Rosa chinensis RcFULL gene and application thereof in regulation and control of floral organ development

Abstract

The invention relates to a gene RcFULL for regulating and controlling organ development of China rose. The sequence of the gene RcFULL is as shown in SEQ ID NO. 1. The RcFULL gene is cloned in'calyx ', heterologous expression of the RcFULL gene causes precocious flowering of an arabidopsis thaliana positive strain, and compared with an empty vector transfected plant, the pistil form of a flower of the RcFULL positive plant is prominent and longer, the number and the form of stamens are not changed, but the condition that the stamens and the pistils cannot be self-pollinated occurs. The color of petals of the RcFULL positive plant is green, the color close to the main vein is deepest, the edge is white, the shape is similar to a leaf shape, the petals become smaller, the whole plant is in a narrow oval shape, the color and the shape of sepals are not greatly different, but the number of the sepals is increased. The result of the invention provides an important clue for further understanding the function of the RcFULL gene in China rose development.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to the rose RcFULL gene and its application in regulating floral organ development. Background Technology

[0002] The rose (Rosa chinensis) is a semi-evergreen shrub or vine belonging to the genus Rosa in the family Rosaceae. Renowned as the "Queen of Flowers," it ranks among China's top ten famous flowers, its blossoms exhibiting diverse value in ornamental, edible, medicinal, and cultural fields. Currently, research on rose flower morphology largely focuses on the mechanism of petal number regulation. However, research on the developmental mechanism of the popular European cut flower variety 'Green Calyx', due to its leaf-like characteristics in its stamens, pistils, and petals (i.e., the "flower-to-leaf" phenomenon), is still in its early stages.

[0003] While the molecular regulatory mechanisms of floral organ development are complex, scientists have established the classic "ABCDE model" to explain the developmental patterns of floral organs through systematic research on regulatory genes. In practical applications, because class D genes mainly participate in the development and regulation of ovules (carpel derivatives), the "ABCE model," which focuses more on core floral organs such as sepals, petals, stamens, and pistils, is widely used in plant floral organ development research. This model reveals that class A genes alone determine sepal morphology; class A and class B genes synergistically regulate petal formation; class B and class C genes jointly drive stamen development; class C genes dominate pistil development; and class E genes act as "global regulators," participating in the developmental process of all floral organs. In the model plant Arabidopsis thaliana, simultaneous overexpression of class ABCE genes can induce homeomorphic transformation of leaves, directly developing into floral organs, thus confirming the regulatory mechanism of this model. In the field of rose research, Han et al.'s study on the ancient variety 'Yueyuefen' found that the class A gene RcAP1 was not expressed in the early stage of flower bud differentiation, but was specifically highly expressed only in the sepals in the later stage of development. Moreover, the expression pattern of this gene in the sepals of 'Yueyuefen' and the variant 'Lü'e' was similar (Han Y, Tang A, Yu J, et al. RcAP1, a homolog of APETALA1, is associated with flower bud differentiation and floral organmorphogenesis in Rosa chinensis[J]. International Journal of Molecular Sciences, 2019, 20(14):3557). Similar homologous transformation phenomena also exist in petunias—silencing the expression of the class C gene pMADS3 can cause the stamens to transform into petal-like structures. These findings indicate that the ABCE model has a conserved regulatory mechanism in different species, but also suggest that there may be a unique gene regulatory network for the variation of floral organs in ornamental plants such as roses. Research on rose flower shapes can provide scientific support for the protection of traditional germplasm resources, innovation in the flower industry, and efficiency improvement in flower cultivation.

[0004] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the first aspect of the present invention provides a gene RcFULL that regulates the development of rose flower organs, the sequence of which is shown in SEQ ID NO.1.

[0006] According to a preferred embodiment, the rose variety is 'Green Calyx'.

[0007] A second aspect of the present invention provides a method for altering the morphology of rose flower organs, the method comprising:

[0008] Overexpression of the rose RcFULL gene, and / or

[0009] Overexpression of the rose RcFULL protein, wherein the RcFULL gene sequence is shown in SEQ ID NO.1, and the RcFULL protein is encoded by the RcFULL gene. The amino acid sequence of the RcFULL protein is shown in SEQ ID NO.2.

[0010] The third aspect of this invention provides the application of the gene RcFULL provided in the first aspect of this invention and the method provided in the second aspect of this invention in altering the flowering time of roses.

[0011] According to a preferred embodiment, the flowering time of roses is changed to promote earlier flowering of roses.

[0012] The fourth aspect of this invention provides the application of the gene RcFULL provided in the first aspect of this invention and the method provided in the second aspect of this invention in altering the morphology of rose pistils.

[0013] According to a preferred embodiment, the morphology of the rose pistil is altered to promote pistil elongation.

[0014] The fifth aspect of the present invention provides the application of the gene RcFULL provided in the first aspect of the present invention and the method provided in the second aspect of the present invention in altering the morphology of petals.

[0015] According to a preferred embodiment, the petal shape is changed to reduce the petal area.

[0016] The sixth aspect of the present invention provides the application of the gene RcFULL provided in the first aspect of the present invention and the method provided in the second aspect of the present invention in increasing the number of sepals.

[0017] The seventh aspect of the present invention provides an expression vector containing the RcFULL gene provided in the first aspect of the present invention.

[0018] The eighth aspect of the present invention provides a strain containing the RcFULL gene provided in the first aspect of the present invention.

[0019] Technical effects of the present invention:

[0020] The RcFULL gene belongs to the MADS-box gene family and primarily affects the development of petals and sepals. In the results of this invention, among Arabidopsis thaliana plants heterologously overexpressing RcFULL, positive seedlings selected from T1 generation seeds showed two plants exhibiting leaf-like petal morphology, increased sepals, smaller petals, elongated stigmas, and sterile stamens and pistils. One of the RcFULL-expressing Arabidopsis thaliana seedlings, with slightly lower expression levels, showed a small number of flowers with normal morphology. It is speculated that this may be because the overexpression vector used had a 35S promoter, preventing the gene from being concentrated in the floral organs.

[0021] Furthermore, this invention cloned the RcFULL gene from 'Green Calyx' rose. Bioinformatics analysis showed it to be a hydrophilic protein containing two conserved domains, exhibiting varying degrees of phylogenetic relationship with other species in the genus Rosa, with the closest phylogenetic relationship to the rose. Predictive analysis revealed the gene is located in the cell nucleus. Heterologous overexpression experiments in Arabidopsis thaliana showed that the petals of RcFULL transgenic Arabidopsis exhibited a greening effect, a leaf-like morphology, multiple sepals, and inability to produce fruit. The results of this invention provide important clues for further understanding the function of the RcFULL gene in rose flower development, offer a theoretical basis for elucidating the genetic regulatory network of rose flower morphology, and thus lay a solid foundation for molecular breeding improvement of high-quality rose varieties. Attached Figure Description

[0022] Figure 1 Results of RcFULL gene cloning;

[0023] Figure 2 This is the RcFULL phylogenetic tree;

[0024] Figure 3 Summary of bioinformatics analysis results for the RcFULL gene;

[0025] Figure 4 Subcellular localization results of RcFULL protein;

[0026] Figure 5 The results of the identification of positive Arabidopsis thaliana seedlings;

[0027] Figure 6 Results of expression level analysis in RcFULL transgenic Arabidopsis thaliana;

[0028] Figure 7 The results of the RcFULL gene transgenic phenotype in Arabidopsis thaliana;

[0029] Figure 8 The results are for verification of RcFULL positive plants;

[0030] Figure 9 The results show the comparison of pistil length. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of this disclosure. In the following detailed description, numerous specific details are set forth to provide a comprehensive understanding of the embodiments of this disclosure for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having meanings consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0032] In the preliminary research, this invention identified the RcFULL gene as a key candidate gene for floral organ development by comparing transcriptome data from different developmental stages of floral organs in 'Yuyuefen' and 'Lü'e'. Furthermore, this application cloned the RcFULL gene from 'Lü'e', and used phylogenetic tree construction and bioinformatics analysis techniques to clarify its sequence characteristics and evolutionary position; it explored the action site of the RcFULL protein using subcellular localization technology; and it elucidated the function of this gene in the floral organ development process through heterologous overexpression experiments in Arabidopsis thaliana. Specifically, this invention utilizes the NCBI online website to search for homologous sequences of the RcFULL gene in Arabidopsis thaliana; constructs a phylogenetic tree using MEGA 11.0; predicts the isoelectric point, adipose coefficient, and other basic physicochemical properties of the protein encoded by the RcFULL gene using the ProtParam online website; predicts the affinity of the protein using the ProtScale online website; predicts the secondary and tertiary structures of the protein using the SOPMA and SWISS-MODEL online tools, respectively; predicts the signal peptide sites and transmembrane regions of the protein sequence using SignalP 4.1 and TMHMM 2.0 online software; performs a BLAST procedure on the RcFULL protein sequence on the NCBI website to search for homologous sequences in other species, and subsequently performs amino acid multiple sequence alignment analysis using DNAMAN software; performs structural conserved domain prediction analysis of the target protein using the CD-Search online website of NCBI; and predicts the subcellular localization of the protein using the WoLF-PSORT online website.

[0033] In the following examples, 'Green Calyx' (Rosa chinensis 'Viridiflora') was used as the material, which was provided by the Beijing Academy of Landscape Architecture and Forestry Sciences. Flower buds of 'Green Calyx' at different stages were collected in a greenhouse from March to July 2023. All samples were flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80°C.

[0034] Example 1

[0035] This embodiment provides a test method, which specifically includes the following steps.

[0036] 1. Total RNA extraction and cDNA synthesis were performed on flower buds of 'Green Calyx' at different stages.

[0037] Following the instructions of the plant total RNA extraction kit (Novizan, Nanjing), total RNA was extracted from flower buds at different stages of 'Green Calyx'.

[0038] According to the instructions of the cDNA reverse transcription kit (Novizan, Nanjing), 0.1 μg of total RNA sample was used as a template to reverse transcribe and synthesize cDNA. After synthesis, the cDNA was stored at -20℃ for later use.

[0039] 2. Gene cloning

[0040] Using the 'green calyx' cDNA obtained in the above steps as a template, PCR amplification was performed using the full-length primers RcFULL-F and RcFULL-R of the RcFULL gene to obtain the full-length CDS sequence of RcFULL. The sequence information of the full-length primers RcFULL-F and RcFULL-R is shown in Table 1.

[0041] Table 1

[0042]

[0043] PCR was performed to purify the target band product. The purified DNA fragment was ligated into the pCE2-TA vector, and the recombinant vector was then transformed into E. coli Fast-T1 competent cells. After LB solid culture, positive plaques were selected and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared, and if the sequencing results were correct (positive), the plasmid was extracted and stored at -20℃.

[0044] 3. Carrier Construction

[0045] The pCE2-TA vector and pCAMBIA2300 vector carrying the RcFULL gene were double-digested with enzymes and then recovered via gel electrophoresis. The linearized vector pCAMBIA2300 was ligated with the RcFULL gene using T4 ligase, and the resulting compound was transformed into *E. coli* Fast-T1. After confirmation by colony PCR, the recombinant plasmid pCAMBIA2300-RcFULL was extracted. The recombinant plasmid was then digested with enzymes to confirm the correct target band and stored at -20°C for subsequent transformation with *Agrobacterium tumefaciens* GV3101.

[0046] Following the vector construction steps described above, pRTL2-RcFULL-GFP and pTRV2-RcFULL recombinant plasmids were constructed and stored at -20℃ for later use. Specific primer information is shown in Table 1.

[0047] 4. Subcellular localization

[0048] Agrobacterium containing the recombinant plasmid pRTL2-RcFULL-GFP was cultured with shaking until the OD value was greater than 1.0. The bacteria were harvested at 6000 rpm for 8 min, and the bacterial cells were washed with resuspension solution. The OD value of the bacterial solution was adjusted to 0.6-0.8.

[0049] The prepared infection solution was incubated in the dark for 3 hours. Native tobacco plants that had grown for 3–4 weeks were selected, and the solution was injected from the underside of the leaves until the entire leaf was submerged. The plants were then bagged and cultured in the dark for 2–3 days. Temporary sections of the lower epidermis were prepared and placed upside down on the stage of a laser confocal microscope to observe the luminescence of the GFP fluorescent protein.

[0050] 5. Agrobacterium-mediated genetic transformation and detection in Arabidopsis thaliana

[0051] Arabidopsis thaliana was transformed using the inflorescence inoculation method. Healthy wild-type Arabidopsis thaliana were selected for inoculation, and any existing pods and flower buds were removed before inoculation. T0 generation Arabidopsis thaliana seeds were sterilized in a clean bench and sown in 1 / 2 MS medium containing Kansas antibiotic. After vernalization at 4°C for 2–3 days, the culture medium was placed in a sterile culture room. Plants with normal growth and green true leaves were selected and transplanted into the substrate, where they were cultured in an artificial climate chamber under a 16-hour light / 8-hour dark cycle. DNA was extracted from the leaves of the transgenic Arabidopsis thaliana for positive detection, and the phenotypic characteristics of the positive plants were subsequently observed. The expression level of the heterologous gene RcFULL was detected using wild-type Arabidopsis thaliana as a control, employing a 2... -△△Ct The relative expression level of genes was calculated using a method with three replicates.

[0052] Example 2

[0053] In this embodiment, the corresponding gene sequence was found using the existing 'Yueyuefen' genome. Specific primers were designed based on the sequence, and the full-length CDS sequence of the gene was cloned using cDNA from 'Yueyuefen' and 'Lüe' as templates. The final PCR and sequencing results showed that the CDS sequence length of RcFULL was 774 bp (encoding 257 amino acids). Figure 1 As shown. Figure 1 In the diagram, M indicates the marker (2000bp); 1–8 indicate positive clones; Figure A shows the RcFULL gene cloning results; Figure B shows the colony PCR screening results.

[0054] Figure 2 This is an RcFULL phylogenetic tree. The gene sequences of 'Green Calyx' and 'Moonlight Pink' are identical. According to... Figure 2 As a result, protein sequences from 11 species with high amino acid similarity to RcFULL were screened out. Among them, the closest relative was rose. In addition, it also has certain kinship with species such as Chinese white pear (Pyrus bretschneideri), hydrangea (Hydrangea macrophyla), red honeysuckle (Rhododendron viali), and jujube (Ziziphus jujuba).

[0055] This embodiment analyzed the physicochemical properties of the protein encoded by the RcFULL gene and found that it has a molecular weight of 29188.24, a theoretical isoelectric point (pI) of 9.21, an instability coefficient of 61.39, and a total average hydrophobicity index of -0.761, indicating it is a basic protein. To further understand the mechanism of action of RcFULL, this embodiment selected the gene with the highest homology to Arabidopsis thaliana using the TAIR online website and performed interaction analysis on the protein with the highest similarity using STRING. The results showed that RcFULL may interact with LFY, which promotes early floral meristem, SPL3, which affects leaf epidermal differentiation and flowering time, and TFL1, which controls the identity of inflorescence meristem.

[0056] In this embodiment, the subcellular localization of RcFULL was predicted using prediction websites such as Cell-PLoc 2.0 (plant subcellular localization). The prediction results showed that the gene was located in the cell nucleus. The transmembrane region prediction analysis of the gene was performed using the TMHMM online software. Figure 3 This is a summary diagram of the bioinformatics analysis of the RcFULL gene. Figure A shows the predicted results of protein hydrophilicity / hydrophobicity; Figure B shows the predicted results of protein signal peptide; Figure C shows the predicted results of protein tertiary structure; Figure D shows the predicted results of protein transmembrane domains; Figure E shows the predicted results of protein secondary structure; and Figure F shows the predicted results of protein conserved domains. Based on... Figure 3The results showed that the protein encoded by the RcFULL gene is located outside the membrane and lacks a transmembrane domain. Gene hydrophilicity prediction results indicated that all RcFULL genes are hydrophilic proteins (Figure A). Gene signal peptide prediction analysis showed that the max.S value of the RcFULL gene was less than 0.5 (Figure B), indicating that the RcFULL gene lacks a signal peptide sequence, is not a secretory protein, and cannot transport proteins. NCBI analysis revealed that all conserved domains of the RcFULL protein contain K-boxes and MADS_MEF2_like structures, differing only in position (Figure F). Analysis of the secondary structure of the RcFULL protein using the SOPMA online software revealed that the protein contains 132 α-helices (51.36%), 21 extended strands (8.17%), 59 random coils (38.13%), and 6 β-turns (2.33%) (Figure E).

[0057] The online website WoLF-PSORT predicts that the RcFULL protein is located in the cell nucleus. Subcellular localization further confirms its location within the cell, and the results are as follows: Figure 4 As shown in the figure. These results indicate that the empty GFP vector exhibits green fluorescence in both the cell membrane and nucleus under 488 nm excitation light, while the RcFULL-GFP vector shows green fluorescence only in the nucleus. The observations are consistent with online predictions; RcFULL is localized within the nucleus, indicating typical subcellular localization characteristics of transcription factors.

[0058] Example 3

[0059] This embodiment investigated the promoter cis-acting elements of the RcFULL gene. As shown in Table 2, the 2000bp upstream sequence of RcFULL was taken as the RcFULL promoter region, and the selected promoter sequences were used for cis-acting element analysis of RcFULL using the Plantcare online tool. A total of 16 cis-acting elements with relatively clear functions were screened.

[0060] Table 2

[0061]

[0062]

[0063] This analysis revealed that RcFULL contains TCCC-motif, TGA-box, ARE, and TC-rich repeats, which are light-responsive elements, hormone-responsive elements, anaerobic-inducible response elements, and defense and stress-responsive elements, respectively. Among these, many are related to light-responsive elements, such as GT1-motif, TCCC-motif, TCT-motif, and ATC-motif, indicating that RcFULL may be induced by external light, affecting plant growth and development. RcFULL's unique cis-acting elements include ARE (a cis-regulatory element essential for anaerobic induction), O2-site (a cis-acting element in the zeatin metabolism pathway), and MSA-like (a cis-acting element involved in cell cycle regulation). RcFULL plays a certain role in cell cycle regulation, contributing to the plant's strong survival ability under stress. The presence of TC-rich repeats cis-acting elements in the RcFULL promoter suggests that RcFULL may play a role in abiotic stress.

[0064] Example 4

[0065] To determine the function of the RcFULL gene, this embodiment constructed the overexpression vector pCAMBIA2300-RcFULL and transformed wild Arabidopsis thaliana using the inflorescence infection method to obtain transgenic plants. DNA was first extracted for PCR verification, yielding 6 positive seedlings. Figure 5 As shown. Figure 5 In the diagram, OE1–OE6 represent transgenic plants, indicating RcFULL-positive plants; M represents Mark 2000; WT represents wild-type plants; and CK represents plants transgenic with an empty vector (Arabidopsis thaliana). Further, RNA was extracted from the flowers of wild-type plants, Arabidopsis thaliana transgenic with an empty vector, and RcFULL-positive plants, and expression levels among different lines were analyzed by qRT-PCR. The results are as follows: Figure 6 As shown. Figure 6 The results showed that the expression level of the RcFULL gene was significantly increased in positive plants (OE1-OE6) compared with plants transformed with empty vector.

[0066] Figure 7The phenotypic results of Arabidopsis thaliana transgenic with the RcFULL gene are shown in the figure (OE in the figure is a disassembled diagram of OE5). CK represents the flower of an empty vector plant, OE represents the flower of a positive vector plant, SE represents disassembled sepals, PE represents disassembled petals, ST represents disassembled stamens, and PI represents disassembled pistils. These results indicate that heterologous expression of the RcFULL gene leads to premature flowering in positive Arabidopsis lines. Compared to empty vector plants, RcFULL positive plants have more prominent and longer pistils, while the number and morphology of stamens remain unchanged. However, they exhibit inability to self-pollinate (failing to obtain Arabidopsis seeds). The petals of RcFULL positive plants show a color change from white to green, with the color being darkest near the midrib and whitish at the edges. The petals are leaf-like in shape, smaller, and narrowly elliptical overall. The color and morphology of the sepals show no significant difference. Figure 9 The results of the pistil length comparison show that the pistil length of the positive strain can reach about 0.5 cm, while that of the empty control group is 0.2-0.3 cm. That is, the pistil morphology of the flowers of the RcFULL positive plants is more prominent and longer than that of the empty control group.

[0067] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; for example, "according to a preferred embodiment" indicates that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the features introduced by "according to a preferred embodiment" are merely optional and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A gene RcFULL that regulates the development of rose flower organs, characterized in that, The sequence of the gene RcFULL is shown in SEQ ID NO.

1.

2. The gene RcFULL according to claim 1, characterized in that, The rose variety is 'Green Calyx'.

3. A method for altering the morphology of rose flower organs, characterized in that, The method includes: Overexpression of the rose RcFULL gene, and / or Overexpression of the rose RcFULL protein, wherein the RcFULL gene sequence is shown in SEQ ID NO.1, and the RcFULL protein is encoded by the RcFULL gene.

4. The application of the gene RcFULL according to claim 1 or 2 and the method according to claim 3 in altering the flowering time of roses.

5. The application according to claim 4, characterized in that, Changing the blooming time of roses is intended to encourage them to bloom earlier.

6. The application of the gene RcFULL according to claim 1 or 2 and the method according to claim 3 in altering the morphology of rose pistils.

7. The application according to claim 6, characterized in that, Changing the morphology of rose pistils can promote pistil elongation.

8. The application of the gene RcFULL according to claim 1 or 2 and the method according to claim 3 in altering petal morphology.

9. The application according to claim 8, characterized in that, Change the shape of the petals to reduce the petal area.

10. The application of the gene RcFULL according to claim 1 or 2 and the method according to claim 3 in increasing the number of sepals.

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