Strigolactone biosynthesis related gene PfD27, expression vector constructed by strigolactone biosynthesis related gene PfD27 and application of strigolactone biosynthesis related gene PfD27

Abstract

The invention relates to a paulownia fortunei PfD27 gene and construction and application of an expression vector of the paulownia fortunei PfD27 gene. The paulownia fortunei strigolactones are screened from a paulownia fortunei genome through a bioinformatics method to synthesize related beta-carotene isomerase DWARF27 (D27) gene. The nucleotide sequence of the PfD27 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the PfD27 gene is as shown in SEQ ID NO. 2. According to the invention, paulownia fortunei PfD27 expression vectors such as pSAK277-PfD27-eGFP, pET-28a (+)-SUMO-PfD27, pCAMBIA3301-PfD27 and the like are constructed, subcellular localization analysis of the paulownia fortunei PfD27 protein, prokaryotic expression and purification of the PfD27 protein and research on the influence of PfD27 gene overexpression on tomato branches are respectively carried out, the function of the paulownia fortunei PfD27 gene is explained in the aspects of strigolactones biosynthesis and plant branch regulation and control, and the application of the paulownia fortunei PfD27 gene is developed. Reference is provided for subsequently revealing the relationship between the paulownia fortunei arbuscular disease pathogenesis and strigolactones hormone, and a molecular basis is provided for cultivation of new varieties of ideal paulownia plant types.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a β-carotene isomerase gene PfD27 related to strigolactone biosynthesis, its constructed expression vector, and its applications. Background Technology

[0002] Strigolactones (SLs) are a class of terpenoid compounds derived from carotenoids, and as a novel plant hormone, they have attracted widespread attention in multiple fields. SLs are mainly synthesized in plant roots. Initially, they were found to promote the germination of parasitic plant seeds, and were subsequently identified as soil signaling molecules secreted by roots, regulating the symbiosis between roots and arbuscular mycorrhizae to enhance nitrogen and phosphorus uptake. Within the plant, SLs are transported from the morphological lower end to the morphological upper end to the axillary buds, regulating the growth of lateral branches. Further research has revealed that SLs also have multiple functions, including shaping plant root structure, accelerating leaf senescence, regulating secondary growth, and mediating plant resistance to abiotic stresses.

[0003] Root hairs (SLs) promote root hair development, increasing root hair length and density, thereby expanding the root system's absorption area and improving the plant's efficiency in absorbing water and nutrients from the soil. Studies show that under low phosphorus stress, plants increase the synthesis and secretion of SLs, which in turn promotes root growth and development, enhancing their phosphorus absorption capacity. Furthermore, under phosphorus starvation conditions, increased root exudation of SLs promotes AMF hyphal branching and symbiosis with the host, further enhancing phosphorus absorption by the plant.

[0004] The β-carotene isomerase encoded by the D27 gene is one of the key enzymes in the biosynthesis of SLs (stemon-like fibroblasts). In the SL biosynthetic pathway, the D27 gene is located upstream, and its main function is to convert all-trans-β-carotene to 9-cis-β-carotene, providing the necessary substrate for subsequent enzymatic reactions. D27, first cloned from rice, is a novel iron-containing protein. Mutations in the D27 gene lead to reduced SL synthesis, which affects the plant's normal branching inhibition mechanism. Compared to the wild type, the d27 mutant exhibits a multi-branching phenotype with an increased number of branches, indicating that D27 plays an important role in controlling tillering and branching.

[0005] Paulownia tomentosa is one of China's important fast-growing tree species, but it is susceptible to witches' broom disease caused by phytoplasma infection. This disease leads to the proliferation of axillary and adventitious buds, shortened internodes, and broom-like or bird's nest-like branchlets, eventually resulting in death. This disease phenomenon has a complex regulatory mechanism, in which plant hormones auxin and cytokinin play a key role. Studies have shown that SLs, as a new type of plant hormone, can synergistically work with auxin and cytokinin to inhibit the growth of branching and lateral buds, thereby regulating the number of branches in the plant.

[0006] Currently, no research has been found on the synthesis of branching stem cells (SLs) in *Paulownia tomentosa* and the related gene PfD27. The expression of the PfD27 gene in *Paulownia tomentosa* may participate in SL biosynthesis, thereby regulating the number of branches and affecting the overall morphology of the plant. Therefore, constructing a *Paulownia tomentosa* PfD27 expression vector and using this vector to study the function and regulatory mechanism of the *Paulownia tomentosa* SL synthesis gene PfD27 is of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide an SLs biosynthetic gene PfD27, its constructed expression vector, and its applications.

[0008] The technical solution of the present invention is as follows:

[0009] This invention provides a PfD27 gene from Paulownia tomentosa, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2. This invention constructs an expression vector for the PfD27 gene, studies the subcellular localization of the PfD27 protein using this expression vector, expresses and purifies the PfD27 protein, and analyzes the role of the PfD27 gene in plant branching regulation.

[0010] The amino acid sequence of the protein encoded by the white-flowered paulownia PfD27 gene is shown in SEQ ID NO.2.

[0011] The expression vector constructed from the white-flowered paulownia PfD27 gene.

[0012] The application of the white-flowered Paulownia tomentosa PfD27 gene in regulating plant branching.

[0013] In the aforementioned application, the white-flowered paulownia PfD27 gene or a vector expressing the white-flowered paulownia PfD27 gene is transferred into plants for expression, including tobacco, white-flowered paulownia, and tomato.

[0014] Beneficial effects of the present invention

[0015] This invention demonstrated the inhibitory effect of SLs on branching in Paulownia tomentosa seedlings infected with witches' broom disease by treating them with the SLs analog GR24. The gene PfD27, one of the key enzymes in SLs synthesis, was screened out. Through further analysis, expression, and purification, and overexpression of the PfD27 gene in tomato, its function was preliminarily clarified. Details are as follows:

[0016] (1) The β-carotene isomerase gene PfD27 in *Paulownia tomentosa* was identified and analyzed. After clarifying the inhibitory effect of SLs on axillary buds and branches of *Paulownia tomentosa*, the D27 gene sequences of *Arabidopsis thaliana*, rice, and sesame were selected as seed sequences. The candidate sequence of the PfD27 gene was obtained through homology alignment in the *Paulownia tomentosa* genome sequence. Based on this, primers were designed, and the PfD27 gene sequence was cloned. Expression pattern analysis showed that the expression level of the PfD27 gene in roots was significantly higher than in other parts, suggesting that the PfD27 gene may be a key gene regulating SL synthesis in *Paulownia tomentosa*.

[0017] (2) The pSAK277-PfD27-eGFP plasmid was constructed to realize the expression of PfD27-GFP fusion protein in tobacco leaves, and to verify that the PfD27-GFP fusion protein is located on chloroplasts.

[0018] (3) A prokaryotic expression vector was constructed by homologous recombination to induce the expression of PfD27 protein, and PfD27 protein with high purity was obtained.

[0019] (4) This invention verifies that the PfD27 gene participates in the synthesis of SLs and has a branching regulation function. By overexpressing the PfD27 gene in tomatoes, it was found that the number of branches in transgenic tomato plants decreased, the root length increased, and the number of lateral roots decreased; the strigophenol content in transgenic tomato plants was higher than that in wild type, indicating that the PfD27 gene can increase the synthesis of SLs hormones and inhibit the production of lateral branches. Attached Figure Description

[0020] Figure 1 This is the cloning result of the PfD27 gene coding sequence of the present invention;

[0021] In this table, A represents the PCR amplification result of the PfD27 gene coding region; B represents the colony identification result of E. coli transformed from the PfD27 gene coding region; and M represents the 5000bp DNA maker.

[0022] Figure 2 Predicted results of the higher-order structure of PfD27 protein;

[0023] In this diagram, A represents the predicted secondary structure of the PfD27 protein; blue represents α-helices, purple represents extended strands, and yellow represents random coils; B represents the tertiary structure model of the PfD27 protein.

[0024] Figure 3 Comparison of PfD27 gene expression levels in different tissues of healthy and wilted branch diseased Paulownia tomentosa seedlings;

[0025] Figure 4 Subcellular localization of the PfD27 protein in this invention;

[0026] Figure 5 The results of prokaryotic expression and purification of the PfD27 protein of this invention;

[0027] Where A represents the prokaryotic expression of PfD27 protein; 1 represents the bacterial culture before induction; and 2 represents the bacterial culture after induction.

[0028] B represents the purification of PfD27 protein; 1 represents the supernatant after lysis; 2 represents the precipitate after lysis; 3 represents the supernatant after inclusion body refolding; 4 represents the solution after passing through a nickel column; 5 represents the elution solution with 30 mM imidazole; 6 represents the elution solution with 300 mM imidazole; and M represents the 180 KD protein marker.

[0029] Figure 6 Molecular identification results of tomato plants that are positive for the PfD27 gene;

[0030] Among them, A is the PCR detection of the resistance tag hygromycin gene; B is the detection of PfD27 gene amplification; and C is the detection of PfD27 gene expression level.

[0031] Figure 7 The results show the identification and phenotypic determination of genetically modified tomatoes.

[0032] Among them, A is the plant phenotypic observation (front); B is the plant phenotypic observation (top); C is the root phenotypic observation; and D is the detection result of endogenous strigophenol content in transgenic tomatoes. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all materials and reagents used are obtained from commercial sources.

[0034] The plant materials that may be involved in the examples are: healthy white-flowered paulownia (Paulownia fortunei, PF), white-flowered paulownia with witches' broom disease (Phytoplasma-infected P. fortunei, PFI), and Nicotiana benthamiana, all of which were obtained from the Paulownia Biology Laboratory of Henan Agricultural University.

[0035] Example 1. Cloning of the SLs biosynthesis gene PfD27

[0036] (1) The inhibitory effect of SLs analogue GR24 on axillary bud growth in Paulownia tomentosa seedlings with wisteria vine disease

[0037] After treating white-flowered paulownia plants infected with witches' broom disease with the synthetic SLs analog GR24 (Kulaibo Technology) at a concentration of 15 μM for 20 days, 15 plants with basically the same growth were selected from each group, and the number of axillary buds and lateral branches was counted.

[0038] Count the number of axillary buds for each plant; axillary buds that develop into branches (stems containing buds and leaves) are called lateral branches. Lateral branches growing from the stem of the paulownia are called first-order lateral branches, lateral branches sprouting from the lateral branches are called second-order lateral branches, and so on. Count the number of branches at different levels and calculate the average.

[0039] The statistical results are shown in Table 1. The number of axillary buds decreased from 42 to 2 after GR24 treatment, significantly fewer than in diseased seedlings; the number of secondary branches decreased from 42 to 4, and the treated seedlings with swollen branches did not produce tertiary branches. These results indicate that exogenous GR24 treatment can slow down the growth rate and number of axillary buds, and SLs have a significant inhibitory effect on the lateral branches of Paulownia tomentosa. It is preliminarily determined that SLs participate in the branching development of Paulownia tomentosa.

[0040] Table 1. Number of axillary buds and branches of Paulownia tomentosa before and after SLs treatment.

[0041]

[0042] (2) Obtaining the candidate sequence of the PfD27 gene

[0043] Using the sequences of the Arabidopsis thaliana AtD27 gene (AT1G03055), rice OsD27 gene (Os11g0587000), and sesame SiD27 gene (SIN_1012364) as seed sequences, the whole genome sequence of Paulownia tomentosa was compared, yielding the candidate sequence Pfo05g003890 for the Paulownia tomentosa PfD27 gene. This gene sequence showed 84%, 55%, and 60% identity with sesame, Arabidopsis thaliana, and rice, respectively, and 92%, 75%, and 78% similarity, respectively.

[0044] (3) Extraction of total RNA from Paulownia tomentosa

[0045] Following the instructions of the TIANGEN Total RNA Extraction Kit, RNA was extracted from the roots, stems, leaves, and terminal buds of healthy *Paulownia tomentosa* seedlings and seedlings suffering from wilting disease. Agarose gel electrophoresis and integrity testing showed that the extracted RNA was structurally intact and undegraded, suitable for subsequent reverse transcription.

[0046] (4) Preparation of cDNA from Paulownia tomentosa

[0047] The extracted RNA from *Paulownia tomentosa* was reverse transcribed using the PrimerScript™ RT regent kit (TaKaRa), following the instructions. The cDNA was identified by agarose gel electrophoresis, indicating good quality, and was used for subsequent experiments.

[0048] (5) Cloning of the PfD27 coding region

[0049] ① Primer design: Based on the CDS sequence of the PfD27 gene in the genome of Paulownia tomentosa, specific primers D27-F / R were designed using Primer Primer 5. The specific sequences are shown in the table. The primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. (hereinafter referred to as Qingke Biotechnology).

[0050] Table 2 Primer sequences

[0051]

[0052] ② Fragment amplification: Using cDNA obtained from the stem of white-flowered paulownia obtained by reverse transcription as a template, the PfD27 gene was amplified using D27-F / R primers. The PCR reaction system is shown in Table 3.

[0053] Table 3 PCR reaction system

[0054]

[0055] Amplification program: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 55℃ annealing for 5s, 72℃ extension for 5s (35 cycles of denaturation, annealing, and extension); 72℃ final extension for 1min.

[0056] ③ Gel electrophoresis: The PCR amplification products were separated into target bands by 1% agarose gel electrophoresis, and the results showed that the positions were correct. Figure 1 A) The DNA was recovered using an agarose gel DNA recovery kit (TIANGEN). The specific operating steps are as per the instructions.

[0057] ④ Ligation reaction: Using the Clone JET PCR cloning kit, follow the instructions to ligate an appropriate amount of the recovered product into the PJET1.2 cloning vector. Ligation was carried out at 22℃ for 30 min to obtain the recombinant vector PJET1.2-D27. The reaction system is shown in Table 4.

[0058] Table 4 Connection System

[0059]

[0060] ⑤ Transformation reaction: Remove DH5α competent cells from the ultra-low temperature freezer, thaw them on ice, and gently place 50 μL of competent cells into the bottom of a 2 mL EP tube. Add 5 μL of ligation product to the competent cells, gently stir the bottom of the tube, incubate on ice for 30 min, heat shock at 42℃ for 45 s, immediately insert into ice and incubate for 2 min, add 500 μL of sterile LB medium (antibiotic-free) to the EP tube, and culture at 37℃ and 220 rpm for 1 h with shaking. Take 30 μL of competent cells, spread them evenly on LB solid medium (containing 100 mg / L ampicillin), and incubate upside down for 12–16 h.

[0061] ⑥ Colony identification: Six single colonies were randomly selected and cultured in 1 mL LB liquid medium (containing 100 mg / L ampicillin) for 4 h. Then, 4 μL of the bacterial culture was used as a template for colony PCR reaction. The reaction system is shown in Table 5.

[0062] Table 5 Colony PCR Reaction System

[0063]

[0064] Reaction program: 94℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 68℃ annealing for 20 s, 72℃ extension for 1 min (35 cycles of denaturation, annealing, and extension); 72℃ final extension for 5 min.

[0065] Figure 1 B represents the colony identification results of E. coli transformed with the PfD27 gene coding region; positive clones were selected and sent to Qingke Biotechnology for sequencing, and the length of the PfD27 gene was found to be 762 bp, located on chromosome 5 of Paulownia tomentosa. The sequence of this gene is 90% identical to the genome sequence of Paulownia tomentosa.

[0066] Figure 1 In B, M is a 5000bp DNA maker, and 1-6 represent different colonies.

[0067] Example 2. Preliminary analysis of the role of the PfD27 gene in SLs biosynthesis

[0068] (1) Prediction of the physicochemical properties of the protein encoded by the PfD27 gene

[0069] The PfD27 protein consists of 253 amino acids with a theoretical molecular weight of 28.9 kDa and an isoelectric point of 9.04. Its instability coefficient is 77.00, indicating a relatively unstable structure. Its hydrophilicity / hydrophobicity ratio is -0.317, classifying it as a hydrophilic protein, while its isoelectric point is greater than 7, classifying it as a basic protein. Analysis of the conserved domains of the PfD27 protein from the NCBI Conserved Domain Database (CDD) reveals the presence of a D27-Like-C domain (DUF4033). Secondary structure prediction using the SOPMA online website indicates that random coils account for approximately 60% of the protein, while α-helices account for approximately 30%. Further structural prediction of the PfD27 protein was performed using the online tool Swiss Model.

[0070] See results Figure 2 , Figure 2 A: Predicted secondary structure of PfD27 protein; blue represents α-helix, purple represents extended strand, and yellow represents random coil; Figure 2 B: Tertiary structure model of PfD27 protein.

[0071] (2) Tissue expression pattern analysis of PfD27 gene

[0072] To analyze the role of the PfD27 gene in the growth and development of Paulownia tomentosa, its expression level in various tissues of healthy Paulownia tomentosa seedlings and seedlings with wilting disease was analyzed by quantitative real-time PCR.

[0073] Based on the CDS sequence of the cloned PfD27 gene, quantitative PCR primer sequences were designed using NCBI-Primier BLAST (see table for details). The PCR reaction program was 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing / extension for 30 s, for a total of 40 cycles; the melting curve was set according to the instrument's default settings. Each sample contained three biological replicates, using qrt-PfGadph-R / F as the internal control primer, referring to step 2. -ΔΔCt Relative expression levels were calculated using Excel 2010, and the experimental results were visualized and significance analysis was performed using Graph Pad 8.0.

[0074] Using the cDNA obtained from the above reverse transcription as a template, a real-time PCR experiment was performed using a dye method. The amplification system is as follows:

[0075] Table 6. Quantitative PCR Amplification System

[0076]

[0077] Quantitative real-time PCR results showed that the expression level of the PfD27 gene in roots was significantly higher than in other parts, exhibiting obvious tissue specificity. The expression level of the PfD27 gene in the roots and stems of healthy seedlings was generally higher than that in seedlings with wilting disease, indicating that it is involved in the formation of multibranching symptoms in *Paulownia tomentosa*. Figure 3 ).

[0078] Figure 3 In the table, PF represents healthy Paulownia tomentosa seedlings, PFI represents Paulownia tomentosa seedlings with wilting disease; error bars represent the average of three biological replicates, * represents the significance level, and the calculation method is multiple t-test.

[0079] Based on the above analysis results, it is inferred that the PfD27 gene plays an important role in the synthesis of white-flowered Paulownia tomentosa SLs, and it has been decided to conduct further research on it.

[0080] The nucleotide sequence of the PfD27 gene of Paulownia tomentosa is (SEQ ID NO.1):

[0081] ATGGATGCAACGCTCGTGCAGCCATACAAGAGGCTATTTCTGCCACCGCGAATCAACCGGAGAGTTACCGTGAAAAATCATAGACGCTCTCCCTTCATTCTGTCGGTGCTCACAGACAACATTTCAGATACATCGTCCCCTCATGAACCCAAAAATGTTTACAAAGACAACTGGTTTGATCGTCTCATCATTAACCATGTCTCCAACAGCTTACAAACTTCAACAGGGCTGAGAAGCAAAAAGAGTGGGTATGACGGGATGGTGGAAGCAGCAACAGTGGCGTCTCGTCACTTCACTCCAACCCAACAAAGAGAACTTGTTCTTGAAACTCTTGATAGGGCCTTTCCTAGGCCTATAGTTAACCTGATCAAGACACTGCTGCCACAATCTAAATTTGCAAGAGAATACTACGCCGCCTTCACAAAAATTTGCTTAGGTTGGCTGGTTGGTCCGTCCGAGGTAATAGAGTCAGAGTTCGAAGGAAGAAAAGAGAAAAATGTCGTGCATATACAAAAATGCAGGTTTTTGGAGGAGGCAAATTGTGTAGGAATGTGCACTAATCTTTGCAAGATGCCGTCTCAAGTGTTCATTAAGGAGGCTTTTGGAATGCCAGTCAATATGGTCCCCAATTTTGATGATATGAGCTGTGTAGTGACATTTGGTGAGGAGCCTCTACCACCATCTTTAGATCCAGCATTCACACAGCCATGTTACAAGCAATGCAAGGCCACCCAAAGACACCAGAAGAACTGCACAAACTAA

[0082] The amino acid sequence of the PfD27 gene-encoded protein of Paulownia fortunei (SEQ ID NO.2) is as follows:

[0083] MDATLVQPYKRLFLPPRINRRVTVKNHRRSPFILSVLTDNISDTSSPHEPKNVYKDNWFDRLIINHVSNSLQTSTGLRSKKSGYDGMVEAATVASRHFTPTQQRELVLETLDRAFPRPIVNLIKTLL PQSKFAREYYAAFTKICLGWLVGPSEVIESEFEGRKEKNVVHIQKCRFLEEANCVGMCTNLCKMPSQVFIKEAFGMPVNMVPNFDDMSCVVTFGEEPLPPSLDPAFTQPCYKQCKATQRHQKNCTN*

[0084] Example 3. Subcellular localization analysis of PfD27 protein

[0085] To determine the specific location of the PfD27 protein in cells, a recombinant vector expressing the PfD27 protein was constructed. This recombinant vector was introduced into tobacco leaf cells via Agrobacterium transformation for transient expression. After 72 hours, the expression of green fluorescent protein and the PfD27 fusion protein was observed to determine the location of the PfD27 protein within the cells. The specific steps are as follows:

[0086] (1) Construction of subcellular localization vectors

[0087] To obtain the recombinant vector, the pSAK277-eGFP vector was first digested with the restriction endonuclease EcoRI to obtain a linearized vector. Then, based on the pSAK277-eGFP vector sequence and the PfD27 gene sequence, homologous recombination primers pSAK277-PfD27-F / R, with EcoRI as the restriction site and without a stop codon, were designed using Primer 5.0 software. The primer sequences are shown in the table. Using these primers and the PJET1.2-D27 plasmid as a template, the target fragment was amplified by PCR. The target fragment was ligated to the linearized vector using the CE II one-step cloning kit (Novizan) to obtain the recombinant vector pSAK277-PfD27-eGFP. The transformation and colony identification of the ligation product were performed as in Example 1.

[0088] (2) Agrobacterium transformation and positive clone screening

[0089] Take 50 μL of GV3101 (pSoup-p19) competent cells into a sterile 1.5 mL EP tube, add 500 ng of plasmid pSAK277-PfD27-eGFP, gently tap the tube wall to mix, and incubate on ice for 30 min. Quickly transfer the EP tube to liquid nitrogen for 5 min, and immediately transfer it to a 37°C water bath for heat shock for 5 min. After heat shock, quickly transfer it back to ice and incubate for 5 min. Add 300 μL of antibiotic-free YEP liquid medium to the EP tube and place it in a shaker at 28°C for 4 h at 220 rpm. Take 30 μL of the transformed bacterial culture and spread it evenly on YEP solid medium containing antibiotics (50 μg / mL spectinomycin and 50 μg / mL rifampin) using a sterile spreader. Incubate at 28°C for 2–3 days until clear single colonies appear. Single colonies with good growth were randomly selected for colony PCR identification, and positive colonies were stored at -80°C. The colony identification procedure was the same as in Example 1.

[0090] (3) Transient transformation and subcellular localization analysis of tobacco

[0091] Eight-week-old Nicotiana benthamiana seedlings were selected as experimental materials. The tobacco plants were water-controlled the day before the experiment, and watering was resumed 1 hour before the experiment to allow the leaves to fully absorb water, which would facilitate Agrobacterium infection.

[0092] Agrobacterium tumefaciens culture containing the target plasmid pSAK277-PfD27-eGFP or the control plasmid pSAK277-eGFP was cultured to OD. 600 =0.8, then centrifuge at 3000 rpm at room temperature for 10 minutes to collect the bacterial cells, discard the supernatant, and resuspend the bacterial cells using resuspension solution. The resuspension solution formula is shown in Table 7.

[0093] Adjust the concentration of the resuspended bacterial culture to OD. 600 =0.6, and let the bacterial suspension stand in the dark for 2-3 hours. Then, slowly inject the bacterial suspension into the lower epidermal intercellular space of the tobacco leaves, ensuring that the bacterial solution is evenly distributed throughout the entire leaf, and mark it. After injection, water the tobacco thoroughly and incubate in the dark for 24 hours, after which transfer the plants to normal light conditions for continued cultivation.

[0094] The expression and localization of the PfD27-GFP fusion protein in tobacco leaves were observed using laser confocal microscopy within 60–72 hours post-injection. Agrobacterium tumefaciens culture containing the pSAK277-eGFP plasmid was used as a control group for synchronous analysis.

[0095] The results showed that the PfD27-eGFP fusion protein was successfully expressed in leaf cells, emitting clear green fluorescence. Red autofluorescence of chloroplasts was also observed in the Cy5 chloroplast channel. In the fluorescence superposition field, the green fluorescence (PfD27-eGFP) and red fluorescence (chloroplasts) overlapped, indicating that the PfD27 protein is localized in the chloroplasts. In the control group, free eGFP showed a diffuse distribution in the cytoplasm and nucleus. Figure 4 ).

[0096] Figure 4 In the study, pSAK277-eGFP served as a control, containing an empty vector without the target gene fusion, while pSAK277-PfD27-eGFP was a recombinant vector fused with the protein encoded by the PfD27 gene.

[0097] Table 7 Resuspension Formulation

[0098]

[0099] Example 4. Prokaryotic expression and purification of PfD27 protein

[0100] To confirm that the PfD27 gene coding region can express the complete protein, prokaryotic expression of the PfD27 protein was performed in the E. coli system, and the expressed protein was purified.

[0101] (1) Construction of prokaryotic expression vector

[0102] ① Extraction of PJET1.2-PfD27 recombinant plasmid: The PJET1.2-PfD27 recombinant plasmid was extracted using a plasmid miniprep kit (TIANGEN) according to the instructions. The plasmid concentration and purity were detected using NanoDrop 2000.

[0103] ② Vector linearization and purification: The pET-28a(+)-SUMO empty vector was digested with the restriction endonuclease XhoI. The digestion system is shown in Table 8. Reaction conditions: incubation at 37℃ for 1 h. After digestion, 5 μL of the digestion product was analyzed by 1% agarose gel electrophoresis to confirm whether the digestion product was the expected linearized vector. After confirmation, the remaining digestion product was purified and recovered using a DNA purification kit (TIANGEN). The purification steps were the same as in Example 1.

[0104] Table 8 Enzyme digestion system

[0105]

[0106] ③ PfD27 gene amplification: Using the PJET1.2-PfD27 recombinant plasmid as a template, PCR amplification was performed using primers pET28a-SUMO-PfD27-F / R (Table 2) and a high-fidelity enzyme (Novizan).

[0107] The product was analyzed by 1% agarose gel electrophoresis to confirm the amplification of a single target band (PfD27 gene fragment) of the expected size. The target band was then purified and recovered using a DNA purification kit (TIANGEN), following the same procedures as in Example 1.

[0108] ④ Homologous recombination: Using a seamless cloning kit (Novizan), the purified PfD27 gene fragment was ligated into the linear pET-28a(+)-SUMO vector to obtain the recombinant plasmid pET-28a(+)-SUMO-PfD27. The reaction system is shown in Table 9, and the reaction conditions were: 37℃, 30 min.

[0109] Table 9 Homologous recombination reaction system

[0110]

[0111] ⑤ Transformation and identification of positive clones: 10 μL of the homologous recombination reaction product was transformed into *E. coli* BL21(DE3) cells (Full Gold) using the heat shock method. After transformation, the cells were evenly spread on LB agar (containing 50 μg / mL kanamycin) and incubated at 37°C for 12–16 h. Single colonies with good growth were randomly selected for colony identification, following the same steps as in Example 1. Clones containing the correct sequence were selected for subsequent protein expression induction experiments.

[0112] (2) Induced expression of PfD27 protein

[0113] ① Activation: 50 μL of Escherichia coli BL21(DE3) bacterial suspension containing recombinant plasmid pET-28a(+)-SUMO-PfD27 was inoculated into 10 mL of LB medium containing kanamycin (50 μg / mL) and cultured at 37℃ and 220 rpm for 4–6 h with shaking.

[0114] ② Inoculation: Take an appropriate amount of activated bacterial solution and inoculate it into 1L of LB medium containing kanamycin (50μg / mL). Incubate at 37℃ and 180rpm for 4 hours.

[0115] ③Induction: Add 250 μL of 1 mol / L IPTG, incubate at 25℃ and 180 rpm for 16 h with shaking.

[0116] ④ Collection of bacteria: Transfer the induction product to a 500mL centrifuge bottle, centrifuge at 8000rpm and 4℃ for 13min, and retain the precipitate.

[0117] ⑤ Disruption: Add PB buffer (1g precipitate to 10mL PB) to the bacterial pellet, add 400μL of 1% nuclease solution, and stir at room temperature for 10min. Disrupt the cells using an ultrasonic cell disruptor (Xinzhi) for 2min. After disruption, centrifuge the sample at 9000rpm for 30min at 4℃, and collect the supernatant and pellet for subsequent experiments.

[0118] The supernatant and precipitate were separately analyzed for protein expression using SDS-PAGE. The results showed a protein band at approximately 50 kDa in the lane corresponding to the precipitate. Figure 5 A) Almost no obvious bands were observed at the corresponding positions in the supernatant sample.

[0119] This result indicates that PfD27 protein expression was successfully induced. The recombinant protein was mainly present in the precipitate, suggesting that it may exist in the form of inclusion bodies.

[0120] The inclusion bodies were then dissolved and refolded, followed by elution and purification. The specific steps are as follows:

[0121] ① Dissolving inclusion bodies: Add dissolving buffer (20mM phosphate buffer, pH 8.0, 0.5M NaCl, 8M urea) to the collected precipitate containing inclusion bodies, and stir overnight (12 hours) at room temperature until the solution becomes clear. Centrifuge the solution at 9500 rpm for 30 minutes at 4°C, collect the supernatant, and discard the insoluble precipitate.

[0122] ② Protein refolding (chromatographic method): Filter the supernatant through a 0.8 μm sterile filter membrane to remove undissolved particles. Load the filtered supernatant onto a pre-equilibrated Ni-NTA affinity chromatography column, and then equilibrate the column sequentially with phosphate buffer (20 mM phosphate buffer, pH 8.0, 0.5 M NaCl) containing 8 M, 6 M, 4 M, 2 M, 1 M, and 0 M urea. After column equilibration with the 0 M urea gradient, wash away impurities with buffer containing 30 mM imidazole, and finally elute the target protein with buffer containing 300 mM imidazole, collecting the eluent.

[0123] A small amount of the eluted fraction was analyzed by SDS-PAGE. The results showed a single, clear main band at the expected size (approximately 50 kDa), indicating that the high-purity, renatured PfD27-SUMO fusion protein was successfully obtained. Figure 5 B).

[0124] Example 5. Effects of PfD27 gene overexpression on tomato branching

[0125] (1) Preparation of sterile tomato seedlings and explants

[0126] First, the tomato seeds were pretreated. The fuzz on the seed surface was gently rubbed off, and the seeds were rinsed three times with distilled water. The seeds were then surface-sterilized in 75% alcohol for 30-60 seconds, followed by rinsing three times with sterile distilled water; then sterilized with 10% (v / v) NaClO solution for 10 minutes, and finally rinsed three times with sterile distilled water. After the above treatment, the seeds were sown on 1 / 2 MS solid medium. The sown seeds were cultured in the dark for 3 days, followed by light culture for 4 days. When the plants reached 8-10 cm in height, hypocotyls approximately 1.0 cm long were cut from the sterile seedlings and placed in pre-medium (MS basal medium + 1.0 mg / L 6-BA + 0.3 mg / L IAA) for 2 days in the dark, in preparation for subsequent transformation experiments.

[0127] (2) Construction and transformation of PfD27 gene overexpression vector

[0128] Using Primer Primer 5 software, homologous recombination primers pCAMBIA-PfD27-F / R (primer sequences are shown in Table 2) with EcoRI as the single restriction enzyme site and excluding the stop codon were designed. Using these primers, and with the recombinant plasmid PJET1.2-PfD27 extracted in Example 4 as a template, the recombinant plasmid pCAMBIA3301-PfD27 was obtained by PCR amplification and product ligation. The ligation, transformation, and colony identification steps were the same as in Example 1.

[0129] Subsequently, Agrobacterium transformation and colony identification were performed. The correctly sequenced pCAMBIA3301-PfD27 plasmid was transformed into Agrobacterium EHA105 competent cells (Weidi Bio). The operation steps are as follows: 2 μL of recombinant plasmid and 20 μL of competent cells were mixed and incubated on ice for 30 min; flash-frozen in liquid nitrogen for 5 min; incubated in water at 37℃ for 5 min, and then quickly incubated on ice for 5 min; 140 μL of antibiotic-free YEP liquid medium was added, and the cells were cultured at 28℃ and 200-220 rpm for 2-3 h with shaking; an appropriate amount of bacterial cells were evenly spread on YEP solid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated upside down at 28℃ for 2-3 days. Single colonies were picked for identification, and the specific method was the same as in Example 1.

[0130] Finally, the recombinant plasmid pCAMBIA3301-PfD27 was successfully constructed and transformed into Agrobacterium strain EHA105 to obtain an Agrobacterium strain containing the target gene PfD27 for subsequent experiments.

[0131] (3) Molecular identification of positive PfD27 transgenic tomato plants

[0132] Agrobacterium strains containing the pCAMBIA3301-PfD27 plasmid were activated on YEP solid medium and then subjected to colony PCR detection. Positive colonies were placed in 5 mL of YEP liquid medium and incubated at 220 rpm and 28°C with shaking for 16 h until OD600 ≈ 1.0. 5 mL of the bacterial culture was transferred to 100 mL of YEP liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated at 28°C with shaking for 6 h until OD600 ≈ 0.6. The cells were then collected by centrifugation at 4000 rpm. The Agrobacterium cells were resuspended in MS liquid medium to prepare recombinant bacterial culture.

[0133] Tomato hypocotyls cultured in the dark were co-cultured with a prepared Agrobacterium-infected bacterial solution (OD600 value 0.6-0.8) for 15 min, after which the inoculum was discarded. Explants were removed, and any remaining bacterial solution on the surface was carefully blotted dry with filter paper. The explants were then transferred to MS medium lined with filter paper. Afterward, they were incubated in the dark at 25°C for 48 h. The explants were then transferred to MS selection medium (composition: 6-BA 1.0 mg / L, IAA 0.3 mg / L, kanamycin 80 mg / L, termethin 100 mg / L) and cultured for 60 days, changing the medium every 15 days to induce shoot emergence.

[0134] After the regenerated shoots are induced, they are inoculated onto 1 / 2 MS rooting medium (containing IAA 0.6 mg·L⁻¹, Kan 80 mg·L⁻¹, and Tm 100 mg·L⁻¹) to induce rooting. Roots are induced in about 30 days. Then, the vent membrane of the culture bottle is opened, sterile water is added to the medium, and the tissue culture seedlings are transferred to the cultivation substrate (humus and vermiculite mixed in a 1:1 ratio), and the vent bag is attached. The vent bag is removed after the tissue culture seedlings have fully adapted to the external environment.

[0135] Tomato plants transfected with the PfD27 gene were hardened off and transplanted. Seven days after transplanting, identification was performed at the DNA and RNA levels. The specific steps are as follows:

[0136] ① DNA level identification: DNA was extracted from tomato plant leaves using a plant DNA extraction kit (TIANGEN). Untransformed wild-type (WT) tomatoes were selected as negative controls, and recombinant plasmids were used as positive controls. The extracted DNA was identified by PCR using primers consisting of HYG-F / R (sequences shown in Table 2). At the same time, the PfD27 gene was amplified using primers D27-F / R (Table 2) to preliminarily determine whether the exogenous gene had been integrated into the tomato genome.

[0137] ②Detection of PfD27 gene expression level: Total RNA was extracted from tomato leaves, roots, and stems using a plant total RNA extraction kit (TIANGEN). The expression level was then analyzed using the PrimerScript kit.TM Reverse transcription was performed using the RT regent kit (TaKaRa). Using cDNA as a template, the expression level of the PfD27 gene was detected by qRT-PCR. The primers used are listed in Table 2: qrt-PfD27-F / R. Wild-type tomato was used as a control, and the tomato Slactin gene was used as an internal control. The Slactin F / R primer pair was used as the internal control primer. Each sample was tested in triplicate. (Refer to Table 2) -ΔΔCt Relative expression levels were calculated using Excel 2010, and the experimental results were visualized and significance analysis was performed using Graph Pad 8.0.

[0138] The results of resistance gene testing showed that, compared with the wild type, positive plants tested positive for the hygromycin resistance gene ( Figure 6 A); Amplification of the PfD27 gene revealed a band at approximately 750 bp in positive plants, consistent with the expected sequence. Sequencing results showed this band was also consistent with the original sequence. A fragment of approximately 300 bp was amplified in both wild-type and transgenic tomato plants, suggesting this might be a homologous gene in tomatoes. Figure 6 B).

[0139] Gene expression analysis showed that in the overexpressing tomato lines, the expression level of the PfD27 gene was significantly higher than that of the wild type. Figure 6 C). This indicates that the PfD27 gene has been integrated into the tomato genome and has been successfully overexpressed.

[0140] (4) Phenotypic identification and SLs detection of PfD27 gene-transgenic tomato plants that are positive

[0141] After hardening off, the positive overexpression lines were transplanted into flowerpots. After 30 days of growth, the number of axillary bud branches, root morphology, and SLs content of the transgenic tomato plants were measured. The specific steps are as follows:

[0142] ① Branching count: T0 generation transgenic positive plants (OE1, OE2, OE3) and wild-type (WT) tomatoes were transplanted to grow under uniform conditions. To evaluate the branching characteristics of transgenic and wild-type tomatoes, the number of lateral branches and axillary buds in both groups were counted (15 plants per group). Four weeks after transplanting, the aboveground and underground phenotypes of the transgenic tomato lines and wild-type tomatoes were observed, and the number of branches and axillary buds was counted.

[0143] Observations revealed that, starting from the third node from the stem base, the transgenic lines did not exhibit axillary bud growth, while wild-type tomatoes generally did. (See results below.) Figure 7Images A and B show that the average number of branches in the transgenic tomato lines was 5.82, while the average number of branches in the wild-type tomatoes was 7.58. The transgenic plants had significantly fewer branches than the wild-type. This indicates that overexpression of the PfD27 gene inhibits axillary bud germination and branching in tomatoes.

[0144] ② Root morphology observation: The root morphology of transgenic tomatoes and wild-type tomatoes was observed separately, and the morphological differences between the two groups of roots were compared. Results are shown below. Figure 7 C shows that, compared to the wild type, the transgenic tomato plants exhibit longer taproots and fewer lateral roots.

[0145] ③ Determination of endogenous SLs content

[0146] Stem tissues (approximately 0.5 g FW) from 4-week-old transgenic lines (OE1, OE2, OE3) and wild-type (WT) tomatoes were flash-frozen in liquid nitrogen and stored at -80°C. The content of endogenous SLs in the overexpressing tomato lines was determined by liquid chromatography-mass spectrometry, as follows:

[0147] a. Preparation of standard solutions: Strigol (CAS: 51820-11-2) standard was prepared into a stock solution of 5 μg / mL with methanol. The stock solution was then diluted with methanol to prepare standard curves with final concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL and 100 ng / mL.

[0148] b. Sample extraction: Weigh 0.5g of fresh tomato stem sample into a 5mL grinding tube, quick-freeze in liquid nitrogen, grind into powder using a refrigerated centrifuge, add 5mL of acetonitrile, and mix well; extract overnight at 4℃, centrifuge at 12,000rpm for 5min at 4℃, and collect the supernatant; extract the precipitate with 5mL of acetonitrile, and combine the two supernatants; add 200mg of C18 packing material, shake vigorously for 30s, centrifuge at 12,000rpm for 5min, and collect the supernatant; concentrate the sample to dryness using a vacuum concentrator, redissolve in 150μL of methanol, filter through a 0.22μm organic phase membrane, and analyze using an instrument.

[0149] Chromatographic conditions: Column: Agilent Poroshell 120SB-C18 (2.1×150mm, 2.7μm); Column temperature: 30℃; Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile; Flow rate: 0.3mL / min; Injection volume: 2μL; Gradient elution.

[0150] Mass spectrometry conditions: Scan type: MRM; Curtain gas: 25 psi; Spray voltage: +5000 V; Nebulizer gas pressure: 55 psi; Auxiliary gas pressure: 65 psi; Nebulization temperature: 350 °C.

[0151] The results show ( Figure 7 D) Compared with the wild type, the content of strigophenol in the stem tissue of transgenic lines (OE1, OE2, OE3) was significantly increased, with an average increase of about 50%. This indicates that the PfD27 protein plays the role of β-carotene isomerase in tomato and participates in regulating the initiation step of the biosynthesis of SLs (especially strigophenol). Its overexpression can increase the endogenous strigophenol level in tomato, thereby inhibiting axillary bud germination and reducing branching.

[0152] Figure 7 In the text, WT represents the wild type, and OE1, OE2, and OE3 represent three transgenic lines, respectively.

Claims

1. A PfD27 gene for white-flowered Paulownia tomentosa, characterized by: The nucleotide sequence of this gene is shown in SEQ ID NO.

1.

2. A protein encoded by the PfD27 gene of Paulownia tomentosa as described in claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. An expression vector constructed from the PfD27 gene of Paulownia tomentosa as described in claim 1.

4. The application of the white-flowered paulownia PfD27 gene as described in claim 1 in regulating plant branching.

5. The application according to claim 4, characterized in that, The PfD27 gene of Paulownia tomentosa or a vector expressing the PfD27 gene of Paulownia tomentosa can be transferred into plants for expression.

6. The application according to claim 4, characterized in that, The plants mentioned include tobacco, white-flowered paulownia, and tomato.

Images