Application of QmWOX4 gene in improving radial growth of arabidopsis

Abstract

The application discloses application of a QmWOX4 gene in improving radial growth of Arabidopsis thaliana and belongs to the field of plant genetic engineering.The application discloses application of the QmWOX4 gene or a protein coded by the QmWOX4 gene in improving radial growth of Arabidopsis thaliana, wherein the sequence of the QmWOX4 gene is shown as SEQ ID NO:1, and the sequence of the protein coded by the QmWOX4 gene is shown as SEQ ID NO:2.It is found by the application that the QmWOX4 gene of Quercus mongolica controls radial growth of Arabidopsis thaliana, and the transgenic Arabidopsis thaliana containing the QmWOX4 gene obtained by the application has significantly improved radial growth, which is helpful to improve secondary growth of Arabidopsis thaliana, provides a reference for radial thickening and resistance enhancement of Arabidopsis thaliana, and has important scientific significance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving the application of the QmWOX4 gene or the protein encoded by the QmWOX4 gene in improving the radial growth of Arabidopsis thaliana. Background Technology

[0002] Vascular tissue, through continuous secondary growth, leads to radial thickening of plant organs and is crucial for various aspects of plant growth and physiology, such as water transport capacity and resistance to mechanical stress. It is driven by the vascular cambium, producing inward secondary xylem and outward secondary phloem. The herbaceous plant Arabidopsis thaliana exhibits weak growth, insufficient secondary stem growth, and an inability to grow upright after bolting, resulting in poor resistance.

[0003] The WUSCHEL-related homeobox (WOX) family is conserved and contains a ZIP-HD domain. This domain contains specific spatial structures that can bind to specific DNA sequences, encoding genes that play prominent biological roles in plant growth and development, such as organogenesis, embryonic stem cell morphogenesis and maintenance. Currently, 15 WOX genes have been identified in Arabidopsis thaliana, named WUS, WOX1-14. Among them, the WOX4 gene plays an important role in primary growth, secondary growth, and root development.

[0004] With the continuous development of molecular biology, the use of increasingly mature genetic engineering technology to cultivate new crop varieties with superior traits has become one of the important means of modern crop improvement. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. To this end, embodiments of this invention propose the application of the QmWOX4 gene or a protein encoded by the QmWOX4 gene in enhancing radial growth in Arabidopsis thaliana.

[0006] This invention provides the application of the QmWOX4 gene or the protein encoded by the QmWOX4 gene in improving the radial growth of Arabidopsis thaliana. The sequence of the QmWOX4 gene is shown in SEQ ID NO: 1, and the sequence of the protein encoded by the QmWOX4 gene is shown in SEQ ID NO: 2.

[0007] atgggaagcatgaaggtgcatcagttcgcacgtggattctgggagcacgaaccatccctcacgcttggctgcaagcgcttacgccctcttgctcccaagctggcc

[0008] aacacagacacttctggtgtcactgctttcgacctcaagagcttcatcagacctgaaagtggacccagaaaactcggttcctctgaagacaaagacaagagagatc

[0009] cacctcaggtggaaacacacccaggagggacacgatggaatccaacacaagaacaaatagggatattggagatgctgtataggggaggaatgcgaactccta

[0010] atgcacagcagatagaacaaatcactgcacagcttgggaagtacggcaaaatcgaagggaagaatgtgttttattggttccaaaatcacaaagcgcgcgagagg

[0011] cagaagcagaagcgcaacagtcttggtcttagtcattgtccaagaaccccgactgccattaccaccattactttggactccaggggggaattggaaagagaggaa

[0012] gatagtccatacaaaagaaagtgcaggagctggggatttgaaaccttagaagaagaagaagaagaaagaagatcatgtaaagaggagggagatagaactctagagctgttcccattacatccggaaagcagatga(SEQ IDNO:1)

[0013] MGSMKVHQFARGFWEHEPSLTLGCKRLRPLAPKLANTDTSGVTAFDLKSFIRPESGPRKLGSSEDKDK

[0014] RDPPQVETHPGGTRWNPTQEQIGILEMLYRGGMRTPNAQQIEQITAQLGKYGKIEGKNVFYWFQNHKA

[0015] RERQKQKRNSLGLSHCPRTPTAITTITLDSRGELEREEDSPYKRKCRSWGFETLEEEEEERRSCKEEGDRTLELFPLHPESR(SEQ ID NO:2)

[0016] In some embodiments, the application is to enhance the radial growth of Arabidopsis thaliana by overexpressing the QmWOX4 gene.

[0017] In some embodiments, the Arabidopsis thaliana is wild-type Arabidopsis thaliana or a wox4 deletion mutant of Arabidopsis thaliana.

[0018] This invention also provides an application of a recombinant vector containing the above-mentioned QmWOX4 gene in improving the radial growth of Arabidopsis thaliana.

[0019] In some embodiments, the recombinant vector is the QmWOX4 gene recombined into the expression vector pRI101.

[0020] This invention also provides an application of recombinant bacteria containing the above-mentioned QmWOX4 gene in improving the radial growth of Arabidopsis thaliana.

[0021] In some embodiments, the recombinant bacteria are obtained by recombining the QmWOX4 gene into the expression vector pRI101 to construct a recombinant vector, and then transforming Agrobacterium GV3101 competent cells.

[0022] This invention also provides a method for cultivating transgenic Arabidopsis thaliana, comprising the following steps:

[0023] Recombinant bacteria containing the QmWOX4 gene were used to infect inflorescences of wild-type Arabidopsis thaliana or Arabidopsis thaliana wox4 deletion mutants, in an environment containing 50 mg·L⁻¹ of [unclear text - likely a specific antibiotic or treatment]. -1 On Kan's 1 / 2 MS medium, positive transgenic plants of generation T1 were screened to obtain T1 generation transgenic Arabidopsis overexpression plants and T1 generation transgenic Arabidopsis functional restorer plants. After two generations of culture and screening, homozygous T3 generation Arabidopsis seeds were obtained from the T1 generation transgenic lines. The T3 generation transgenic Arabidopsis seeds were directly sown and grew normally to obtain transgenic Arabidopsis.

[0024] In some embodiments, the recombinant bacteria containing the QmWOX4 gene are obtained by recombining the QmWOX4 gene into the expression vector pRI101 to construct a recombinant vector, and then transforming Agrobacterium GV3101 competent cells to obtain the recombinant bacteria.

[0025] Advantages and beneficial effects of the present invention.

[0026] Radial growth is a developmental characteristic of great interest to plants. This invention discovered that the QmWOX4 gene of Mongolian oak regulates radial growth in Arabidopsis thaliana. The transgenic Arabidopsis thaliana containing the QmWOX4 gene obtained in this invention showed significantly enhanced radial growth, which helps to improve the secondary growth of Arabidopsis thaliana and provides a reference for radial thickening and enhanced resistance in Arabidopsis thaliana, which has important scientific significance. Attached Figure Description

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0028] Figure 1 Electrophoresis diagram of Mongolian oak genome RNA;

[0029] Among them, 1, 2, 3, 4, and 5 represent the root, stem, leaf, stem tip, and vascular cambium of Mongolian oak, respectively;

[0030] Figure 2 This is an amplified fragment of the QmWOX4 gene;

[0031] Figure 3 The results of PCR identification of the QmWOX4 gene in bacterial culture;

[0032] Figure 4 Multiple sequence alignment for QmWOX4 protein;

[0033] Figure 5 The phylogenetic tree of QmWOX4;

[0034] Figure 6 Predicting the hydrophilicity / hydrophobicity and transmembrane structure of the QmWOX4 protein;

[0035] Figure 7 For QmWOX4 protein transmembrane prediction;

[0036] Figure 8 This is a conserved domain of the QmWOX4 protein;

[0037] Figure 9 Prediction of the secondary structure of the QmWOX4 protein;

[0038] Figure 10 Prediction of the tertiary structure of the QmWOX4 protein;

[0039] Figure 11 The QmWOX4 protein interaction network was constructed with reference to the AtWOX4 (AT1G46480) protein;

[0040] Figure 12 Electrophoresis diagrams of RNA from different tissues of *Quercus mongolica*.

[0041] Among them: 1, 2, 3, and 4 are RNAs from the root tip, stem tip, basal lignified stem segment, and leaf of one-year-old Mongolian oak, respectively; 5, 6, 7, 8, 9, 10, 11, and 12 are RNAs from the stem tip and basal lignified stem segment of one-year-old Mongolian oak outdoors from July to October, respectively; 13, 14, 15, 16, and 17 are RNAs from the vascular cambium at five different heights of three-year-old Mongolian oak, respectively; 18, 19, and 20 represent RNAs from the vascular cambium at different stages of 40-year-old Mongolian oak, respectively.

[0042] Figure 13 This is a semi-quantitative identification result of the Actin internal reference gene;

[0043] Wherein: A: 1-4 represent different tissues of one-year-old Mongolian oak (root, old stem, stem tip, leaf); B: 1-3 represent the vascular cambium at different stages (April, August, October); C: 1-5 represent the vascular cambium of different internodes; D: 1-8 represent the old stem and stem tip of one-year-old seedlings from July to October.

[0044] Figure 14 Analysis of the expression levels of QmCLE41, QmPXY, and QmWOX4 in different tissues of one-year-old Mongolian oak;

[0045] Note: Different letter labels indicate significant differences; the same letter labels indicate no significant differences; where p < 0.05;

[0046] Figure 15 Analysis of QmCLE41, QmPXY, and QmWOX4 gene expression in the old stems and shoot tips of one-year-old Mongolian oak seedlings in different months;

[0047] Where O represents old stem; A represents stem tip; different lowercase letters indicate significant differences; the same lowercase letter indicates no significant difference; significance p<0.05; 7-10 represent different months;

[0048] Figure 16 Expression analysis of QmCLE41, QmPXY, and QmWOX4 in the vascular cambium of 3-year-old Mongolian oak at different heights;

[0049] Wherein: 1-5 represent cambium layers at different heights; different lowercase letters indicate significant differences; the same lowercase letter indicates no significant differences; where the significance p<0.05;

[0050] Figure 17 A graph showing the gene expression analysis of QmCLE41, QmPXY, and QmWOX4 at different stages of a 40-year-old Mongolian oak.

[0051] Where: different lowercase letters indicate significant differences; the same lowercase letter indicates no significant differences; and the significance p < 0.05.

[0052] Figure 18 Electrophoresis image of pRI101-GFP vector plasmid digested with SamⅠ single enzyme;

[0053] Where: M: 10000bp DNAMaker; 1: vector plasmid before enzyme digestion; 2: vector plasmid after 2-9 enzyme digestion.

[0054] Figure 19Image of PCR electrophoresis detection of a seamlessly cloned gene fragment;

[0055] Wherein: M: 1000bp DNAMaker; 2-8: QmWOX4;

[0056] Figure 20 PCR electrophoresis image of E. coli colonies transformed with the recombinant expression vector;

[0057] Wherein: M: 1000bp DNAMaker; 2-8: pRI101-QmWOX4-GFP;

[0058] Figure 21 PCR electrophoresis image of Agrobacterium tumefaciens colonies transformed with the recombinant expression vector;

[0059] Wherein: M: 10000bp DNAMaker; 1-4: pRI101-QmWOX4-GFP.

[0060] Figure 22 Subcellular localization map;

[0061] Figure 23 This is a PCR electrophoresis image of a homozygous Arabidopsis thaliana mutant.

[0062] Wherein: M: 1000bp Maker; 1-6: col wild type; 7-14: wox4 mutant; 1, 3, 5, 7, 9, 8, 10, 12, 14: LP+RP; 2, 4, 6, 7, 9, 11, 13: LP+RP

[0063] Figure 24 For resistance screening of T0 generation seeds;

[0064] Among them: A: Transgenic seeds of QmWOX4-functional restored plants; B: Transgenic seeds overexpressing QmWOX4.

[0065] Figure 25 The image shows a PCR electrophoresis diagram of T1 generation transgenic plants. A: QmWOX4 functionally restored transgenic plant; B: QmWOX4 overexpressing transgenic plant; M: 1000bp DNA Marker; 1: Negative control; 2-5: Positive controls; 6-9: Transgenic plants.

[0066] Figure 26 This is a diagram showing the resistance screening of T1 generation transgenic plant seeds;

[0067] Wherein: A: QmWOX4 overexpressing transgenic plants; B: QmWOX4 function restored transgenic plants.

[0068] Figure 27 This is a resistance screening diagram for T2 generation transgenic plant seeds;

[0069] Wherein: A: QmWOX4 overexpressing transgenic plants; B: QmWOX4 function restored transgenic plants.

[0070] Figure 28 A diagram showing the growth of Arabidopsis thaliana;

[0071] Where: RE: indicates QmWOX4 functional recovery plant; wox4: indicates wox4 deletion mutant; OE: indicates QmWOX4 overexpression plant; Col: indicates wild-type plant; different lowercase letters indicate significant differences; the same letter indicates no significant differences; p<0.05.

[0072] Figure 29 Image of a paraffin section of Arabidopsis thaliana;

[0073] Where: A, functional recovery type; B, wox4 deletion mutant; C, overexpression; D, wild type; ph, phloem; xy, xyne; ca, vascular cambium; pi, pith; scale bar 100μm. Detailed Implementation

[0074] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0075] Example 1: Cloning and Bioinformatics Analysis of the QmWOX4 Gene from Mongolian Oak

[0076] I. Total RNA was extracted from *Quercus mongolica* using a modified CTAB method. Sampling sites included the roots, shoot tips, old stems, and leaves of 1-year-old *Quercus mongolica*; old stems of 1-year-old *Quercus mongolica* from different months (July to October); 5 internodes were divided at 20 cm intervals from the ground in 3-year-old *Quercus mongolica*, and samples were taken from each internode; vascular cambium samples were taken from 40-year-old *Quercus mongolica* at different time points (April, August, and October) at a depth of 1.3 meters from the ground. The specific method for qRT-PCR expression analysis is as follows:

[0077] (1) Prepare the lysis buffer by adding 4 mL of 2×CTAB and 150 μL of β-mercaptoethanol to a centrifuge tube, vortexing and mixing, and preheating at 65°C for later use.

[0078] (2) The vascular cambium samples of Mongolian oak were taken out from the -80℃ ultra-low temperature freezer, placed in a mortar, and ground quickly with liquid nitrogen. After grinding, the samples were quickly transferred to centrifuge tubes and returned to liquid nitrogen for flash freezing. 1 mL of lysis buffer was added to the samples, vortexed to mix, and then placed in a metal bath at 65℃ for 30 min.

[0079] (3) Place the sample in a centrifuge and centrifuge at a maximum speed of 10 min. Use the Tiangen plant RNA extraction kit to extract the supernatant and insert it into a CS column. Centrifuge at 12000 rpm for 2 min. Collect the supernatant from the bottom layer of the CS column into a centrifuge tube and place it on ice. Add 0.5 times the volume of ice-cold anhydrous ethanol to the centrifuge tube and vortex to mix.

[0080] (4) Transfer the sample into the CR3 column, 12000 rpm, 10 s, discard the lower layer, leave the column, add 350 μL of protein removal solution (RW1) to the CR3 column, 12000 rpm, 30 s, discard the lower layer, leave the column.

[0081] (5) Pipette 80 μL of DNase-removing buffer onto the CR3 column membrane, cap it, and incubate at room temperature for 15 min. Repeat this step by adding 500 μL of wash buffer (RW) to the CR3 column, incubating at room temperature for 2 min, and centrifuging at 12000 rpm for 30 s. Repeat this step, generally up to 3 times, until the lower layer is colorless. Discard the lower layer and centrifuge at 12000 rpm for 2 min.

[0082] (6) Insert the CR3 upper column into a clean 1.5 mL centrifuge tube, place it on a sterile workbench and blow for 1 min to remove ethanol. Add 50 μL ddH2O dropwise to the CR3 upper column, place at room temperature for 2 min, and centrifuge at 13000 rpm for 2 min.

[0083] The RNA samples obtained in the experiment were tested for concentration using a nucleic acid concentration meter, and then tested for fragment integrity using 1.2% agarose gel electrophoresis. After being flash-frozen in liquid nitrogen, they were immediately stored in a -80°C freezer for later use.

[0084] II. Reverse transcription synthesis of Mongolian oak cDNA

[0085] Using TaKaRa's Prime Script TM The 1st Strand cDNA Synthesis Kit (6110A) uses reverse transcription to synthesize the first strand of cDNA. The specific method is as follows:

[0086] (1) Add the following mixture to a 200 μL centrifuge tube:

[0087]

[0088] (2) After instantaneous centrifugation, keep the temperature at 65℃ for 5 minutes, and then place it on ice.

[0089] (3) Add the following mixture to the 200 μL centrifuge tube, bringing the total volume to 20 μL.

[0090]

[0091] (4) After instantaneous centrifugation, the sample was placed in a PCR instrument for amplification. The reaction program was as follows: 42℃, 45min; 72℃, 15min.

[0092] Part of the inversion product was used for gene cloning, and the other part was diluted to a concentration of 200 ng / μL. -1 This is used for quantitative real-time PCR. III. PCR cloning of the QmWOX4 gene from *Quercus mongolica*.

[0093] (1) Primers designed based on the Mongolian oak genome are shown in Table 1.

[0094] Table 1 Primer sequences used in the experiment

[0095]

[0096] (2) Using the Mongolian oak vascular cambium cDNA synthesized via reverse transcription as a clone, Prime was used. The full-length fragment of the QmWOX4 gene was amplified using HS (Premix) high-fidelity enzyme.

[0097] The system is as follows:

[0098]

[0099] The amplification procedure is as follows:

[0100]

[0101]

[0102] (3) Add the PCR product obtained by the above reaction to a gel block containing 1.3% agarose using a pipette. Add 2 μL to each well and detect the size of the PCR product bands using a gel imaging system (BIO-RAD).

[0103] (4) PCR product recovery. Using a pipette, add 20 μL of the PCR product to a 2.5% agarose gel block for each well. After completion, purify the target gene fragment using a DNA purification kit by gel extraction. The specific experimental procedures are as follows:

[0104] ① Add 4 times the weight of the gel to the centrifuge tube, then heat in a metal bath at 37°C for about 5-10 minutes, shaking intermittently to mix until the gel is completely dissolved.

[0105] ② Place the spin column onto the collection tube, transfer the solution from step ① into the spin column, centrifuge at 12000 rpm for 1 min, transfer the filtrate back into the spin column, centrifuge at 12000 rpm for 1 min to improve the recovery rate of the PCR product, and then discard the waste liquid.

[0106] ③ Add 700 μL of Buffer WB to the Spin column, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, repeat once, place the Spin column on the collection tube, centrifuge at 12000 rpm for 1 minute at room temperature, place the Spin column on a new centrifuge tube, add 30 μL of sterile water dropwise to the Spin column membrane, and let stand at room temperature for 1 minute.

[0107] ④ Centrifuge at 12,000 rpm for 1 min at room temperature to elute the PCR products, and store the recovered pure PCR product solution at -20℃ for later use.

[0108] (5) pMD TM 18-T-QmWOX4 vector connection construction

[0109] After attaching the A tail to the end of the fragment, the vector is ligated.

[0110] The system is as follows:

[0111]

[0112] ① After vortexing the above reaction system briefly, place it in a PCR instrument, set the PCR instrument to 72℃ for extension for 20 min, and then let it stand on ice for 2 min.

[0113] ②A-tailed linkage product and pMD TM 18-T carrier connection. The system is as follows:

[0114]

[0115] Solution Ⅰ: pMD TM The 18-T Vector Cloning Kit contains a high-efficiency ligation solution for linking fragments and linear 18-T vectors.

[0116] ③16℃, 12h.

[0117] (6) Escherichia coli transformation and positive detection

[0118] TaKaRa E. coli DH5α competent cells were taken from an ultra-low temperature freezer at -80℃, rapidly thawed in ice, and then transformed after thawing on ice. The specific experimental method is as follows:

[0119] ① Take 2 μL of the ligation product from step (5) and add it to a centrifuge tube containing 50 μL of LDH5α competent cells. Incubate on ice for 30 min. ② Heat shock at 42℃ for 90 s, then incubate on ice for 1-2 min.

[0120] ③ Add 500 μL of antibiotic-free LB medium to a sterile container, seal the container, and incubate at 37°C with shaking (200 rpm) for 40 min.

[0121] ④ Spread the turbid bacterial solution evenly on LB (50 mg·mL) -1 On solid culture medium (Amp), seal and incubate overnight at 37°C. ⑤ The next day, select 8 single colonies to prepare bacterial suspensions; use one portion for culture shaking and the other for colony PCR identification.

[0122] The system is as follows:

[0123]

[0124] The amplification procedure is as follows:

[0125]

[0126] ⑦ Add the positive bacterial solution to the culture medium, mix, and send for testing.

[0127] IV. Bioinformatics Analysis

[0128] (1) Physicochemical properties of proteins

[0129] The genome data of *Quercus mongolica* from DNAMAN was compared with the sequencing data of QmWOX4, and bioinformatics analysis was performed using an online website.

[0130] (2) Homologous evolution analysis

[0131] The amino acid sequence of QmWOX4 in *Quercus mongolica* is compared with that of *Populus euphratica* PeWOX4 (XP_011029533.1), *Populus trichocarpa* PtrWOX4 (XP_006374903.1), *Populus alba* PaWOX4 (XP_034897679.1), *Ricinus communis* RcWOX4 (XP_002510184.1), *Hevea brasiliensis* HbWOX4 (XP_021689235.1), and *Jatropha curcas* JcWOX4 (XP_0120683). 10.1) The amino acid sequences of *Quercus suber* QsWOX4 (XP_023876315.1), *Quercus lobata* QlWOX4 (XP_030955768.1), *Manihotesculenta* MeWOX4 (XP_021600785.1), *Mangiferaindica* MiWOX4 (XP_044510789.1), *Duriozibethinus* DzWOX4 (XP_022766471), *Theobromacacao* TcWOX4 (XP_007017177.1), and *Gossypium hirsutum* GhWOX4 (XP_016749311.1) were subjected to multiple sequence alignment.

[0132] Experimental results:

[0133] 1.1 RNA quality detection of Mongolian oak stem segments

[0134] RNA extracted from the cambium vascularis of *Quercus mongolica* was analyzed by 1.2% gel electrophoresis, revealing two bands in the *Quercus mongolica* RNA. Figure 1 The bands in the image are clear and bright. The concentration of the extracted RNA was determined using an ultra-micro UV spectrophotometer, revealing the OD values ​​of RNA from the vascular cambium of different parts of *Quercus mongolica*. 260 / OD 280 The ratios were all between 1.90 and 2.00 (Table 2), indicating that the extracted RNA from the vascular cambium tissue of *Quercus mongolica* met the requirements for subsequent experiments in terms of both quality and integrity.

[0135] Table 2. Detection of RNA concentration in *Quercus mongolica*

[0136]

[0137] 1.2 PCR amplification of the QmWOX4 gene and colony PCR identification

[0138] Using cDNA from the vascular cambium of *Quercus mongolica* as a template, RT-PCR was performed to amplify the full-length gene. Gel electrophoresis revealed a specific amplification product of approximately 700 bp in length. Figure 2 The amplification products were recovered by gel electrophoresis and reacted with pMD. TM The 18-T vector was ligated and successfully transformed into TaKaRa's E. coli DH5α competent cells. Colony PCR was used to detect positive results for selected single colonies, yielding a single specific band approximately 700 bp in length. Figure 3 Sequencing results from Genewiz Biotechnology showed that the gene sequence is 657 bp in length, as shown in SEQ ID NO: 1 (NCBI website, ID: 2702148). ORF Finder analysis revealed that the cloned target gene has a relatively complete open reading frame, capable of fluent translation into 228 amino acids. This indicates successful cloning of the Mongolian oak WOX4 gene, which has been named QmWOX4.

[0139] 1.3QmWOX4 Bioinformatics Analysis

[0140] (1) Homology alignment of QmWOX4 protein sequence

[0141] To investigate the function and characteristics of the QmWOX4 gene in *Quercus mongolica*, the sequencing results of QmWOX4 were compared with the transcriptome sequence of *Quercus mongolica* using the DNAMAN online software, and the nucleotide sequences were translated into amino acid sequences encoding the protein.

[0142] Studies have found that the WOX4 gene exhibits structural conservation, with all tree species containing a homeomorphic HD domain, and it belongs to the typical WOX family member. Figure 4 )

[0143] (2) Construction of phylogenetic tree

[0144] Using MEGA 7.0 bioanalysis software, the homology between QmWOX4 protein and Arabidopsis thaliana WUS and WOX1-WOX14 proteins was further analyzed, and a phylogenetic tree was constructed using the Neighbor-Joining method. Figure 5 The results showed that the QmWOX4 protein and the AtWOX4 protein from Mongolian oak clustered closely together in one branch, indicating that they are the most closely related homologous proteins and therefore may have similar functions.

[0145] (3) Analysis of the structure and physicochemical properties of the QmWOX4 protein from Mongolian oak

[0146] Analysis of the physicochemical parameters of the amino acid sequence of the QmWOX4-encoded protein using the ProtParam online software allowed for a preliminary prediction of the gene's biological function. The results showed that the chemical formula of the Mongolian oak QmWOX4 protein is C0.05. 1095 H 1734 N 330 O 336 S8 has a total of 3503 atoms, a theoretical molecular weight of 25154.30 Da, and a theoretical isoelectric point (pI) of 8.86. The most abundant amino acid in the QmWOX4 protein is glutamic acid (Glu) at 11.5%, while cysteine ​​(Cys), methionine (Met), tryptophan (Trp), tyrosine (Tyr), and valine (Val) are the least abundant at 1.8% each. This protein does not contain pyrrolidone (Pyl) or selenocysteine ​​(Sec). The aliphatic amino acid index of the QmWOX4 protein is 55.50, with 33 negatively charged residues and 37 positively charged residues, indicating that the protein is positively charged. Its instability coefficient is 61.36, which is greater than 50, suggesting that QmWOX4 is an unstable protein.

[0147] (4) Predictive analysis of hydrophobicity / hydrophilicity and transmembrane structure of proteins encoded by the QmWOX4 gene.

[0148] Protein folding is primarily driven by the hydrophilicity and hydrophobicity of amino acids. Therefore, the folding pattern of a protein can be determined by analyzing the hydrophilicity and hydrophobicity of its amino acids. This study used the online software Prot Scale to obtain the distribution curves of the hydrophilicity and hydrophobicity of Mongolian oak QmWOX4. Figure 6 As can be clearly seen from the figure, more than half of the images are located on the negative coordinates, and the protein exhibits significant hydrophilicity and hydrophobicity. The highest score (1.133) is found at position 160, while the lowest score (-3.922) is at position 141, indicating that the Mongolian oak QmWOX4 protein is a hydrophilic protein. The transmembrane structure of the QmWOX4 protein was predicted using TMHMM analysis software. Figure 7 It is known that amino acids 1 to 218 of the Mongolian oak QmWOX4 protein are located outside the cell membrane, so the QmWOX4 protein does not have transmembrane function.

[0149] (5) Prediction of conserved domains of QmWOX4 protein

[0150] Predicting conserved domains of the QmWOX4 protein using the CDD database in NCBI revealed that the Mongolian oak QmWOX4 protein contains a conserved HOMEOBOX domain, belonging to the homeodomain superfamily. Figure 8 ).

[0151] (6) Prediction of the secondary and tertiary structures of the QmWOX4 protein from Mongolian oak

[0152] SOPMA online analysis software predicted the secondary structure of the QmWOX4 protein. The results showed that the QmWOX4 protein is composed of secondary structure elements in four states. The largest proportion was random coils (62.39%), followed by α-helices (28.90%), extended strands (5.96%), and the smallest proportion was β-turns (2.75%). Figure 9 A model of the QmWOX4 protein was constructed using the online database SWISS-MODEL. The predicted results showed a high degree of agreement between the tertiary and secondary structures of the protein, indicating that the three-dimensional structure of the QmWOX4 protein is mainly composed of helices and random coils. Figure 10 ).

[0153] (7) Prediction of QmWOX4 protein interaction network

[0154] Predicting target proteins that interact with the QmWOX4 protein in *Quercus mongolica* is of great significance for in-depth exploration of the molecular regulatory mechanism of QmWOX4 in the growth and development of *Quercus mongolica*. This study used the STRING database and referenced the At WOX4 protein to construct a network diagram of QmWOX4 interacting proteins in *Quercus mongolica*. Figure 11 The results showed that 10 proteins interact with QmWOX4. Among them, CLV3 / CLE41 / 44-PXY-WOX4 protein constitutes a signaling pathway, which plays a crucial role in the development of vascular meristems during secondary growth; MOL1 (MORE LATERAL GROWTH1) antagonizes the CLE41-PXY-WOX4 signaling pathway, affecting the inward and outward differentiation of vascular cambium by regulating its activity; HAM3 (HAIRYMERISTEM) and SCL27 (SCARECROW-LIKE) belong to the GRAS family of proteins. HAM3 is a conserved cofactor of WUS / WOX proteins, while SCL27 and HB-8 (HOMEOBOX-8) belong to the Homeodomain-Leucine Zipper family and synergistically regulate plant growth and development with WOX4; RUL1 (REDUCED IN LATERAL GROWTH1) also interacts with QmWOX4. The strong interaction between GROWTH1 and PXY suggests that RUL1 may have a significant regulatory effect on the CLE41-PXY-WOX4 signaling pathway, jointly regulating plant growth; TPL belongs to the TOPLESS gene family and has an inhibitory effect on WOX regulation of meristematic growth.

[0155] Example 2: Analysis of the expression pattern of the QmWOX4 gene in Mongolian oak.

[0156] I. Semi-quantitative analysis of internal reference genes

[0157] Based on the sequencing results, semi-quantitative primers were designed (Table 3). Diluted cDNA from various tissues of *Quercus mongolica* was used as amplification template, and PCR amplification was performed using Premix Taq™ DNA polymerase. Gel electrophoresis was used to determine if the product was a single band and if the product fragment length was between 100-250 bp.

[0158] Table 3 Primer sequences for qRT-PCR of different tissues of *Quercus mongolica*

[0159] Primers Sequence 5'-3' QmWOX4-F TGGTTCCAAAATCACAAAGCG (SEQ ID NO: 5) QmWOX4-R AAAGTAATGGTGGTATGGCAGTC (SEQ ID NO: 6) QmCLE41-F ATCCTCTTCATTTTCTTCCTGCTC (SEQ ID NO: 7) QmCLE41-R TCCTGGTTTGCTTTGGGTG (SEQ ID NO: 8) QmPXY-F TCCATCCGTCGTCGTTTTC (SEQ ID NO: 9) QmPXY-R CTGTTGCTTATTATTCGCCTCC (SEQ ID NO: 10) Actin-F CTGAGGCACCACTCAACCCTA (SEQ ID NO: 11) Actin-R GGCATCAGTTAGATCACCGACCA (SEQ ID NO: 12)

[0160] The system is as follows:

[0161]

[0162] The amplification procedure is as follows:

[0163]

[0164]

[0165] II. Real-time quantitative PCR

[0166] Using TBGreen from Takara TM PremixExTaq TM II. qRT-PCR experiments were performed on the vascular cambium of different tissues and at different stages of *Quercus mongolica*. Specific experimental procedures were followed according to the manufacturer's instructions, with each sample tested in triplicate. The relative expression level of QmWOX4 was calculated using the calculation method, and the data were analyzed using the Duncan method in SPSS software.

[0167] The system is as follows:

[0168]

[0169] The amplification procedure is as follows:

[0170]

[0171] Experimental results

[0172] 2.1 RNA extraction from different tissues of *Quercus mongolica*

[0173] Total RNA was extracted from various tissues of *Quercus mongolica*, and its concentration was determined using an ultra-micro UV spectrophotometer. The OD values ​​of RNA from different tissues of *Quercus mongolica* were then analyzed. 260 / OD 280The ratios were all between 1.90 and 2.00. 1.2% gel electrophoresis was used to detect RNA from the vascular cambium of different tissues and at different stages of *Quercus mongolica*. The results showed that each *Quercus mongolica* RNA sample had two clear and bright bands, indicating that the extracted *Quercus mongolica* RNA met the requirements for subsequent experiments in terms of both quality and integrity. Figure 12 ).

[0174] 2.2 Semi-quantitative analysis of internal reference genes

[0175] A semi-quantitative experiment was performed using Actin internal control primers, and the PCR amplification products were detected by agarose gel electrophoresis. The results are as follows: Figure 13 As shown, the length and band brightness of the Actin gene fragments amplified in different tissues (roots, old stems, leaves, and shoot tips), at different internode heights, and at different stages of the vascular cambium of Mongolian oak are basically consistent, which meets the experimental requirements of subsequent real-time quantitative PCR.

[0176] 2.3 Analysis of gene expression distribution of QmCLE41, QmPXY, and QmWOX4 in different tissues of one-year-old Mongolian oak

[0177] To investigate the expression patterns of various genes in the CLE41-PXY-WOX4 signaling pathway in *Quercus mongolica*, qRT-PCR expression analysis was performed on the roots, shoot tips, old stems, and leaves of *Quercus mongolica*. Figure 14 The results showed that QmWOX4 and QmPXY genes were expressed in all tissues. QmPXY expression was significantly higher in the shoot tip than in older stems and leaves, but not significantly different from its expression in the root. QmWOX4 expression was significantly higher in older stems than in the root, shoot tip, and leaves, but not significantly different from its expression in the root and shoot tip, and lowest in the leaf. QmCLE41 was expressed only in older stems, shoot tips, and leaves, with significantly higher expression in older stems than in the shoot tip and leaves (p<0.05). This indicates that QmWOX4 and QmPXY genes are involved in the entire growth and development process of *Quercus mongolica*, and may play an important role, especially in root and stem growth, while QmCLE41 is not expressed in the root.

[0178] The gene sequence of QmCLE41 is shown in SEQ ID NO: 13, and the gene sequence of QmPXY is shown in SEQ ID NO: 14.

[0179] 2.4 Expression analysis of QmCLE41, QmPXY, and QmWOX4 genes at different time points

[0180] To investigate the role of genes in the CLE41-PXY-WOX4 signaling pathway in the growth of Mongolian oak seedlings, qRT-PCR expression analysis was performed on the QmCLE41, QmPXY, and QmWOX4 genes in the old stems (lignified stem segments at the base of the ground) and shoot tips of one-year-old Mongolian oak seedlings at different months. Figure 15 The results showed that the expression level of QmWOX4 in the shoot tip was significantly higher than that in the old stem in July. There was no significant difference between the shoot tip and the old stem in August, September, and October. The highest expression level was observed in both the old stem and the shoot tip in July (p<0.05). From July to October, the expression level of QmWOX4 in both the old stem and the shoot tip showed a decreasing trend. The expression level of QmCLE41 in the old stem was significantly higher than that in the shoot tip in July and August. In September, the expression level in the old stem was significantly lower than that in the shoot tip. In October, there was no significant difference in expression between the old stem and the shoot tip (p<0.05). QmPXY showed a similar expression pattern to QmCLE41. In July and August, the expression level in the old stem was significantly higher than that in the shoot tip. In September, the expression level in the old stem was significantly lower than that in the shoot tip. In October, there was no significant difference in expression between the old stem and the shoot tip (p<0.05).

[0181] 2.5 Expression analysis of QmCLE41, QmPXY, and QmWOX4 genes at different trunk heights of Mongolian oak

[0182] To further investigate the roles of QmCLE41, QmPXY, and QmWOX4 in the vascular cambium of *Quercus mongolica*, three-year-old *Quercus mongolica* trees in their vigorous growth phase were divided into five internodes at 20 cm above the ground, with the first internode serving as a control. qRT-PCR expression analysis was performed on the vascular cambium at different heights, and relative expression bars were plotted. Figure 16 The results showed that the expression levels of QmCLE41, QmPXY, and QmWOX4 genes in the 4th and 5th internodes were significantly higher than those in the 1st, 2nd, and 3rd internodes, with the lowest expression level in the 3rd internode (p < 0.05). The expression pattern of the QmWOX4 gene was similar in the vascular cambium of three-year-old Mongolian oak and in the old stems and shoot tips of one-year-old Mongolian oak, with higher expression in the shoot tips than in the old stems. The 1st, 2nd, and 3rd internodes of three-year-old Mongolian oak are in a lignified state, while the 4th and 5th internodes are in a semi-lignified state during secondary growth. At this stage, tree growth is accompanied by a gradual transition from semi-lignification to lignification. Therefore, it can be inferred that the signaling pathway composed of QmCLE41, QmPXY, and QmWOX4 may positively regulate the inward and outward differentiation of the vascular cambium into secondary xylem and secondary phloem, thereby promoting the secondary growth of Mongolian oak.

[0183] 2.640-year-old Mongolian oak gene expression analysis at different stages: QmCLE41, QmPXY, and QmWOX4.

[0184] qRT-PCR was performed on the vascular cambium at 1.3 meters above the ground in 40-year-old Mongolian oak at different time points. Figure 17 The results showed that the expression levels of QmPXY and QmWOX4 were significantly higher in October than in April and August. QmCLE41 was not expressed in October, while QmCLE41, QmPXY, and QmWOX4 were all expressed in April and August. Furthermore, the expression levels of QmCLE41 and QmPXY were significantly higher in April than in August, while the expression level of QmWOX4 did not differ significantly between April and August (p<0.05). Therefore, it is speculated that QmPXY and QmWOX4 mainly regulate the radial growth of *Quercus mongolica* by periclinal division of the vascular cambium in October, and their high expression levels in October may have a positive regulatory effect on radial thickening of *Quercus mongolica*.

[0185] Example 3. Functional analysis of QmWOX4 gene related to vascular cambium development in *Quercus mongolica*

[0186] I. Extraction of QmWOX4 plasmid and construction of plant recombinant expression vector

[0187] (1)pMD TM Extraction of 18-T-QmWOX4 plasmid

[0188] Propagate and sequence the correctly sequenced QmWOX4 bacterial culture and pRI101-GFP vector bacterial culture, extract plasmids and store at -20℃ for later use.

[0189] (2) Seamless cloning of gene fragments PCR amplification

[0190] SmaⅠ restriction enzyme sites were introduced at both ends of the QmWOX4 gene (with stop codons removed), and the corresponding linearized pRI101-GFP vector was inserted, followed by seamless cloning fusion. Primers are shown in Table 4.

[0191] Table 4 Primer sequences for seamless cloning vector construction

[0192] Primers Sequence 5'-3' pRI101-QmWOX4-GFP-F TACATATGCCCGTCGACCCCATGGGAAGCATGAAGGTGCA (SEQ ID NO: 15) pRI101-QmWOX4-GFP-R AATTCGGATCCGGTACCCCCTCATCTGCTTTCCGGATGTA (SEQ ID NO: 16) 35S GACGCACAATCCCACTATCC (SEQ ID NO: 17)

[0193] The system for obtaining a seamless clone of the QmWOX4 gene fragment is as follows:

[0194]

[0195] The procedure for obtaining a seamless clone of the QmWOX4 gene fragment is as follows:

[0196]

[0197] The reaction system for obtaining the linearized pRI101-GFP vector is as follows:

[0198]

[0199] (3) Recovery of the QmWOX4 gene fragment and the linearized pRI101-GFP vector fragment

[0200] The product of SamⅠ single enzyme digestion of the pRI101-GFP vector fragment and the QmWOX4 gene fragment were recovered using the TaKaRa Mini BEST DNA Fragment Purification Kit Ver.4.0, following the instructions.

[0201] (4) Seamless cloning

[0202] Using full gold -The Basic Seamless Cloning and Assembly Kit enables seamless cloning and ligation. The specific reaction system is as follows:

[0203]

[0204] After incubating at 50℃ for 1 hour, the product was placed on ice for 3 minutes and then stored at 20℃ for later use.

[0205] II. Transformation and Identification of GV3101 Agrobacterium

[0206] The pRI101-GFP (empty vector) and pRI101-QmWOX4-GFP expression vectors were transformed into Agrobacterium GV3101.

[0207] The method is as follows:

[0208] ① Add 2 μL of the plasmid of the pRI101-QmWOX4-GFP expression vector to Agrobacterium competent cells in an ice-water mixture and mix gently.

[0209] ② Ice bath for 5 minutes, liquid nitrogen for 5 minutes, 37℃ for 5 minutes.

[0210] ③ In a clean bench, add 600 μL of antibiotic-free LB culture to ② and incubate at 28°C with shaking (170 rpm) for 2 h.

[0211] ④ Spread the turbid bacterial solution evenly on YEP solid medium (50 mg / mL) -1 Kan and Rif) were incubated at 28℃ inverted for 2 days. ⑤ The next day, 8 single colonies were selected to prepare bacterial suspensions; one part was used for shaking and the other part for colony PCR identification.

[0212] III. Extraction of DNA from Arabidopsis thaliana leaves and identification of homozygous mutants

[0213] Five fresh leaves were selected from each of the Arabidopsis thaliana species, the wox4 mutant (purchased from AraShare) and the wild-type col-0. The leaves were placed in clean centrifuge tubes and rapidly frozen in liquid nitrogen. DNA was extracted from the Arabidopsis leaves using the Tiangen Biotech Plant Genomic DNA Extraction Kit (DP320). The wox4 deletion mutant was identified using a three-primer method. The PCR amplification products were detected by 1.2% agarose gel electrophoresis to observe the presence of the target band, thus determining whether the wox4 deletion mutant and the wild-type Arabidopsis plants were homozygous. Primers are shown in Table 4.

[0214] Table 4 Primer sequences for identifying homozygous Arabidopsis mutants

[0215]

[0216] The system is as follows:

[0217]

[0218] The amplification procedure is as follows:

[0219]

[0220]

[0221] IV. Arabidopsis genetic transformation

[0222] (1) Preparation of inflorescence infection transformation solution

[0223] The Agrobacterium expression vector culture was incubated overnight at 28°C with shaking (200 rpm). 1 mL of the activated culture was then added to a centrifuge tube containing 50 mL of LYEP (containing Kan and Rif antibiotics), and incubated with shaking for 36 hours to allow the culture to reach OD500. 600 =0.8-1.2. Centrifuge the bacterial culture at 4000 rpm for 15 min, discard the supernatant, add 50 mL of Arabidopsis thaliana genetic transformation suspension, mix well, and adjust the OD of the suspension to 0.8-1.2. 600 =0.4-0.6.

[0224] (2) Agrobacterium infection of Arabidopsis thaliana inflorescences

[0225] Soak all the non-fruiting inflorescences in the suspension for 30 seconds, then lay them flat and keep them in the dark for 12 hours before transferring them to normal culture. Infect them once every 3 days, for a total of 3-4 times.

[0226] (3) Obtaining transgenic Arabidopsis seeds

[0227] Collect mature seeds by lineage and store at 4℃.

[0228] V. Screening of transgenic positive Arabidopsis plants

[0229] After sterile vernalization, the seeds were sown in an environment containing 1 / 2 MS (50 mg·L⁻¹). -1 The resistant seedlings were cultured in Kan solid medium under suitable conditions. After the seedlings developed robust leaves, they were transplanted into nutrient soil, and leaves were taken for positive identification. After the PCR reaction was completed, the negative control (col-0) product bands were simultaneously detected by 1.2% agarose gel electrophoresis.

[0230] The system is as follows:

[0231]

[0232] The amplification procedure is as follows:

[0233]

[0234] VI. Physiological Trait Analysis of Arabidopsis Transgenic Plants

[0235] The seedling height of T3 generation transgenic Arabidopsis thaliana functional restorer plants, overexpression plants, and concurrently sown wox4 deletion mutant and wild-type plants was observed and photographed at 3, 6, and 9 days. Paraffin section analysis was performed on stem segments of the four Arabidopsis thaliana lines after 15 days of growth (calculated from the emergence of four true leaves).

[0236] VII. Analysis of Arabidopsis thaliana paraffin sections

[0237] (1) Fixation and rinsing: Stem segments of four Arabidopsis thaliana strains at the same growth height, which had grown for 15 days (calculated from the emergence of four true leaves), including the QmWOX4 functional restorer, QmWOX4 overexpressor, wild type, and wox4 deletion mutant, were selected and fixed in FAA fixative. The fixation time was no less than 48 hours. After removing the Arabidopsis thaliana stem segments from the fixative, they were rinsed with 50% alcohol for 2-3 seconds to remove any residual fixative from the surface of the stem segments.

[0238] (2) Sampling: Use a scalpel to cut Arabidopsis thaliana stem segments horizontally. Cut into small segments with a length of about 5 mm and place them in 5 mL centrifuge tubes.

[0239] (3) Dehydration and clearing: The cut Arabidopsis stem segments were sequentially dehydrated in 60%-100% ethanol solutions (each 10% increment), with each stage lasting 1.5 hours, and the final dehydration treatment in anhydrous ethanol lasting 40 minutes. The material was then removed from the anhydrous ethanol and quickly transferred to a mixture of anhydrous ethanol and xylene for clearing treatment, with each stage lasting 1.5 hours. The clearing time for pure xylene was halved, with the first stage lasting 60 minutes and the second stage lasting 30 minutes.

[0240] (4) Wax impregnation: Pour out 2 / 3 of the pure xylene from the last step of the transparent process, and gradually add wax scraps (melting point 52-54℃) until the xylene is saturated. Impregnate in a constant temperature oven at 30℃ for 24 hours, then add a small amount of wax scraps, raise the temperature to 40℃ and impregnate for 2 hours. Then change to a mixed wax solution (made by fusing sliced ​​paraffin with melting point 58-60℃ and melting point 50-52℃) and adjust the temperature to 60℃ for constant temperature impregnation. Change the mixed wax solution twice a day. Embedding begins after 6 days of wax impregnation.

[0241] (5) Embedding: Embedding was performed using a KD-BM biological tissue embedding machine (70℃). The material to be observed was adjusted downwards using heated forceps to facilitate sectioning and observation. The embedding cassette was then smoothly transferred to the freezing stage of the KD-BL embedding machine. After the paraffin solution had completely solidified, it was removed for use.

[0242] (6) Sectioning, mounting, and baking: Sections were prepared using a manual rotary microtome, maintaining a section thickness of 5-6 μm. Glass slides were soaked in 75% alcohol solution and then air-dried. Using 2% gelatin as an adhesive, the wax strips were gently placed on the slides. The slides were then spread using a KD-T computerized biological tissue spreader (37℃). After the wax strips were flattened, the slides were baked in a 37℃ oven for 3-5 days.

[0243] (7) Dewaxing and rehydration: Transfer the dried slides into a xylene solution and dewax them in the following gradient: 2 times pure xylene → 1 / 2 anhydrous ethanol + 1 / 2 xylene → anhydrous ethanol. Each step takes 2-3 minutes. After dewaxing, rehydrate the slides in the following order: anhydrous ethanol → distilled water (each 10% gradient), for the same time as above.

[0244] (8) Safranin-Fix Green Staining: Place the rehydrated slide in a 1% safranin aqueous solution (1g safranin + 100mL distilled water) and stain at room temperature for 24 hours. After staining, perform Fast Green staining, sequentially adding distilled water → 30% anhydrous ethanol → 50% anhydrous ethanol → 60% anhydrous ethanol → 70% anhydrous ethanol → 80% anhydrous ethanol → 1% Fast Green alcohol solution (1g Fast Green + 100mL 95% ethanol). Each step takes 2-3 minutes, and Fast Green staining takes 1-3 seconds.

[0245] (9) Dehydration: The stained slides were dehydrated by passing them through a gradient of anhydrous ethanol → anhydrous ethanol 1 / 2 anhydrous ethanol + 1 / 2 xylene → pure xylene → pure xylene.

[0246] (10) Mounting: In a fume hood, use a 1:1 mixture of xylene and neutral resin as the mounting medium. Place the mounting medium on a glass slide, cover with a coverslip, and remove any excess air bubbles. Place the mounted slide in a constant temperature of 46°C for 3 days, then remove it and air dry in a well-ventilated area for observation.

[0247] Experimental results:

[0248] 3.1 Digest pRI101-GFP vector plasmid with SamⅠ single enzyme.

[0249] The pRI101-GFP vector plasmid was digested with SamI endonuclease, and the results were detected by agarose gel electrophoresis. Figure 18 The results are as follows: both the bands before and after enzyme digestion are above 10000bp, while the full length of the pRI101-GFP vector sequence is 12041bp, indicating that this plasmid is the pRI101-GFP vector plasmid; there is a significant difference in the height of the bands before and after enzyme digestion, indicating that the pRI101-GFP vector plasmid has been successfully digested by SamI restriction enzyme.

[0250] 3.2 Seamless Cloning of Target Gene Fragments

[0251] Using the target gene plasmid as a template, the target gene (with the stop codon removed) was inserted into the SamⅠ restriction site of the PRI101-GFP vector. Specific seamless cloning fusion primers (20 bp restriction site + 20 bp target gene) were designed for PCR amplification. The amplified products were detected by agarose gel electrophoresis. Figure 19 The results showed that the seamless clone gene fragment of the pRI101-QmWOX4-GFP plant recombinant expression vector was in the range of 700bp-1000bp, indicating that the seamless clone gene fragment of the QmWOX4 gene in the plant recombinant expression vector had been successfully amplified.

[0252] 3.3 Positive detection of recombinant expression vectors

[0253] The pRI101-QmWOX4-GFP plant recombinant expression vector was transformed into Escherichia coli DH5α. After screening with LB solid medium containing Kan resistance, positive single colonies were selected. Colony PCR verification was performed using 35S vector primers and seamless cloning gene fusion primer R, followed by agarose gel electrophoresis detection. Figure 20 The results showed that the PCR amplification band of the pRI101-QmWOX4-GFP plant recombinant expression vector was approximately 750 bp, indicating that the target gene had been successfully constructed into pRI101-GEP. Sequencing of positive clones revealed that the sequencing results were completely consistent with the recombinant expression vector sequence, confirming the successful construction of the pRI101-QmWOX4-GFP plant recombinant expression vector.

[0254] 3.4 GV3101 Agrobacterium transformation positive detection

[0255] The pRI101-QmWOX4-GFP plant recombinant expression vector was transformed into GV3101 Agrobacterium competent cells. Positive single colonies were selected on LB solid medium containing Kan and Rif resistance. Colony PCR verification was performed using 35S vector primers and seamless cloning gene fusion primers, and the colonies were detected by agarose gel electrophoresis. Figure 21 The results showed that the PCR amplification band of the pRI101-QmWOX4-GFP plant recombinant expression vector was around 750bp, indicating that the GV3101 Agrobacterium had been successfully transformed.

[0256] 3.5 Subcellular localization analysis

[0257] The lower epidermis of tobacco leaves was peeled off to prepare a slide, which was then observed using a laser confocal microscope (Zeiss). Figure 22 The results showed that the cell structure was clearly visible in the bright field; in the GFP field, QmWOX4 showed green fluorescent dots; in the Dapi field, the cell nuclei of QmWOX4 were successfully stained and showed blue fluorescence; in the superimposed field, the large green fluorescent dots and the blue fluorescent cell nuclei were superimposed and located inside the cell, indicating that QmWOX4 was located in the cell nucleus.

[0258] 3.6 Homozygous identification of Arabidopsis mutants

[0259] Using Arabidopsis leaf DNA as a template, PCR amplification was performed using the three-primer method, and the results were detected by agarose gel electrophoresis. Figure 23 The results showed that in wild-type (Col-0) Arabidopsis thaliana, QmWOX4 amplified a band of approximately 700 bp using LP and RP primers, while no band was amplified using LB and RP primers, indicating that wild-type Arabidopsis thaliana does not have T-DNA insertion and is homozygous. In the wox4 Arabidopsis thaliana mutant, no band was amplified using LP and RP primers, while a band of approximately 400 bp was amplified using LB and RP primers, indicating that all four identified wox4 Arabidopsis thaliana mutants were homozygous. In conclusion, the identified wild-type Arabidopsis thaliana and Arabidopsis thaliana wox4 mutants are all homozygous.

[0260] 3.7 Screening of transgenic homozygous plants

[0261] (1) Positive screening of T0 generation seeds

[0262] T0 generation seeds harvested from T0 generation overexpression and functionally restored transgenic plants were sown on 1 / 2 MS medium containing Kan resistance. The results showed that most seedlings from QmWOX4 overexpression T0 generation transgenic plants on Kan-resistant 1 / 2 MS medium were light green with only two leaves and could not grow normally, eventually turning white and dying, indicating a lack of Kan resistance. However, a small number of seedlings developed four dark green leaves and grew normally. Figure 24 The results (A) indicate that these plants possess resistance to Kan, and they are named QmWOX4 overexpression T1 generation transgenic plants; T0 generation seeds of QmWOX4 function restored T0 generation transgenic plants, on Kan-resistant 1 / 2 MS medium, only a small portion of the seeds from the QmWOX4 function restored T0 generation transgenic plants grew four dark green leaves and were able to grow normally. Figure 24 (B) indicates that this type of plant has the ability to resist Kan, and it is named QmWOX4 functionally restored T1 generation transgenic plant. In summary: QmWOX4 overexpressing T1 generation transgenic plants and QmWOX4 regenerating T1 generation transgenic plants have been obtained.

[0263] (2) PCR identification of T1 generation transgenic plants

[0264] Overexpressing transgenic plants were used with wild-type Arabidopsis as a negative control, and functionally restored transgenic plants were used with the target gene mutant as a negative control. The target gene plasmid was used as a positive control. PCR amplification was performed using DNA as a template, with 35S vector primers and seamless cloning gene fusion primers. Results were detected by 1.2% agarose gel electrophoresis. Figure 25 As shown.

[0265] In both QmWOX4 overexpressing T1 generation transgenic plants and positive controls, a band of approximately 750 bp was amplified, while no band was amplified in the negative control. Figure 25 Figure A) indicates that the T1 generation transgenic plants overexpressing QmWOX4 contain the QmWOX4 gene. In both the QmWOX4-function-restored T1 generation transgenic plants and the positive control, a band of approximately 750 bp was amplified, while no band was amplified in the negative control, indicating that the T1 generation transgenic plants with restored QmWOX4 function contain the QmWOX4 gene. In summary, QmWOX4-overexpressing T1 generation transgenic plants and QmWOX4-function-restored T1 generation transgenic plants have been obtained.

[0266] (3) Positive screening of T1 generation seeds

[0267] T1 generation seeds harvested from T1 generation transgenic plants were sown on 1 / 2 MS medium containing Kan resistance. Figure 26(A) The results showed that only a small portion of the seedlings of the T1 generation transgenic plants overexpressing QmWOX4 were light green on 1 / 2 MS medium with Kan resistance, and they could not grow normally until they turned white and died, indicating that they did not have the ability to resist Kan. However, the vast majority of seedlings grew four dark green leaves and were able to grow normally, indicating that the T1 generation transgenic plants overexpressing QmWOX4 were heterozygous and their resistance to Kan could not be stably inherited by their offspring. The progeny plants of the QmWOX4-overexpressing T1 generation transgenic plants that possessed Kan resistance were divided into 8 lines, named QmWOX4-overexpressing T2 generation transgenic plants 1-8; T1 generation seeds of the QmWOX4-restored T1 generation transgenic plants, when grown on Kan-resistant 1 / 2 MS medium, showed that only a small portion of the seedlings were light green and failed to grow normally, eventually turning white and dying, indicating a lack of Kan resistance. The vast majority of the seedlings, however, developed four dark green leaves and grew normally. Figure 26 (B) indicates that the QmWOX4 functional recovery T1 generation transgenic plants are heterozygous, and their resistance to Kan cannot be stably inherited by their offspring. The offspring plants with Kan resistance from the QmWOX4 functional recovery T1 generation transgenic plants were divided into 8 lines, named QmWOX4 functional recovery T2 generation transgenic positive plants 1-8.

[0268] (4) Positive screening of T2 generation seeds

[0269] T2 generation seeds harvested from lines 1-8 of the T2 generation transgenic plants were sown on 1 / 2 MS medium containing Kan resistance. The results showed that T2 generation seeds from line 5, a T2 generation transgenic plant overexpressing QmWOX4, produced progeny plants with four dark green leaves and normal growth on 1 / 2 MS medium containing Kan resistance. Figure 27 (A) This indicates that the resistance to Kan in the QmWOX4 overexpressing T2 generation transgenic plant line 7 can be stably inherited by its offspring, and the offspring are named the QmWOX4 overexpressing T3 generation homozygous transgenic plant.

[0270] QmWOX4 functional recovery T2 generation transgenic plant line 5's T2 generation seeds, on K-resistant 1 / 2 MS medium, produced progeny plants that all grew four dark green leaves and were able to grow normally. Figure 27 (B) indicates that the resistance to Kan of the QmWOX4 functional recovery T2 generation transgenic plant line 5 can be passed on to its offspring, and the offspring are named QmWOX4 functional recovery T3 generation transgenic homozygous plants.

[0271] (5) Physiological traits analysis of T3 generation transgenic Arabidopsis thaliana

[0272] Transgenic Arabidopsis thaliana of generation T3, including functional restorer, overexpression, wox4 deletion mutant, and wild type, were cultured in 1 / 2 MS medium. After four true leaves emerged, they were sown in nutrient soil. Three days later, the leaves of the functional restorer plants were found to be significantly larger than those of the mutant, overexpression, and wild type. Figure 28 After 7 days, the height of functionally restored plants was significantly greater than that of mutant, overexpressing, and wild-type plants. The overexpressing lines showed better growth than wild-type plants, and the wild-type plants showed better growth than mutants. Figure 28 After 9 days, the functionally restored plants showed the fastest growth rate, bolting earlier than other plants. Furthermore, their stem elongation was superior not only to the mutants but also to the wild-type plants. Their flowering time was similar to the overexpression plants, preceding both the mutants and wild-types. The experimental results also showed that the stems of the QmWOX4 functionally restored plants were significantly thicker than those of the mutants and wild-type plants. Figure 28 In the middle (C), that is, the QmWOX4 gene is superior to the AtWOX4 gene in promoting Arabidopsis growth and radial thickening (NCBI website, ID:841113) (p<0.05).

[0273] (6) Analysis of Arabidopsis thaliana paraffin sections

[0274] To investigate the effect of the QmWOX4 gene on vascular cambium development, this study analyzed paraffin sections of Arabidopsis stem segments from four Arabidopsis lines (wild-type, mutant, functionally restored, and overexpressing) after 15 days of growth (calculated from the emergence of four true leaves). The results showed that the number of vascular cambium cell layers in the four lines was in the following order: functionally restored > overexpressing > wild-type > mutant. Figure 29 The number of vascular cambium cells in stem segments of the wox4 deletion mutant was significantly less than that in the functional restorer and wild-type plants. Notably, the functional restorer plants had a greater number of vascular cambium cells than the wild-type plants, further demonstrating that the QmWOX4 gene, compared to the AtWOX4 gene, promotes the differentiation and division of the Arabidopsis vascular cambium, thereby affecting radial growth and contributing to stem thickening and enhanced resistance in Arabidopsis.

[0275] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. QmWOX4 Gene or QmWOX4 The application of a gene-encoded protein in enhancing the radial growth of Arabidopsis stems is characterized by, The QmWOX4 The gene sequence is shown in SEQ ID NO:

1. QmWOX4 The sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The application is through QmWOX4 Overexpression of the gene increases radial growth of Arabidopsis stems.

3. The application according to claim 1, characterized in that, The Arabidopsis thaliana is wild-type Arabidopsis thaliana or Arabidopsis thaliana. wox4 Deletion mutant.

4. A kind containing QmWOX4 The application of gene recombinant vectors in enhancing the radial growth of Arabidopsis stems is characterized by, The QmWOX4 The gene is in the application according to claim 1 QmWOX4 Gene.

5. The application according to claim 4, characterized in that, The recombinant vector is a QmWOX4 Gene recombination was performed on the expression vector pRI101.

6. A substance containing QmWOX4 The application of recombinant bacteria of genes in enhancing radial growth of Arabidopsis stems is characterized by, The QmWOX4 The gene is in the application according to claim 1 QmWOX4 Gene.

7. The application according to claim 6, characterized in that, The recombinant bacteria is... QmWOX4 The gene was recombined into the expression vector pRI101 to construct the recombinant vector, which was then transformed into competent Agrobacterium GV3101 cells to obtain the recombinant bacteria.

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