Phoebe bournei MDAS33 gene, protein coded by same and application of gene in advancing plant flowering

Abstract

The invention discloses a phoebe bournei MADS33 gene, and the phoebe bournei MADS33 gene has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence which is obtained by deleting, adding and / or substituting one or more nucleotides of the sequence and has an unchanged function. The invention also discloses a protein coded by the phoebe bournei MADS33 gene, and the protein is composed of amino acids as shown in SEQ ID No.2. The invention further discloses a method for advancing plant flowering. The invention also discloses an application of the phoebe bournei MADS33 gene in advancing plant flowering. The phoebe bournei SOC1 gene member MADS33 is obtained by screening based on transcriptome data, the biological function of the phoebe bournei SOC1 gene member MADS33 is verified through heterologous transgenic arabidopsis thaliana, the fact that the phoebe bournei MADS33 gene can advance plant flowering is revealed, and the phoebe bournei MADS33 gene is expected to artificially advance plant flowering.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and relates to a Phoebe bournei MDAS33 gene and a protein encoded by the gene and an application of the gene in advancing plant flowering. Background Art

[0002] Phoebe bournei is an evergreen tree of the genus Phoebe in the family Lauraceae. It is one of the original species of "golden silk nanmu" and is mainly distributed in Fujian, Zhejiang, Jiangxi and Guangdong provinces in China. Due to its high-quality wood, Phoebe bournei is widely used in construction, furniture and handicrafts. Due to its economic value, Phoebe bournei has been over-cut, resulting in a significant reduction in natural forest resources. It has now been listed as a national second-level protected plant.

[0003] Phoebe bournei and other precious tree species have difficulties in tissue culture regeneration and mainly rely on sexual reproduction. Its flowering and fruiting characteristics are the key biological basis for production, but Phoebe bournei has a long juvenile period, usually taking about 15 years to enter the reproductive stage, which has a serious impact on hybrid breeding and poses a challenge to breeding work. Although basic models of flowering regulation pathways have been established in model plants and some crops, related research in perennial trees is still insufficient. Compared with model plants and other forest trees, genetic breeding research on Phoebe bournei is relatively weak. Therefore, studying the mechanism of flowering regulation of Phoebe bournei has important theoretical and practical significance for shortening the breeding cycle.

[0004] Recent studies have shown that MADS-box genes not only have important functions in plants, but also play a key role in evolution and species diversity. The SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) gene is an important gene in Arabidopsis thaliana, responsible for regulating the flowering time and flower development of plants. As the core regulator of the flowering pathway of Arabidopsis, SOC1 is regulated by environmental signals such as photoperiod and temperature to ensure that plants bloom in the appropriate season. It plays an important role in flowering signal transduction and integrates multiple signals such as photoperiod, temperature and plant endogenous hormones to promote flowering. The expression of SOC1 is affected by multiple regulatory factors, including daylight duration and temperature changes. In addition, SOC1 interacts with other flowering regulatory genes such as FT (FLOWERING LOCUS T) and AP1 (APETALA1) to jointly regulate the development of floral organs. Although the functions of SOC1 may vary in different plant species, its core position in the regulation of flowering time is generally recognized.

[0005] As an important member of the MADS-box family, SOC1 is an important flowering integrator. It has been studied in plants such as Arabidopsis, Zea mays, and Nicotiana benthamiana, showing that it plays a role in integrating multiple signaling pathways and promoting the transition of plants from vegetative growth to reproductive growth. SOC1 can form heterodimers or heterotetramers with other MADS-box proteins to jointly regulate the expression of downstream target genes and ultimately guide the development of floral organs. So far, no studies have reported the function of the MDAS33 gene in Phoebe bournei. Summary of the invention

[0006] To solve the above problems, the present invention provides a Phoebe bournei MADS33 gene, which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted but the function remains unchanged. Preferably, the nucleotide sequence of the Phoebe bournei MADS33 gene is SEQ ID NO: 1.

[0007] The invention also provides a protein encoded by the Phoebe bournei MADS33 gene, which is composed of the amino acids shown in SEQ ID No.2.

[0008] The invention also provides a recombinant vector, a transgenic cell line or a recombinant bacterium containing the Phoebe bournei MADS33 gene.

[0009] The present invention also provides a method for advancing plant flowering, the method comprising preparing a modified plant containing the Phoebe bournei MADS33 gene, wherein the expression of the Phoebe bournei MADS33 gene is increased in the modified plant relative to the wild-type plant; wherein the nucleotide sequence of the Phoebe bournei MADS33 gene is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased.

[0010] The present invention also provides the application of Phoebe bournei MADS33 gene in advancing plant flowering, and preparing modified plants containing Phoebe bournei MADS33 gene, wherein the expression of Phoebe bournei MADS33 gene is increased in the modified plants relative to wild-type plants; wherein the nucleotide sequence of the Phoebe bournei MADS33 gene is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the increase in the product obtained by transcription and the product obtained by translation can be an increase.

[0011] In one embodiment, the plant is Arabidopsis thaliana or Phoebe chinensis. Preferably, the plant is Arabidopsis thaliana.

[0012] In one embodiment, the B. minnan MADS33 gene is connected to a vector, transformed into Arabidopsis thaliana through Agrobacterium-mediated transformation, and then screened, cultured, and transgenic strains are obtained.

[0013] Based on transcriptome data, the applicant screened out MADS33, a member of the B. minnan L. SOC1 gene, and verified its biological function through heterologous transformation into Arabidopsis thaliana, revealing that the B. minnan L. MADS33 gene can advance plant flowering. The application of the B. minnan L. MADS33 gene is expected to artificially advance plant flowering, which will help regulate the flowering time of B. minnan L. through molecular biological means and provide new strategies for plant breeding and protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0015] Figure 1 The total RNA electrophoresis diagram of the leaves and flower organs of Phoebe bournei of the present invention;

[0016] Figure 2 This is the electrophoresis diagram of PCR detection of the bacterial solution of the expression vector of Phoebe bournei MADS33 gene of the present invention;

[0017] Figure 3 qPCR detection of Arabidopsis thaliana overexpressing MADS33 gene of the present invention;

[0018] Figure 4 The flowering phenotypes of wild-type WT and P. minnan MADS33 transgenic Arabidopsis thaliana OE observed in the present invention. DETAILED DESCRIPTION

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] 1. Materials

[0023] 1.1 Experimental Materials

[0024] In May 2023, leaves and flower organs of 20-year-old Phoebe bournei were collected from the Experimental Forest Farm in Qingyuan County, Lishui City, Zhejiang Province. The samples were immediately stored in liquid nitrogen and placed in a -80°C refrigerator for future use. Arabidopsis wild-type seeds used the Columbia ecotype (Columbia-0).

[0025] 1.2 Experimental reagents and instruments

[0026] DNA polymerase, various restriction endonucleases, T4 ligase, Marker and TRIzol reagents were purchased from Bao Biotechnology (Dalian) Co., Ltd.; plasmid extraction kit and DNA gel recovery kit were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The PCR instrument was a US PE9700 PCR instrument, and the clean bench was purchased from Suzhou Chengjing Purification Technology Co., Ltd.

[0027] 1.3 Primer synthesis and sequencing

[0028] Primer synthesis and sequencing were completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0029] 2. Methods

[0030] 2.1 Extraction of total RNA from leaves and flowers of Phoebe bournei

[0031] Total RNA was extracted using the Novezan kit as follows:

[0032] (1) Rapidly transfer plant tissue frozen at -80°C to a pre-cooled liquid nitrogen mortar and grind without adding liquid nitrogen to maintain low temperature until it is ground into fine powder.

[0033] (2) Add approximately 100 mg of ground plant tissue to 600 μL of Buffer PSL and vortex vigorously for 30 seconds to ensure thorough mixing with the lysis buffer. Then centrifuge at 12,000 rpm for 5 minutes and proceed immediately to the next step.

[0034] (3) Transfer about 500 μL of the supernatant to FastPure gDNA-Filter Columns III (already placed in a collection tube), centrifuge at 12,000 rpm for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate.

[0035] (4) Add 0.5 times the volume of the filtrate to the collection tube and shake to mix for 15 seconds.

[0036] (5) Transfer the above mixed solution to FastPure RNA Columns V (already placed in a collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0037] (6) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm (13,400 x g) for 30 seconds, and discard the filtrate.

[0038] (7) Add 500 μL of Buffer RWB to FastPure RNA Columns V (make sure 48 mL of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Repeat this step once.

[0039] (8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm (13,400 x g) for 2 minutes.

[0040] (9) Transfer FastPure RNA Columns V to a new RNase-free collection tube (1.5 mL), add 30-100 μL of RNase-free ddH2O to the center of the membrane, and centrifuge at 12,000 rpm for 1 minute.

[0041] (10) The extracted RNA was stored at -85 to -65°C.

[0042] 2.2 Gene cloning

[0043] The RNA (Total RNA) of Phoebe bournei was diluted with an appropriate amount of DEPC ddH2O, and reverse transcribed according to the instruction manual of PrimeScriptTM RTReagent Kit (Perfect Real Time) (TaKaRa) to obtain cDNA.

[0044] Prepare the following gene cloning PCR system (50 μL) and clone the MADS33 sequence:

[0045]

[0046] The reaction procedure was: 95°C, 2 min; 95°C, 30 sec; 55°C, 30 sec; 72°C, 1 min, 37 cycles; 72°C, 10 min.

[0047] Primers used for gene cloning (Table 1).

[0048]

[0049] 2.3 Target fragment recovery

[0050] Utilizing AxyPrep TM The target fragments were recovered using DNA gel recovery kit (AXYGEN).

[0051] (1) Under long-wave ultraviolet light, use a clean blade to carefully cut out the desired DNA band, and try to avoid cutting into the gel portion that does not contain DNA.

[0052] (2) First weigh an empty 1.5 mL centrifuge tube, then put the gel block in and weigh it. Calculate the difference between the two weights to get the weight of the gel.

[0053] (3) Add 3 times the volume of the sol solution. If the gel weight is 100 mg, its volume is considered to be 100 μL, so add 300 μl of the sol solution.

[0054] (4) Place in a 56°C water bath for 10 minutes (or until the gel is completely dissolved), swirling gently every 2-3 minutes to accelerate dissolution.

[0055] (5) Add the dissolved liquid to the EC adsorption column (placed in a collection tube), let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

[0056] (6) Add 600 μL of washing buffer (WB) (make sure anhydrous ethanol has been added), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0057] (7) Add 600 μL of washing buffer WB again, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0058] (8) Place the EC adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 min to remove as much rinse solution as possible to prevent residual ethanol from inhibiting subsequent reactions.

[0059] (9) Remove the EC column and place it in a clean centrifuge tube. Add 50 μL of elution buffer EB (preheated in a 65-70°C water bath for best results) to the center of the membrane. Leave at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. If more DNA is needed, add the resulting solution back to the column and centrifuge for 1 minute.

[0060] 2.4 Target fragment ligation and transformation into entry vector

[0061] Refer to the instructions of pEASy-Blunt Zero Cloning Kit, mix the following solutions, mix gently, and centrifuge briefly. PCR: 37℃5-30min (depending on the size of the target gene).

[0062]

[0063] (1) Thaw the competent cells on ice (or wait for a while in the palm of your hand or at room temperature, and quickly put them in ice when the cells are in an ice-water mixture state), add the ligation product, gently stir the bottom of the EP tube with your hand to mix, and let it stand on ice for 25 minutes.

[0064] (2) Heat shock in a 42°C water bath for 45 seconds, then quickly return to ice and let stand for 2 minutes.

[0065] (3) Add 700 μL of LB medium without antibiotics to the centrifuge tube, mix well, and resuscitate at 37°C and 200 rpm for 60 min.

[0066] (4) Collect the cells by centrifugation at 5000 rpm for 1 minute, retain about 100 μL of supernatant, gently pipette to resuspend the cells, and spread them on a solid culture medium containing kanamycin (50 mg / L Kan), and culture them upside down at 37°C overnight.

[0067] 2.5 PCR verification of target fragment

[0068] (1) Pick up a single white colony on the solid culture medium from step 4, place it in 500 μL of liquid LB medium containing the same concentration of antibiotics, culture it at 37°C for 3-6 hours, and then take 1 μL for bacterial liquid PCR.

[0069] (2) Gene cloning primers were used to perform bacterial liquid PCR detection. The reaction system (10 μL) was as follows:

[0070]

[0071] (3) The PCR program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min (adjusted according to the length of the inserted fragment), for 34 cycles; and final extension at 72°C for 10 min.

[0072] (4) Detect PCR products using agarose gel electrophoresis, select positive clones and send them to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Compare the sequencing results with the original sequence, mix the bacterial solution containing the correct target gene fragment with an equal volume of glycerol and store it at -80°C. Use the MADS33 gene cloned above to construct a MADS33 overexpression vector.

[0073] 2.6 Construction of overexpression vector

[0074] 100 μL of the bacterial solution stored in method 2.5 was taken and added to 20 mL of LB medium containing kanamycin, and cultured overnight at 37°C and 200 rpm.

[0075] Refer to the EasyPure Plasmid MiniPrep Kit instructions to extract the plasmid containing the target fragment. The steps are as follows:

[0076] (1) Centrifuge the overnight culture at 10,000 x g for 1 minute and remove the supernatant (aspirate as much as possible).

[0077] (2) Add 250 μL of colorless RB solution (containing RNase A) and gently shake to suspend the bacterial precipitate, making sure no small bacterial clumps are left.

[0078] (3) Add 250 μL of blue solution LB and gently mix by inverting the tube upside down 4-6 times to fully lyse the bacteria and form a blue transparent solution. The color changes from semi-transparent to transparent blue, indicating that the lysis is complete (the time should not exceed 5 minutes).

[0079] (4) Add 350 μL of yellow solution NB and mix gently for 5-6 times until the color changes completely from blue to yellow, indicating uniform mixing and complete neutralization, forming a compact yellow agglomerate, and let stand at room temperature for 2 minutes.

[0080] (5) Centrifuge at 12,000 x g for 5 minutes, carefully aspirate the supernatant and add it to the centrifuge column. Centrifuge at 12,000 x g for 1 minute and discard the flow-through. If the volume of the supernatant is greater than 800 μL, add it to the column several times and repeat the centrifugation, discarding the flow-through.

[0081] (6) Add 650 μL of solution WB, centrifuge at 12,000 x g for 1 minute, and discard the flow-through.

[0082] (7) Centrifuge again at 12,000 x g for 1-2 minutes to completely remove residual WB.

[0083] (8) Place the centrifuge column in a clean centrifuge tube, add 30-50 μL of EB or deionized water (pH>7.0) to the center of the column, and let it stand at room temperature for 1 minute.

[0084] (9) Centrifuge at 10,000 x g for 1 min to elute the DNA, and store the eluted DNA at -20°C.

[0085] The obtained plasmid is used as a template, the primers used for cloning genes are added to the linker of the overexpression vector, and PCR amplification is performed to obtain the target gene with the linker of the overexpression vector. The primers and system required for amplification are as follows: The obtained plasmid is used as a template, the primers used for cloning genes are added to the linker of the overexpression vector, and PCR amplification is performed to obtain the target gene with the linker of the overexpression vector.

[0086] The adapter primers are as follows:

[0087]

[0088] Amplification system:

[0089]

[0090] The amplified fragments were recovered using the method in 2.3 and stored at -20°C.

[0091] The fragment with the adapter is homologously recombined with the overexpression vector as follows:

[0092] (1) Calculate the amount of DNA required for the recombination reaction according to the formula.

[0093] The optimal amount of cloning vector used = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol),

[0094] The optimal amount of insert fragment used = [0.04 x insert base pairs] ng (0.06 pmol).

[0095] To ensure the accuracy of sample addition, the linearized vector and the inserted fragment can be appropriately diluted before preparing the recombination reaction system, and the sample addition volume of each component should not be less than 1 μL.

[0096] (2) Prepare the following reaction system on ice:

[0097]

[0098] Incubate at 37°C for 30 min; cool to 4°C or immediately cool on ice.

[0099] Transform to E. coli according to the method in 2.4, and check whether the target fragment is connected to the plasmid according to the method in 2.5. Extract the plasmid from the bacterial solution containing the correct target fragment and store it at -20℃ for subsequent experiments.

[0100] 2.7 Transformation of Agrobacterium

[0101] (1) Take out the competent Agrobacterium from -80℃, place it on ice until it partially melts, and immediately return it to ice when it is in an ice-water mixture state.

[0102] (2) Add 1 μg of plasmid DNA per 100 μL of competent medium (the volume should not exceed 10 μL), gently stir the bottom of the tube to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 2.28°C water bath for 5 minutes, and then return to the ice bath for 5 minutes.

[0103] (3) Add 700 μL of LB or YEB liquid medium without antibiotics and shake culture at 28°C for 2-3 hours. Collect bacteria by centrifugation at 6000 rpm for 1 minute, retain about 100 μL of supernatant, gently blow to resuspend the bacterial mass, and spread it on LB or YEB plates containing corresponding antibiotics, and culture in a 28°C incubator for 2-3 days.

[0104] Next, the plasmid extracted from E. coli containing the overexpression vector MADS33 was transformed into Agrobacterium GV3101. After the plasmid was extracted from the Agrobacterium, it was tested using gene primers to verify the correct Agrobacterium containing the MADS33 plasmid and stored at -80°C.

[0105] 2.8 Operation steps of Arabidopsis thaliana transformation by inflorescence dip method

[0106] (1) Wild-type Arabidopsis seeds (Col) were sown in 1 / 2MS medium and transferred to sterilized soil pots when they grew to four cotyledons. The culture room conditions were 24°C (16 hours) during the day and 24°C (8 hours) at night. After about 3 weeks, the main stem of Arabidopsis was cut to inhibit apical dominance and promote reproductive growth. After it had grown multiple side branches and entered the flowering period, it was infected by the inflorescence dipping method. The soil needed to be soaked before infection.

[0107] (2) Prepare Arabidopsis infiltration medium (1 / 2MS liquid medium, pH 5.8). Activate Agrobacterium containing the target expression vector on YEP medium containing antibiotics, pick a single clone and inoculate it into 200 mL YEP liquid medium (50 mg / L Rif and 50 mg / L Kan) with antibiotics, and culture it at 200 rpm on a 28°C constant temperature shaker for 2 days until the OD600 reaches 0.8-1.0.

[0108] (3) Collecting strains: Pour the cultured bacterial solution into a 50 mL sterile centrifuge tube and centrifuge at 4000-6000 rpm for 10 min. Discard the supernatant and resuspend the bacteria in an equal volume of infiltration medium and add 0.02% of the surfactant Silwet L-77.

[0109] (4) Soak the Arabidopsis inflorescence in full bloom in the resuspension solution for 40 seconds, and then gently wipe off the excess liquid on the leaves of the plant.

[0110] (5) After infection, cover the plants with plastic bags and incubate them in the dark for 12-24 hours, then continue incubating for 7-10 days for secondary infection.

[0111] (6) After infection, the plants were cultured normally. The amount of watering was reduced 3 days before the seeds matured. T0 seeds were collected from individual plants, placed in 1.5 mL centrifuge tubes, and stored in a 4°C refrigerator.

[0112] 2.9 Screening of Arabidopsis transgenic positive strains

[0113] (1) The experimental reagents include: 75% ethanol, anhydrous ethanol, sterile water and 1 / 2MS solid culture medium containing antibiotics (30 mg / L Kan).

[0114] (2) Take an appropriate amount of transgenic Arabidopsis seeds to be screened, place them in a 1.5 mL centrifuge tube, add 75% ethanol, and shake thoroughly for 10 minutes. After pouring off the upper liquid, add anhydrous ethanol and shake for 1 minute. Repeat this operation three times. Finally, pour the seeds onto sterilized filter paper and blow dry on a clean bench.

[0115] (3) The dried Arabidopsis seeds were evenly sown on the solid culture medium, the culture dish was sealed, and vernalization was carried out at 4°C for 1 day, and then transferred to a long-day incubator (16 hours of light / 8 hours of darkness).

[0116] (4) Observe the germination of Arabidopsis thaliana in the culture dish. When the seedlings grow 4-5 cotyledons, select the seedlings with good root systems and green leaves and transplant them into the substrate for growth.

[0117] (5) After Arabidopsis thaliana grows to a certain biomass, extract leaf RNA for qPCR verification.

[0118] (6) Compare the growth of the control group (wild-type Col-0 Arabidopsis thaliana) and the transgenic Phoebe MADS33 Arabidopsis thaliana, and count the number of rosette leaves.

[0119] 3. Experimental results

[0120] 3.1 Total RNA extraction and analysis of Phoebe bournei leaves and flowers

[0121] Total RNA extracted from leaves and flowers of Phoebe bournei (see Figure 1 ) The absorbance at 260nm and 280nm was measured by UV spectrophotometer to calculate the concentration and purity of RNA. The results showed that the optical density ratio of all RNA samples was between 2.0-2.2, indicating that the RNA samples were almost free of sugar, phenol and protein contamination. At the same time, the results of 1% agarose gel electrophoresis showed that the 18S and 28S rRNA bands were clear, indicating that the RNA was not degraded and met the requirements of subsequent experiments.

[0122] 3.2 Cloning of the gene sequence of Phoebe bournei MADS33

[0123] According to the designed primer sequences, the MADS33 gene of Phoebe bournei was amplified using PCR technology and detected by bacterial detection PCR (see Figure 2 ). Subsequently, the amplified product was sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing to confirm the presence of the P. minnan MADS33 gene.

[0124] 3.3 Functional verification of MADS33 gene in Phoebe bournei

[0125] The obtained Phoebe bournei MADS33 gene sequence was constructed into the PK2GW7 expression vector and transformed into DH5α competent cells. The successfully connected monoclonal plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101 for Arabidopsis transgenic experiments. The transgenic Arabidopsis seeds were screened for resistance, and the RNA of the selected transgenic plants was confirmed by qPCR detection (see Figure 3 , the expression level of MADS33 gene in OE overexpressing plants was significantly higher than that in WT plants). After 20 days of seedling culture, it was observed that OE overexpressing MADS33 plants bolted at 9-11 rosette leaves, while wild-type WT bolted at 14-16 rosette leaves. Compared with WT plants, OE plants overexpressing MADS33 gene in Phoebe chinensis flowered earlier and bore fruit normally (see Figure 4 ).

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Phoebe bournei MADS33 gene, characterized in that: The Phoebe bournei MADS33 gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

1.

3. The protein encoded by the gene according to claim 1 or 2, characterized in that The protein consists of the amino acids shown in SEQ ID No.

2.

4. A recombinant vector, transgenic cell line or recombinant bacterium containing the gene according to claim 2.

5. A method for advancing plant flowering, the method comprising preparing a modified plant containing the Phoebe bournei MADS33 gene, characterized in that: Compared with the wild-type plant, in the modified plant, the expression of the Phoebe bournei MADS33 gene is increased; wherein the nucleotide sequence of the Phoebe bournei MADS33 gene is shown in SEQ ID NO:

1.

6. Application of Phoebe bournei MADS33 gene in advancing plant flowering, characterized in that: A modified plant containing the Phoebe bournei MADS33 gene is prepared, wherein the expression of the Phoebe bournei MADS33 gene is increased in the modified plant relative to the wild-type plant; wherein the nucleotide sequence of the Phoebe bournei MADS33 gene is shown in SEQ ID NO:

1.

7. The use according to claim 6, characterized in that: The plant is Arabidopsis thaliana or Phoebe bournei.

8. The use according to claim 7, characterized in that: The plant is Arabidopsis thaliana.

9. The use according to claim 6, characterized in that: The Phoebe bournei MADS33 gene is connected to a vector, transformed into Arabidopsis thaliana through Agrobacterium-mediated transformation, and then screened, cultured, and transgenic strains are obtained.

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