Coding sequence of flowering regulatory gene SEP1 in Cymbidium sinense and its application

Abstract

The present invention discloses a coding sequence of a flowering regulatory gene SEP1 of Chinese orchid and its application. The present invention isolates an orchid flower organ development regulatory gene from the flower organ cDNA of the Chinese orchid variety 'Bai Mo'. After increasing its expression level, the flowering period of Arabidopsis thaliana can be significantly advanced, but the flower organ morphological structure is not affected. Through downstream gene expression analysis, it is found that the gene can positively regulate the expression of flowering control genes FT, SOC1, and LFY1, further indicating the role of the gene in promoting plant flowering. The Chinese orchid flowering regulatory gene of the present invention can be used for the study of the molecular mechanism of flowering regulation of orchidaceae plants and the improvement of flowering traits of 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 SEP1 of Cymbidium sinense, its encoded protein and application. Background Art

[0002] Orchids are a globally important flower crop. Terrestrial species of the genus Orchid, mostly native to China, are also known as Chinese orchids. With a cultivated history of over 800 years, they are listed as one of China's ten famous flowers. They primarily include eight species: Jianlan, Molan, Chunlan, Huilan, Lianbanlan, Doubanlan, Hanlan, and Chunjian. However, Chinese orchid varieties currently rely primarily on domestication and selection from wild sources, resulting in a limited number of varieties suitable for large-scale industrialization. Hybrid breeding is difficult and time-consuming, with unpredictable offspring traits. Modern molecular biotechnology breeding methods are severely underdeveloped.

[0003] Significant progress has been made in crop breeding through the construction of efficient molecular breeding technology systems, including large-scale gene discovery, functional molecular markers for important traits, and gene introduction and knockout. Flowering characteristics are key factors in determining the ornamental and economic value of Cymbidium orchids and promoting their industrial development. With the rapid development and widespread application of new-generation molecular breeding technologies, such as multi-omics molecular design breeding and gene editing, in-depth analysis of the key functional genes that determine flowering in Cymbidium orchids will serve as an important basis for molecular breeding and targeted improvement, with broad industrial application prospects and economic value. Summary of the Invention

[0004] 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.

[0005] 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 738 bases, as shown in SEQ ID NO:1.

[0006] The second object of the present invention is to provide a protein encoded by the above-mentioned Cymbidium flowering regulatory gene, which consists of 245 amino acid residues, as shown in SEQ ID NO:2.

[0007] 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.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned Cymbidium flowering regulatory gene in improving the flowering characteristics of plants. Preferably, the improvement of the flowering characteristics of plants is to improve the flowering period of plants in advance.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned Cymbidium flowering regulatory protein in improving the flowering characteristics of plants. Preferably, the improvement of the flowering characteristics of plants is to improve the flowering period of plants in advance.

[0010] The sixth object of the present invention is to provide a method for promoting early flowering of plants, 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.

[0011] Preferably, the recombinant expression vector is transformed into the plant by transforming the recombinant expression vector into Agrobacterium, and then infecting the plant with the obtained recombinant Agrobacterium.

[0012] Through genetic engineering, the present invention discovered that overexpression of the orchid organ development regulatory gene in Arabidopsis thaliana can alter the plant's flowering characteristics, significantly advancing the flowering period without affecting the morphological structure of the floral organs. Therefore, this gene and the protein it encodes can be used to study flowering regulation pathways and improve flowering timing traits in plants.

[0013] The beneficial effects of the present invention are:

[0014] This study isolated a floral organ development regulatory gene from the floral organ cDNA of the Chinese orchid cultivar 'White Ink' (Cymbidium sinense). Increasing its expression significantly advanced the flowering period of Arabidopsis thaliana without affecting floral organ morphology. Downstream gene expression analysis revealed that this gene positively regulates the expression of flowering control genes FT, SOC1, and LFY1, further demonstrating its role in promoting flowering in plants. This flowering regulatory gene can be used to study the molecular mechanisms of flowering regulation in orchidaceae plants and improve flowering traits in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the phylogenetic tree analysis of CsSEP1 and other species in Example 1 of the present invention.

[0016] Figure 2This is the phylogenetic tree of the SEP family of Orchidaceae in Example 1 of the present invention. As: Apostasia shenzhenica; At: Arabidopsis thaliana; Cs: Cymbidium sinense; Dc: Dendrobium catenatum; Pe: Phalaenopsis equestris; Os: Oryza sativa.

[0017] Figure 3 This is an analysis of the tissue expression pattern of the SEP1 gene family at different developmental stages of Cymbidium in Example 2 of the present invention: FD1: floral meristem stage, FD2: early floral primordium stage, FD3: late floral primordium stage, FD4: bud stage, and FD5: anthesis stage.

[0018] Figure 4 This is an analysis of the expression patterns of the SEP1 gene family in different floral organs of Cymbidium sinense in Example 2 of the present invention.

[0019] Figure 5 This is the phenotypic analysis of transgenic Arabidopsis thaliana in Example 3 of the present invention. WT represents the wild type; 35S:CsSEP1 represents the transgenic Arabidopsis thaliana plant.

[0020] Figure 6 This is a comparative analysis of the bolting time of transgenic Arabidopsis thaliana in Example 3 of the present invention. WT represents the wild type; line 2, line 3, and line 9 represent three independent T2 transgenic plant lines, respectively.

[0021] Figure 7 This is the expression analysis of flowering-related genes in transgenic Arabidopsis thaliana in Example 3 of the present invention. CK represents wild-type control plants, and line 2, line 3, and line 9 represent three independent T2 transgenic plant lines, respectively. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1 Cloning and sequence analysis of the CsSEP1 gene

[0024] 1. RNA Extraction

[0025] 2 g of floral organ tissue of Cymbidium cultivar 'Bai Mo' was collected and total RNA was extracted using plant Trizol reagent (Invitrogen) and reverse transcribed into cDNA (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit).

[0026] 2. Acquisition of the target gene CsSEP1

[0027] PCR was performed using primers CsSEP1-F1: 5'-TTTATGGGGAGGGGGAGAG-3' (SEQ ID NO: 3) and CsSEP1-R1: 5'-GCCTCACATCCAACCTGAAA-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, CsSEP1, consisting of 738 bases. The nucleotide sequence is shown in SEQ ID NO:1 and is named the Cymbidium orchid organ development regulatory gene (CsSEP1 gene). The amino acid sequence of the protein encoded by it is composed of 245 amino acid residues, as shown in SEQ ID NO:2, and is named Cymbidium orchid organ development regulatory protein (CsSEP1 protein). The resulting E. coli containing CsSEP1-pMD19-T is currently stored at the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences.

[0028] The CsSEP1 gene has a nucleotide sequence shown in SEQ ID NO. 1, and the specific sequence is as follows: ATGGGGAGGGGGAGAGTGGAGTTGAGGAGGATTGAGAACAAGATAAACAGGCAAGTGACTTTTGCGAAGAGGAGGAATGGGTTGTTGAAGAAGGCTTATGAGCTATCGGTGCTC TGCGATGCGGAGGTTGCGCTGATCGTCTTCTCCAACCGCGGCAGACTATTTGAGTTCTGCAGCAGCAGTAGCATGGCAACAACCCTTGAGAGGTACCAAAAGTGCAGCTATAATGCATCTGACTCCATGGTTCCATCCAAGGATACACAGCACAGTTATCAGGAGTACTTAAAGCTGAAAGCAAAAGTGGAATATCTACAACTGTCTCAAAGGAATCTTCTAGGAGAGGACC TAGTTGAATTAAGCAGCAAGGAACTGGAGGAACTTGAGCTCCAATTAGAAATGTCTCT GAAACATATCAGGTCAACAAAGACTCAATTGATGCTCGATCAGCTCTGTGATCTCAAA AGAAAGGAGCAAATCTTGCATGAAGCTAACAGAGCTTTAAAAAGAAAGTTGCAGGAAGATGGACCAGAGATTCCCCTGGAGCTGACCTGGCCTGGTGGAGGCGCTAATGGGTCAT GTGAACGTCACCAGCCTCAACCAGATGAATTATTCTTTCAGCCCCTGCCTTGTGACCCTTCTTTGCAAATTGGGTATAGTCCAATCTATATAGATCAACAGTTGAACACAGGATCAAC ATCCGCACATAATATTAATGGCTTCTTTTCAGGTTGGATGTGA.

[0029] The protein encoded by the CsSEP1 gene has the amino acid sequence shown in SEQ ID NO.2, and the specific sequence is as follows: MGRGRVELRRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIVFSNRGRLF

[0030] EFCSSSSMATTLERYQKCSYNASDSMVPSKDTQHSYQEYLKLKAKVEYLQLSQRNL LGEDLVELSSKELEELELQLEMSLKHIRSTKTQLMLDQLCDLKRKEQILHEANRALKRKLQEDGPEIPLELTWPGGGANGSCERHQPQPDELFFQPLPCDPSLQIGYSPIYIDQQLNTGSTS AHNINGFFSGWM.

[0031] 3. CsSEP1 gene sequence analysis

[0032] Homologous sequences of the CsSEP1 gene from Cynanchum indica were searched in NCBI. The amino acids encoded by the homologous sequences were aligned using MEGA software, and a phylogenetic tree was constructed. The results showed that CsSEP1 is highly conserved across species, with all possessing the MADS domain and K domain conserved by MADS-box genes. Among them, it has 99% homology with Cymbidium MADS1 (KC148540), Chunlan MADS-box (MF46208), Chunlan SEP2 (KX347447), 98.9% homology with AP1 / FUL gene KX347442, 88% homology with Dendrobium DcMADS2 (XM_020822814), Phalaenopsis SEP2 (KF673858), 87% homology with Phalaenopsis CMB1-ike MADS-box gene, and 81% homology with peanut SEP1 gene XM_025799117, 118, etc. ( Figure 1 ).

[0033] Further analysis of the whole genome sequencing data of Cymbidium sinense revealed four SEP genes at the genomic level. Four SEP genes were also found in other Cymbidium species, including Jianlan, Chunlan, and Hanlan, through transcriptome sequencing and gene cloning. Compared with other orchids whose genomes have been sequenced, it was found that Cymbidium pseudophyllum had three SEP genes, and Phalaenopsis and Dendrobium both had five SEP genes ( Figure 2 Sequence analysis revealed sequence differences between different paralogous genes, especially at the C-terminus.

[0034] Example 2 Expression pattern of CsSEP1 in orchids

[0035] 1. RNA Extraction

[0036] Total RNA was extracted using plant Trizol (Invitrogen) reagent, and 2 μL of RNA was reverse transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit.

[0037] 2. Quantitative PCR

[0038] Real-time quantitative PCR was performed to detect CsSEP1 gene expression in different orchid tissues using primers CsSEP1 QRT-F: 5'-TGATCGTCTTCTCCAACCGC-3' (SEQ ID NO: 5) and CsSEP1 QRT-R: 5'-CCTCCAGTTCCTTGCTGCTTA-3' (SEQ ID NO: 6). Actin QRT-F: 5'-ATGTCGCCATCCAAG CTGTT-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) (VazymeBiotech Co., Ltd) kit.

[0039] 3. Expression analysis

[0040] PCR results were analyzed using icycler realtime detection system software (version 7.0). The developmental process of orchids was divided into five stages, from the transition from the shoot apical meristem to the floral meristem to full flowering: the floral meristem stage, the early floral primordium stage, the late floral primordium stage, the bud stage, and the flowering stage. Real-time fluorescence quantitative PCR was used to detect the expression of the CsSEP1 gene at different stages of flower bud development. It was found that the expression levels of the gene did not differ significantly from the beginning of flower bud development to the opening of the flower, remaining at a low level. However, in the fully bloomed flower, the expression level increased significantly, by about 5.5 times ( Figure 3Further expression detection of the four floral organs of Cymbidium sinense during the flowering period, namely sepals, petals, lip and column, revealed that CsSEP1 was expressed at a higher level in the outer sepals and petals, while at a lower level in the inner lip and column ( Figure 4 ).

[0041] Example 3 Functional Analysis of the CsSEP1 Gene in Arabidopsis

[0042] 1. Construction of high expression vector for transformation of Arabidopsis thaliana

[0043] The CDs sequence of the C. cyrtonema CsSEP1 gene (i.e., the nucleotide sequence of the CsSEP1 gene, shown in SEQ ID NO: 1) was amplified using primers CsSEP1-F1: 5'-TTTATGGGGAGGGGGAGAG-3' (SEQ ID NO: 3) and CsSEP1-R1: 5'-GCCTCACATCCAACCTGAAA-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 designated pBI-SEP1.

[0044] 2. Transformation of Arabidopsis plants

[0045] 2.1 Transformation of Agrobacterium GV3101

[0046] 1) Thaw the competent cells stored at -80°C on ice. After the cells are completely lysed, add approximately 1 μg of plasmid (pBI-SEP1), mix gently, and place on ice for 30 minutes.

[0047] 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.

[0048] 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.

[0049] 4) Plate 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 h. Once colonies have grown, re-streak the bacteria to ensure they are single clones, thereby obtaining Agrobacterium transformed with pBI-SEP1.

[0050] 2.2 Arabidopsis transformation using the inflorescence infection method.

[0051] Inoculate Arabidopsis inflorescences with Agrobacterium transformed with pBI-SEP1 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.

[0052] 2.3 Arabidopsis transformant screening

[0053] 1) Prepare a 0.1% mercuric chloride solution.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 3. Analysis of CsSEP1 expression in transgenic Arabidopsis

[0060] To determine the biological function of the CsSEP1 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 CsSEP1QRT-F: 5'-TGATCGTCTTCTCCAACCGC-3' (SEQ ID NO: 5) and CsSEP1QRT-R: 5'-CCTCCAGTTCCTTGCTGCTTA-3' (SEQ ID NO: 6) were used to detect the expression of the CsSEP1 gene in transgenic Arabidopsis. The following protocol was used: initial denaturation at 95°C for 30 s, followed by 40 cycles of (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 procedures were performed according to the instructions for the Hiscript II QRT SuperMix for qPCR (with gDNA wiper) (Vazyme Biotech Co., Ltd.) kit. The results showed that CsSEP1 expression levels were increased in all transgenic plants obtained, and three of these lines were selected for subsequent phenotypic analysis.

[0061] 4. Phenotypic Analysis of Transgenic Arabidopsis

[0062] Harvested T2 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 2 days and then transferred to a light-period of 16 h L / 8 h D at 23°C. Phenotypic analysis of the transgenic plants revealed that overexpression of SEP1 significantly advanced flowering. Under equivalent growth conditions, bolting occurred 13-20 days earlier than in wild-type plants, and organ morphology developed normally, showing no significant changes compared to the wild-type. This suggests that SEP1 primarily affects flowering time and has no effect on floral organ morphology. Figure 5 , 6).

[0063] 5. SEP1 regulates the expression of flowering genes

[0064] To determine the role of the CsSEP1 gene in the flowering regulation pathway, four-week-old leaves of wild-type and transgenic Arabidopsis plants were used as materials. Total RNA was extracted using plant Trizol reagent (Invitrogen), and 2 μL of the total RNA was reverse-transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. Expression of flowering pathway genes FT, SOC1, and LFY1 in transgenic Arabidopsis was determined using the following protocol: initial 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), followed by extension at 72°C for 10 minutes. Amplification was performed using the iCycler IQ Real-time PCR Detection System (Bio-Rad, USA), following the instructions for the Hiscript II QRT SuperMix for qPCR (with gDNA wiper) (Vazyme Biotech Co., Ltd.). The primers used were AtFTQRT-F / R (AtFTQRT-F: 5'-TGGAGACGTTCTTGATCCGTT-3' (SEQ ID NO: 9) and AtFTQRT-R: 5'-TGAGGGTTGCTAGGACTTGG-3' (SEQ ID NO: 10)), AtSOC1QRT-F / R (AtSOC1QRT-F: 5'-TCGCCAGCTCCAATATGCAA-3' (SEQ ID NO: 11) and AtSOC1QRT-R: 5'-TCTGTTGCAGCTCCTCGATT-3' (SEQ ID NO: 12)), AtLFYQRT-F / R (AtLFYQRT-F: 5'-AGACGCCGTCATTTGCTACT-3' (SEQ ID NO: 13) and AtLFYQRT-R: 5'-CTGCGTCCCAGTAACCACTT-3' (SEQ ID NO: 14)). NO:14)) The results showed that the expression levels of flowering positive regulatory factors FT, SOC1, and LFY1 were significantly increased in the transgenic lines, further verifying the role of the SEP1 gene in promoting flowering ( Figure 7 ).

[0065] 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 flowering trait of plants is improved by advancing the flowering period of plants, 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 Cymbidium flowering regulatory protein in improving flowering traits of plants, wherein the improvement of flowering traits of plants is to advance the flowering period of plants, the amino acid sequence of Cymbidium flowering regulatory protein is shown in SEQ ID NO: 2, and the plant is Arabidopsis thaliana.

3. A method for promoting early flowering of Arabidopsis thaliana, characterized in that: The following steps are involved: A recombinant expression vector of the flowering regulatory gene of Cymbidium wilfordii with an overexpression sequence as shown in SEQ ID NO: 1 was constructed, and the recombinant expression vector was transformed into 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 Arabidopsis.

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