Pinus massoniana pmfip37 gene, encoded protein and application thereof

Abstract

The present application discloses a Pinus massoniana PmFIP37 gene, its coding protein and application, relates to the technical field of plant genetic engineering.The nucleotide sequence of the Pinus massoniana PmFIP37 gene is shown in SEQ ID NO.2; the nucleotide sequence of the coding protein is shown in SEQ ID NO.1.The expression vector of the Pinus massoniana PmFIP37 gene is constructed and transformed into '84k' poplar; the transgenic plants with significantly increased plant height, increased stem node quantity, thickened stem, and increased leaf area are obtained through cultivation and screening.The Pinus massoniana PmFIP37 protein and PmFIP37 gene not only improve the growth efficiency of trees, but also further enhance the function and value in the ecological system, and provide strong support for the optimized utilization of forestry resources and environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the Masson pine PmFIP37 gene and its encoded protein and its applications. Background Technology

[0002] Masson pine is an important afforestation tree species unique to my country. Its bark is generally reddish-brown, turning grayish-brown at the base. Branches are spreading or obliquely spreading, with a broad pyramidal or umbrella-shaped crown. Branches are pale yellowish-brown and hairless. Leaf sheaths are grayish-black, with sparsely serrated, bristle-like margins. Male cones are pale reddish-brown, cylindrical, and drooping. Annual cones are spherical or ovoid, and seeds are oblong-ovoid. Besides afforestation, Masson pine can also be used as building material, fuel, and industrial raw material, bringing significant economic benefits to my country and occupying an important position in my country's forestry. Masson pine possesses excellent traits such as upright trunk, strong adaptability, strong wind resistance, and tolerance to smoke and dust. Therefore, studying the influence of Masson pine proteins and genes on tree growth and development is crucial.

[0003] FIP37 (FKBP12 Interacting Protein 37kDa) has been shown to regulate growth and development in plants. The expression of its encoding gene, FIP37, is involved throughout the entire life cycle of plants, including Arabidopsis thaliana and poplar, encompassing vegetative growth, reproductive growth, and development. 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. FIP37 also participates in inhibiting blue light-mediated hypocotyl elongation. However, research on the function of the PmFIP37 gene in Pinus massoniana remains lacking. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the PmFIP37 protein from *Pinus massoniana*. Another technical problem this invention aims to solve is to provide the encoding gene for the PmFIP37 protein from *Pinus massoniana*. A further technical problem this invention aims to solve is to provide applications for the PmFIP37 protein from *Pinus massoniana*, used to regulate the early growth rate of trees.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

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

[0007] The protein encoded by the PmFIP37 gene of Pinus massoniana has the amino acid sequence shown in SEQ ID NO. 1.

[0008] Vectors and recombinant bacteria containing the PmFIP37 gene of Pinus massoniana.

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

[0010] The regulation of plant growth is to promote plant growth, including:

[0011] 1) Construct an expression vector for the PmFIP37 gene of Pinus massoniana;

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

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

[0014] Application of the PmFIP37 gene in regulating the number of stem nodes in Masson pine.

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

[0016] Application of the PmFIP37 gene in regulating plant leaf area (Pinus massoniana).

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

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1) This invention is the first to discover and successfully isolate the homologous protein PmFIP37 of AtFIP37 from Masson pine. Its 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) This invention constructs an expression vector encoding the PmFIP37 protein of *Pinus massoniana* and transforms it into '84k' poplar. Transgenic plants with significantly increased height, number of stem nodes, thicker stems, and larger leaf area were cultivated, screened, and obtained. The growth rate of the transgenic lines was significantly enhanced. The results indicate that the PmFIP37 gene not only improves tree growth efficiency but also further enhances its function and value in the ecosystem, providing strong support for the optimal utilization of forestry resources and environmental protection. Attached Figure Description

[0021] Figure 1 This is a multi-species FIP37 amino acid sequence alignment diagram;

[0022] Figure 2 The secondary structure prediction diagram of PmFIP37;

[0023] Figure 3 Figure showing the results of hydrophilicity and hydrophobicity analysis of the PmFIP37 protein structure;

[0024] Figure 4 Figure showing the results of transmembrane structure analysis of PmFIP37 protein;

[0025] Figure 5 Agarose gel electrophoresis image of the cloned PmFIP37 gene;

[0026] Figure 6 The graph shows the relative expression levels of the PmFIP37 gene in different tissues of Pinus massoniana.

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

[0028] Figure 8 Agarose gel electrophoresis results of gDNA detection in WT and OE-PmFIP37 strains;

[0029] Figure 9 The relative expression levels of the PmFIP37 gene in the WT and OE-PmFIP37 lines are shown in the diagram.

[0030] Figure 10 Phenotypic observation diagrams of transgenic lines (A is a growth diagram 30 days after transplanting to soil; B is a statistical diagram of plant height, number of stem nodes, and stem diameter 30 days after transplanting to soil; C is a growth diagram of the 1st to 10th leaves from the top 30 days after transplanting to soil; D is a statistical diagram of leaf area 30 days after transplanting to soil). Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

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

[0033] Example 1

[0034] The FIP37 protein identified from the transcriptome of *Pinus massoniana* was compared with the FIP37 protein sequences of other species, including *Cryptomeria japonica*, *Populus trichocarpa*, *Arabidopsis thaliana*, *Vitis vinifera*, *Cucumis sativus*, and *Solanumlycopersicum*. The results showed that the FIP37 protein exhibits extremely high conservation across multiple species, particularly its core WTAP domain, which showed sequence identity exceeding 95%. This strongly suggests the importance and functional stability of this domain in biological evolution. Figure 1 Sequence alignment analysis also identified two highly conserved coiled-coil domains, which play a crucial role in protein-protein interaction networks. These domains not only participate in complex metabolic pathways within cells but are also essential for maintaining protein structural stability and can regulate gene expression at multiple levels. Figure 2-4 ).

[0035] Example 2

[0036] 1. Total RNA extraction and cDNA acquisition

[0037] Clean and dry the mortar and pestle, grinding rod, small steel spoon, tweezers, and worktable with 75% alcohol and nucleic acid removal agent; prepare the pipettes, pipette tips, and sterile enzyme-free centrifuge tubes in advance. Preheat RNase-free ddH2O to 65℃. Add approximately 100mg of pine needles ground in liquid nitrogen to a 1.5ml centrifuge tube and immediately add 600μL of Buffer PSL. Vortex vigorously for 30s to ensure thorough mixing of the sample and lysis buffer. Centrifuge at 12,000rpm for 5min and proceed with subsequent operations immediately. Transfer 500μL of the supernatant to a FastPure gDNA-Filter Columns III (already placed in a collection tube), centrifuge at 12,000rpm for 30s, discard the FastPure gDNA-Filter Columns III, and collect the filtrate. Add 0.5 times the volume of the filtrate to the collection tube and vortex for 15s to mix. Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V is already in the collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 500 μL of Buffer RWB (with anhydrous ethanol added before use) to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Repeat once. Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm for 2 minutes. Transfer FastPure RNA Columns V to a new 1.5 ml RNase-free Collection Tube centrifuge tube. Add 50 μL of RNase-free ddH2O dropwise to the center of the adsorption column membrane and centrifuge at 12,000 rpm for one minute. Store the extracted RNA at -80°C. After measuring the temperature with a spectrophotometer, it can be directly used for cDNA synthesis.

[0038] Using Hifair from Shanghai Yisheng Biotechnology Co., Ltd. ® III. 1st Strand cDNA SynthesisSuperMix for qPCR Reverse Transcription Kit: Extracted RNA from pine needles is reverse transcribed to obtain cDNA. Follow the instructions in the manual for specific procedures.

[0039] 2. Cloning of the target gene

[0040] Specific primers were designed based on the PmFIP37 gene sequence identified from the genome of *Pinus massoniana*. The cloning primer sequences for the PmFIP37 gene are shown below:

[0041] PmFIP37-F:

[0042] 5'-ATGGCCAGCTCCCCTAATTTGG-3',

[0043] PmFIP37-R:

[0044] 5'-TCACCTACTACCCTTTGCTCCATC-3'.

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

[0046] The PCR reaction conditions were as follows: 98℃ reaction for 1 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 3 min.

[0047] The PCR products were mixed with 6× loading buffer and spotted into a gel. Electrophoresis was then performed at 180V for 15 min along with the marker. After electrophoresis, the gel was placed in a gel imaging system for observation. Figure 5 The product was purified using the Novizan product purification kit (DC301) to obtain the target gene.

[0048] The final sequencing yielded an ORF of 978 bp for the PmFIP37 gene, whose 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 the PmFIP37 gene

[0050] Design specific primers for real-time quantitative PCR, with the internal reference gene being TUA from *Pinus massoniana*. The primer sequences are shown below:

[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 2× real-time quantitative PCR amplification pre-solution from Shanghai Yisheng Biotechnology Co., Ltd. was used for experiments via the StepOnePlus Real-Time PCR System. The reaction mixture was prepared on ice, including 10 μL of Hieff UNICONUniversal Blue qPCR SYBR Green Master Mix, 2 μL of template cDNA from each tissue, 0.4 μL of primers (10 μM each), and 7.2 μL of sterile, enzyme-free ultrapure water. Samples were individually spotted onto 96-well plates in an ice-cold container, centrifuged for 1 min, and the program was started. The program settings were: 95℃ pre-denaturation for 2 min; 40 cycles of 95℃ denaturation for 10 s, 60℃ annealing for 30 s; finally, the melting curve was acquired using the instrument's default program.

[0056] The results are as follows Figure 6 As shown, PmFIP37 is expressed in all organs of *Pinus massoniana*, especially in vegetative organs, including cones and both male and female cones, where the relative expression level is significantly higher than in reproductive organs. PmFIP37 expression is most prominent in female cones, while expression in the roots is relatively low. Furthermore, the relative expression level of PmFIP37 is higher in current-year stems compared to perennial stems. These results indicate that PmFIP37 plays a crucial role in regulating reproductive development and early growth stages of *Pinus massoniana*.

[0057] Example 3

[0058] 1. Carrier Construction

[0059] Primers were designed based on the nucleotide sequence of the purified *Pinus massoniana* PmFIP37 gene, the vector used, and the sequences of the restriction enzyme sites. The primer sequences are shown below:

[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] The pCAMBIA1305::35S-3×Flag plasmid was linearized using restriction endonucleases XbaⅠ and PstⅠ. The specific steps were as follows:

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

[0070] Then, the pET28a-MBP-6×His vector plasmid was linearized using reverse PCR. The specific steps are as follows:

[0071] The primer sequences for reverse PCR are shown below:

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

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

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

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

[0076] The purified products from the reverse PCR were ligated to two different linear vectors, and the specific reaction systems and procedures are as follows.

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

[0078] The ligation products of pCAMBIA1305::35S-3×Flag and pET28a-MBP-6×His vectors were transformed into *E. coli* DH5α competent cells. The cells were then cultured overnight on LB agar plates containing kanamycin (50 mg / mL) and ampicillin (50 mg / mL), respectively. Single colonies were picked and cultured for 5–6 h in LB liquid medium containing the corresponding antibiotics. Colonies were identified by PCR and sequencing. The constructed recombinant vectors were named pCAMBIA1305::35S-PmFIP37-3×Flag and pET28a-PmFIP37-MBP-6×His, and 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 competent Rosetta (DE3) E. coli cells and incubate on ice for 30 min. Then, heat shock the centrifuge tubes in a 42°C water bath for 45 s and quickly transfer them to ice for 2 min. Next, add 700 μL of antibiotic-free liquid LB medium to the centrifuge tubes and incubate at 37°C with a shaker at 200 rpm for 1 h. Finally, collect the bacteria by high-speed instantaneous centrifugation, discard 680 μL of supernatant, and spread the remaining bacterial culture evenly on LB solid medium containing Amp resistance. After overnight incubation, pick single colonies for colony PCR and agarose gel electrophoresis, and retain the bacterial cultures with correctly positioned bands for subsequent experiments.

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

[0083] Add 1 mL of Amp-resistant liquid LB medium to the selected bacterial culture and incubate overnight at 37°C and 200 rpm until saturation. Transfer the bacterial culture to 20 mL of Amp-resistant liquid LB medium (in a 50 mL EP tube) and incubate overnight at the same conditions until saturation. Pour the bacterial culture into a conical flask containing 2 L of Amp-resistant liquid LB medium and incubate at 37°C and 200 rpm until OD reaches 0.5. 600After mixing with 0.5-0.7, cool to room temperature and take a 100 μL sample. Add 10 mL of the prepared 0.1 M IPTG stock solution to make a final concentration of 0.5 mM, and induce protein expression at 18 °C, 200 rpm, for 16 h.

[0084] 3) Ultrasonic disruption and protein purification

[0085] The 2L bacterial culture after expression was completed was centrifuged at 4500rpm for 15min and transferred to four 50mL EP tubes. The cells were resuspended in 25mL of prepared cell lysis buffer (10mM Tris-HCl, 500mM NaCl, and 5% glycerol were added, then deionized water was added to bring the volume to 1L; after autoclaving, 3mM DTT and 1mM PMSF were added before use). After resuspending, the cells were disrupted using an ultrasonic cell disruptor. The program was as follows: total working time 20min, power 300W, working / rest time 10s / 15s, all performed in an ice-water bath. After disruption, the centrifuge tubes were centrifuged at 4℃ and 12000rpm for 1h. The cells were filtered through a 0.45μm filter membrane, and 100μL of supernatant and precipitate were retained separately.

[0086] 4) Purification of the target protein

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

[0088] Wash with water: Wash the resin with 5 to 10 column volumes of pure water at a flow rate of 50 to 150 cm⁻¹ / h to remove ethanol.

[0089] Equilibration: Equilibrate the medium with 5-10 column volumes of binding buffer at 150-600 cm⁻¹ / h 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] Washing: Use 10-20 column volumes of washing buffer at 150 cm / h to wash away non-specifically adsorbed proteins, and collect 100 μL of washing buffer for subsequent analysis.

[0092] Elution: Elute with 5-10 column volumes of elution buffer at a low flow rate, and collect the elution buffer as the target protein solution.

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

[0094] The formula for making polyacrylamide gel is as follows:

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

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

[0097] Then, take 100 μL each of the retained samples (pre-induction, post-induction, post-disruption supernatant, precipitate, flow-through, washing buffer, and elution buffer) and mix with 20 μL of 6× Protein Loading Buffer. Incubate at 95°C for 10 min, centrifuge at 1,4000 rpm for 10 min, carefully remove approximately 5 μL of supernatant, and spot it sequentially after the Protein Marker. After 1.2 h of electrophoresis at 180 V, stain with Coomassie Brilliant Blue for 20 min, destain with deionized water, and observe the band positions.

[0098] The results are as follows Figure 7 As shown, comparing the bands before and after induction, as well as the location of the thickened band, the results indicate that the complex protein product of PmFIP37-MBP-6×His (approximately 81.8 kDa) was successfully obtained through prokaryotic expression in Escherichia coli. Furthermore, the eluted bands clearly demonstrate that the complex protein was successfully purified in vitro using a gravity-packed Ni-TED agarose purification resin 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 two days of incubation at 28°C, single colonies were picked for PCR detection. 1 mL of the correctly detected Agrobacterium culture was placed in a shaker at 28°C for further incubation. 1 mL of the prepared Agrobacterium culture was then added to 200 mL of LB broth (containing 50 mg / mL Rif and 100 mg / mL Kana) and incubated at 28°C with shaking at 200 rpm until OD (Organic Degrees Per Count) was reached. 600The value is 0.6-0.8. The centrifuge was pre-cooled, and the bacterial culture was centrifuged at 3500 rpm for 10 minutes at 4℃, discarding the supernatant. The cells were then resuspended in an inoculation solution (composed of 4.43 g / L MS, 30 g / L sucrose, and 50 μL / L AS, adjusted to pH 5.8 after preparation). Healthy '84k' poplar tissue culture seedlings (approximately 4 weeks old) were selected. Large, tender green leaves were taken, the leaf margins and veins were removed, and some wounds were carefully made on the leaves. The leaves were then placed in water (to prevent drying and inactivation). The leaves were then removed and immersed in the inoculation solution containing Agrobacterium, agitated, for approximately 10 minutes. After inoculation, the leaves were placed in a glass dish (with sterile filter paper already placed) using sterile tweezers and allowed to air dry. Spread the undersides of the leaves flat on dark culture medium (MS 4.43g; sucrose 30g; agar 8g; 1mg / mL 6-BA 500μL; 1mg / mL NAA 50μL; 100μM AS 1mL deionized water to a final volume; pH=5.8), about 5 leaves per medium (do not overlap), seal and label, and incubate in the dark for 3 days. Prepare sterilized forceps and culture dishes in advance, and prepare 150g / L termetidine. After the dark incubation, remove the leaves from the dark medium and wash them three times with termetidine solution for about 10 seconds each time, then wash them with ddH2O for 10 seconds each time. After washing, place the leaves on filter paper in a glass dish to absorb the moisture, and then place them on light culture medium for about 10 days. After the light incubation, remove the leaves from the light culture medium and observe them. If bacteria are present on the leaves, wash them once with termetidine solution and once with ddH2O before transferring them; if no bacteria are present, transfer them directly to selection medium. Culture until clusters of buds differentiate on the leaves. When the young buds in the selection 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 or more, the adventitious buds can be transferred to the rooting medium to induce rooting.

[0102] 2. Identification of transgenic plants

[0103] gDNA was extracted from transgenic and wild-type poplar leaves using the Novizan Genome Extraction Kit (DC104). Using the gDNA as a template, agarose gel electrophoresis was performed with specific primers PmFIP37-F and 1305-PmFIP37-R for detection.

[0104] The results are as follows Figure 8 As shown, the target gene band was amplified in the transgenic plants, while no band was observed in the wild-type plants, indicating that the transgenic lines were successfully constructed. Then, from the 10 positive lines selected, the 3 lines with the brightest PCR product bands (i.e., Figure 8 Subsequent experiments were conducted on plants that overexpressed OE-PmFIP37 at bands 1, 2, and 10.

[0105] Total RNA was extracted from leaves of transgenic and wild-type poplar trees and reverse transcribed into cDNA. RT-qPCR was performed using the cDNA as a template with PmFIP37-qF / R. Primer sequences are shown below:

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

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

[0108] The results are as follows Figure 9 As shown, the relative expression level of the PmFIP37 gene in transgenic poplar was significantly higher than that in wild-type poplar. This indicates that the PmFIP37 gene has been successfully transferred into '84k' poplar.

[0109] 3. Observation of transgenic phenotypes

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

[0111] The results are as follows Figure 10 As shown, overexpression of the PmFIP37 gene significantly increased poplar height, stem node number, stem thickness, and leaf area. The results indicate that overexpression of PmFIP37 promotes the early growth of '84k' poplar.

[0112] In summary, PmFIP37 protein plays an important role in promoting early plant growth, resulting in increased height, stem diameter, and leaf size in transgenic plants during the early growth stages.

[0113] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

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

2.

2. The Masson pine as described in claim 1 PmFIP37 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.

1.

3. Containing the Masson pine as described in claim 1 PmFIP37 Gene encoding vectors and recombinant bacteria.

4. The Masson pine as described in claim 1 PmFIP37 Application of genes in promoting the growth of '84k' poplar.

5. The application according to claim 4, characterized in that, include: 1) Constructing Masson pine PmFIP37 Gene expression vectors; 2) The constructed Masson pine PmFIP37 The gene expression vector was transformed into '84k' poplar; 3) Cultivate, screen, and obtain transgenic plants with significantly increased plant height.

6. The Masson pine as described in claim 1 PmFIP37 Application of genes in promoting the number of stem nodes in '84k' poplar.

7. The Masson pine as described in claim 1 PmFIP37 Application of genes in promoting the thickness of '84k' poplar stems.

8. The Masson pine as described in claim 1 PmFIP37 Application of genes in promoting leaf area of ​​'84k' poplar.

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