A Masson pine PmSND4 gene and its expression protein and application

Abstract

The invention discloses a Masson pine PmSND4 gene and its expression protein and application, and belongs to the field of plant genetic engineering. The Masson pine PmSND4 gene of the invention has a nucleotide sequence as shown in SEQ ID NO.1. The invention uses 2-year-old Masson pine leaves as materials, obtains the Masson pine PmSND4 gene by cloning, constructs its overexpression vector pCAMBIA1301-PmSND4 on this basis, and transfers it into Populus dasyphylla to obtain transgenic plants. Compared with the wild-type Populus dasyphylla, the transgenic Populus dasyphylla strain grows faster; the leaf area of ​​the 3rd, 6th and 9th leaves is larger; the stem height of the strain and the stem diameter of the ninth internode are significantly increased; the cell wall is significantly thickened; the thickness of the stem xylem and phloem are increased; the content of lignin, cellulose and hemicellulose is significantly increased.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and more specifically relates to a Masson pine PmSND4 gene and its expression protein and application. Background Art

[0002] Masson pine (Pinus massoniana Lamb.) is an important strategic tree species in the timber industry in southern China. Masson pine wood is of high quality and is widely used in construction, road and bridge engineering, etc.; its wood cellulose content is high, it is a high-quality pulp and paper raw material, and is also an important raw material for man-made fiberboard. Masson pine wood has a wide range of uses, so using advanced technology to cultivate and select excellent germplasm materials, increase the yield of Masson pine wood, and improve the wood properties of Masson pine wood has huge social and economic benefits.

[0003] The formation of wood is mainly achieved by the deposition of plant secondary wall materials, including lignin, hemicellulose and cellulose. The three are cross-linked and combined to form the basic skeleton of plant cells and play the role of mechanical support for plant cells. Existing studies have shown that the formation and deposition of plant secondary walls are controlled by an extremely complex cell regulatory network composed of multiple different transcription factor families. There are three layers of transcription factors in this regulatory network. The top regulatory factor is mainly NAC transcription factor, which can regulate downstream transcription factors; secondary transcription factors can not only be directly activated or inhibited by the top transcription factor, but also regulate downstream transcription factors or directly bind to the cis-acting elements of structural genes to promote or inhibit gene expression; the three-layer transcription factors are regulated by the upstream top and secondary transcription factors and bind to the cis-acting elements of structural genes. Secondary transcription factors and the third-layer transcription factors are mainly MYB transcription factors, which can bind to the promoter cis-acting elements of SCW synthesis genes. NAC transcription factors and MYB transcription factors play different roles and jointly regulate the deposition of lignin, cellulose and hemicellulose.

[0004] NAC transcription factor family members are top regulatory factors for xylem cell development and deposition of plant secondary cell wall components, and play an important role in the transcriptional regulatory network of wood formation. The present invention discloses the sequence and function research of Masson pine PmSND4, which is helpful for in-depth research on the function of Masson pine PmSND4 gene, and provides a molecular means and basis for the directional breeding work of increasing Masson pine wood yield and improving Masson pine wood properties. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Masson pine PmSND4 gene to meet the use requirements. Another technical problem to be solved by the present invention is to provide a Masson pine PmSND4 gene encoding protein. Another technical problem to be solved by the present invention is to provide an application of the Masson pine PmSND4 gene for plant improvement and breeding.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A Masson pine PmSND4 gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the Masson pine PmSND4 gene is shown in SEQ ID NO.2.

[0009] The application of the Masson pine PmSND4 gene in improving the growth rate of Populus davidianus includes the following steps:

[0010] (1) Constructing a vector for the Masson pine PmSND4 gene;

[0011] (2) Transforming the constructed vector of Masson pine PmSND4 gene into Populus davidianus leaves;

[0012] (3) Cultivate and screen transgenic Populus shanxinensis with accelerated growth rate.

[0013] The application of the Masson pine PmSND4 gene in increasing the thickness of xylem and phloem in the stem of Populus dasyphylla, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

[0014] The application of the Masson pine PmSND4 gene in increasing the height of Populus davidianus plants or the diameter of the ninth internode or the leaf area, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

[0015] Application of the Masson pine PmSND4 gene in thickening the cell wall of the eighth internode of Populus davidianus, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

[0016] The application of the Masson pine PmSND4 gene in increasing the lignin content of Populus sutchuenensis, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

[0017] The application of the Masson pine PmSND4 gene in increasing the cellulose content of Populus dasyphylla, the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

[0018] The application of the Masson pine PmSND4 gene in increasing the hemicellulose content of Populus davidianus, the Masson pine PmSND4 gene nucleotide sequence is shown in SEQ ID NO.1.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention uses 2-year-old Masson pine leaves as materials, obtains the Masson pine PmSND4 gene by cloning, constructs its overexpression vector pCAMBIA1301-PmSND4 on this basis, and transfers it into Populus dasyphylla to obtain transgenic plants. Compared with the wild-type Populus dasyphylla, the transgenic Populus dasyphylla strain grows faster; the leaf areas of the 3rd, 6th and 9th leaves are larger; the stem height and the stem diameter of the ninth internode of the strain are significantly increased; the cell wall is significantly thickened; the thickness of the stem xylem and phloem are increased; the content of lignin, cellulose and hemicellulose is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the tissue-specific expression diagram of PmSND4 gene in Masson pine;

[0022] Figure 2 This is a GUS staining analysis of PmSND4 promoter transgenic Arabidopsis;

[0023] Figure 3 This is a diagram of the PmSND4 transcriptional self-activation assay;

[0024] Figure 4 This is the electrophoresis detection diagram of the genomic DNA of the PmSND4 transgenic Populus shanxinensis;

[0025] Figure 5 This is the expression analysis diagram of PmSND4 transgenic Populus shanxinensis;

[0026] Figure 6 This is a plant phenotype analysis diagram of PmSND4 transgenic Populus shanxinensis;

[0027] Figure 7 This is a scanning electron microscope image of the stem cross section of the PmSND4 transgenic Populus shanxinensis;

[0028] Figure 8 This is the tissue staining picture of PmSND4 transgenic Populus shanxinensis and wild-type plants;

[0029] Fig. 9 This is the determination diagram of lignin, cellulose and hemicellulose content in PmSND4 transgenic Populus shanxinensis;

[0030] Fig.10 Yeast one-hybrid map of PmSND4 and PmMYB4 promoters;

[0031] Fig.11 Dual luciferase fluorescence intensity analysis of PmSND4 and PmMYB4 promoters. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0033] The two-year-old Masson pine used in this experiment was collected from the Arboretum of Nanjing Forestry University in Xuanwu District, Nanjing City, Jiangsu Province.

[0034] Example 1

[0035] 1. The coding reading frame sequence of the PmSND4 gene was obtained by screening according to the Masson pine transcriptome data (PRJNA655997), and specific primers were designed using Oligo 6 to amplify the PmSND4 gene. The primer sequences were:

[0036] PmSND4-F: 5'-ATGGAGTGGTTTATCCATGGAA-3',

[0037] PmSND4-R: 5'-TCAAATCAGTCCTCCATAGTGG-3'.

[0038] 2. Target gene amplification

[0039] Total RNA from two-year-old Pinus massoniana was extracted using the EASYspinPlus Plant RNA Rapid Extraction Kit from Aidlab. One-Step RT-PCR SuperMix Reverse Transcription Kit reverse transcribes the extracted RNA into cDNA.

[0040] PCR was performed using cDNA extracted from RNA reverse transcribed from leaves of two-year-old Masson pine as a template and specific primers of the PmSND4 gene. The open reading frame cloning reaction system of the PmSND4 gene was as follows:

[0041]

[0042]

[0043] The reaction procedure is:

[0044]

[0045] The PCR product was purified to obtain the PmSND4 gene, and the target fragment was connected to the Blunt vector using the pClone007 Blunt Simple Vector Kit. The above vector was transformed into Escherichia coli, and positive monoclonal colonies were screened for sequencing. The nucleotide sequence of the PmSND4 gene was measured as shown in SEQ ID NO.1, and the amino acid sequence was shown in SEQ ID NO.2.

[0046] Example 2

[0047] 1. qRT-PCR technology was used to analyze the specific expression of PmSND4 gene in eight tissues of two-year-old Masson pine, including root (R), old stem (OS), young stem (YS), old needle (ON), young leaf (YN), terminal bud (TB), xylem (X) and phloem (P). Xylem (X) was used as the control group. Masson pine PmTUA (KM496535.1) was selected as the internal reference gene, and Primer 5.0 software was used to design specific primers for PmSND4 gene. The primer sequences are as follows:

[0048] PmTUA-F: 5'-CAAACTTGGTCCCGTATCCTC-3',

[0049] PmTUA-R: 5'-CACAGAAAGCTGCTCATGGTAA-3';

[0050] PmSND4-QF: 5'-ATTCCACTCCTCATCCTTTA-3',

[0051] PmSND4-QR:5'-TGCCTGTATTTTCCTTCGT-3'.

[0052] Add the following reaction system to a sterile, enzyme-free 96-well PCR plate (operate on ice):

[0053]

[0054]

[0055] After adding the samples, the 96-well plate was sealed with a sealing film, centrifuged at 3000 rpm at 4°C for 5 min, and placed in a real-time fluorescence quantitative PCR instrument.

[0056] The reaction procedure was the standard procedure, i.e., 95°C pre-denaturation for 2 min, 95°C denaturation for 10 s, 60°C annealing / extension for 30 s, 40 cycles, with a total volume of 10 μL. The rest of the settings were based on the Hieff Universal Blue qPCR SYBR Green Master Mix Instructions. -△△CT The relative expression levels were calculated using the method of , and data were analyzed using Excel and GraphPadPrism 9.0.

[0057] The results are as follows Figure 1 As shown, the PmSND4 gene is expressed in terminal buds (TB), roots (R), old stems (OS), young stems (YS), old leaves (ON), young leaves (YN), xylem (X), and phloem (P), indicating that the gene is widely expressed in various tissues of Masson pine plants, but the expression levels are slightly different. The expression level in old leaves is slightly higher than that in other parts, followed by the stems.

[0058] Example 3

[0059] 1. Refer to the Pinus tabulaeformis genome data (doi: https: / / doi.org / 10.1016 / j.cell.2021.12.006) to screen and obtain the promoter sequence of the SND4 gene, and use Oligo 6 to design specific primers to amplify the PmSND4 gene. The primer sequence is:

[0060] PmSND4-F: 5'-ATGGAGTGGTTTATCCATGGAA-3',

[0061] PmSND4-R: 5'-TCAAATCAGTCCTCCATAGTGG-3'.

[0062] 2. Cloning of PmSND4 gene promoter

[0063] The genomic DNA (gDNA) of 2-year-old Masson pine seedlings was extracted using the FastPure Plant DNA Isolation Mini Kit. The specific steps were referred to the FastPure Plant DNA Isolation Mini Kit instruction manual.

[0064] Using Pinus massoniana gDNA as a template, PCR was performed using specific primers for the PmSND4 gene promoter. The cloning reaction system for the PmSND4 gene promoter was:

[0065] PmSND4-F 2.5μL PmSND4-R 2.5μL gDNA template 2.5μL 2×Taq PCR Master Mix 25μL <![CDATA[ddH2O]]> Up to 50μL

[0066] The reaction procedure is:

[0067]

[0068] After PCR product purification, the PmSND4 gene promoter fragment was obtained, and the target fragment was connected to the Blunt vector. The vector connected with the target fragment was transformed into Escherichia coli, and positive monoclonal colonies were screened for sequencing, and the nucleotide sequence of the PmSND4 gene promoter was measured as shown in SEQ ID NO.3.

[0069] 3. GUS staining analysis of PmSND4 promoter

[0070] The pBI121-ProPmSND4::GUS vector was constructed using homologous recombination. The successfully constructed recombinant vector was transformed into Agrobacterium tumefaciens EHA105. The correctly sequenced bacterial solution was recovered and purified, and then the Arabidopsis inflorescence was infected using the inflorescence infection method. In order to improve the transformation efficiency, each plant can be infected 2-3 times.

[0071] The T0 generation Arabidopsis seeds were collected, sterilized and planted in MS culture medium containing Hyg, and then grown for two weeks before being transferred to soil for planting to obtain T1 generation Arabidopsis; T1 generation Arabidopsis seeds were collected and planted in the same way to obtain T2 generation Arabidopsis; T2 generation Arabidopsis seedlings were fully immersed in culture medium containing GUS dye, and cultured overnight at 37°C in the dark; then the plants were treated with a mixture of 70% ethanol and 30% glacial acetic acid for 10 minutes, and then the plants were decolorized with 75% ethanol; after the decolorization was completed, sections were made for observation.

[0072] The results are as follows Figure 2 As shown, there is no staining in wild-type Arabidopsis seedlings (WT), while transgenic Arabidopsis seedlings are stained blue, with deeper staining in stems and leaf veins. This indicates that the PmSND4 gene promoter is widely expressed in various plant tissues, and is highly expressed in leaves and stems.

[0073] 4. Transcriptional activation activity of PmSND4

[0074] The pGBKT7-PmSND4 vector was constructed by homologous recombination. The sequences of the homologous recombination primers are:

[0075] pGBKT7-PmSND4-F:

[0076] 5'-aggccgaattcccggggatccATGGAGTGGTTTATCCATGGAAA-3',

[0077] pGBKT7-PmSND4-R:

[0078] 5'-ggttatgctagttatgcggccgcTCAAATCAGTCCTCCATAGTGGG-3'.

[0079] The homologous recombination vector pGBKT7-PmSND4 was transformed into AH109 yeast competent cells (Shanghai Weidi Biotechnology Co., Ltd.). The clones were randomly selected from the SD / -Trp medium and plated on the SD / -Trp / -Leu / -His medium. The pGBKT7 empty plasmid was used as a negative control. The cells were cultured at 28°C for 72 hours, and the plaques of the experimental group and the control group were observed to see if they grew.

[0080] The results are as follows Figure 3 As shown, yeast expressing the recombinant plasmid of pGBKT7-PmSND4 can only grow on SD / -Leu medium, while its growth state on SD / -Leu / -Trp / -His medium is consistent with that of the negative control pGBKT7 vector. Moreover, the color of the bacterial plaques did not change after the addition of X-α-gal to SD / -Leu / -Trp / -His. Therefore, the experiment proves that PmSND4 does not have self-activation.

[0081] Example 4

[0082] 1. Construction of overexpression vector of PmSND4 gene

[0083] The pCAMBIA1301-PmSND4 plant overexpression vector was constructed by homologous recombination, and the successfully constructed recombinant vector was transformed into Agrobacterium tumefaciens EHA105, and positive monoclonal colonies were screened for subsequent transformation experiments. The homologous recombination primer sequences are:

[0084] pCAMBIA1301-PmSND4-F:

[0085] 5'-ggtacccggggatcctctagaATGGAGTGGTTTATCCATGGAAA-3',

[0086] pCAMBIA1301-PmSND4-R:

[0087] 5'-ttaccctcagatctaccatggTCAAATCAGTCCTCCATAGTGGG-3'.

[0088] 2. Stable transformation of Populus davidianus by leaf disc method

[0089] The positive monoclonal colony containing the pCAMBIA1301-PmSND4 vector was inoculated into a 15 mL sterile enzyme-free centrifuge tube, and 5 mL of liquid LB medium containing Kan and Rif antibiotics was added. The culture was incubated overnight at 220 rpm in a shaker at 28 ° C to obtain a bacterial solution; 3 mL of the bacterial solution was transferred to a 250 mL conical flask and 50 mL of liquid LB medium containing Kan and Rif antibiotics was added. When the OD value was 0.4, the solution was centrifuged at 5000 rpm for 30 min at room temperature and the supernatant was discarded; an equal volume of MS liquid medium containing AS was used to resuspend the Agrobacterium tumefaciens precipitate to obtain a resuspended Agrobacterium liquid; take a Populus shanxin tissue culture seedling that has grown for about 30 days and is in good growth condition, cut the edge of its leaf with scissors to make a wound, and cut it into a square leaf of about 1cm×1cm, put it into the resuspended Agrobacterium liquid, seal it, and leave it at room temperature for 20 minutes; use sterilized tweezers to take out the leaf in the Agrobacterium liquid, use filter paper to absorb the liquid on the surface of the leaf, place the leaf with the front side facing up in a dark culture medium (differentiation medium + AS), and culture it in the dark for 2 days; after the dark culture is completed, wash the leaf twice with sterile water in the clean bench, and wash it with sterile water containing Tim for the third time, and wash it dry After cleaning, use sterilized absorbent paper to absorb the water, and use light culture medium (differentiation medium + Tim) to culture for 7 days; after the light culture, wash the leaves twice with sterile water in the clean bench, and wash them with sterile water containing Tim for the third time. After washing, use sterilized absorbent paper to absorb the water, and continue to culture with screening medium (leaf differentiation medium + Tim (150mg / L) + Kan (25mg / L)) until the leaves grow callus tissue, and wash the leaves once every 7 days; when the leaves grow green buds, transfer the buds to the seedling medium (elongation medium + Tim (150mg / L )+Kan(25mg / mL)) for 20-25d. If Agrobacterium colonies appear in the culture medium, clean the plant materials in time and replace with new seedling medium; after seedling culture, transfer the adventitious buds to rooting medium containing Kan resistance (rooting medium+Tim(150mg / L)+Kan(25mg / mL)) to induce rooting; take the seedlings of the wild type and overexpression strains cultured in the rooting medium for about 40d, select three seedlings from each strain, take out the plants after opening the lid for 3d of culture, wash off the agar at the roots of the plants with clean water, transplant them into pots, and place them in a greenhouse for culture.

[0090] 3. Positive detection of genetically modified Populus shanxin

[0091] The FastPure Plant DNA Isolation Mini Kit was used to extract gDNA of transgenic Populus davidiana (Ⅰ, Ⅱ, Ⅲ) and wild-type Populus davidiana (negative control, WT). The overexpression of the PmSND4 gene was identified by PCR using the gDNA of wild-type Populus davidiana and transgenic Populus davidiana and the overexpression vector plasmid (successfully constructed pCAMBIA1301 recombinant plasmid, CK) as templates.

[0092] The results are as follows Figure 4 As shown, the three transgenic Populus shanxinensis lines (Ⅰ, Ⅱ, Ⅲ) were all positive plants. After 3 days of acclimatization and hardening, the seedlings were transferred to soil and continued to grow in the greenhouse.

[0093] The RNA of transgenic Populus davidianus was extracted by Trizol method and reverse transcribed into cDNA. The cDNA obtained by reverse transcription was diluted 20 times and used for expression analysis of PmSND4 gene in transgenic Populus davidianus.

[0094] The results are as follows Figure 5 As shown, the expression levels of transgenic Populus davidiana lines (Ⅰ, Ⅱ, Ⅲ) were higher than those of wild-type Populus davidiana (WT), and the highest expression level of the line (Ⅱ) was 60 times that of the wild-type.

[0095] 4. Phenotypic analysis of transgenic Populus shanxinensis

[0096] Three transgenic Populus davidiana lines (Ⅰ, Ⅱ, Ⅲ) were selected as the experimental group and the untransformed wild-type Populus davidiana (WT) as the control. After 90 days of growth, the growth rate, leaf area (3rd, 6th, and 9th leaves), plant height, and the diameter of the ninth internode were observed and statistically analyzed.

[0097] The results are as follows Figure 6 As shown in Figure 2, compared with the wild-type P. shanxinensis (WT), the transgenic P. shanxinensis lines (Ⅰ, Ⅱ, Ⅲ) grew faster ( Figure 6 A); the 3rd, 6th and 9th leaves were observed respectively, and the results also showed that the leaf area of ​​the transgenic Populus shanxin strain was larger ( Figure 6 B); Statistical analysis of plant height and the diameter of the ninth internode also showed that the stem height of the transgenic Populus shanxinensis lines ( Figure 6 C) and stem diameter ( Figure 6 D) all showed significant increases.

[0098] The 8th internode of the 90-day-old wild-type and transgenic Populus dahliae strains was removed and quickly placed in FAA fixative (5 mL each of 38% formaldehyde solution and glacial acetic acid solution, added to 90 mL of 70% alcohol solution, and mixed by inversion) and fixed for more than 24 h. After drying, the slices were cut and the morphology of each xylem cell was quantitatively analyzed using scanning electron microscopy (SEM) and image analysis software IMAGEJ (https: / / imagej.nih.gov / ij / ).

[0099] The results are as follows Figure 7 As shown, the cell walls of transgenic Populus davidianus were significantly thickened.

[0100] 5. Toluidine blue tissue staining

[0101] The transgenic Populus shanxin stem segments were used as materials for toluidine blue tissue staining, and then washed with water and observed under a microscope. The specific method is referred to the instructions of Solarbio's plant tissue staining solution (toluidine blue method).

[0102] The results are as follows Figure 8 As shown, compared with the wild type, the thickness of stem xylem and phloem of transgenic Populus shanxinensis were increased.

[0103] 6. Determination of secondary wall component content in positive transgenic Populus shanxin strains

[0104] The lignin content was determined using the sulfuric acid method. The specific steps are as follows:

[0105] Take the complete stem trunk of the wild-type and positive transgenic Shanxin Populus strains grown for 90 days as materials, use an oven to dry them to constant weight, grind them into powder, take 0.1g and place them in a centrifuge tube. Set up three replicates for each sample; add 10mL of acetic acid (CH3COOH) solution (1%) to the above centrifuge tube, shake and mix, then centrifuge, discard the supernatant, and wash the precipitate once with 5mL of CH3COOH solution; add 3.5mL of ethanol (CH3CH2OH) and ether (C2H5OC2H5) of equal volume, let stand for 3min and discard the supernatant, repeat this step three times; dry the precipitate in a centrifuge tube in a boiling water bath, add 72% sulfuric acid (H2SO4) solution to the centrifuge tube after drying, stir and mix with a glass rod, let stand at room temperature for 16h, and wait for the cellulose to be completely dissolved; add 10mL of sterile water and mix with a glass rod, then boil in a water bath for 5min, cool to room temperature, add 5mL of sterile water and 0.5mL of barium chloride (BaCl2) solution (10%), shake and mix for 300 The mixture was centrifuged at 0 rpm for 5 min, and the precipitate was washed twice with sterile water after the centrifugation. 10 mL of 10% sulfuric acid solution and 0.1 mol / L potassium dichromate (K2Cr2O7) solution were added respectively, and the mixture was placed in a boiling water bath for 15 min. During the boiling water bath, a glass rod was used to stir continuously, and the mixture was cooled for use. The cooled sample was transferred into a beaker and 5 mL of 20% potassium iodide solution (KI) and 1 mL of 0.5% starch solution were added, and the mixture was titrated with 0.2 mol / L sodium thiosulfate (Na2S2O3). The control group was titrated separately with 10 mL of 10% sulfuric acid solution and 0.1 mol / L potassium dichromate solution.

[0106] The calculation formula of lignin content (%) is:

[0107]

[0108] In this calculation formula, k represents the concentration of Na2S2O3 (mol / L); a represents the volume of Na2S2O3 consumed in the blank titration (mL); b represents the volume of Na2S2O3 consumed in the titration (mL); 48 represents 1 mol of lignin (in C 11 H 12 O4 as a representative) is equivalent to the titer of Na2S2O3; n is the mass of the sample powder (g).

[0109] The cellulose content was determined by the anthrone sulfate colorimetric method, and the hemicellulose content was determined by the hydrochloric acid hydrolysis method.

[0110] In the above content determination, three technical replicates and three biological replicates were taken for each positive transgenic line, and data analysis was performed using GraphPad Prism 9.0. The results were tested for significance using T-test.

[0111] The results are as follows Fig. 9 As shown, compared with the wild-type Populus shanxin strain, the lignin and hemicellulose contents in the positive transgenic Populus shanxin strain were significantly increased, and the cellulose content also increased. The results showed that the PmSND4 gene can promote the synthesis of lignin, cellulose and hemicellulose.

[0112] 7. Yeast one-hybrid interaction verification

[0113] PGADT7-PmSND4 and pHis2-ProPmMYB4 vectors were constructed by homologous recombination, and the successfully constructed recombinant vectors were transformed into yeast competent Y187 to screen for positive clones. Single colonies of positive clones were picked from the plates, resuspended with 1 mL of sterile water, and spotted on the corresponding SD / -His but 3-AT deficient medium with different concentrations (10, 20, 30, 40, 50, 60, 70 mM), and cultured at 28°C for 3 days. Observe their growth status; resuspend the single colonies successfully verified above in the same way, and take 5 μL and spot them in SD / -Trp / -Leu / -His and corresponding concentrations of 3-AT medium, culture at 28°C for 4 days, and observe their growth status. PGADT7-PmSND4+pHis2 was used as a negative control.

[0114] The results are as follows Fig.10 As shown, the growth state of the negative control PGADT7-PmSND4+pHis2 on SD / -Trp / -Leu / -His medium was completely inhibited, while PGADT7-PmSND4+pHis2-Pro-PmMYB4 could grow normally on SD / -Trp / -Leu / -His medium. The results showed that PmSND4 can bind to the promoter of PmMYB4.

[0115] 8. Dual luciferase assay

[0116] The pGreenⅡ-0800-LUC-ProPmMYB4 and pGreenⅡ-62-SK-PmSND4 vectors were constructed by homologous recombination, and the successfully constructed recombinant vectors were transformed into Agrobacterium tumefaciens EHA105, and positive clones were screened to obtain single colonies; the transient expression of the dual luciferase test was referenced to the Yisheng Dual Luciferase Reporter Gene Assay Kit. The control group of ProPmMYB4-LUC+SK was used as the standard and set to 1.

[0117] The results are as follows Fig.11As shown, the promoter of the PmMYB4 gene can bind to PmSND4, and the fluorescence intensity of ProPmMYB4-LUC+PmSND4-SK is 6 times that of the control group ProPmMYB4-LUC+SK.

Claims

1. A Masson Pine PmSND4 The gene has a nucleotide sequence as shown in SEQ ID NO.

1.

2. The Masson pine of claim 1 PmSND4 The amino acid sequence of the expressed protein of the gene is shown in SEQ ID NO.

2.

3. Masson pine PmSND4 Application of genes in increasing the growth rate of Pinus massoniana PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. Masson pine according to claim 3 PmSND4 The application of the gene in improving the growth rate of Populus serrata is characterized in that: The following steps are involved: (1) Construction of Masson pine PmSND4 Gene carrier; (2) The constructed Masson pine PmSND4 The gene vector was transformed into the leaves of Populus dasyphylla; (3) Cultivate and screen transgenic Populus dasyphylla with accelerated growth rate.

5. Masson pine PmSND4 Application of genes in increasing the thickness of xylem and phloem in the stem of Populus serrata, the Masson pine PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. Masson pine PmSND4 Application of genes in increasing the height of mountain poplar plants or the diameter of the ninth internode or the area of ​​leaves, the Masson pine PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. Masson pine PmSND4 Application of genes in thickening the cell wall of the eighth internode of Populus serrata, Pinus massoniana PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. Masson pine PmSND4 Application of genes in increasing the lignin content of mountain poplar, the masson pine PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. Masson Pine PmSND4 Application of genes in increasing the cellulose content of mountain poplar, the masson pine PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. Masson pine PmSND4 Application of genes in increasing the hemicellulose content of Pinus massoniana PmSND4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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