The coding sequence of the flowering regulatory gene AP1 in Cymbidium sinense and its application

Abstract

The present invention discloses a coding sequence for the AP1 flowering regulatory gene in Chinese orchids and its application. This orchid floral organ development regulatory gene was isolated from the floral organ cDNA of the Chinese orchid cultivar 'Bai Mo'. Increasing its expression can cause loss of apical dominance in Arabidopsis thaliana, an increase in lateral inflorescences, irregular arrangement of florets on the inflorescence axis, and abnormal petal development in flowers, with the occurrence of fewer petals or petals transforming into sepals. In Chinese orchids, a significant positive correlation was found between gene expression and an increase in sepals, and expression was significantly elevated in variants with a tree-forked flower pattern, indicating that this gene promotes sepal development in Chinese orchids. The Chinese orchid flowering regulatory gene disclosed herein can be used to study the molecular mechanisms of floral organ development in orchidaceae plants and improve flowering traits in plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a flowering regulatory gene AP1 of Cymbidium orchid, its encoded protein and application. Background Art

[0002] Orchids, with their highly specialized floral organs and rich biodiversity, are valuable ornamental flowers. Their floral organs differ significantly from the traditional perianth model of angiosperms. Six tepals are arranged in two whorls, with petals in the inner whorl and sepals in the outer whorl. The central petal is specialized into the labellum. Furthermore, the stamens and pistils fuse into a single column, demonstrating a post-ovary developmental pattern that only initiates after pollination. This represents a peak in seed plant phylogeny within monocots, making them ideal materials for studying floral development and the evolution of floral organ systems in monocots. Whole-genome sequencing has annotated a number of MADS-related genes, and studies of Phalaenopsis and Oncidium have proposed a molecular hypothesis for orchid perianth formation based on a tetrad model. However, the petal variation of Cymbidium orchids is far richer than that of tropical aerial orchids such as Phalaenopsis and Oncidium. Through artificial domestication and breeding, a variety of sepal and petal morphological variations have been cultivated, including plum-petal, lotus-petal, narcissus-petal, and butterfly-petal varieties. Varieties such as fewer or no petals, multiple petals, and tree-shaped flowers have also been developed, significantly enhancing their ornamental and economic value. However, Cymbidium orchids currently rely primarily on domestication and breeding from wild resources, resulting in a limited number of varieties suitable for large-scale and industrialized production. Hybrid breeding is difficult and time-consuming, and the progeny traits are difficult to predict. Modern molecular biotechnology breeding methods are seriously lagging behind. Therefore, how to improve ornamental traits and efficiently breed new varieties through modern molecular biotechnology has become a pressing scientific challenge. Summary of the Invention

[0003] The purpose of the present invention is to overcome the problems of long cycle and unpredictable traits in traditional hybrid breeding. The present invention uses the latest molecular biological methods to explore genetic resources that can be used to improve plant flower types and achieve improvement of flowering traits of Chinese orchids.

[0004] To achieve the above object, the present invention provides a flowering regulatory gene of Cymbidium sinense, which is isolated from the floral organ cDNA of the Chinese orchid variety 'Bai Mo' (Cymbidium sinense), and its nucleotide sequence consists of 744 bases, as shown in SEQ ID NO:1.

[0005] The second object of the present invention is to provide a protein encoded by the above-mentioned Cymbidium flowering regulatory gene, which consists of 247 amino acid residues, as shown in SEQ ID NO:2.

[0006] The third object of the present invention is to provide an expression vector, a transgenic cell line, a host bacteria or a transgenic material comprising the above-mentioned Cymbidium flowering regulatory gene.

[0007] A fourth object of the present invention is to provide the use of the aforementioned flowering regulatory gene for improving flowering traits in plants. Preferably, the improved flowering traits of plants are to increase the number of lateral inflorescences, and / or reduce the number of petals, and / or promote the transformation of petals into sepals.

[0008] A fifth object of the present invention is to provide the use of the above-mentioned Cymbidium flowering regulatory protein in improving flowering traits of plants. Preferably, the improved flowering traits of plants are to increase the number of lateral inflorescences, and / or reduce the number of petals, and / or promote the transformation of petals into sepals.

[0009] The sixth object of the present invention is to provide a method for increasing the number of lateral inflorescences of plants, and / or having fewer petals, and / or promoting the transformation of petals into sepals, which comprises the following steps: constructing a recombinant expression vector that overexpresses the above-mentioned Cymbidium flowering regulatory gene, and transforming the recombinant expression vector into the plant.

[0010] Preferably, the said transforming the recombinant expression vector into the plant is to transform the recombinant expression vector into Agrobacterium, and then infect the plant with the obtained recombinant Agrobacterium.

[0011] The present invention uses genetic engineering technology to discover that overexpression of the orchid organ development regulatory gene in Arabidopsis thaliana can change the flowering characteristics of the plant, resulting in loss of apical dominance, increased lateral branches, abnormal petal development, and transformation of petals into sepals. Therefore, the gene and the protein it encodes can be used to study the regulatory pathways of flowering characteristics such as floral organ development and the number of lateral branches, as well as to improve the flowering characteristics of plants.

[0012] The beneficial effects of the present invention are:

[0013] The present invention isolated a floral organ development regulatory gene from the floral organ cDNA of the Chinese orchid cultivar 'White Ink' (Cymbidium sinense). Increasing its expression can cause loss of apical dominance in Arabidopsis thaliana, an increase in lateral inflorescences, irregular arrangement of florets on the inflorescence axis, and abnormal petal development in flowers, with the occurrence of fewer petals or petals transforming into sepals. In Cymbidium sinense, a significant positive correlation was found between gene expression and an increase in sepals, and expression was significantly elevated in variants with a tree-forked flower pattern, indicating that this gene promotes sepal development in Cymbidium sinense. The Cymbidium sinense flowering regulatory gene disclosed herein can be used to study the molecular mechanisms of floral organ development in orchidaceae plants and improve flowering traits in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the phylogenetic tree analysis of CsAP1 from Example 1 of the present invention and its origin from other species.

[0015] Figure 2 This is the analysis of the expression pattern of CsAP1 in different tissues of Cymbidium sinensis in Example 2 of the present invention.

[0016] Figure 3 This is the analysis of the tissue expression pattern of CsAP1 at different developmental stages of Cymbidium sinensis in Example 2 of the present invention.

[0017] Figure 4 This is the phenotypic analysis of transgenic Arabidopsis thaliana in Example 3 of the present invention.

[0018] Figure 5 This is the AP1 gene expression profile in different petal-type variants of Cymbidium in Example 4 of the present invention. WT_1 to WT_4 represent the standard flower type; LaPV1_1 to LaPV1_4 represent the butterfly-petal flower variety 'Huaxi Hedie'; LaPV2_1 to LaPV2_4 represent the butterfly-petal flower variety 'Sanxingdie'; MPV_1 to MPV_4 represent the double-petal flower variety 'Yushizi'; and NLV_1 to NLV_4 represent the six-petal flower variety 'Fucui'.

[0019] Figure 6 This is the verification of AP1 gene expression in different petal-shaped variants of Cymbidium in Example 4 of the present invention. WT represents the common variety 'Yinzhen'; Var1 represents the tree Y flower mutant material 'Cui Yu Mudan'. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and beneficial technical effects of the present invention clearer, the present invention is further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are only for the purpose of explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc. of the embodiments can be selected according to local conditions without substantial impact on the results.

[0021] Example 1 Cloning and sequence analysis of the CsAP1 gene

[0022] 1. RNA Extraction

[0023] 2 g of floral organ tissue from the cultivar 'Bai Mo' of Cymbidium sinense was used to extract total RNA using plant Trizol reagent (Invitrogen) and reverse transcribed into cDNA (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit).

[0024] 2. Acquisition of the target gene CsAP1

[0025] PCR was performed using primers CsAP1-F1: 5'-GTCAATAGAGAAAGTACAATG-3' (SEQ ID NO: 3) and CsAP1-R1: 5'-TTATCCATTCATATGAGTGAGC-3' (SEQ ID NO: 4) and the cDNA obtained in step 1 above as a template using Ex-Taq enzyme (TaKaRa Biotechnology Co.) under the following conditions: 94°C for 4 min, followed by 34 cycles (94°C for 40 s, 59.5°C for 40 s, 72°C for 1.5 min, and 72°C for 10 min). The PCR product was recovered from an agarose gel, ligated into the pMD19-T vector (TaKaRa Biotechnology Co.), and sent to BGI for sequencing. Sequencing analysis revealed that the amplified fragment contained the complete CDs sequence of the target gene, CsAP1, consisting of 744 bases. The nucleotide sequence is shown in SEQ ID NO:1 and is designated the Cymbidium orchid organ development regulatory gene (CsAP1 gene). The amino acid sequence of the protein encoded by it consists of 247 amino acid residues, as shown in SEQ ID NO:2 and is designated Cymbidium orchid organ development regulatory protein (CsAP1 protein). The resulting E. coli containing CsAP1-pMD19-T is currently stored at the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences.

[0026] The described CsAP1 gene has the nucleotide sequence shown in SEQ ID NO.1, and the specific sequence is as follows: ATGGGAAGAGGGAGGGTTCAGTTGAAGCGAATTGAGAATAAAATTAACCGGCAGGTGACTTTCTCTAAGCGAAGATCGGGGTTGCTTAAGAAGGCTCACGAGATATCGGTGCTCTGCGACGCTGAGGTCGCTCTAATCGTGTTTTCTAACAAGGGAAAACTCTATGAGTATTCCACCGAAGCCAGTATGGAGAAGATTCTTGAACGGTATGAGCGCCATTCATACGCTGAAAGAGCATTATTTTCCAATGAGGCTAACTCACAGGCTGATTGGCGCCTTGAATATAATAAATTGAAGGCAAGGGTCGAAAGCTTACAGAAGAGCAAACGGCACCTTATGGGGGAGCAACTTGATTCCTTGAGCACTAAAGAACTTCAACATCTAGAGCAACAGCTTGAAAGTTCCTTGAAACATATACGATCTAGAAAGACCCAGCTCATGCTCGATTCAATTTCCGAGCTACAAAAAAAGGAAAAATTATTGCTGGATCAAAACAAGACCTTAGAGAAAGAGATTATGGCTAAAGAGAAGGCCAAAGCTTTGGTGCAGAATGCACCTTGGGAAAAGCAAAACCAATGCCAGTATAGCTCTGCTCCATCTCATGCCGAAATTTCGAATTTTGGTTCAACTCCTGCCAGCAGAACTCTTCGAGCAAGAGCCAGTGAAGAAGAATCACCCCAGCCACAGTTAAGATTAGGCAACACTTTACTGCCGCCATGGATGCTCACTCATATGAATGGATAA。

[0027] The protein encoded by the CsAP1 gene has the amino acid sequence shown in SEQ ID NO.2, and the specific sequence is as follows: MGRGRVQLKRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSNKGKLYEYSTEASMEKILERYERHSYAERALFSNEANSQADWRLEYNKLKARVESLQKSKRHLMGEQLDSLSTKELQHLEQQLESSLKHIRSRKTQLMLDSISELQKKEKLLLDQNKTLEKEIMAKEKAKALVQNAPWEKQNQCQYSSAPSHAEISNFGSTPASRTLRARASEEESPQPQLRLGNTLLPPWMLTHMNG.

[0028] 3. CsAP1 gene sequence analysis

[0029] Homologous sequences of the Cymbidium orchid CsAP1 gene were searched in NCBI. The amino acid sequences were aligned using MEGA software, and a phylogenetic tree was constructed. The results showed that CsAP1 is highly conserved across species, possessing the MADS and K domains conserved by MADS-box genes. It shares 99% homology with Cymbidium orchid MADS1 (KC148540) and Cymbidium orchid MADS-box (MF474256), and 98.9% homology with Cymbidium orchid / FUL gene KX347442. The sequence identity is 82.7-88.0% with the DoMADS2 (AF198175) in Dendrobium orchid, CAL (XM_020737650) in Phalaenopsis (DQ104327), and MADS gene in Orchidaceae (KF914211), and 80% identity with CAL gene (XM_024167505, XM_024167506) in Morus alba. Figure 1 ).

[0030] Example 2 Expression pattern of CsAP1 in orchids

[0031] 1. RNA Extraction

[0032] Total RNA was extracted from 2 g of plant tissue from different parts of the Cymbidium orchid cultivar 'Bai Mo' during flowering, including sepals, petals, labellum, four whorls of floral organs, inflorescence axis, pedicel, outer bracts, and inner bracts. Trizol (Invitrogen) was used to extract total RNA, and 2 μL of the RNA was reverse transcribed into cDNA using the Thermo Scientific RevertAid FirstStrand cDNA Synthesis Kit.

[0033] 2. Quantitative PCR

[0034] Real-time quantitative PCR was performed to detect CsAP1 gene expression in different orchid tissues using primers CsAP1QRT-F: 5'-GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R: 5'-AGGAATCAAGTTGCTCCCCC-3' (SEQ ID NO: 6). Actin QRT-F: 5'-ATGTCGCCATCCAAGCTGTT-3' (SEQ ID NO: 7) and Actin QRT-R: 5'-CACGTCCAGCAAGGTCAAGA-3' (SEQ ID NO: 8) were used as primers, and Actin was used as an internal control. The following protocol was used: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 59.5°C for 10 seconds, and 72°C for 30 seconds, followed by extension at 72°C for 10 minutes. Amplification was performed using the iCycler IQ Real-time PCR Detection System (Bio-Rad, USA), and the operation was performed according to the instructions of the HiscriptⅡQRT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd) kit.

[0035] 3. Expression analysis

[0036] The PCR results were analyzed using icycler realtime detection system software (version 7.0). It was found that the expression level of CsAP1 gene in the four floral organs of Cymbidium sinense at the flowering stage was low, including sepals, petals, lip and column, while the expression level in the inflorescence axis, pedicels, outer bracts and inner bracts was significantly increased. Figure 2 The expression levels of the above tissues were further detected during the development of black orchids, from the semi-dormant period of flower buds in September, the rapid elongation period of pedicels in December, and the opening period of flowers in January. From September 8 to January 14 of the following year, 9 samples were collected to detect the changes in CsAP1 expression. It was found that the expression level of CsAP1 gradually decreased with the development of flower buds and the opening of flowers ( Figure 3 ).

[0037] Example 3 Functional analysis of the CsAP1 gene in Arabidopsis

[0038] 1. Construction of high expression vector for transformation of Arabidopsis thaliana

[0039] The CDs sequence of the CsAP1 gene from Cynanchum chinense (CsAP1 gene, nucleotide sequence shown in SEQ ID NO: 1) was amplified using primers CsAP1-F1: 5'-GTCAATAGAGAAAGTACAATG-3' (SEQ ID NO: 3) and CsAP1-R1: 5'-TTATCCATTCATATGAGTGAGC-3' (SEQ ID NO: 4) and cloned into the pMD19-T vector (Takara) and sent to BGI for sequencing. After sequencing, the fragment was purified using EcoRI and SacI double digestion (Thermo Fisher Scientific) and ligated into the pBI121 vector, which had been digested and purified using the same two enzymes and named pBI-AP1.

[0040] 2. Transformation of Arabidopsis plants

[0041] 2.1 Transformation of Agrobacterium GV3101

[0042] 1) Thaw the competent cells stored at -80°C on ice. After the cells are completely lysed, add approximately 1 μg of plasmid (pBI-AP1), mix gently, and place on ice for 30 minutes.

[0043] 2) Quickly freeze the cells in liquid nitrogen for 1 minute, then thaw at 37°C for 5 minutes. Repeat this process. Add 1 mL of LB and incubate at 28°C with shaking at 200 rpm for 4-6 hours.

[0044] 3) After shaking the bacterial suspension to concentrate, centrifuge at 4000 rpm for 5 minutes at room temperature. Discard the supernatant and resuspend the Agrobacterium pellet in 100 μL of LB medium.

[0045] 4) Spread the entire mixture onto LB plates containing 50 μg / mL kanamycin and 25 μg / mL gentamicin and incubate in a 28°C incubator for 48-72 hours. Once colonies have grown, re-streak the bacteria to ensure they are single clones, thereby obtaining Agrobacterium transformed with pBI-AP1.

[0046] 2.2 Arabidopsis transformation using the inflorescence infection method.

[0047] Inoculate Arabidopsis inflorescences with Agrobacterium transformed with pBI-AP1 at an OD of 0.3-0.5 for 2-3 seconds, remove from the inflorescence, seal in the dark for 16 hours, and then transfer to a growth chamber for further growth. After approximately 3-4 weeks, harvest the seeds and store them at 4°C in the dark.

[0048] 2.3 Arabidopsis transformant screening

[0049] 1) Prepare a 0.1% mercuric chloride solution.

[0050] 2) Place the collected seeds in a 1.5 mL Eppendorf tube, add 1 mL of 75% alcohol, shake well, and rotate on a Blood Tube Rotator for 5 minutes. Sterilize with 0.1% mercuric chloride for 5 minutes.

[0051] 3) Centrifuge at 6000 rpm for 2 minutes. Discard the supernatant, add 1 mL of sterile water, mix thoroughly, and rotate on a Blood Tube Rotator for 2 minutes. Repeat three times.

[0052] 4) Remove the supernatant, resuspend the seeds in 1 mL of sterile water, and spread them onto the surface of 1 / 2 MS medium containing 50 μg / mL kanamycin. Seal the plate and vernalize in the dark at 4°C for 3 days. Afterwards, move the plate to a tissue culture room (16 h L / 8 h D) at 23°C with light.

[0053] 5) After the seedlings grow green cotyledons, observe and count the number of seedlings with green cotyledons. Remove the culture medium adhering to the roots and transfer them to the substrate for culture.

[0054] 6) After a life cycle of approximately 60 days, the first generation of transgenic Arabidopsis seeds were obtained. The second generation homozygous seeds (transgenic plants) were harvested for subsequent experiments.

[0055] 3. Analysis of CsAP1 expression in transgenic Arabidopsis

[0056] To determine the biological function of the CsAP1 gene in Arabidopsis, cDNA from wild-type Arabidopsis and transformants obtained in step 2 (transgenic plants grown from homozygous T2 seeds) was used as templates. Primers CsAP1QRT-F: 5'-GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R: 5'-AGGAATCAAGTTGCTCCCCC-3' (SEQ ID NO: 6) were used to detect the expression of the CsAP1 gene in transgenic Arabidopsis. The following protocol was used: initial denaturation at 95°C for 30 s, followed by 40 cycles (95°C for 10 s, 59.5°C for 10 s, and 72°C for 30 s), and extension at 72°C for 10 min. Amplification was performed using the iCycler IQ Real-time PCR Detection System (Bio-Rad, USA) and the Hiscript II QRT SuperMix for qPCR (with gDNA wiper) (Vazyme Biotech Co., Ltd.) kit instructions. The results showed that CsAP1 expression levels were increased in all transgenic plants obtained, and three of these lines were selected for subsequent phenotypic analysis.

[0057] 4. Phenotypic Analysis of Transgenic Arabidopsis

[0058] Harvested T3 generation homozygous transgenic plant seeds were surface-sterilized and plated on MS medium containing 50 μg / mL kanamycin. They were cultured in the dark at 4°C for two days and then transferred to a light cycle of 16 hours per day (8 hours per day) at 23°C. During the flowering stage of Arabidopsis, abnormal morphological structures of floral organs were observed in the transgenic plants, including abnormal petal development, transformation of petals into sepals, and curling of one petal per flower. Furthermore, all transgenic lines exhibited a loss of apical dominance, increased lateral branches, and the development of more numerous inflorescences ( Figure 4 ).

[0059] Example 4 Correlation Analysis between CsAP1 Gene Expression and Different Flower Petal Types in Cymbidium

[0060] 1. Materials of different petal shapes of orchids

[0061] Using the Chinese orchid, a species of perianth, the study selected varieties with a standard, small, fragrant flower shape and different petal-shaped variants. These included the perianth varieties 'Huaxi Hedie' (labellum-like perianth variety 1, LaPV1) and 'Sanxingdie' (labellum-like perianth variety 2, LaPV2), which exhibit a transition from petals to lip petals; the multi-perianth variety 'Yushizi' (double-petaled perianth variety, MPV); and the null-lip variety 'Fucui' (null-lip variety, NLV), which lacks both lip petals and stigma. Total RNA was extracted from 2g of each of the four floral organs (sepals, petals, lip petals, and stigma) from newly opened flowers of each variety using the plant Trizol reagent (Invitrogen). The RNA samples were then tested for purity, concentration, and integrity. Once the samples passed the test, library construction was performed. The main process is as follows:

[0062] (1) Enrichment of eukaryotic mRNA using magnetic beads with Oligo(dT);

[0063] (2) Add Fragmentation Buffer to randomly fragment the mRNA;

[0064] (3) Using mRNA as a template, the first cDNA chain was synthesized using random hexamers. Then, buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second cDNA chain. The cDNA was purified using AMPure XP beads.

[0065] (4) The purified double-stranded cDNA was then end-repaired, A-tailed, and ligated to sequencing adapters, and then size-selected using AMPure XP beads;

[0066] (5) Finally, the cDNA library was obtained by PCR enrichment.

[0067] After the library construction was completed, the concentration and insert size of the library were detected using Qubit2.0 and Agilent 2100, respectively, and the effective concentration of the library was accurately quantified using the Q-PCR method to ensure the quality of the library.

[0068] 2. Transcriptome Sequencing

[0069] Illumina Hiseq TM The resulting raw image data files were converted into raw sequenced reads through CASAVA base calling analysis. After base composition and quality analysis, the data were filtered based on the raw data analysis results to remove adapter sequences and contaminants. Sequences containing excessive "N" bases (>10%) and excessive low-quality bases (bases with a quality score below 20 accounting for more than 20% of the entire sequence) were also removed.

[0070] 3. Gene Expression Analysis

[0071] The quality-controlled sequencing reads were aligned to the reference genome using HISAT2. RSeQC statistical alignment results were used to group the different sequencing samples. Gene expression levels in different groups were compared using expression distribution plots and box plots for all genes. For replicate samples within the same group, the final expression level was the average of all replicates. Sequencing reads were aligned to the Unigene library using Bowtie. Expression levels were estimated based on the alignment results and combined with RSEM. FPKM values ​​were used to represent the expression abundance of the corresponding Unigene.

[0072] FPKM (Fragments Per Kilobase of transcript per Million mapped reads) is the number of reads per kilobase of length mapped to a gene per million reads. It is a commonly used method for estimating gene expression levels in transcriptome sequencing data analysis. FPKM can eliminate the effects of differences in gene length and sequencing load on calculated gene expression. The FPKM calculation formula is as follows:

[0073]

[0074] In the formula, cDNA Fragments represents the number of fragments mapped to a certain transcript, that is, the number of paired-end reads; Mapped Fragments (Millions) represents the total number of fragments mapped to the transcript, in units of 10^6; Transcript Length (kb): transcript length, in units of 10^3 bases. The results showed that the expression level of CsAP1 was low in all whorls of floral organs, with a slightly higher expression level in sepals. However, in six-petal flowers with sepalized tepals and lacking normal petals, lip and syncytium, the expression level increased, especially in the outermost whorl of sepals, where the expression level was the highest, indicating that AP1 gene expression may be positively correlated with sepal development ( Figure 5 ).

[0075] 4. Gene expression verification

[0076] To further characterize the expression pattern of the AP1 gene in Cymbidium orchids, whole-flower cDNA from the common Cymbidium 'Yinzhen' and the Y-flower mutant 'Cuiyu Mudan' was used as templates. Primers CsAP1QRT-F: 5'-GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R: 5'-AGGAATCAAGTTGCTCCCCC-3' (SEQ ID NO: 6) were used to detect AP1 gene expression in Cymbidium orchids. The following protocol was used: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 57°C for 10 seconds, and 72°C for 26 seconds. The same cDNA was also used as a template to amplify Actin as an internal control using Actin QRT-F: 5'-ATGTCGCCATCCAAGCTGTT-3' (SEQ ID NO: 7) and Actin QRT-R: 5'-CACGTCCAGCAAGGTCAAGA-3' (SEQ ID NO: 8). The following protocol was used: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles (95°C for 10 seconds, 59.5°C for 10 seconds, and 72°C for 30 seconds), and extension at 72°C for 10 minutes. Amplification was performed using the iCycler IQ Real-time PCR Detection System (Bio-Rad, USA) according to the instructions for the HiscriptⅡQRT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd.) kit. The results showed that AP1 expression was significantly elevated in the truncation flower type, which only developed sepal-like tepals without petals, labellum, or glyptostachys. Figure 6 These results suggest that AP1 in Cymbidium sinense may control the development of sepals and may also play an important regulatory role in the structure of flower branches.

[0077] Based on the disclosure and teachings of the above description, those skilled in the art may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and any modifications and variations of the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although certain specific terms are used in this description, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. Application of a flowering regulatory gene of Cymbidium orchid in improving flowering traits of plants, wherein the improved flowering traits of plants are to increase the number of lateral inflorescences, and / or reduce the number of petals, and / or promote the transformation of petals into sepals; the nucleotide sequence of the flowering regulatory gene of Cymbidium orchid is shown in SEQ ID NO: 1, and the plant is Arabidopsis thaliana.

2. Application of a flowering regulatory protein of Cymbidium orchid in improving flowering traits of plants, wherein the improvement of flowering traits of plants is to increase the number of lateral inflorescences, and / or reduce the number of petals, and / or promote the transformation of petals into sepals; the amino acid sequence of the flowering regulatory protein of Cymbidium orchid is shown in SEQ ID NO: 2, and the plant is Arabidopsis thaliana.

3. A method for increasing the number of lateral inflorescences of a plant, and / or reducing the number of petals, and / or promoting the transformation of petals into sepals, characterized in that: The following steps are involved: A recombinant expression vector for overexpressing the flowering regulatory gene of Cymbidium orchid is constructed and the recombinant expression vector is transformed into a plant. The nucleotide sequence of the flowering regulatory gene of Cymbidium orchid is shown in SEQ ID NO: 1, and the plant is Arabidopsis thaliana.

4. The method according to claim 3, characterized in that The recombinant expression vector is transformed into Agrobacterium, and then the obtained recombinant Agrobacterium is used to infect plants.

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