Application of Peony PoLEC1 Gene and Protein Encoded Thereby

Abstract

The present invention relates to the field of gene editing, and particularly to the application of the peony PoLEC1 gene and the protein encoded thereby. The peony PoLEC1 gene was cloned from the cultivated peony 'Fengdan' in the present invention. After transforming it into Arabidopsis thaliana, through phenotypic comparison, it was found that the peony PoLEC1 gene can play a role in the bolting, flowering, growth and development of plants, specifically manifested in that overexpression of the peony PoLEC1 gene can increase the number of leaves, delay the bolting and flowering time and reduce the plant height. Taking Arabidopsis thaliana as a model plant, by exploring the functional characteristics of the peony PoLEC1 gene and its expression characteristics in the whole process of direct somatic embryogenesis and various tissues of overexpressed Arabidopsis thaliana, a certain theoretical basis is provided for establishing a complete plant regeneration and genetic transformation system for peonies.

Description

Technical Field

[0001] The present invention relates to the field of gene editing, and particularly to the application of the peony PoLEC1 gene and the protein encoded thereby. Background Art

[0002] The LEAFY COTYLEDON (LAFL) regulatory network consists of four genes (LEC1, ABI3, FUS3, and LEC2), and the transcription factors encoded by these genes are the main regulators during seed development, including oil accumulation and embryo development.

[0003] LEC1 belongs to the NF-YB subfamily of the NF-Y nuclear transcription factor family, and this family is a conserved heterotrimeric complex that is ubiquitous in eukaryotes. The NF-Y transcription factor is composed of three main subunits, NF-YA, NF-YB, and NF-YC, which bind to the CCAAT box in the gene promoter region and function by forming heterodimers, forming diverse subfamilies. As a member of the NF-YB subfamily, LEC1 itself does not directly bind to DNA, but interacts with NF-YA and NF-YC that have the ability to bind DNA, jointly forming a complex that can recognize and bind to the CCAAT cis-element at the transcription start point. The NF-YB and NF-YC subunits have a unique histone fold domain (HFD / HFM), which is formed by three α-helices and two loops, and this is crucial for protein-DNA and protein-protein interactions. LEC1 is also a homolog of the HAP3 (Heme-activated protein 3) subunit, and HAP3 is particularly conserved in region B, which is a decisive factor for the function of LEC1.

[0004] LEC genes are involved in plant growth and development, somatic embryogenesis, and the accumulation of storage substances, etc. The induction of spontaneous somatic embryogenesis is due to the ectopic expression of transcription factor genes. Among them, LEC, as a key regulator of plant cell totipotency, controls the initiation and regulation of the maturation stage during embryonic development, thereby promoting the formation of somatic embryos. LEC1 was first isolated and identified from Arabidopsis thaliana and is an important regulator of embryonic development. It can activate the gene transcription required for embryonic morphogenesis and cell differentiation. Ectopic overexpression of AtLEC1 can induce the formation of embryoid structures on Arabidopsis leaves and abnormal seedling development. Stone et al. found that LEC1 promotes embryonic development by forming an environment conducive to the co-expression of genes related to embryonic morphological development and maturation. DcLEC1 was isolated from carrot (Daucus carota). In situ hybridization analysis showed that it was expressed in embryogenic cells and somatic embryos, but not in endosperm, indicating that DcLEC1 plays a key regulatory role in zygotic embryo and somatic embryo development. The research by Zhang et al. showed that the expression pattern of ZmLEC1 in maize (Zea mays L.) somatic embryo development was similar to that of Arabidopsis thaliana zygotic embryo, confirming its important role in inducing zygotic embryo and somatic embryo formation. Cassava (Manihot esculenta) overexpressing MeLEC1 showed morphological structure changes such as fleshy cotyledons, swollen hypocotyls, abnormally shaped leaves, and green roots. MeLEC1 had high-level expression at the initial and later stages of somatic embryo induction. Transferring Selaginella moellendorffii SmLEC1 into Arabidopsis thaliana lec1 mutants showed that it mainly accumulated in sexual and asexual reproductive structures, indicating its involvement in cell processes similar to those in the seed development and maturation period. In soybean (Glycine max) embryos, GmLEC1 transcriptionally activated the genes encoding the light reaction components of photosystem I and II and another group of genes involved in photosynthesis and chloroplast biogenesis. In rice (Oryza sativa L.), OsLEC1 controls endosperm development through interaction with the AP2 transcription factor. However, there has been no report on LEC1 in peony yet.

[0005] To further understand the regulatory network during peony somatic embryogenesis, on the basis of inheriting the previous research on key genes of peony and other plant somatic embryos, the present invention further explores in depth the expression characteristics of peony LEC genes during embryonic development and their roles in plant growth and development, and at the same time reveals the molecular regulatory network among these key genes. Through these studies, it aims to provide theoretical support and technical guidance for constructing an efficient and stable peony regeneration system and genetic transformation system. Summary of the Invention

[0006] The object of the present invention is to provide the application of the peony PoLEC1 gene and the protein encoded thereby, so as to solve the problems existing in the above-mentioned prior art. Taking Arabidopsis thaliana as a model plant, by exploring the expression characteristics of the peony PoLEC1 gene in various tissues of overexpressing Arabidopsis thaliana, a certain theoretical basis is provided for establishing a complete plant regeneration and genetic transformation system for peony.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the application of the peony PoLEC1 gene and the protein encoded thereby in delaying the bolting and flowering of Arabidopsis thaliana. The nucleotide sequence of the peony PoLEC1 gene is as shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is as shown in SEQ ID NO.12.

[0009] The present invention provides a method for delaying the bolting and flowering of Arabidopsis thaliana, including introducing the peony PoLEC1 gene into Arabidopsis thaliana to obtain a transgenic Arabidopsis thaliana plant stably expressing the peony PoLEC1 gene, so that the transgenic Arabidopsis thaliana plant delays bolting and flowering; the nucleotide sequence of the peony PoLEC1 gene is as shown in SEQ ID NO.11.

[0010] The present invention provides the application of the peony PoLEC1 gene and the protein encoded thereby in reducing the plant height of Arabidopsis thaliana. The nucleotide sequence of the peony PoLEC1 gene is as shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is as shown in SEQ ID NO.12.

[0011] The present invention provides a method for reducing the plant height of Arabidopsis thaliana, including introducing the peony PoLEC1 gene into Arabidopsis thaliana to obtain a transgenic Arabidopsis thaliana plant stably expressing the peony PoLEC1 gene, so that the transgenic Arabidopsis thaliana plant has a reduced plant height; the nucleotide sequence of the peony PoLEC1 gene is as shown in SEQ ID NO.11.

[0012] The present invention provides the application of the peony PoLEC1 gene and the protein encoded thereby in increasing the number of leaves of Arabidopsis thaliana. The nucleotide sequence of the peony PoLEC1 gene is as shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is as shown in SEQ ID NO.12.

[0013] The present invention provides a method for increasing the number of Arabidopsis leaves, comprising introducing a peony PoLEC1 gene into Arabidopsis to obtain a transgenic Arabidopsis plant stably expressing the peony PoLEC1 gene, so that the number of leaves of the transgenic Arabidopsis plant is increased; the nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11.

[0014] The present invention provides an application of a peony PoLEC1 gene and a protein encoded by the gene in promoting Arabidopsis callus differentiation into buds. The nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is shown in SEQ ID NO.12.

[0015] The present invention provides a method for promoting Arabidopsis callus to differentiate into buds, comprising overexpressing a peony PoLEC1 gene in Arabidopsis to promote the Arabidopsis callus to differentiate into buds; the nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11.

[0016] The present invention discloses the following technical effects:

[0017] The present invention clones the peony PoLEC1 gene from the cultivated peony 'Fengdan', transforms it into Arabidopsis, and finds through phenotypic comparison that the peony PoLEC1 gene can play a role in plant bolting, flowering, growth and development, which is specifically reflected in that overexpression of the peony PoLEC1 gene can increase the number of leaves, delay bolting and flowering time, and reduce plant height. The present invention uses Arabidopsis as a model plant, explores the functional characteristics of the peony PoLEC1 gene and its expression characteristics in the entire somatic embryo direct generation process and in various tissues of overexpressed Arabidopsis, and provides a certain theoretical basis for establishing a complete plant regeneration and genetic transformation system for peony. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 is the map of the overexpression plasmid pHG;

[0020] Figure 2 This is the PCR electrophoresis diagram of the tree peony PoLEC1 gene; M is the DL2000 maker; 1 is the target band;

[0021] Figure 3This is the phylogenetic tree of peony PoLEC1 protein and LEC1 of other species;

[0022] Figure 4 This is the amino acid sequence alignment of the peony PoLEC1 protein and LEC1 of other species; among them, the amino acid sequence similarity represented by dark blue is 100%, the amino acid sequence similarity represented by pink is ≥75%, and the amino acid sequence similarity represented by sky blue is ≥50%;

[0023] Figure 5 The wild-type tree peony (WT) and T 3 Phenotypes of transgenic Arabidopsis thaliana (OE-PoLEC1) overexpressing the gene; a is a picture taken at 20 days after planting; b is a picture taken at 28 days after planting; c is the number of leaves counted at 20 days after planting; d is the height of the plant counted at 40 days after planting; e is a picture taken at 40 days after planting; f and g are pictures taken at 15 days and 25 days after seeding, respectively;

[0024] Figure 6 The PoLEC1 gene in peony is expressed in T 3 Expression patterns in overexpressing transgenic Arabidopsis thaliana. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Experimental materials and methods

[0032] 1.1 Test Materials

[0033] The experimental material is the cultivar 'Fengdan', which has been cultivated and evolved for a long time and has ornamental, medicinal, oil-use characteristics and stable genetic traits. The seeds of 'Fengdan' that matured that year were selected, and the seeds came from Heze, Shandong. The peony tissue culture seedlings are developed from the embryo as explants. After 7 days of dark treatment, the explants are transferred to the tissue culture room for normal culture. The normal culture conditions in the tissue culture room are: alternating light for 16 hours and dark for 8 hours, and the light intensity is 40μmol·m -2 ·s -1 , temperature 24±1℃. Subculture once every 20 days. The roots, stems, leaves and callus of peony seeds and tissue culture seedlings were taken for RNA extraction for real-time fluorescence quantitative analysis. The Arabidopsis thaliana used in the present invention were Columbia-0 (Col-0) wild type, planted in the laboratory artificial climate room, with a light intensity of 40μmol·m -2 ·s -1 , humidity 60-70%, temperature 24±1℃.

[0034] 1.2 Methods

[0035] 1.2.1. Total RNA and cDNA synthesis

[0036] RNA was extracted from sterile peony tissue culture seedlings using a Quick RNA Isolation Kit (Huayueyang, China). The RNA concentration was determined by spectrophotometer. The integrity of the RNA band was confirmed by 1% agarose gel electrophoresis and the RNA was stored in a -80°C ultra-low temperature refrigerator for later use. The extracted RNA was used as a template and the TAKARA reverse transcription kit PrimeScript was used as a reference. TM The first strand of cDNA was synthesized by reverse transcription using the procedure of RT reagent Kit (Perfect Real Time) (RR047A).

[0037] 1.2.2 Cloning of the Paeonia suffruticosa PoLEC1 gene

[0038] According to the primer design principle, the primers were designed using SnapGene (V2.3.2) software (Table 1). The target sequence was amplified by using a high-fidelity enzyme (Vazyme, Nanjing, China) in Max Master Mix (P515). The target sequence was detected by 1% agarose gel electrophoresis. When the target band was consistent with the corresponding length of the marker sequence, it indicated that the target sequence was obtained and the CDS sequence of the peony PoLEC1 gene was cloned. The gel was cut on a UV gel cutting table, and the product was recovered according to the steps of the DNA purification and recovery kit (Tiangen, Beijing, China). The gel recovery product was connected to the cloning vector using the pCE2TA / Blunt-Zero vector (C601) (Vazyme, Nanjing, China) kit for transformation. Take Escherichia coli DH5α competent cells and transform them according to the steps. Then, evenly spread them on LB solid agar medium containing AMP and culture them upside down at 37°C for 14 hours. Then, select single clones for colony PCR, shake the positive single clones, extract the plasmid using TIANGEN Tiangen Plasmid Mini Extraction Kit (TIAN prep Mini Plasmid Kit, DP103-03, Beijing, China), and take 10 μL of the plasmid solution to send to Anshengda (Anshengda, Beijing, China) for sequencing to verify that the target sequence was obtained correctly.

[0039] Table 1 Detailed information of primers

[0040]

[0041] 1.2.3 Bioinformatics analysis

[0042] The basic physicochemical properties of the amino acid sequence of PoLEC1 were predicted and analyzed online using the ExPASy-ProtParam website. The secondary structure of the PoLEC1 protein was analyzed using SOPMA. TMHMM-v.2.0 and SignalP-v.6.0 were used to predict the presence or absence of transmembrane domains and signal peptides in PoLEC1, respectively. Plant mPLoc was used to predict the subcellular localization of the PoLEC1 protein. The PlantTFDB plant transcription factor database (https: / / planttfdb.gao-lab.org / ) and the NCBI public database (https: / / www.ncbi.nlm.nih.gov / ) were used to search for homology to the LEC1 protein sequences of other species. Clustal Wv.2.0.11 software was used to perform multiple sequence alignment of the amino acid sequence of peony LEC1 with the amino acid sequences of LEC1 of six species, including Arabidopsis thaliana, rapeseed, grape, corn, poplar, and cocoa. The phylogenetic tree was constructed by combining the LEC1 protein sequences of other species and the PoLEC1 protein sequences using the NJ (Neighbor-Joining) method of MEGA 11, with 1000 bootstrap replicates and other parameters set to default values. The tree was beautified online using iTOL (https: / / itol.embl.de / ).

[0043] DNAMAN 9.0 software was used to perform multiple sequence alignment of LEC1 protein sequences of peony, Arabidopsis, rapeseed, grape, cocoa, poplar and maize. MEME online software was used to perform motif analysis of PoLEC1 and LEC1 protein sequences of other species, and the number of motifs was set to 10. The saved XML file and the Newick file obtained in the evolutionary tree construction were input into the Gene Structure View (Advanced) window of TBtools v.2.4.0.119028 software for visualization and analysis.

[0044] 1.2.4 Functional verification of peony PoLEC1 gene transfection into Arabidopsis thaliana

[0045] The peony PoLEC1 gene was cloned into the plant expression vector pHG (pHG map as shown in Figure 1 The plant vector used in this example is pHG, and the 35S::PoLEC1 overexpressing transgenic Arabidopsis plants with stable inheritance were obtained by Agrobacterium-mediated inflorescence infection. 3 The overexpressing transgenic Arabidopsis thaliana and wild-type controls were planted and cultured under the same culture conditions, and observed and recorded.

[0046] 1.2.4.1. Observation of transgenic plant growth phenotype

[0047] Combine wild type and T 3 The seeds of the overexpressing transgenic Arabidopsis were sterilized and sown in a culture dish containing MS medium. After vernalization for 7 days, they were transplanted into planting soil and cultured in an artificial climate chamber. The number of leaves of Arabidopsis thaliana was counted at the same time after germination. The biological and technical replicates were repeated three times, and the growth was observed and photographed.

[0048] 1.2.4.2 Phenotypic observation of transgenic plant roots

[0049] Combine wild type and T 3 The seeds of the transgenic Arabidopsis thaliana overexpressing were sterilized and sown neatly side by side in a culture dish containing MS medium and cultured for 30 days after vernalization. The root development of Arabidopsis thaliana was observed and photographed.

[0050] 1.3 Fluorescence quantitative analysis

[0051] Seven stages of peony somatic embryo development (day 0 of peony somatic embryo development; day 5 of peony somatic embryo development; day 10 of peony somatic embryo development; day 15 of peony somatic embryo development; day 20 of peony somatic embryo development; day 30 of peony somatic embryo development; day 60 of peony somatic embryo development) and five different peony tissues (seed, root, stem, leaf and callus) were selected from the cultured peony in step 1.1. 3 The roots, stems, leaves, flowers, and fruits of the transgenic Arabidopsis thaliana were used to extract RNA and reverse transcribe the first strand of cDNA. 2 O was diluted 10 times as a template, and the primers are shown in Table 1. The reaction was completed on a QTOWER real-time fluorescence quantitative PCR instrument (analytikjena, Germany) using the TB Green Premix Ex TaqⅡ fluorescent quantitative kit (Tli RNaseH Plus, TaKaRa). The PCR reaction system was TB Green Premix Ex Taq 5μL; Template 1μL; Primer (F+R) 0.4μL; ddH 2 O was added to 10 μL, and a total of 3 biological replicates were set up. The reaction conditions were 95℃ pre-denaturation for 90s; 95℃ denaturation for 5s, 60℃ melting for 30s, 40 cycles; melting curve from 60 to 95℃, 1℃ increase every 15s. Peony ubiquitin ligase and AtActin were used as internal reference genes, and the expression levels of peony somatic embryos on day 0, peony root tissues, and Arabidopsis root tissues were used as control groups, respectively. The relative expression level was measured using 2 -ΔΔCTThe relative expression of PoLEC1 in various tissues was calculated and analyzed.

[0052] 2. Results and Analysis

[0053] 2.1 Cloning and sequence analysis of the tree peony PoLEC1 gene

[0054] Using the transcriptome data of somatic embryos from 10 different stages of peony as a reference, cDNA from the stage with higher expression of the target gene was selected as a template for gene cloning. The electrophoresis diagram showed that an obvious specific fragment appeared at the position of about 500 bp ( Figure 2 ), the length of the peony PoLEC1 gene is 582 bp, which is basically consistent with the expected target gene length. The target fragment is then connected to the cloning vector and sequenced to obtain 100% correct sequence information, and the desired target gene is cloned.

[0055] The nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11, specifically:

[0056] Analyze the basic physical and chemical properties of the obtained Paeonia ostii PoLEC1 gene. The results show that the Paeonia ostii PoLEC1 protein encoded by this gene has a total of 193 amino acids, with a relative molecular weight of approximately 20.5 kDa, a theoretical isoelectric point of 5.98, an instability coefficient II of 29.10, a total average hydrophilicity of -0.865, and a fat coefficient of 50.05. It belongs to a stable hydrophilic protein (instability coefficient < 40, hydrophilicity index is negative). Subcellular localization prediction shows that the LEC1 protein is localized in the nucleus.

[0057] The amino acid sequence of the Paeonia ostii PoLEC1 protein encoded by the Paeonia ostii PoLEC1 gene is shown in SEQ ID NO.12, specifically: MAESDDESGGHNTSGNANGELSQREQDRLLPIANVSRIMKKALPANAKISKDAKETVQECVSEFISFITGEASDKCQREKRKTINGDDLLWAMTTLGFEEYVEPLKVYLQKFREMEGEKTSVGRQGENSKDGSSGHGGSLVNSGNSGGGYGGGEMYGGMMLGNPHHPHPHGQMYGSGSYNHSGGGGASTGRPR*.

[0058] The secondary structure of the Paeonia ostii PoLEC1 protein is composed of a large number of α-helices and random coils, including 3 α-helices and 2 L-loops, and contains the highly conserved B domain in the HAP3 subunit, without a transmembrane domain and no signal peptide.

[0059] 2.2 Phylogenetic tree analysis of Paeonia ostii PoLEC1 protein

[0060] To further understand the evolution and phylogenetic relationship of the Paeonia ostii PoLEC1 protein with members of other species, the obtained Paeonia ostii PoLEC1 protein was used to construct a phylogenetic tree together with the LEC1 proteins of Arabidopsis thaliana, Brassica napus, Theobroma cacao, Vitis vinifera, Zea mays, Glycine max, Brassica oleracea var. acephala, Jatropha curcas, and Selaginella moellendorffii ( Figure 3 ). The results show that the PoLEC1 protein has a relatively close phylogenetic relationship with Theobroma cacao and Vitis vinifera.

[0061] 2.3 Multiple sequence alignment analysis of Paeonia ostii PoLEC1 protein

[0062] To analyze the similarity between Paeonia ostii PoLEC1 and LEC1 of other species, the obtained amino acid sequence of Paeonia ostii PoLEC1 was aligned with the LEC1 protein sequences of other closely related species. The results show that the amino acid sequence of the Paeonia ostii PoLEC1 protein contains three α-helices connected by two loops, forming the B3 conserved domain of the NF-YB subunit, and contains the conserved B region of the HAP3 subunit ( Figure 4 ).

[0063] 2.4. Phenotypic analysis of Arabidopsis thaliana overexpressing the Paeonia ostii PoLEC1 gene

[0064] 2.4.1 Growth and development phenotypes of Paeonia ostii PoLEC1 transgenic plants

[0065] To explore the role of the Paeonia ostii PoLEC1 gene in plant growth and development, the phenotypes of wild-type and T 3 -generation overexpressing transgenic Arabidopsis thaliana were observed. It was found that at 20 days after planting, there was no significant difference in leaf size between the T 3 -generation overexpressing transgenic Arabidopsis thaliana and the wild type, and the number of leaves was slightly more than that of the wild type ( Figure 5 a and c in); at 28 days after planting, it was observed that the bolting of the T 3 -generation overexpressing transgenic Arabidopsis thaliana was later than that of the wild type ( Figure 5 b in); continuing to observe and culture, when the plant heights of the two were measured at about 40 days after planting, it was found that the plant height of the T 3 -generation overexpressing transgenic Arabidopsis thaliana was shorter than that of the wild type ( Figure 5 d and e in). The seeds of the T 3 -generation overexpressing transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana seeds were surface-sterilized and sown on MS medium containing hygromycin, and the growth status of the roots was observed. It was found that there was no significant difference in the root length and density between the two ( Figure 5 f and g in). The above results indicate that the Paeonia ostii PoLEC1 gene can play a role in plant bolting, flowering, growth and development. Specifically, overexpressing the Paeonia ostii PoLEC1 gene can increase the number of leaves, delay the bolting and flowering time, and reduce the plant height.

[0066] 2.5 Expression pattern analysis

[0067] The RT-qPCR method was used to analyze the expression levels of the Paeonia ostii PoLEC1 gene in various tissues of Arabidopsis thaliana: root, stem, leaf, flower, and fruit in the T 3 -generation overexpressing transgenic Arabidopsis thaliana ( Figure 6 ).

[0068] When the T 3 -generation overexpressing transgenic Arabidopsis thaliana plants were cultured for about 45 days, their roots, stems, leaves, flowers, and pods were collected respectively to extract RNA and reverse-transcribe it into cDNA. The Paeonia ostii PoLEC1 gene was quantified in the roots, stems, leaves, flowers, and pods, and three biological replicates and three technical replicates were performed for each tissue. The results showed that the PoLEC1 gene was expressed in all five tissues of transgenic Arabidopsis thaliana, and the highest expression level was in the leaves. This is consistent with the expression pattern of the PoLEC1 gene in Paeonia ostii, suggesting that it may play a role in leaf development.

[0069] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of the peony PoLEC1 gene and the protein encoded therein in delaying the bolting and flowering of peony, characterized in that: The nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is shown in SEQ ID NO.

12.

2. A method for delaying the bolting and flowering of peony, characterized in that: The method comprises introducing the peony PoLEC1 gene into peony to obtain a transgenic peony plant stably expressing the peony PoLEC1 gene, so that the transgenic peony plant delays bolting and flowering; the nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.

11.

3. Application of the peony PoLEC1 gene and the protein encoded by it in reducing the high blood count of peony plants, characterized in that: The nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is shown in SEQ ID NO.

12.

4. A method for reducing peony plant height, characterized in that: The method comprises introducing the peony PoLEC1 gene into peony to obtain a transgenic peony plant stably expressing the peony PoLEC1 gene, so that the plant height of the transgenic peony plant is reduced; the nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.

11.

5. Use of the peony PoLEC1 gene and the protein encoded therein for increasing the number of peony leaves, characterized in that: The nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11; the amino acid sequence of the protein encoded by the peony PoLEC1 gene is shown in SEQ ID NO.

12.

6. A method for increasing the number of peony leaves, characterized in that: The method comprises introducing the peony PoLEC1 gene into peony to obtain a transgenic peony plant stably expressing the peony PoLEC1 gene, so that the number of leaves of the transgenic peony plant is increased; the nucleotide sequence of the peony PoLEC1 gene is shown in SEQ ID NO.11.

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