Pbjaz1 gene and application thereof in regulating plant flowering

Abstract

The application discloses a pear PbJAZ1 gene and application thereof in regulating plant flowering. The amino acid sequence of the pear PbJAZ1 protein is shown as SEQ ID NO:1; and the nucleotide sequence of the pear PbJAZ1 protein coding gene is shown as SEQ ID NO:2. Researches show that increasing the activity or / and content of the pear PbJAZ1 protein in target plants, or overexpressing the pear PbJAZ1 gene coding the pear PbJAZ1 protein can promote early flowering of plants or / and cultivate new germplasm of transgenic plants with advanced flowering time and apply the new germplasm in production. The results show that the pear PbJAZ1 gene plays a role in regulating plant flowering.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to the pear PbJAZ1 gene and its application in regulating plant flowering. Background Technology

[0002] Pear (Pyrus L.) is a global fruit, and my country is the world's largest producer of pears, ranking first in both cultivated area and yield. Pear flowering is a decisive factor in yield formation, a crucial point in the transition from vegetative to reproductive growth, and is regulated by multiple factors, including external environment and internal factors. Therefore, identifying and studying genes that regulate pear flowering is of great significance for controlling flowering time, increasing yield, and breeding new varieties.

[0003] The JAZ (Jasmonate ZIM-domain) protein, as a core negative regulator of the jasmonic acid (JA) signaling pathway, is a key component of the plant flowering time regulation network, precisely regulating the flowering process by integrating hormonal and environmental signals. Studies have shown that the regulation of flowering by JAZ proteins is species-specific, and its function depends on interactions with key transcription factors and its own signal response characteristics.

[0004] The JAZ protein family all contain conserved ZIM and Jas domains, with the Jas domain being the core mediator of flowering regulation, responsible for binding to flowering-related transcription factors. In Arabidopsis thaliana, JAZ proteins regulate the expression of the floriculture hormone gene FT through interaction with TOE1 / TOE2. Full-length JAZ proteins maintain flowering rhythm balance, while overexpression of the JAZ1ΔJas truncated form accelerates flowering. In rice, knockout of OsJAZ9 advances flowering in japonica rice, which is related to the regulation of pulp cell wall rigidity. Overexpression of ScJAZ15-1 in sugarcane leads to early flowering, reflecting species-specific functional differentiation.

[0005] JA signaling activation promotes COI1-dependent ubiquitination and degradation of JAZ proteins, thereby relieving their regulatory role on flowering genes. When plants encounter stress, increased JA levels induce JAZ degradation, which in turn downregulates FT expression, delaying flowering and ensuring reproductive success. Furthermore, JAZ proteins can interact across pathways, such as with auxin signaling proteins, integrating multiple signals to regulate the flowering process. In different species such as Arabidopsis, rice, and chrysanthemum, JAZ homologs are involved in flowering regulation, and their core functions are somewhat conserved.

[0006] Currently, there are no reports on the function of the PbJAZ1 gene in pear and its effect on the flowering process. Given the role of the JA signal transduction pathway gene JAZ1 in regulating flowering in other plants, studying the characteristics and function of the PbJAZ1 gene in pear using molecular biology methods is of great significance and practical application value for regulating pear flowering and improving pear yield and quality. Summary of the Invention

[0007] The purpose of this invention is to provide the pear PbJAZ1 gene and its encoded protein and its applications.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks protection for at least one use of the pear PbJAZ1 protein in the following (a1)-(a3):

[0010] (a1) To make plants bloom earlier;

[0011] (a2) To prepare products that make plants flower earlier;

[0012] (a3) Cultivating new transgenic plant germplasm with earlier flowering time;

[0013] The pear PbJAZ1 protein is the protein shown in (b1) or (b2) below:

[0014] (b1) A protein having the amino acid sequence shown in SEQ ID NO:1;

[0015] (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0016] Secondly, the present invention seeks protection for the use of biomaterials related to the pear PbJAZ1 protein in at least one of the following (a1)-(a3):

[0017] (a1) To make plants bloom earlier;

[0018] (a2) To prepare products that make plants flower earlier;

[0019] (a3) Cultivating new transgenic plant germplasm with earlier flowering time;

[0020] The biomaterial associated with the pear PbJAZ1 protein is at least one of the following (c1)-(c11):

[0021] (c1) The pear PbJAZ1 gene encoding the pear PbJAZ1 protein;

[0022] (c2) Primer pair used to clone the pear PbJAZ1 gene described in (c1);

[0023] (c3) An expression cassette containing the pear PbJAZ1 gene described in (c1);

[0024] (c4) A recombinant vector containing the pear PbJAZ1 gene described in (c1), or a recombinant vector containing the expression cassette described in (c3);

[0025] (c5) A recombinant microorganism containing the pear PbJAZ1 gene described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4);

[0026] (c6) A transgenic plant cell line containing the pear PbJAZ1 gene described in (c1), or a transgenic plant cell line containing the expression cassette described in (c3), or a transgenic plant cell line containing the recombinant vector described in (c4);

[0027] (c7) Transgenic plant tissue containing the pear PbJAZ1 gene described in (c1), or transgenic plant tissue containing the expression cassette described in (c3), or transgenic plant tissue containing the recombinant vector described in (c4);

[0028] (c8) A transgenic plant organ containing the pear PbJAZ1 gene described in (c1), or a transgenic plant organ containing the expression cassette described in (c3), or a transgenic plant organ containing the recombinant vector described in (c4);

[0029] (c9) A transgenic plant containing the pear PbJAZ1 gene described in (c1), or a transgenic plant containing the expression cassette described in (c3), or a transgenic plant containing the recombinant vector described in (c4);

[0030] (c10) Tissue cultures produced from regenerative cells of the transgenic plant described in (c9);

[0031] (c11) Protoplasts produced from the tissue culture described in (c10).

[0032] Furthermore, the above applications include: increasing the activity and / or content of the pear PbJAZ1 protein in target plants, or overexpressing the pear PbJAZ1 gene encoding the pear PbJAZ1 protein, which can promote earlier flowering of plants and / or cultivate new transgenic plant germplasm with earlier flowering time for production application.

[0033] Thirdly, the present invention claims protection for a method, which is at least one of the following (d1) to (d2):

[0034] (d1) Methods to induce plants to flower earlier;

[0035] (d2) Methods for cultivating new germplasm of transgenic plants with earlier flowering time;

[0036] The method involves increasing the activity and / or content of the pear PbJAZ1 protein as described in claim 1 in a target plant, or overexpressing the gene encoding the pear PbJAZ1 protein.

[0037] Fourthly, the present invention claims protection for a pear PbJAZ1 protein, which is a protein as shown in (b1) or (b2) below:

[0038] (b1) A protein having the amino acid sequence shown in SEQ ID NO:1;

[0039] (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0040] Fifthly, the present invention seeks protection for biological materials related to the aforementioned pear PbJAZ1 protein, wherein the biological material is at least one of the following (c1)-(c5):

[0041] (c1) The pear PbJAZ1 gene encoding the pear PbJAZ1 protein;

[0042] (c2) Primer pair used to clone the pear PbJAZ1 gene described in (c1);

[0043] (c3) An expression cassette containing the pear PbJAZ1 gene described in (c1);

[0044] (c4) A recombinant vector containing the pear PbJAZ1 gene described in (c1), or a recombinant vector containing the expression cassette described in (c3);

[0045] (c5) A recombinant microorganism containing the pear PbJAZ1 gene described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4).

[0046] In the technical solution of the present invention, the nucleotide sequence of the pear PbJAZ1 gene encoding the pear PbJAZ1 protein is shown in SEQ ID NO:2.

[0047] In the technical solution of this invention, the nucleotide sequences of the primer pairs used to clone the pear PbJAZ1 gene are shown in SEQ ID NO:15 and SEQ ID NO:16.

[0048] In the technical solution of this invention, the plant is pear or Arabidopsis thaliana.

[0049] This invention uses the Arabidopsis thaliana JAZ1 protein sequence as a reference and employs the NCBI BLASTP tool to perform homology comparison with the pear database to screen and obtain the PbJAZ1 protein with the highest homology in pear. PbJAZ1 gene-specific primers were designed (sequences shown in SEQ ID NO:15 and SEQ ID NO:16), and the full-length 840 bp CDS (coding sequence) of the PbJAZ1 gene, encoding a 280-amino acid protein, was cloned using pear leaf cDNA as a template (the sequences of the PbJAZ1 gene and its encoded protein are shown in SEQ ID NO:2 and SEQ ID NO:1).

[0050] The expression of the PbJAZ1 gene in different tissues and flower bud induction stages of pear was analyzed using real-time PCR. The results showed that the gene was most highly expressed in the stem, followed by the roots and leaves, and expressed at lower levels in the style, anthers, and fruit. The gene was expressed in all stages of flower bud induction, with the highest expression level during morphological transformation, followed by a gradual decrease.

[0051] The full-length CDS sequence of the PbJAZ1 gene was constructed into the overexpression vector pCAMBIA1300-35S:GFP. The constructed recombinant expression vector was transformed into wild-type Arabidopsis thaliana (Col-0) using the Agrobacterium-mediated flower immersion method. Transgenic T1 and T2 generation positive seedlings were obtained after hygromycin resistance selection. Under long-day conditions, compared with the control plants, the PbJAZ1 transgenic lines exhibited significantly earlier first flower opening and a significantly reduced number of rosette leaves. These results indicate that the pear PbJAZ1 gene plays a role in regulating plant flowering.

[0052] For more detailed technical solutions, please refer to the "Specific Implementation Plan".

[0053] The sequence listing SEQ ID NO:1 is the protein sequence encoded by the pear PbJAZ1 gene cloned in this invention.

[0054] The sequence listing SEQ ID NO:2 is the CDS sequence of the pear PbJAZ1 gene cloned in this invention.

[0055] The sequence listing SEQ ID NO:3 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ1 gene.

[0056] The sequence listing SEQ ID NO:4 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ2 gene.

[0057] The sequence listing SEQ ID NO:5 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ3 gene.

[0058] The sequence listing SEQ ID NO:6 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ4 gene.

[0059] The sequence listing SEQ ID NO:7 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ5 gene.

[0060] The sequence listing SEQ ID NO:8 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ6 gene.

[0061] The sequence listing SEQ ID NO:9 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ7 gene.

[0062] The sequence listing SEQ ID NO:10 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ8 gene.

[0063] The sequence listing SEQ ID NO:11 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ9 gene.

[0064] The sequence listing SEQ ID NO:12 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ10 gene.

[0065] The sequence listing SEQ ID NO:13 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ11 gene.

[0066] The sequence listing SEQ ID NO:14 is the protein sequence encoded by the Arabidopsis thaliana AtJAZ12 gene.

[0067] The sequence listings SEQ ID NO:15 and SEQ ID NO:16 are the nucleotide sequences of the primers used in this invention to clone the full-length CDS sequence of the PbJAZ1 gene.

[0068] The sequence listing SEQ ID NO:17 is a specific nucleotide sequence of the overexpression vector.

[0069] The sequence listings SEQ ID NO:18 and SEQ ID NO:19 are the nucleotide sequences of the specific primers for detecting the expression level of the PbJAZ1 gene using the real-time PCR technique in this invention.

[0070] The sequence listings SEQ ID NO:20 and SEQ ID NO:21 are the nucleotide sequences of the primers for detecting the expression level of the PbUBQ internal reference gene using real-time PCR technology in this invention.

[0071] The sequence listings SEQ ID NO:22 and SEQ ID NO:23 are the nucleotide sequences of the AtActin internal reference gene primers for detecting expression levels using the real-time PCR technique in this invention.

[0072] The beneficial effects of this invention are:

[0073] 1. The gene function was clarified, and the function of the pear PbJAZ1 gene in regulating flowering was identified and clarified for the first time. This enriched the molecular regulatory network theory of flowering in woody fruit trees and provided a reference for subsequent research on related gene functions.

[0074] 2. Agricultural production value: By regulating the expression level of the pear JAZ1 gene, the efficiency of flower bud differentiation in pear and other Rosaceae fruit trees can be targeted, which helps to improve the quality of flower bud differentiation and has important practical application value in ensuring high and stable yields and improving fruit yield and quality.

[0075] 3. Great potential for cross-species application: It reveals the cross-functional role of pear JAZ1 in the jasmonic acid signaling pathway and flowering regulation. Its function is somewhat conserved in different plant species, providing a technical approach that can be referenced for the study of flowering regulation in other crops. Attached Figure Description

[0076] Figure 1 Phylogenetic tree of PbJAZ1 protein and JAZ1 and its homologs in Arabidopsis thaliana.

[0077] Figure 2 This is a clone of the full-length CDS of the PbJAZ1 gene. Figure label explanation: DNA molecular weight standard marker (100~2000 bp).

[0078] Figure 3 The relative expression levels of the PbJAZ1 gene in different tissues of pear are shown.

[0079] Figure 4 This shows the expression pattern of the PbJAZ1 gene at different developmental stages of pear flower buds.

[0080] Figure 5 This is a comparison of the flowering phenotypes of PbJAZ1 transgenic plants and control plants. Figure labels: PbJAZ1-OE3, PbJAZ1-OE5, and PbJAZ1-OE6 are Arabidopsis lines overexpressing the PbJAZ1 gene; Vector control is an Arabidopsis control line overexpressing the empty vector.

[0081] Figure 6 This is a comparison of the relative expression levels of the PbJAZ1 gene in PbJAZ1 transgenic plants and control plants.

[0082] Figure 7 This figure shows a comparison of the flowering time of PbJAZ1 transgenic plants and control plants. Figure label explanation: **** represents p<0.001.

[0083] Figure 8This figure shows a comparison of the number of rosette leaves between PbJAZ1 transgenic plants and control plants. Figure label explanation: **** represents p<0.001. Detailed Implementation

[0084] The following examples are provided to better understand the present invention, but are not intended to limit the scope of the invention.

[0085] Example 1: Cloning and Sequence Structure Analysis of the PbJAZ1 Gene in Pear

[0086] Twelve proteins labeled as Jasmonate-zim-domain proteins were obtained by comparing and screening the sequences of Arabidopsis JAZ1 and JAZ2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 in the NCBI pear database. Phylogenetic trees were constructed for these 12 Arabidopsis and 12 pear JAZ family members, and the candidate member in pear with the highest homology to AtJAZ1 was named PbJAZ1. Figure 1 ).

[0087] Using leaves of 'Dangshan Crisp Pear' as material, RNA samples were extracted using a polysaccharide and polyphenol plant total RNA extraction kit (purchased from Chengdu Fujie Biotechnology, China) following the manufacturer's instructions. First-strand cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (purchased from Beijing TransGen Biotech, China), with each 20 µL reaction mixture containing 1 μg of total RNA. The entire reaction was performed on ice, and the system and steps are as follows:

[0088]

[0089] Mix gently, incubate at 65°C for 5 min, and then stand on ice for 2 min.

[0090] The following components were added to the above reaction solution in sequence:

[0091]

[0092] Mix gently, incubate at 42°C for 30 min, incubate at 85°C for 5 sec, and store at -20°C.

[0093] Specific primers for amplifying the full-length CDS of the PbJAZ1 gene (with the stop codon removed) were designed using SnapGene software. The forward primer is PbJAZ1-GFP-F (SEQ ID NO:15), and the reverse primer is PbJAZ1-GFP-R (SEQ ID NO:16). The specific primer sequences are as follows:

[0094] PbJAZ1-GFP-F: gagaacacgggggactctagaATGTCGACTTCGTCGGAGAC

[0095] PbJAZ1-GFP-R: gcccttgctcaccatggatccTTGGGTTTGCTGGGCAG

[0096] Using pear leaf cDNA as a template, and PbJAZ1-GFP-F / R as primers, the PbJAZ1 gene was amplified using 2×Hieff CanaceGold PCR Master Mix (purchased from Shanghai Yisheng Biotechnology, China). The PCR reaction system (50 μL) consisted of: 25 μL of 2×Hieff Canace PCR Master Mix, 2.5 μL each of 10 μM forward and reverse primers, 1 μL of cDNA template, and ddH2O to a final volume of 50 μL. The preferred PCR program was: 98℃ pre-denaturation for 3 min; 30 amplification cycles, including 98℃ denaturation for 10 sec, 63℃ annealing for 50 sec, and 72℃ extension for 15 sec; after each cycle, 72℃ extension for 10 min and incubation at 20℃ for 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis, and the results were observed using a gel imaging system. Figure 2 The image shows a single, clear band. The target fragment was recovered using the FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novizan Biotechnology, China), following the instructions.

[0097] Based on the characteristics of the multiple cloning site of the plant overexpression vector pCAMBIA1300-35S:GFP and in accordance with the general principles of homologous recombination primer design, homologous sequences from both ends of the linearized vector were added to the 5' end of the specific primers amplifying the full length of the PbJAZ1 gene CDS. The upstream vector end homologous sequence is “gagaacacgggggactctaga” (SEQ ID NO:15), and the downstream vector end homologous sequence is “gcccttgctcaccatggatcc” (SEQ ID NO:16). This ensures that the 5' and 3' ends of the amplified target fragment contain homologous sequences that correspond to the ends of the linearized vector. The plant overexpression vector pCAMBIA1300-35S:GFP plasmid (Wang et al., Plant Cell, https: / / doi.org / 10.1093 / plcell / koad067) was digested with restriction endonucleases Xba I and BamHI (Thermo Scientific, USA). The linearized vector fragment was recovered using the FastPureGel DNA Extraction Mini Kit. The recovered gene fragment was homologously recombinated into the overexpression vector pCAMBIA1300-35S:GFP using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology, China). The required amounts for the recombination reaction were calculated according to the formula in the manufacturer's instructions. The entire reaction was performed on ice. The system and steps are as follows:

[0098]

[0099] Gently mix and incubate at 37°C for 30 min, then let stand on ice for 2 min. Transform the recombinant product into DH5α Escherichia coli competent cells using the heat shock method. Positive clones were screened on LB agar plates containing kanamycin and confirmed by sequencing using the overexpression vector-specific reverse primer GFP-R (SEQ ID NO:17). The sequencing results are shown in SEQ ID NO:2.

[0100] Example 2: Expression characteristics analysis of the pear PbJAZ1 gene

[0101] The material used in this experiment was the white pear variety 'Dangshan Crisp Pear', which was naturally grown in the pear resource nursery of Nanjing Agricultural University and managed using conventional methods. Different pear materials were collected, and total RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (purchased from Nanjing Novizan Biotechnology, China). cDNA was then synthesized by reverse transcription using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (purchased from Nanjing Novizan Biotechnology, China).

[0102] Fluorescent quantitative PCR-specific primers for the target gene PbJAZ1 and the internal reference gene PbUBQ were designed using the NCBI primer design tool Primer-BLAST, and named PbJAZ1-qPCR-F / R (SEQ ID NO:18 and SEQ ID NO:19) and PbUBQ-qPCR-F / R (SEQ ID NO:20 and SEQ ID NO:21), respectively. The specific primer sequences are as follows:

[0103] PbJAZ1-qPCR-F: TGTCGACTTCGTCGGAGACT

[0104] PbJAZ1-qPCR-R: GGGCTTTCGGACGAAACATCT

[0105] PbUBQ-qPCR-F: CCCTTCACTTGGTTCTCCGT

[0106] PbUBQ-qPCR-R: TAATCAGCAAGCGTGCGACC

[0107] The relative expression level of the PbJAZ1 gene in the collected samples was detected using real-time quantitative PCR. Using cDNA from different pear materials as templates, PbJAZ1-qPCR-F / R and PbUBQ-qPCR-F / R primers were used, respectively, and amplification was performed using 2×SYBR Green Master Mix (purchased from Roche, Switzerland). The PCR reaction system (20 μL) consisted of: 0.1 µL cDNA template, 5.0 µL forward and reverse primer premix (0.05 µM), 10 µL 2×SYBR Green Master Mix, and 4.9 µL ddH2O. A Light Cycler 480 II (Roche) real-time quantitative PCR instrument was used, with the optimal reaction program being: 95℃ for 5 min; 95℃ for 3 sec, 63℃ for 50 sec, 72℃ for 30 sec, for a total of 45 cycles. To ensure specific amplification of each primer, each melting curve of the experimental reaction was analyzed. Data were used in 2...-ΔΔCt The relative expression levels of the target gene were calculated and plotted and analyzed using Graph Pad Prism 8 software.

[0108] (1) Analysis of expression characteristics of PbJAZ1 gene in different tissues of pear

[0109] Using cDNA from roots, stems, leaves, styles, anthers, and fruits as templates, the expression characteristics of the PbJAZ1 gene in various pear tissues were analyzed using real-time quantitative PCR. The results are as follows: Figure 3 As shown, the PbJAZ1 gene was found to be expressed in all tissues, with the highest expression level in the stem, followed by the roots and leaves, and lower expression levels in the style, anthers, and fruit.

[0110] (2) Analysis of the expression characteristics of the PbJAZ1 gene during the flower bud induction stage

[0111] Pear blossom buds under natural growth conditions in the field were selected and sampled six times consecutively, with each sampling 15 days apart. The sampling start date was May 11th, and the specific sampling dates were May 11th, May 26th, June 10th, June 25th, July 11th, and July 26th. Using cDNA from the pear blossom buds at these six sampling points as templates, the expression characteristics of the PbJAZ1 gene at different developmental stages were analyzed using real-time quantitative PCR. The results are as follows: Figure 4 As shown, the PbJAZ1 gene was expressed throughout the flower bud induction stage, with the highest expression level observed during morphological transformation, indicating that the PbJAZ1 gene plays a role in the flower bud differentiation preparation stage.

[0112] Example 3: Transformation and Functional Analysis of PbJAZ1 Gene in Arabidopsis thaliana

[0113] Positive clones with correct sequencing from Example 1 were selected and cultured overnight with shaking in LB medium containing kanamycin resistance. The 35S:PbJAZ1-GFP recombinant plasmid was extracted using the FastPure Plasmid Mini Kit (purchased from Nanjing Novizan Biotechnology, China), following the manufacturer's instructions. The recombinant plasmid was transformed into GV3101 Agrobacterium competent cells using the heat shock method. Positive clones were screened on LB agar plates containing kanamycin and rifampin, and the positive Agrobacterium strains were confirmed by colony PCR. The correct Agrobacterium strains were selected and infected with wild-type Arabidopsis thaliana (Col-0) using the Agrobacterium flower immersion method. Infected plants continued to grow under long-day conditions (16 h light / 8 h dark) at 22°C and 70% humidity until maturity and seed harvest.

[0114] After harvesting, Arabidopsis seeds were dried in silica gel for one week, and a suitable amount was used to screen for transgenic positive seedlings. The seeds were sterilized with anhydrous ethanol and sodium hypochlorite of a certain concentration and evenly sown on MS medium containing hygromycin and termethin antibiotics. They were placed in darkness at 4°C for 3 days, then transferred to normal long-day conditions for 12 days of growth. Arabidopsis successfully inoculated with the target gene grew normally on MS selection medium containing antibiotics, while those that were not successfully inoculated turned yellow and died at the cotyledon stage. Seedlings that had normally developed 4 leaves were transplanted into nutrient pots containing substrate (nutrient soil: vermiculite = 1:2, w / w) to continue growth. Starting from the date the Arabidopsis seeds were exposed to normal light, the number of days required for each Arabidopsis plant to flower its first flower after bolting was recorded, along with the number of rosette leaves at that time. Two rosette leaves were collected as materials, and DNA was extracted from Arabidopsis leaves using the CTAB method. Positive seedlings were identified by PCR using 2 × Taq Master Mix (purchased from Nanjing Novizan Biotechnology, China), the specific forward primer PbJAZ1-GFP-F (SEQ ID NO:15) for the PbJAZ1 gene, and the specific reverse primer GFP-R (SEQ ID NO:17) for the overexpression vector. Seeds from correctly identified Arabidopsis positive seedlings were harvested individually after maturity, stored in absorbent silica gel, and dried for one week. The above steps were repeated to harvest seeds from T2 generation positive plants.

[0115] Based on PCR identification and phenotypic statistics, the PbJAZ1 transgenic representative line was selected. The harvested T2 generation seeds were then subjected to the same MS screening culture, phenotypic statistics, and PCR identification process. Under long-day conditions, the phenotypic results of the PbJAZ1 transgenic plants relative to the unloaded control plants are as follows: Figure 5 As shown.

[0116] First, the expression level of the PbJAZ1 gene in representative plants was detected using real-time quantitative PCR. Using the PbJAZ1-qPCR-F / R primers (SEQ ID NO:18 and SEQ ID NO:19) for quantitative PCR of the PbJAZ1 gene from Example 2 and the AtActin-qPCR-F / R primers (SEQ ID NO:22 and SEQ ID NO:23) for the Arabidopsis thaliana internal reference gene AtActin, cDNA from control plants, PbJAZ1-OE3, PbJAZ1-OE5, and PbJAZ1-OE6 plants were used as templates, and the reaction system and optimized procedure for quantitative PCR in Example 2 were followed. The results are as follows: Figure 6 As shown, no expression of the PbJAZ1 gene was detected in the control plants, while the expression level of the PbJAZ1 gene was high in the PbJAZ1-OE3, PbJAZ1-OE5 and PbJAZ1-OE6 transgenic plants, indicating that the PbJAZ1 gene was overexpressed in wild-type Arabidopsis thaliana.

[0117] Secondly, statistical analysis was conducted on the time of the opening of the first flower and the corresponding number of rosette leaves. The results are as follows: Figure 7 and Figure 8 As shown. The average time for the first flower to bloom in the control plants was 30.4 days, while the PbJAZ1 transgenic lines OE3, OE5, and OE6 required 26.7, 26.2, and 26.3 days, respectively. Figure 7 The control plant had 12.4 rosette leaves, while the PbJAZ1 transgenic lines OE3, OE5, and OE6 had 7.3, 6.9, and 8.2 leaves, respectively. Figure 8 Compared with the control plants, the PbJAZ1 transgenic lines flowered significantly earlier. Based on the above results, the pear PbJAZ1 gene has the function of promoting flowering in plants such as Arabidopsis thaliana.

[0118] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0119] sequence list

[0120] SEQ ID NO:1 Protein encoded by the PbJAZ1 gene

[0121] MSTSSETAEVSGQRGMRAAEKPSNFTQTCSMLCQYIKEKASFGDLNLDMACNNMQQSNRGTPEMFRPKAPPMNFFPFMENSRNMPTAATDFKSMDLFPQQAGFGPSAPTPREEVPKMADSSVKKSAPGEPQKAQMTIFYG GQVIVYNDFPADKAKEVMLLASKESSQSHTAPASPPANTNNAFASHLGKSPINSSSPVPPSANMFPNHGNQAIQEGVKPPPRPVVCDLPIARKASLHRFLEKRKDRLNTLAPYQTSSPASGPAKPTENKSWLGLAAQQTQ

[0122] CDS of the PbJAZ1 gene (SEQ ID NO:2)

[0123] ATGTCGACTTCGTCGGAGACTGCGGAGGTTTCAGGCCAGAGAGGGATGAGGGCGGCGGAGAAGCCGTCGAACTTCACTCAGACATGCAGTATGCTTTGCCAGTACATCAAGGAGAAAGCCAGCTTTGGAGATCTGAATCTTGACATGGCATGCAACAACATGCAACAATCTAATCGAGGAACACCTGAGATGTTTCGTCCGAAAGCCCCACCCATGAACTTCTTTCCATTTATGGAAAATTCTCGGAACATGCCAACGGCGGCTACAGACTTCAAGTCCATGGATTTGTTTCCTCAACAAGCTGGTTTTGGTCCCTCTGCCCCCACTCCCAGAGAAGAGGTCCCCAAGATGGCTGACTCTAGTGTGAAGAAATCTGCCCCTGGAGAGCCTCAGAAGGCGCAAATGACTATCTTTTATGGTGGACAAGTAATCGTGTACAACGATTTTCCGGCGGACAAGGCCAAGGAAGTCATGCTCTTAGCAAGCAAGGAGAGTTCTCAGAGCCACACCGCTCCGGCTTCACCTCCGGCCAATACTAACAATGCCTTTGCCTCTCATTTGGGCAAGAGTCCCATTAATTCCAGCAGTCCAGTTCCTCCGAGCGCCAACATGTTTCCTAACCATGGCAATCAAGCGATTCAGGAGGGTGTCAAACCACCCCCTCGACCCGTTGTTTGTGATCTTCCGATTGCAAGGAAAGCTTCGCTTCATCGATTCCTCGAGAAGAGAAAGGATAGGCTCAACACCCTAGCACCGTACCAAACGAGCAGCCCTGCATCCGGCCCGGCGAAGCCAACCGAAAACAAGTCATGGTTAGGGTTGGCTGCCCAGCAAACCCAATGA

[0124] SEQ ID NO:3 Protein encoded by AtJAZ1 gene

[0125] MSSSMECSEFVGSRRFTGKKPSFSQTCSRLSQYLKENGSFGDLSLGMACKPDVNGTLGNSRQPTTTMSLFPCEASNMDSMVQDVKPTNLFPRQPSFSSSSSSLPKEDVLKMTQTTRSVKPESQTAPLTIFYAGQVIVFNDFSAEKAKEVINLASKGTANSLAKNQTDIRSNIATIANQVPHPRKTTTQEPIQSSPTPLTELPIARRASLHRFLEKRKDRVTSKAPYQLCDPAKASSNPQTTGNMSWLGLAAEI

[0126] SEQ ID NO:4 Protein encoded by AtJAZ2 gene

[0127] MSSFSAECWDFSGRKPSFSQTCTRLSRYLKEKGSFGDLSLGMTCKPDVNGGSRQPTMMNLFPCEASGMDSSAGQEDIKPKTMFPRQSSFSSSSSSGTKEDVQMIKETTKSVKPESQSAPLTIFYGGRVMVFDDFSAEKAKEVIDLANKGSAKSFTCFTAEVNNNHSAYSQKEIASSPNPVCSPAKTAAQEPIQPNPASLACELPIARRASLHRFLEKRKDRITSKAPYQIDGSAEASSKPTNPAWLSSR

[0128] SEQ ID NO:5 Protein encoded by AtJAZ3 gene

[0129] MERDFLGLGSKNSPITVKEETSESSRDSAPNRGMNWSFSNKVSASSSQFLSFRPTQEDRHRKSGNYHLPHSGSFMPSSVADVYDSTRKAPYSSVQGVRMFPNSNQHEETNAVSMSMPGFQSHHYAPGGRSFMNNNNNSQPLVGVPIMAPPISILPPPGSIVGTTDIRSSSKPIGSPAQLTIFYAGSVCVYDDISPEKAKAIMLLAGNGSSMPQVFSPPQTHQQVVHHTRASVDSSAMPPSFMPTISYLSPEAGSSTNGLGATKATRGLTSTYHNNQANGSNINCPVPVSCSTNVMAPTVALPLARKASLARFLEKRKERVTSVSPYCLDKKSSTDCRRSMSECISSSLSSAT

[0130] SEQ ID NO:6 Protein encoded by AtJAZ4 gene

[0131] MERDFLGLGSKLSPITVKEETNEDSAPSRGMMDWSFSSKVGSGPQFLSFGTSQQETRVNTVNDHLLSSAAMDQNQRTYFSSLQEDRVFPGSSQQDQTTITVSMSEPNYINSFINHQHLGGSPIMAPPVSVFPAPTTIRSSSKPLPPQLTIFYAGSVLVYQDIAPEKAQAIMLLAGNGPHAKPVSQPKPQKLVHHSLPTTDPPTMPPSFLPSISYIVSETRSSGSNGVTGLGPTKTKASLASTRNNQTAAFSMAPTVGLPQTRKASLARFLEKRKERVINVSPYYVDNKSSIDCRTLMSECVSCPPAHHLH

[0132] SEQ ID NO:7 Protein encoded by AtJAZ5 gene

[0133] MSSSNENAKAQAPEKSDFTRRCSLLSRYLKEKGSFGNIDLGLYRKPDSSLALPGKFDPPGKQNAMHKAGHSKGEPSTSSGGKVKDVADLSESQPGSSQLTIFFGGKVLVYNEFPVDKAKEIMEVAKQAKPVTEINIQTPINDENNNNKSSMVLPDLNEPTDNNHLTKEQQQQQEQNQIVERIARRASLHRFFAKRKDRAVARAPYQVNQNAGHHRYPPKPEIVTGQPLEAGQSSQRPPDNAIGQTMAHIKSDGDKDDIMKIEEGQSSKDLDLRL

[0134] Protein encoded by SEQ ID NO:8 AtJAZ6 gene

[0135] MSTGQAPEKSNFSQRCSLLSRYLKEKGSFGNINMGLARKSDLELAGKFDLKGQQNVIKKVETSETRPFKLIQKFSIGEASTSTEDKAIYIDLSEPAKVAPESGNSQLTIFFGGKVMVFNEFPEDKAKEIMEVAKEANHVAVDSKNSQSHMNLDKSNVVIPDLNEPTSSGNNEDQETGQQHQVVERIARRASLHRFFAKRKDRAVARAPYQVNQHGSHLPPKPEMVAPSIKSGQSSQHIATPPKPKAHNHMPMEVDKKEGQSSKNLELKL

[0136] Protein encoded by SEQ ID NO:9 AtJAZ7 gene

[0137] MIIIIKNCDKPLLNFKEMEMQTKCDLELRLLTSSYDSDFHSSLDESSSSEISQPKQESQILTIFYNGHMCVSSDLTHLEANAILSLASRDVEEKSLSLRSSDGSDPPTIPNNSTRFHYQKASMKRSLHSFLQKRSLRIQATSPYHRYR

[0138] Protein encoded by SEQ ID NO:10 AtJAZ8 gene

[0139] MKLQQNCDLELRLFPTSYDSDSSDTTSVVESTSSGNPQPNEESQRITIFYNGKMCFSSDVTHLQARSIISIASREMKTKSSSNGSDPPNKSTSFHHNQLPNPKASMKKSLQSFLQKRKIRIQATSPYHSRR

[0140] SEQ ID NO:11 Protein encoded by AtJAZ9 gene

[0141] MERDFLGLSDKQYLSNNVKHEVNDDAVEERGLSTKAAREWGKSKVFATSSFMPSSDFQEAKAFPGAYQWGSVSAANVFRRCQFGGAFQNATPLLLGGSVPLPTHPSLVPRVASSGSSPQLTIFYGGTISVFNDISPDKAQAIMLCAGNGLKGETGDSKPVREAERMYGKQIHNTAATSSSSATHTDNFSRCRDTPVAATNAMSMIESFNAAPRNMIPSVPQARKASLARFLEKRKERFDFVFFFFIENFEVFQLNLKELMLLVQAYECNAIQEDAS

[0142] SEQ ID NO:12 Protein encoded by AtJAZ10 gene

[0143] MSKATIELDFLGLEKKQTNNAPKPKFQKFLDRRRSFRDIQGAISKIDPEIIKSLLASTGNNSDSSAKSRSVPSTPREDQPQIPISPVHASLARYFCLSGKVFFFFLTFLALPEKTKKFEIQIFKTFIFLRSSTELVSGTVPMTIFYNGSVSVFQVSRNKAGEIMKVANEAASKKDESSMETDLSVILPTTLRPKLFGQNLEGDLPIARRKSLQRFLEKRKER

[0144] SEQ ID NO:13 Protein encoded by AtJAZ11 gene

[0145] MAEVNGDFPVPSFADGTGSVSAGLDLLVERSIHEARSTEPDASTQLTIIFGGSCRVFNGVPAQKVQEIIRIAFAGKQTKNVTGINPALNRALSFSTVADLPIARRRSLQRFLEKRRDRSTKPDGSMILPSQLTIIFGGSFSVFDGIPAEKVQEILHIAAAAKATETINLTSINPALKRAISFSNASTVACVSTADVPIARRRSLQRFFEKRRHRFVHTKPYSATTSEADKNETSPIVT

[0146] SEQ ID NO:14 Protein encoded by AtJAZ12 gene

[0147] MTKVKDEPRASVEGGCGVADGDGGAAEIGGTGSVEKSINEVRSTEIQTAEPTVPPNQLTIFFGGSVTVFDGLPSEKVQEILRIAAKAMETKNSTSISPVSSPALNRAPSFSSTSNVASPAAQPFPIQPISFCRSTADLPIARRHSLQRFLEKRRDRLVNKNPYPTSDFKKTDVPTGNVSIKEEFPTA

[0148] SEQ ID NO:15

[0149] PbJAZ1-GFP-F: gagaacacgggggactctagaATGTCGACTTCGTCGGAGAC

[0150] SEQ ID NO:16

[0151] PbJAZ1-GFP-R: gcccttgctcaccatggatccTTGGGTTTGCTGGGCAG

[0152] SEQ ID NO:17

[0153] GFP-R: CGTCGTCCTTGAAGAAGATG

[0154] SEQ ID NO:18

[0155] PbJAZ1-qPCR-F: TGTCGACTTCGTCGGAGACT

[0156] SEQ ID NO:19

[0157] PbJAZ1-qPCR-R: GGGCTTTCGGACGAAACATCT

[0158] SEQ ID NO:20

[0159] PbUBQ-qPCR-F: CCCTTCACTTGGTTCTCCGT

[0160] SEQ ID NO:21

[0161] PbUBQ-qPCR-R: TAATCAGCAAGCGTGCGACC

[0162] SEQ ID NO:22

[0163] AtActin-qPCR-F: GGTGTCATGGTTGGTATGGGTC

[0164] SEQ ID NO:23

[0165] AtActin-qPCR-R: CCTCTGTGAGTAGAACTGGGTGC。

Claims

1. Application of pear PbJAZ1 protein in at least one of the following (a1)-(a3): (a1) To make plants bloom earlier; (a2) To prepare products that make plants flower earlier; (a3) Cultivating new transgenic plant germplasm with earlier flowering time; The pear PbJAZ1 protein is the protein shown in (b1) or (b2) below: (b1) A protein having the amino acid sequence shown in SEQ ID NO:1; (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

2. The use of biomaterials related to the pear PbJAZ1 protein of claim 1 in at least one of the following (a1)-(a3): (a1) To make plants bloom earlier; (a2) To prepare products that make plants flower earlier; (a3) Cultivating new transgenic plant germplasm with earlier flowering time; The biomaterial associated with the pear PbJAZ1 protein is at least one of the following (c1)-(c11): (c1) The pear PbJAZ1 gene encoding the pear PbJAZ1 protein; (c2) Primer pair used to clone the pear PbJAZ1 gene described in (c1); (c3) An expression cassette containing the pear PbJAZ1 gene described in (c1); (c4) A recombinant vector containing the pear PbJAZ1 gene described in (c1), or a recombinant vector containing the expression cassette described in (c3); (c5) A recombinant microorganism containing the pear PbJAZ1 gene described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4); (c6) A transgenic plant cell line containing the pear PbJAZ1 gene described in (c1), or a transgenic plant cell line containing the expression cassette described in (c3), or a transgenic plant cell line containing the recombinant vector described in (c4); (c7) Transgenic plant tissue containing the pear PbJAZ1 gene described in (c1), or transgenic plant tissue containing the expression cassette described in (c3), or transgenic plant tissue containing the recombinant vector described in (c4); (c8) A transgenic plant organ containing the pear PbJAZ1 gene described in (c1), or a transgenic plant organ containing the expression cassette described in (c3), or a transgenic plant organ containing the recombinant vector described in (c4); (c9) A transgenic plant containing the pear PbJAZ1 gene described in (c1), or a transgenic plant containing the expression cassette described in (c3), or a transgenic plant containing the recombinant vector described in (c4); (c10) Tissue cultures produced from regenerative cells of the transgenic plant described in (c9); (c11) Protoplasts produced from the tissue culture described in (c10).

3. The application according to claim 2, characterized in that, The nucleotide sequence of the pear PbJAZ1 gene encoding the pear PbJAZ1 protein in (c1) is shown in SEQ ID NO:

2.

4. The application according to claim 2, characterized in that, The nucleotide sequences of the primer pair described in (c2) are shown in SEQ ID NO:15 and SEQ ID NO:

16.

5. The application according to claim 1 or 2, characterized in that, Increasing the activity and / or content of the pear PbJAZ1 protein in target plants, or overexpressing the pear PbJAZ1 gene encoding the pear PbJAZ1 protein, can promote earlier flowering of plants and / or cultivate new transgenic plant germplasm with earlier flowering time for production application.

6. The application according to claim 1, 2, or 5, characterized in that, The plant in question is either pear or Arabidopsis thaliana.

7. A method, characterized in that, The method is at least one of the following (d1) to (d2): (d1) Methods to induce plants to flower earlier; (d2) Methods for cultivating new germplasm of transgenic plants with earlier flowering time; The method involves increasing the activity and / or content of the pear PbJAZ1 protein as described in claim 1 in a target plant, or overexpressing the gene encoding the pear PbJAZ1 protein.

8. A pear PbJAZ1 protein, characterized in that, The pear PbJAZ1 protein is the protein shown in (b1) or (b2) below: (b1) A protein having the amino acid sequence shown in SEQ ID NO:1; (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

9. A biomaterial related to the pear PbJAZ1 protein of claim 8, characterized in that, The biomaterial is at least one of the following (c1)-(c5): (c1) The pear PbJAZ1 gene encoding the pear PbJAZ1 protein; (c2) Primer pair used to clone the pear PbJAZ1 gene described in (c1); (c3) An expression cassette containing the pear PbJAZ1 gene described in (c1); (c4) A recombinant vector containing the pear PbJAZ1 gene described in (c1), or a recombinant vector containing the expression cassette described in (c3); (c5) A recombinant microorganism containing the pear PbJAZ1 gene described in (c1), or a recombinant microorganism containing the expression cassette described in (c3), or a recombinant microorganism containing the recombinant vector described in (c4).

10. The biomaterial according to claim 9, characterized in that, The nucleotide sequence of the pear PbJAZ1 gene encoding the pear PbJAZ1 protein in (c1) is shown in SEQ ID NO:2; The nucleotide sequences of the primer pair described in (c2) are shown in SEQ ID NO:15 and SEQ ID NO:16.

Images