Rosa chinensis RcAG2 gene and application thereof in regulation and control of floral organ development

Abstract

The invention relates to a gene RcAG2 for regulating and controlling organ development of China rose. The sequence of the gene RcAG2 is as shown in SEQ ID NO. 1. According to the invention, the RcAG2 gene is cloned in'calyx ', through heterologous overexpression of the RcAG2 gene in arabidopsis thaliana, it is found that the RcAG2 gene can significantly change the form of petals, the gene can cause gradual degradation of the petals, and the size of the petals is significantly reduced compared with a no-load arabidopsis thaliana positive strain. The result of the invention provides an important clue for further understanding the function of the RcAG2 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 RcAG2 gene and its application in regulating floral organ development. Background Technology

[0002] The 'Green Calyx' rose (Rosa chinensis 'Viridiflora') is an ancient Chinese rose variety, named for its petals which are completely degenerated into green sepals. In the normal rose flower organs (sepia-petals-stamens-pistil), the petals and stamens of the Green Calyx are replaced by sepal-like structures, forming a "fully sepalized" flower shape.

[0003] Previous studies have found that, using 'Yueyuefen' as a control, the expression levels of RcFUL2, RcFUL3, and RcFUL5 were significantly increased during the carpel differentiation stage of 'Lv'e'; RcAP3.3 was not expressed during the vegetative meristem stage and sepal primordium differentiation stage of 'Lv'e'; RcAP1.2, RcAG2, and RcSEP4.3 were expressed only in the leaves of 'Lv'e' and not in the leaves of 'Yueyuefen' (Liu J, Fu X, Dong Y, et al. MIKCC-type MADS-boxgeneS in Rosa chinensis: the remarkable expansion of ABCDE model genes and their roles in floral organogenesis[J]. Horticulture research, 2018, 5). Other studies have constructed, sequenced, and analyzed miRNA libraries for 'Green Calyx' and 'Monthly Pink', finding that miR172 expression was significantly downregulated in the petals, stamens, and pistils of 'Green Calyx' (P<0.01), while the predicted target gene RcAP2 was significantly upregulated in all organs. This suggests that miR172 may cause the "flower-to-leaf" phenomenon in 'Green Calyx' by negatively regulating RcAP2 expression (Sui Mengjie, Yan Huijun, Wang Zhenzhen, et al. Identification and analysis of microRNAs related to flower organ development in 'Green Calyx' rose [J]. Acta Phytoscientiae Sinica, 2019, 37(01): 37-46). Furthermore, recent research has found that RhMYB123 in VIGS silent roses can increase the number of malformed petal stamens. It is speculated that RhMYB123 can regulate the development of malformed petal stamens by regulating the expression of certain MADS-box family member genes and auxin signaling pathway members (Li K, Li Y, Wang Y, et al. Disruption of transcription factor RhMYB123 causes the transformation of stamen to malformed petal in rose (Rosa hybrida)[J]. Plant Cell Reports, 2022, 41(12): 2293-2303).

[0004] Currently, there are few reports on the molecular mechanisms of the 'green calyx' rose flower type. Research on genes involved in the developmental regulation of floral organs is of great significance for the cut flower quality and economic value of roses in later stages.

[0005] 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

[0006] In view of the shortcomings of the prior art, the first aspect of the present invention provides a gene RcAG2 that regulates the development of rose flower organs, the sequence of which is shown in SEQ ID NO.1.

[0007] According to a preferred embodiment, the rose variety is 'Green Calyx'.

[0008] A second aspect of the present invention provides a method for altering the morphology of rose flower organs, the method comprising:

[0009] Overexpression of the rose RcAG2 gene, and / or

[0010] Overexpression of the rose RcAG2 protein, wherein the RcAG2 gene sequence is shown in SEQ ID NO.1, and the RcAG2 protein is encoded by the RcAG2 gene. The amino acid sequence of the RcAG2 protein is shown in SEQ ID NO.2.

[0011] The third aspect of this invention provides the application of the gene RcAG2 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 petals.

[0012] According to a preferred embodiment, the morphology of rose petals is changed to petal degeneration or petal area reduction.

[0013] The third aspect of this invention provides the application of the gene RcAG2 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.

[0014] According to a preferred embodiment, the flowering time of roses is changed so that the roses bolt and bloom earlier.

[0015] The fourth aspect of the present invention provides an expression vector containing the RcAG2 gene provided in the first aspect of the present invention.

[0016] The fifth aspect of the present invention provides a strain containing the RcAG2 gene provided in the first aspect of the present invention.

[0017] Technical effects of the present invention:

[0018] This invention cloned the RcAG2 gene from 'Green Calyx' rose. Bioinformatics analysis revealed 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 showed the gene is located in the cell nucleus. Heterologous overexpression experiments in Arabidopsis thaliana showed that RcAG2 transgenic Arabidopsis exhibited smaller and degenerated petals. The results of this invention provide important clues for further understanding the function of the RcAG2 gene in rose development. Attached Figure Description

[0019] Figure 1 Results of RcAG2 gene cloning;

[0020] Figure 2 This is the phylogenetic tree of the RcAG2 system;

[0021] Figure 3 Summary of bioinformatics analysis results for the RcAG2 gene;

[0022] Figure 4 Subcellular localization results of RcAG2 protein;

[0023] Figure 5 The results of the identification of positive Arabidopsis thaliana seedlings;

[0024] Figure 6 Results of expression level analysis in RcAG2 transgenic Arabidopsis thaliana;

[0025] Figure 7 The results of the RcAG2 gene transformation in Arabidopsis thaliana;

[0026] Figure 8 The results are for the validation of RcAG2 positive plants;

[0027] Figure 9 This is a statistical result of the number of days the flowers bloom. Detailed Implementation

[0028] 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.

[0029] In the following examples, 'Green Calyx' 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.

[0030] This invention utilizes the NCBI online database to search for homologous sequences of the RcAG2 gene in Arabidopsis thaliana and constructs a phylogenetic tree using MEGA 11.0. It uses the ProtParam online database to predict the isoelectric point, adipose coefficient, and other basic physicochemical properties of the protein encoded by the RcAG2 gene; the ProtScale online database to predict the protein's hydrophilicity and hydrophobicity; the SOPMA and SWISS-MODEL online tools to predict the protein's secondary and tertiary structures, respectively; the SignalP 4.1 and TMHMM 2.0 online software to predict the signal peptide sites and transmembrane regions of the protein sequence; the RcAG2 protein sequence is subjected to a BLAST procedure on the NCBI website to search for homologous sequences in other species, followed by amino acid multiple sequence alignment analysis using DNAMAN software; the CD-Search online database on the NCBI website is used to predict conserved domains of the target protein; and the WoLF-PSORT online database is used to predict the protein's subcellular localization.

[0031] Example 1

[0032] This embodiment provides a test method, which specifically includes the following steps.

[0033] 1. Total RNA extraction and cDNA synthesis were performed on flower buds of 'Green Calyx' at different stages.

[0034] 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'.

[0035] 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.

[0036] 2. Gene cloning

[0037] Using the 'green calyx' cDNA obtained in the above steps as a template, PCR amplification was performed using the full-length primers RcAG2-F and RcAG2-R of the RcAG2 gene to obtain the full-length CDS sequence of RcAG2. The sequence information of the full-length primers RcAG2-F and RcAG2-R is shown in Table 1.

[0038] Table 1

[0039]

[0040] 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, the plasmid was extracted and stored at -20℃.

[0041] 3. Carrier Construction

[0042] The pCE2-TA vector and pCAMBIA2300 vector carrying the RcAG2 gene were double-digested with enzymes and then recovered by gel electrophoresis. The linearized vector pCAMBIA2300 was ligated with the RcAG2 gene using T4 ligase, and the resulting compound was transformed into *E. coli* Fast-T1. After confirmation by colony PCR, the recombinant plasmid pCAMBIA2300-RcAG2 was extracted. The recombinant plasmid was further analyzed by enzyme digestion to confirm the correct target band, and then stored at -20°C for subsequent transformation with *Agrobacterium tumefaciens* GV3101.

[0043] Following the vector construction steps described above, recombinant plasmids pRTL2-RcAG2-GFP and pTRV2-RcAG2 were constructed and stored at -20℃ for later use. Specific primer information is shown in Table 1.

[0044] 4. Subcellular localization

[0045] Agrobacterium containing the recombinant plasmid pRTL2-RcAG2-GFP was cultured with shaking until the OD value was greater than 1.0. The bacteria were harvested at 6000 rpm for 8 min. The bacteria were washed with resuspension solution and the OD value of the bacterial solution was adjusted to 0.6-0.8 to obtain the infection solution.

[0046] 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.

[0047] 5. Agrobacterium-mediated genetic transformation and detection in Arabidopsis thaliana

[0048] 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 positive plants were subsequently observed. The expression level of the heterologous gene RcAG2 was detected using wild-type Arabidopsis thaliana as a control. -△△Ct The relative expression level of genes was calculated using a method with three replicates.

[0049] Example 2

[0050] In this embodiment, the corresponding gene sequence was found through 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 RcAG2 was 756 bp. Figure 1 As shown. Figure 1 In the diagram, M indicates the Marker (2000bp); 1–8 indicate positive clones; Figure A shows the RcAG2 gene cloning results; Figure B shows the colony PCR screening results.

[0051] Figure 2 This is the RcAG2 phylogenetic tree. The gene sequences of 'Green Calyx' and 'Moonlight Pink' are identical. According to... Figure 2 As a result, protein sequences from 10 species with high amino acid sequence similarity to RcAG2 were screened out. Among them, the closest relative was rose, followed by almond (Prunus dulcis), plum (Prunus mume), and European wild apple (Malus sylvestris).

[0052] Physicochemical property analysis of the protein encoded by the RcAG2 gene revealed that it has a molecular weight of 28529.19, a theoretical isoelectric point (pI) of 9.40, an instability coefficient of 50.17, and a total average hydrophobicity index of -0.785, classifying it as a basic protein. To further understand the mechanism of action of RcAG2, this study selected the gene with the highest homology to Arabidopsis thaliana using the TAIR online database and performed interaction analysis on the protein with the highest similarity using STRING. The results showed that RcAG2 may interact with SEP3, which affects inflorescence development and floral organ formation, CRC, which regulates cell division, and KUN, which is related to cell proliferation.

[0053] In this embodiment, the subcellular localization of RcAG2 was predicted using prediction websites such as Cell-PLoc 2.0, and the prediction results showed that the gene was located in the cell nucleus; the transmembrane region prediction analysis of the gene was performed using TMHMM online software. Figure 3 This is a summary diagram of the bioinformatics analysis of the RcAG2 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. Figure 3 The results showed that the RcAG2 gene protein is located outside the membrane and lacks a transmembrane domain. Gene hydrophilicity prediction results indicated that all RcAG2 genes are hydrophilic proteins (Figure A). Gene signal peptide prediction analysis showed that the max.S value of the RcAG2 gene was less than 0.5 (Figure B), indicating that the RcAG2 gene lacks a signal peptide sequence, is not a secretory protein, and cannot transport proteins. NCBI analysis revealed that all conserved domains of the RcAG2 protein contain K-boxes and MADS_MEF2_like structures, differing only in position (Figure F). Analysis of the secondary structure of the RcAG2 protein using the SOPMA online software revealed that the protein contains 139 α-helices (56.05%), 8 β-turns (3.23%), 22 extended strands (8.87%), and 79 random coils (31.85%) (Figure E).

[0054] The online website WoLF-PSORT predicted that the RcAG2 protein was located in the cell nucleus. Subcellular localization was used to further confirm its location within the cell, and the results were as follows: Figure 4 As shown in the figure. The results indicate that the empty GFP vector exhibits green fluorescence in both the cell membrane and nucleus under 488 nm excitation light, while the RcAG2-GFP vector exhibits green fluorescence in the nucleus. The observation results are consistent with the online prediction results, and RcAG2 is located in the nucleus, indicating that it has the typical subcellular localization characteristics of a transcription factor.

[0055] Example 3

[0056] This embodiment investigated the promoter cis-acting elements of the RcAG2 gene. As shown in Table 2, the 2000bp upstream sequence of RcAG2 was taken as the RcAG2 promoter region, and the selected promoter sequences were used for cis-acting element analysis of RcAG2 using the Plantcare online tool. A total of 13 promoter cis-acting elements with relatively clear functions were screened.

[0057] Table 2

[0058]

[0059] Analysis of this embodiment revealed that the promoter of RcAG2 contains elements such as GTL-motif, P-box, and TC-rich repeats, which are involved in light response, hormone response, stress response, and defense response elements, respectively. In addition, the promoter of RcAG2 contains multiple hormone-like response elements such as P-box, ABRE, and AuxRR-core, suggesting that RcAG2 can be influenced by natural plant hormones and regulate the growth and development of roses.

[0060] Example 4

[0061] To determine the function of the RcAG2 gene, this embodiment constructed the overexpression vector pCAMBIA2300-RcAG2 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 RcAG2-positive plants; M represents Marker 2000; WT represents wild-type; and CK represents Arabidopsis thaliana transgenic with empty vector. Further, RNA was extracted from the flowers of wild-type, transgenic Arabidopsis thaliana, and RcAG2-positive lines, 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 RcAG2 gene was significantly increased in positive plants (OE1–OE6); on the other hand, compared with the empty vector control, transgenic Arabidopsis OE4, OE5, and OE6 exhibited abnormal phenotypes. Specific results are shown below. Figure 7As shown, the positive lines bolted and flowered slightly earlier than the unsupported control. A notable difference was that when the flowers of the positive seedlings were just forming (18 days), the floral organs of the stamens and pistils were exposed, while the sepals of the normally growing unsupported Arabidopsis positive seedlings were tightly closed. Furthermore, the pistils of the RcAG2 positive lines showed no significant changes, and the morphology and number of stamens also showed no significant differences. However, the petal morphology of the OE4, OE5, and OE6 positive seedlings showed significant changes. The petals of OE4 were significantly smaller and nearly elliptical fan-shaped, while the petals of OE5 and OE6 resembled curled stamens and were a lighter yellow color. The morphology and number of sepals were not significantly different from the unsupported control. Figure 7 In the text, CK, OE4, OE5, and OE6 are explained as before; SE indicates disassembling the sepals; PE indicates disassembling the petals; ST indicates disassembling the stamens; and PI indicates disassembling the pistil.

[0062] In this embodiment, a transient gene silencing experiment was conducted on 'Green Calyx' rose cuttings using rootless cuttings. The gene silencing was performed by infecting the 'Green Calyx' rose plant with Agrobacterium tumefaciens using a vacuum pump. Leaves from each rose line were randomly selected (three replicates). The pTRV2 vector was detected by PCR amplification. The results are as follows: Figure 8 As shown. Figure 8 The results showed that one RcAG2 positive seedling was obtained, the RcAG2 positive line was OE3, and the empty positive lines were CK1 and CK2. Figure 9 The results show that the RcAG2 positive strain can flower 4-5 days earlier than the control group (CK).

[0063] 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 RcAG2 that regulates the development of rose flower organs, characterized in that, The sequence of the gene RcAG2 is shown in SEQ ID NO.

1.

2. The gene RcAG2 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 RcAG2 gene, and / or Overexpression of the rose RcAG2 protein, wherein the RcAG2 gene sequence is shown in SEQ ID NO.1, and the RcAG2 protein is encoded by the RcAG2 gene.

4. The method according to claim 3, characterized in that, The amino acid sequence of the RcAG2 protein is shown in SEQ ID NO.

2.

5. The application of the gene RcAG2 according to claim 1 or 2 and the method according to claim 3 or 4 in altering the morphology of rose petals.

6. The application according to claim 5, characterized in that, The shape of rose petals is altered to result in petal degeneration or a reduction in petal area.

7. The application of the gene RcAG2 according to claim 1 or 2 and the method according to claim 3 or 4 in altering the flowering time of roses.

8. The application according to claim 7, characterized in that, The timing of rose blooming is altered to induce the rose to bolt and bloom earlier.

9. An expression vector containing the RcAG2 gene as described in claim 1.

10. A strain containing the RcAG2 gene as described in claim 1.

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