Pinus massoniana PmFIP37 gene as well as encoding protein and application thereof

Abstract

The invention discloses a pinus massoniana PmFIP37 gene as well as an encoding protein and application thereof, and relates to the technical field of plant genetic engineering. The invention discloses a pinus massoniana PmFIP37 gene. The nucleotide sequence of the pinus massoniana PmFIP37 gene is as shown in SEQ ID NO. 2; the nucleotide sequence of the encoded protein of the gene is shown as SEQ ID NO. 1. The method comprises the following steps: constructing an expression vector of the PmFIP37 gene of the pinus massoniana, and transforming the expression vector into a '84k' poplar; and cultivating and screening to obtain a transgenic plant with obviously increased plant height, increased number of stem nodes, thickened stem and enlarged leaf area. The pinus massoniana PmFIP37 protein and the pinus massoniana PmFIP37 gene not only improve the growth efficiency of trees, but also further enhance the function and value of the pinus massoniana PmFIP37 protein and the pinus massoniana PmFIP37 gene in an ecological system, thereby providing powerful support for optimal utilization of forestry resources and environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Masson pine PmFIP37 gene and its encoded protein and application. Background Art

[0002] Masson pine is an important afforestation tree species unique to my country. Its bark is generally reddish brown and gray-brown at the bottom. The branches are flat or oblique, the crown is wide tower-shaped or umbrella-shaped, and the branches are light yellow-brown and hairless. The leaf sheath is gray-black, and the leaf margins have sparse spiny serrations. The male cones are light reddish brown, cylindrical, and drooping. The annual cones are spherical or oval, and the seeds are long oval. In addition to afforestation, Masson pine can also be used as building materials, fuel, industrial raw materials, etc., which has brought huge economic benefits to my country and occupies an important position in my country's forestry. Masson pine has excellent traits such as upright trunks, strong adaptability, strong wind resistance, and smoke resistance. Therefore, it is very important to study the effects of Masson pine proteins and genes on the growth and development of forests.

[0003] FIP37 (FKBP12 Interacting Protein 37kDa) has been shown to regulate growth and development in plants. The expression of its encoding gene FIP37 can be seen throughout the entire life process, including the vegetative growth, reproductive growth and development of plants such as Arabidopsis and poplar. Further studies in model plants have shown that overexpression of FIP37 affects trichome development and increases plant height; knockout leads to premature cessation of seed development and excessive proliferation of stem meristems. At the same time, FIP37 is also involved in inhibiting hypocotyl elongation mediated by blue light. However, research on the function of the PmFIP37 gene in Masson pine is still blank. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Masson pine PmFIP37 protein. Another technical problem to be solved by the present invention is to provide a gene encoding a Masson pine PmFIP37 protein. Another technical problem to be solved by the present invention is to provide an application of a Masson pine PmFIP37 protein for regulating the early growth rate of trees.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The nucleotide sequence of the Masson pine PmFIP37 gene is shown in SEQ ID NO. 2.

[0007] The amino acid sequence of the protein encoded by the Masson pine PmFIP37 gene is shown in SEQ ID NO. 1.

[0008] Vectors and recombinant bacteria containing the Masson pine PmFIP37 gene.

[0009] Application of Masson pine PmFIP37 gene in regulating plant growth.

[0010] The regulating plant growth is promoting plant growth, including:

[0011] 1) constructing an expression vector of the Masson pine PmFIP37 gene;

[0012] 2) Transforming the constructed expression vector of Masson pine PmFIP37 gene into poplar;

[0013] 3) Cultivate, screen and obtain transgenic plants with significantly increased plant height.

[0014] Application of Masson pine PmFIP37 gene in regulating plant stem node number.

[0015] Application of the Masson pine PmFIP37 gene in regulating plant stem thickness.

[0016] Application of Masson pine PmFIP37 gene in regulating plant leaf area.

[0017] Application of Masson pine PmFIP37 gene in regulating plant growth.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The present invention is the first to discover and successfully isolate the homologous protein PmFIP37 of AtFIP37 in Masson pine, whose amino acid sequence is shown in SEQ ID NO. 1, and its sequence structure is different from the known homologous sequences in other species.

[0020] 2) The present invention constructs an expression vector of the gene encoding the Masson pine PmFIP37 protein and transforms it into the '84k' poplar; cultivates and screens transgenic plants with significantly increased plant height, increased number of stem nodes, thicker stems, and larger leaf areas; the growth rate of the transgenic strains is significantly improved. The results show that the PmFIP37 gene not only improves the growth efficiency of trees, but also further enhances their functions and values ​​in the ecosystem, providing strong support for the optimal utilization of forestry resources and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison of the amino acid sequences of FIP37 from multiple species;

[0022] Figure 2 The predicted secondary structure of PmFIP37;

[0023] Figure 3 This is the result of the analysis of the hydrophilicity and hydrophobicity of the PmFIP37 protein structure;

[0024] Figure 4 This is the result of the analysis of the transmembrane structure of the PmFIP37 protein;

[0025] Figure 5 is the agarose gel electrophoresis diagram of the cloned PmFIP37 gene;

[0026] Figure 6 This is the relative expression level of PmFIP37 gene in different tissues of Masson pine;

[0027] Figure 7 This is the prokaryotic expression and purification diagram of PmFIP37 protein;

[0028] Figure 8 It is the gDNA agarose gel electrophoresis detection diagram of WT and OE-PmFIP37 strains;

[0029] Fig. 9 is the relative expression level of PmFIP37 gene in WT and OE-PmFIP37 strains;

[0030] Fig.10 These are phenotypic observation diagrams of transgenic strains (A is a growth status diagram 30 days after being transferred to soil for cultivation; B is a statistical diagram of plant height, number of stem nodes, and stem diameter 30 days after being transferred to soil for cultivation; C is a growth status diagram of the 1st to 10th leaves from the top 30 days after being transferred to soil for cultivation; D is a statistical diagram of leaf area 30 days after being transferred to soil for cultivation). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out by referring to the methods listed in the book "Molecular Cloning Laboratory Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0032] The plant materials used in this application are Pinus massoniana needles and '84k' poplar, both of which are preserved in the National Key Laboratory of Tree Genetics and Breeding (60503 Pine Genetic Improvement Laboratory) of Nanjing Forestry University.

[0033] Example 1

[0034] The PmFIP37 identified from the Masson pine transcriptome was compared with the protein sequences of Cryptomeria japonica, Populus trichocarpa, Arabidopsis thaliana, Vitis vinifera, Cucumis sativus and Solanum lycopersicum. The results showed that the FIP37 protein was highly conserved among multiple species, especially the core region WTAP domain, with a sequence identity of more than 95%, which strongly suggests the importance of this domain in the process of biological evolution and the stability of its function ( Figure 1 ). The sequence alignment analysis also identified two highly conserved Coiled-coil domains, which play a key role in the protein interaction network. They are not only involved in complex metabolic pathways within cells, but are also essential for maintaining the structural stability of proteins and can play a regulatory role at multiple levels of gene expression ( Figure 2-4 ).

[0035] Example 2

[0036] 1. Total RNA extraction and cDNA acquisition

[0037] Clean and dry the mortar, grinding rod, small steel spoon, tweezers, operating table, etc. with 75% alcohol and nucleic acid remover; take out the pipette, pipette tip and sterile enzyme-free centrifuge tube in advance. Preheat RNase-free ddH2O at 65℃. Take about 100mg of Masson pine needles ground by liquid nitrogen in a 1.5ml centrifuge tube and immediately add 600μL of Buffer PSL. Vortex vigorously for 30s to mix the sample and lysate thoroughly. Centrifuge at 12,000rpm for 5min in a high-speed centrifuge and proceed with subsequent operations immediately. Take 500μL of the supernatant after centrifugation to FastPure gDNA-Filter Columns Ⅲ (already placed in the collection tube), centrifuge at 12,000rpm for 30s, discard FastPure gDNA-Filter Columns Ⅲ, and collect the filtrate. Add 0.5 times the volume of the above filtrate to the collection tube and oscillate for 15s. Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12,000rpm for 30s, and discard the filtrate. Add 700μL Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000rpm for 30s, and discard the filtrate. Add 500μL Buffer RWB (anhydrous ethanol has been added before use) to FastPure RNA Columns V, centrifuge at 12,000rpm for 30s, and discard the filtrate. Repeat once. Put FastPure RNA Columns V back into the collection tube and centrifuge at 12,000rpm for 2min. Transfer FastPure RNA Columns V to new RNase-free Collection Tubes 1.5ml centrifuge tubes, add 50μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000rpm for one minute. The extracted RNA is stored at -80℃ and can be directly used for cDNA synthesis after measuring the temperature using a spectrophotometer.

[0038] Use Hifair from Shanghai Yisheng Biotechnology Co., Ltd. ® The extracted RNA from Pinus massoniana needles was reverse transcribed using the Ⅲ 1st Strand cDNA SynthesisSuperMix for qPCR reverse transcription kit to obtain cDNA. The specific operation was carried out according to the instruction manual.

[0039] 2. Cloning of target gene

[0040] Specific primers were designed based on the PmFIP37 gene sequence identified from the Masson pine genome. The cloning primer sequence of the PmFIP37 gene is as follows:

[0041] PmFIP37-F:

[0042] 5'-ATGGCCAGCTCCCCTAATTTGG-3',

[0043] PmFIP37-R:

[0044] 5'-TCACCTACTACCCTTTGCTCCATC-3'.

[0045] The PCR reaction system was: cDNA 2.5 μL; primers F / R 2.5 μL each; 2× high-fidelity enzyme 25 μL; ddH2O 17.5 μL.

[0046] The PCR reaction conditions were as follows: 98°C for 1 min; 95°C for 15 s, 55°C for 15 s, and 72°C for 10 s, for 35 cycles; and 72°C for an additional 3 min.

[0047] Mix the PCR product with 6× loading buffer and spot it on the gel. Electrophoresis with the marker at 180V for 15 minutes. After the electrophoresis, place the gel in a gel imaging system for observation ( Figure 5 ). The product was purified using the product purification kit (DC301) of Novozymes to obtain the target gene.

[0048] The final sequencing obtained the ORF of the PmFIP37 gene of 978 bp, and its nucleotide sequence is shown in SEQ ID NO.1; it encodes 326 amino acids, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0049] 3. Analysis of the expression pattern of PmFIP37 gene

[0050] Design specific primers for real-time fluorescence quantitative analysis, and the internal reference gene is TUA of Masson pine. The primer sequences are as follows:

[0051] PmFIP37-qF: 5´-AGGCTGCATTTATCGTAAACTTCGC-3´,

[0052] PmFIP37-qR: 5´-GGCTTCAACTGTGACTTCAAATCCC-3´;

[0053] PmTUA-qF: 5´-CAAACTTGGTCCCGTATCCTC-3´,

[0054] PmTUA-qR: 5´-CACAGAAAGCTGCTCATGGTAA-3´.

[0055] The experiment was conducted using the StepOnePlus Real-Time PCR System using the 2× real-time quantitative PCR amplification pre-solution from Shanghai Yisheng Biotechnology Co., Ltd. The reaction solution was prepared on ice, and 10 μL of Hieff UNICONUniversal Blue qPCR SYBR Green Master Mix, 2 μL of template cDNA from each tissue, 0.4 μL of each primer (concentration of 10 μM), and 7.2 μL of sterile enzyme-free ultrapure water were added. The samples were spotted one by one on a 96-well plate in an ice box, and the program was started after centrifugation for 1 min. The program was set as follows: 95°C pre-denaturation for 2 min; 40 cycles of 95°C denaturation for 10 s, and 60°C annealing for 30 s; finally, the default program of the instrument was used to collect the melting curve.

[0056] The results are as follows Figure 6 As shown in the results, PmFIP37 is expressed in all organs of Masson pine, especially in vegetative organs, including cones and male and female cones, where the relative expression level is significantly higher than that in reproductive organs. The expression of PmFIP37 is most prominent in female cones, while the expression in roots is relatively low. In addition, the relative expression of PmFIP37 is higher in current year stems than in perennial stems. The results indicate that PmFIP37 plays a key role in regulating the reproductive development and early growth stages of Masson pine.

[0057] Example 3

[0058] 1. Vector construction

[0059] Primers were designed based on the nucleotide sequence of the purified Masson pine PmFIP37 gene and the sequences of the vector and restriction sites used. The primer sequences are as follows:

[0060] 1305-PmFIP37-F:

[0061] 5´-TTTGGAGAGAACACGTCTAGAATGGCCAGCTCCCCTAATTT-3´,

[0062] 1305-PmFIP37-R:

[0063] 5´-GTCAAGCTTGCATGCCTGCAGCCTACTACCCTTTGCTCCATCAA-3´;

[0064] pET28a-PmFIP37-F:

[0065] 5´-TTCCAATCCCATATGGCCATGGCCAGCTCCCCTAATTTGGATG-3´,

[0066] pET28a-PmFIP37-R:

[0067] 5´-ATGGGTGATGGTGATGGTGCCTACTACCCTTTGCTCCATCA-3´.

[0068] Linearize the pCAMBIA1305::35S-3×Flag plasmid using restriction endonucleases XbaⅠ and PstⅠ. The specific steps are as follows:

[0069] Take 1μg of plasmid; 5μL of 10×Quick Cut Buffer; 1μL of XbaⅠ; 1μL of PstⅠ and add them to ddH2O to form a 50μL reaction system, and then run it in a PCR instrument with the program of 37℃, 30min; 85℃, 20min; 4℃, ∞.

[0070] Then use inverse PCR to linearize the pET28a-MBP-6×His vector plasmid. The specific steps are:

[0071] The primer sequences for inverse PCR are as follows:

[0072] pET28a-F: 5´CACCATCACCATCACCATTAACTCG-3´

[0073] pET28a-R: 5´-GGCCATATGGGATTGGAAGTAGAGG-3´

[0074] The inverse PCR reaction system was: 2.5 μL of pET28a-MBP-6×His plasmid; 2.5 μL of primers F / R; 25 μL of 2× high-fidelity enzyme; and 17.5 μL of ddH2O.

[0075] The inverse PCR reaction program was as follows: pre-denaturation at 98°C for 1 min; denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for another 5 min.

[0076] The products purified by the above reverse PCR were connected to two linear vectors respectively. The specific reaction system and procedure are as follows.

[0077] The ligation reaction system was: 20 ng of PCR-purified product; 400 ng of linear vector; 4 μL of 5×CEⅡ Buffer; 2 μL of ExnaseⅡ added to ddH2O, and then run in a PCR instrument at 37°C, 30 min; 4°C, ∞.

[0078] The ligation products with pCAMBIA1305::35S-3×Flag vector and the ligation products with pET28a-MBP-6×His vector were transformed into E. coli DH5α competent cells; cultured overnight on LB solid plates containing kanamycin (50 mg / mL) and ampicillin (50 mg / mL). Pick a single clone and culture it in LB liquid medium containing the corresponding resistance for 5-6 hours. PCR and sequencing were performed on the colonies for identification. The constructed recombinant vectors were named pCAMBIA1305::35S-PmFIP37-3×Flag and pET28a-PmFIP37-MBP-6×His and the plasmids were extracted.

[0079] 2. Prokaryotic expression and purification of PmFIP37 protein

[0080] 1) Prokaryotic expression

[0081] Add 2 μL of the correctly sequenced plasmid pET28a-PmFIP37-MBP-6×His to 50 μL of Rosetta (DE3) Escherichia coli competent cells and let it stand on ice for 30 minutes. Then place the centrifuge tube in a 42°C water pot for 45 seconds and quickly transfer it to ice for 2 minutes. Then add 700 μL of liquid LB medium without resistance to the centrifuge tube and resuscitate it at 200 rpm for 1 hour in a 37°C constant temperature shaker. Finally, centrifuge at high speed to collect the bacteria, discard 680 μL of supernatant and evenly spread the remaining bacterial liquid on the LB solid medium containing Amp resistance. After overnight culture, pick a single colony for colony PCR and agarose gel electrophoresis, and retain the bacterial liquid with the correct band position for subsequent experiments.

[0082] 2) IPTG-induced prokaryotic expression in Escherichia coli

[0083] Add 1 mL of liquid LB medium containing Amp resistance to the selected bacterial solution, and propagate overnight to saturation in a 37°C constant temperature shaker at 200 rpm. Transfer the bacterial solution to 20 mL of liquid LB medium containing Amp resistance (50 mL EP tube), and propagate overnight to saturation under the same conditions. Pour the bacterial solution into a conical flask containing 2 L of liquid LB medium containing Amp resistance, and shake at 37°C at 200 rpm until OD 600=0.5-0.7, then cooled to room temperature and sampled 100 μL, added 10 mL of the prepared 0.1 M IPTG stock solution to make the final concentration 0.5 mM, and induced protein expression at 18°C, 200 rpm, 16 h.

[0084] 3) Ultrasonic fragmentation and protein purification

[0085] After the expression, 2L of bacterial solution was centrifuged at 4500rpm for 15min and collected into 4 50mL EP tubes. Resuspend the bacteria with 25mL of prepared cell lysis buffer (first add 10mM Tris-HCl; 500mM NaCl and 5% glycerol, then add deionized water to 1L; add 3mM DTT and 1mM PMSF before use after high pressure sterilization). After resuspension, use an ultrasonic cell disruptor to disrupt the cells. The procedure is: total working time 20min, power 300w, working / resting time 10s / 15s, and the whole process is carried out in an ice water bath. After the disruption, centrifuge the centrifuge tube at 4℃, 1,2000rpm for 1h. Filter with a 0.45μm filter membrane, and retain 100μL of supernatant and precipitate respectively.

[0086] 4) Purification of target protein

[0087] The obtained supernatant was purified using a Ni-TED agarose purification resin gravity pre-packed column from Sangon Biotechnology. The specific steps are as follows:

[0088] Water washing: Wash the resin with 5 to 10 column volumes of pure water at 50 to 150 cm / h to remove ethanol.

[0089] Equilibration: Use 5-10 column volumes of binding buffer at 150-600 cm / h to equilibrate the medium to ensure that the composition and pH of the solution in the medium are consistent with those of the sample.

[0090] Sample loading: Load the supernatant after centrifugation and filtration at a low flow rate. Take 100 μL of the outflowing liquid.

[0091] Wash: Use 10-20 column volumes of washing solution at 150 cm / h to wash non-specifically adsorbed impurities, and collect 100 μL of washing solution for subsequent analysis.

[0092] Elution: Use 5 to 10 times the column volume of eluent at a low flow rate and collect the eluate as the target protein solution.

[0093] 5) Detection of target protein by polyacrylamide gel electrophoresis (SDS-PAGE)

[0094] To make polyacrylamide gel, the formula is as follows:

[0095] Lower separation gel: 30% acrylamide 3.5 mL; 3×tricine gel buffer 3 mL; 10% APS 70 μL; ddH2O 2.5 mL; TEMED 10 μL.

[0096] Upper stacking gel: 30% acrylamide 670μL; 3×tricine gel buffer 1mL; 10% APS 40μL; ddH2O 2.3mL; TEMED 10μL.

[0097] Then take 100μL of the above-retained samples (before induction, after induction, supernatant after disruption, precipitate, flow-through, washing solution and elution) and mix with 20μL 6×Protein Loading Buffer, then place in a 95℃ water bath for 10min, centrifuge at 1,4000rpm for 10min, carefully remove about 5μL of the supernatant and spot it behind the Protein Marker in sequence. Finally, after 1.2h, 180V electrophoresis, use Coomassie Brilliant Blue staining for 20min, and decolorize with deionized water to observe the band position.

[0098] The results are as follows Figure 7 As shown, the positions of the uninduced and induced bands and the bold bands were compared, and the results showed that the complex protein product of PmFIP37-MBP-6×His (size of about 81.8 kDa) was successfully obtained through prokaryotic expression in Escherichia coli, and the eluted bands also clearly showed that the complex protein was successfully purified in vitro by the Ni-TED agarose purification resin gravity pre-packed column.

[0099] Example 4

[0100] 1. Agrobacterium-mediated genetic transformation of poplar leaves

[0101] The recombinant plasmid pCAMBIA1305::35S-PmFIP37-3×Flag was transformed into Agrobacterium GV3101. After culturing at 28°C for two days, a single colony was picked for PCR detection. 1 mL of the correctly detected Agrobacterium culture was placed in a 28°C shaker for shaking and culture. 1 mL of the prepared Agrobacterium culture was added to 200 mL LB liquid culture medium (containing 50 mg / mL Rif and 100 mg / mL Kana) and cultured in a shaker at 28°C and 200 rpm until OD 600The value is 0.6-0.8. Precool the centrifuge in advance, centrifuge the bacterial solution at 3500rpm at 4℃ for 10min, discard the supernatant; then resuspend the bacteria with infection solution (composed of 4.43g / L MS, 30g / L sucrose, 50μL / L AS, adjust pH=5.8 after preparation). Select 4-week-old strong '84k' poplar tissue culture seedlings, take its broad and tender green leaves, cut off the leaf edges and veins, and carefully cut some wounds on the leaves, and then put the leaves in water (to prevent the leaves from drying up and inactivating). Take out the leaves and soak them in the infection solution containing Agrobacterium and shake them for about 10min. After the infection is completed, use sterilized tweezers to pick up the leaves and place them in a glass dish (with sterilized filter paper), and dry the surface of the leaves. Spread the back of the leaf flat on a dark culture medium (MS 4.43g; sucrose 30g; agar 8g; 1mg / mL 6-BA 500μL; 1mg / mL NAA 50μL; 100μM AS 1mL deionized water; pH=5.8), about 5 leaves on each culture medium (cannot be overlapped), seal and mark, and culture in the dark for 3 days. Prepare sterilized tweezers and culture dishes in advance, and prepare 150g / L of Timentin. After the dark culture is completed, remove the leaves from the dark culture medium, wash them with Timentin solution 3 times, about 10s each time, and then wash them with ddH2O for 10s. After washing, place the leaves on filter paper in a glass dish to absorb water, and then place them on the light culture medium for about 10 days. After the light culture is completed, remove the leaves from the light culture medium and observe them. If there are bacteria on the leaves, wash them once with Timentin solution and once with ddH2O before transferring; if there are no bacteria, transfer them directly to the screening culture medium. Cultivate until clusters of buds differentiate on the leaves. When the young buds in the screening medium grow to more than 1 cm, they can be transferred to the bud elongation medium to continue inducing their growth. After 20-30 days, when the buds grow to about 2-3 cm, the adventitious buds can be transferred to the rooting medium to induce rooting.

[0102] 2. Identification of transgenic plants

[0103] The genomic extraction kit (DC104) of Novozymes was used to extract gDNA from transgenic and wild-type poplar leaves. Using gDNA as a template, agarose gel electrophoresis was performed using specific primers PmFIP37-F and 1305-PmFIP37-R.

[0104] The results are as follows Figure 8 As shown in the figure, the target gene band was amplified in the transgenic plants, but not in the wild type, indicating that the transgenic lines were successfully constructed. Then, the three lines with the brightest PCR product bands (i.e. Figure 8 The plants corresponding to the 1st, 2nd and 10th bands overexpressing OE-PmFIP37 were used for subsequent experiments.

[0105] Total RNA was extracted from transgenic and wild-type poplar leaves and reverse transcribed into cDNA. Using cDNA as a template, RT-qPCR was performed using PmFIP37-qF / R. The primer sequences are as follows:

[0106] PmFIP37-qF: 5´-AGGCTGCATTTATCGTAAACTTCGC-3´,

[0107] PmFIP37-qR: 5´-GGCTTCAACTGTGACTTCAAATCCC-3´.

[0108] The results are as follows Fig. 9 As shown, the relative expression level of PmFIP37 gene in transgenic poplars was significantly higher than that in wild type, indicating that PmFIP37 gene had been successfully transferred into '84k' poplars.

[0109] 3. Transgenic phenotype observation

[0110] The plant height, number of stem nodes, stem thickness, stem node length and leaf size of wild-type and transgenic '84k' poplars transplanted into soil for 30 days were measured and counted.

[0111] The results are as follows Fig.10 As shown, the plant height of poplars overexpressing PmFIP37 increased significantly, the number of stem nodes increased, the stem became thicker, and the leaf area became larger. The results showed that overexpression of PmFIP37 promoted the early growth of '84k' poplars.

[0112] In summary, PmFIP37 protein plays an important role in promoting the early growth of plants, promoting the increase in the height, stem thickness and leaf size of transgenic plants in the early growth stage.

[0113] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. The PmFIP37 gene of Masson pine, whose nucleotide sequence is shown in SEQ ID NO.

2.

2. The protein encoded by the Masson pine PmFIP37 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

1.

3. A vector and a recombinant bacterium containing the Masson pine PmFIP37 gene according to claim 1.

4. Use of the Masson pine PmFIP37 gene according to claim 1 in regulating plant growth.

5. The use according to claim 4, characterized in that: The regulating plant growth is promoting plant growth.

6. The use according to claim 5, characterized in that: include: 1) constructing an expression vector of the Masson pine PmFIP37 gene; 2) Transforming the constructed expression vector of Masson pine PmFIP37 gene into poplar; 3) Cultivate, screen and obtain transgenic plants with significantly increased plant height.

7. Use of the Masson pine PmFIP37 gene according to claim 1 in regulating the number of plant stem nodes.

8. Use of the Masson pine PmFIP37 gene according to claim 1 in regulating the thickness of plant stems.

9. Use of the Masson pine PmFIP37 gene according to claim 1 in regulating plant leaf area.

10. Use of the Masson pine PmFIP37 gene according to claim 1 in regulating plant growth.

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