive transformed plants were detected by PCR to verify the systematic diffusion of TRV virus particles in leaves. Primer sequences are detailed in Table 5. Among them, F1 / R1, F1 / R2, and F2 / R1 were used to detect the diffusion of virus particles in leaves of pTRV control, pTRV-CLV3, and pTRV-OE-CLV3 transiently transformed plants, respectively. Floral organ counting was performed after the flowers were fully open, and Student's t-test was used to compare the difference in the number of floral organs between the experimental group (silenced / overexpressed) and the control group (empty vector).

[0043] Table 5 like Figure 5 As shown: To investigate the regulatory mechanism of CLV3 on the number of flower organs in roses, a transient transgenic experiment was conducted on OB cuttings. The total number of flower organs in 10 transgenic positive control plants transformed with the empty vector (pTRV) ranged from 52 to 68 (mean 59.7 ± 6.1), and the number of petals ranged from 21 to 40 (mean 28.4 ± 5.8). Figure 5 A-5C). The total number of floral organs in the CLV3 overexpression line (pTRV-OE-CLV3) decreased to 38-62 (mean 53.9±7.5), and the number of petals decreased to 7-37 (mean 18.7±7.7), significantly lower than the control group (total organs decreased by 5.8, or 9.7%, P=0.04; petals decreased by 9.7, or 34.1%, P=0.001; t-test results). Among the 7 positive plants transformed with the pTRV-CLV3 vector, 3 had a total number of floral organs exceeding 70, but the number of petals was not significantly different from the control (…). Figure 5 B, 5C).

[0044] In summary, the CLV3 gene in roses is involved in regulating the total number of floral organs.

[0045] Example 7: Heterologous transformation of Arabidopsis thaliana (1) The Agrobacterium GV3101 strain transformed into the p30-3XMYC-RwCLV3-GFP vector in Example 3 was taken out from the -80℃ freezer and activated. 5 mL of the culture was picked and added to LB medium containing Rifampicin (purchased from Solarbio), Gentamicin (purchased from Solarbio), and Spectinomycin (purchased from Solarbio). The culture was shaken overnight at 28℃ and 200 rpm. The shaken culture was transferred to 300 mL of the above LB medium and shaken vigorously at 28℃ and 200 rpm until the OD value was 1.2. (2) Take out the bacterial culture after shaking, centrifuge at 5000 rpm for 10 min at room temperature to collect the bacterial cells, discard the supernatant, add an appropriate amount of 5% sucrose solution to resuspend the bacterial cells, and adjust the OD value to about 0.8; then add acetylsuccinone solution with a final concentration of 100 μM to the resuspended bacterial cells and place at room temperature in the dark for 2 h. (3) Add Silwet-77 solution to the bacterial culture to a final concentration of 0.5% and shake well. Select vigorous Arabidopsis plants that are in the flowering period, immerse all the inflorescences of Arabidopsis in the bacterial culture for 20 seconds, then let them stand in the dark for 24 hours before restoring light for culture. Repeat the transformation 2-3 times. In the later stage, collect seeds and screen positive plants to obtain a total of 25 T1 generation transgenic lines. Among these T1 generation lines, some plants died prematurely or developed abnormal flowers that could not produce seeds. Only 5 lines (#17, #18, #20, #24 and #25) obtained a considerable number of seeds. After germination and screening, each line was numbered in the order of 1, 2, 3... for phenotypic observation and statistics.

[0046] like Figure 6 As shown: Some T2 generation plants have only 3-4 flowers developed from their inflorescence meristems. Figure 6 A), and the number of floral organs in the inner two whorls is reduced ( Figure 6 IP, 6Q). Compared to the wild type, most T2 generation plants had reduced shoot apical meristem (SAM). Figure 6 FH), and many flowers failed to develop into normal seed-bearing siliques (FH). Figure 6 E). The apical meristem of wild-type plants continues to produce rosette leaves during the vegetative stage. Figure 6 B), while in some T2 generation seedlings, the apical meristem shows developmental arrest after the emergence of two cotyledons and several normal or malformed leaves. Figure 6 CD). The above results indicate that the rose CLV3 gene is involved in regulating the size of the apical meristem and the number of floral organs in Arabidopsis thaliana.

[0047] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A gene regulating the development of rose flower organs, characterized in that, The gene regulating the development of rose organs is RwCLV3, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A small peptide encoded by a gene regulating the development of rose flower organs, characterized in that, The small peptide is CLV3p, and its amino acid sequence is shown in SEQ ID NO.

2.

3. A vector containing the gene for regulating the development of rose organs as described in claim 1.

4. A recombinant bacterium containing the gene for regulating the development of rose organs as described in claim 1.

5. A cell containing a gene for regulating the development of rose organs as described in claim 1.

6. The application of a gene regulating the development of rose flower organs, characterized in that, Application of the rose flower organ development regulatory gene RwCLV3 as described in claim 1 in improving the traits of plant flower organs.

7. The application of a gene regulating the development of rose organs according to claim 6, characterized in that, The floral organ trait of the plant refers to the number of floral organs.

8. The application of a gene regulating the development of rose organs according to claim 6, characterized in that, The rose organ development regulatory gene RwCLV3 is also used to regulate the size of the plant apical meristem.

9. The application of a gene regulating the development of rose organs according to claim 6, characterized in that, The rose organ development regulatory gene RwCLV3 is also used to regulate plant root length.

10. The application of a rose flower organ development regulatory gene according to any one of claims 6 to 9, characterized in that, The plant in question is either rose or Arabidopsis thaliana.

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