CN117088952A - Pinus massoniana PmSND4 gene and expression protein and application thereof - Google Patents

Abstract

The invention discloses a Pinus massoniana PmSND4 gene, an expression protein and application thereof, and belongs to the field of plant genetic engineering. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1. The invention takes 2-year-old pinus massoniana leaves as materials, obtains the PmSND4 gene of pinus massoniana through cloning, constructs an over-expression vector pCAMBIA1301-PmSND4 on the basis, and transfers the over-expression vector into mountain new poplar to obtain a transgenic plant. Compared with wild mountain fresh poplar, transgenic mountain fresh Yang Zhu line grows faster; the blade areas of the 3 rd blade, the 6 th blade and the 9 th blade are larger; the plant stem height and the ninth internode stem diameter are both increased significantly; the cell wall showed significant thickening; the thicknesses of the stem xylem and phloem are increased; the lignin, cellulose and hemicellulose content is significantly increased.

Description

A Masson pine PmSND4 gene and its expression protein and application

Technical field

The invention belongs to the field of plant genetic engineering, and more specifically, relates to a Pinus massoniana PmSND4 gene and its expression protein and application.

Background technique

Masson pine (Pinus massoniana Lamb.) is an important strategic tree species for the timber industry in southern China. Masson pine wood is excellent and widely used in construction, road and bridge projects, etc. Its wood has high cellulose content and is a high-quality raw material for pulping and papermaking. It is also an important raw material for man-made fiber boards. Masson pine wood has a wide range of uses, so using advanced technology to cultivate and screen excellent germplasm materials, increase masson pine wood production and improve masson pine wood properties has huge social and economic benefits.

The formation of wood is mainly achieved by the deposition of plant secondary wall materials. The deposits include lignin, hemicellulose and cellulose. The three are cross-linked and combined with each other to form the basic skeleton of plant cells and bear the responsibilities of plant cells. The role of mechanical support. Existing studies have proven that the formation and deposition of plant secondary walls are controlled through an extremely complex cellular regulatory network composed of multiple different transcription factor families. There are three layers of transcription factors in this regulatory network. The top-level regulatory factors are mainly NAC transcription factors, which can regulate downstream transcription factors. The secondary transcription factors can not only be directly activated or inhibited by the top-level transcription factors, 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 layer and secondary transcription factors and bind to the cis-acting elements of structural genes. The secondary transcription factors and third-level 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 respectively and jointly regulate the deposition of lignin, cellulose and hemicellulose.

Members of the NAC transcription factor family are top regulators of 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 functional 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 directional breeding work to increase masson pine wood yield and improve masson pine wood properties.

Contents of the invention

In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Pinus massoniana PmSND4 gene to meet the use requirements. Another technical problem to be solved by the present invention is to provide a protein encoded by the PmSND4 gene of Pinus massoniana. The technical problem to be solved by the present invention is to provide an application of the PmSND4 gene of Pinus massoniana for plant improvement and breeding.

In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

A Masson pine PmSND4 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

The amino acid sequence of the expressed protein of Pinus massoniana PmSND4 gene is shown in SEQ ID NO.2.

The application of Pinus massoniana PmSND4 gene in increasing the growth rate of Populus elata includes the following steps:

(1) Construct a vector for the PmSND4 gene of Pinus massoniana;

(2) Transform the constructed vector of Pinus massoniana PmSND4 gene into the leaves of Populus alba;

(3) Breed and screen to obtain transgenic poplar with accelerated growth rate.

The application of the Pinus massoniana PmSND4 gene in increasing the thickness of the xylem and phloem of the stems of Populus elegans. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO.1.

The application of the Pinus massoniana PmSND4 gene in increasing the height of a Populus sanifera plant or increasing the diameter of the ninth internode or increasing the leaf area. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO.1.

The application of the Pinus massoniana PmSND4 gene in thickening the cell wall between the eighth internodes of Populus alpiniae. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO.1.

The application of the Pinus massoniana PmSND4 gene in increasing the lignin content of Populus asparagus. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO. 1.

The application of the Pinus massoniana PmSND4 gene in increasing the cellulose content of Populus asparagus. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO.1.

The application of the Pinus massoniana PmSND4 gene in increasing the hemicellulose content of Populus elegans. The nucleotide sequence of the Pinus massoniana PmSND4 gene is shown in SEQ ID NO.1.

Compared with the existing technology, the beneficial effects of the present invention are:

The present invention uses 2-year-old Masson pine leaves as materials, obtains the Pinus massoniana PmSND4 gene through cloning, constructs its overexpression vector pCAMBIA1301-PmSND4 on this basis, and transfers it into Populus elata to obtain transgenic plants. Compared with the wild-type Populus elata, the transgenic Populus elata 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 have significantly increased; the cell wall Significant thickening occurred; the thickness of stem xylem and phloem increased; the contents of lignin, cellulose and hemicellulose increased significantly.

Description of the drawings

Figure 1 shows the tissue-specific expression diagram of PmSND4 gene in Masson pine;

Figure 2 is a GUS staining analysis of PmSND4 promoter transgenic Arabidopsis;

Figure 3 is a graph of PmSND4 transcriptional autoactivation test;

Figure 4 shows the genomic DNA electrophoresis detection chart of PmSND4 transgenic Populus elata;

Figure 5 is an expression analysis diagram of PmSND4 transgenic Populus elata;

Figure 6 is a plant phenotypic analysis diagram of PmSND4 transgenic Populus elata;

Figure 7 is an electron microscope scanning image of the stem cross-section of PmSND4 transgenic poplar;

Figure 8 is a tissue staining diagram of PmSND4 transgenic Populus elata and wild-type plants;

Figure 9 shows the content determination of lignin, cellulose and hemicellulose in PmSND4 transgenic poplar;

Figure 10 is a yeast one-hybrid diagram of PmSND4 and PmMYB4 promoters;

Figure 11 shows dual-luciferase fluorescence intensity analysis of PmSND4 and PmMYB4 promoters.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified in the following examples, the technical means used are conventional means well known to those skilled in the art.

The 2-year-old Masson pine used in this experiment was collected from the Arboretum of Nanjing Forestry University, Xuanwu District, Nanjing City, Jiangsu Province.

Example 1

1. Screen the coding reading frame sequence of the PmSND4 gene based on the masson pine transcriptome data (PRJNA655997), and use Oligo 6 to design specific primers to amplify the PmSND4 gene. The primer sequence is:

PmSND4-F: 5'-ATGGAGTGGTTTATCCATGGAA-3',

PmSND4-R: 5'-TCAAATCAGTCCTCCATAGTGG-3'.

2. Target gene amplification

Use Aidlab's EASYspinPlus plant RNA rapid extraction kit to extract total RNA from 2-year-old Masson pine. Use full gold One-StepRT-PCRSuperMix Reverse Transcription Kit reverse-transcribes the extracted RNA into cDNA.

PCR was performed using the reverse-transcribed cDNA of RNA extracted from 2-year-old Masson pine leaves as a template and the specific primers of the PmSND4 gene. The PmSND4 gene open reading frame cloning reaction system is:

The reaction procedure is:

After purifying the PCR product, the PmSND4 gene was obtained, and the pClone007 Blunt Simple Vector Kit was used to connect the target fragment to the Blunt vector. The above vector was transferred into Escherichia coli, and positive single clone colonies were screened and sequenced. The nucleotide sequence of the PmSND4 gene was determined as shown in SEQ ID NO. 1, and the amino acid sequence was shown in SEQ ID NO. 2.

Example 2

1. Use qRT-PCR technology to analyze the PmSND4 gene in 2-year-old Masson pine roots (R), old stems (OS), young stems (YS), old needles (ON), young leaves (YN), terminal buds (TB), Specific expression in eight tissues, xylem (X) and phloem (P), with xylem (X) serving as the control group. Pinus massoniana PmTUA (KM496535.1) was selected as the internal reference gene, and Primer 5.0 software was used to design specific primers for the PmSND4 gene. The primer sequences are as follows:

PmTUA-F: 5'-CAAACTTGGTCCCGTATCCTC-3',

PmTUA-R: 5'-CACAGAAAGCTGCTCATGGTAA-3';

PmSND4-Q-F: 5'-ATTCCACTCCTCATCCTTTA-3',

PmSND4-Q-R: 5'-TGCCTGTATTTTCCTTCGT-3'.

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

After adding the sample, seal the 96-well plate with a sealing film, centrifuge at 3000 rpm at 4°C for 5 min, and put it into a real-time fluorescence quantitative PCR instrument.

The reaction program is a standard program, that is, pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 s, annealing/extension at 60°C for 30 s, 40 cycles, the total volume is 10 μL, and the other settings are based on Hieff Universal Blue qPCR SYBR GreenMaster Mix Instructions. After the reaction, the relative expression level was calculated using the 2 ^-ΔΔCT method, and Excel and GraphPad Prism 9.0 were used for data analysis.

The results are shown in Figure 1. 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), It is expressed in phloem (P), indicating that this gene is commonly expressed in various tissues of Pinus massoniana plants, but the expression level is slightly different. The expression level in old leaves is slightly higher than that in other parts, followed by stems.

Example 3

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

PmSND4-F: 5'-ATGGAGTGGTTTATCCATGGAA-3',

PmSND4-R: 5'-TCAAATCAGTCCTCCATAGTGG-3'.

2. Cloning of PmSND4 gene promoter

Use FastPure Plant DNA Isolation Mini Kit to extract genomic DNA (gDNA) from 2-year-old Masson pine seedlings. For specific steps, refer to the FastPure Plant DNA Isolation Mini Kit instructions.

Using Pinus massoniana gDNA as a template and using specific primers of the PmSND4 gene promoter to perform PCR, the cloning reaction system of the PmSND4 gene promoter is:

PmSND4-F 2.5μL PmSND4-R 2.5μL gDNA template 2.5μL 2×Taq PCR Master Mix 25μL ddH ₂ O Up to 50μL

The reaction procedure is:

After purifying the PCR product, the PmSND4 gene promoter fragment was obtained, and the target fragment was connected to the Blunt vector. The vector after connecting the target fragment was transferred into E. coli, and positive single clone colonies were screened and sequenced. The nucleotide sequence of the PmSND4 gene promoter was determined as shown in SEQ ID NO. 3.

3. GUS staining analysis of PmSND4 promoter

The pBI121-ProPmSND4::GUS vector was constructed using the homologous recombination method, and the successfully constructed recombinant vector was transformed into Agrobacterium tumefaciens EHA105. After the correctly sequenced bacterial liquid was recovered and purified, the inflorescence infection method was used to infect Arabidopsis thaliana inflorescences. Infection, in order to improve the transformation efficiency, each plant can be repeatedly infected 2-3 times.

Collect the above-mentioned T0 generation Arabidopsis thaliana seeds, sterilize them and plant them in MS medium containing Hyg. After two weeks of growth, transfer them to soil for planting to obtain T1 generation Arabidopsis thaliana seeds. Collect T1 generation Arabidopsis thaliana seeds and use the same Methods: Plant them to obtain T2 generation Arabidopsis; fully soak the T2 generation Arabidopsis seedlings in the culture solution containing GUS dye, and culture them overnight at 37°C in the dark; then use 70% ethanol and The plants were treated with 30% glacial acetic acid mixture for 10 minutes, and then 75% ethanol was used to decolorize the plants; after decolorization was completed, sections were made for observation.

The results are shown in Figure 2. There is no staining in wild-type Arabidopsis seedlings (WT), while transgenic Arabidopsis seedlings are stained blue, with deeper staining in the stems and leaf veins. This shows that the PmSND4 gene promoter is universally expressed in various plant tissues, and is highly expressed in leaves and stems.

4. Transcriptional activation activity of PmSND4

The pGBKT7-PmSND4 vector was constructed using the homologous recombination method. The homologous recombination primer sequence is:

pGBKT7-PmSND4-F:

5'-aggccgaattcccggggatccATGGAGTGGTTTATCCATGGAAA-3',

pGBKT7-PmSND4-R:

5'-ggttatgctagttatgcggccgcTCAAATCAGTCCTCCATAGTGGG-3'.

The homologous recombination vector pGBKT7-PmSND4 was transformed into AH109 yeast competent cells (Shanghai Weidi Biotechnology Co., Ltd.). Randomly select clone spot plates from SD/-Trp medium to SD/-Trp/-Leu/-His medium, and use pGBKT7 empty plasmid as a negative control. Incubate at 28°C for 72 hours, and observe whether plaque grows in the experimental group and the control group.

The results are shown in Figure 3. The yeast of the recombinant plasmid of pGBKT7-PmSND4 can only grow on SD/-Leu medium, and the growth status on SD/-Leu/-Trp/-His medium is the same as the negative control pGBKT7 The vectors are consistent, and the plaque color does not change after adding X-α-gal to SD/-Leu/-Trp/-His. Therefore, the test proves that PmSND4 does not have self-activation.

Example 4

1. Construction of overexpression vector of PmSND4 gene

The pCAMBIA1301-PmSND4 plant overexpression vector was constructed using the homologous recombination method. The successfully constructed recombinant vector was transformed into Agrobacterium tumefaciens EHA105, and positive single clone colonies were screened for subsequent transformation experiments. The homologous recombination primer sequence is:

pCAMBIA1301-PmSND4-F:

5'-ggtacccggggatcctctagaATGGAGTGGTTTATCCATGGAAA-3',

pCAMBIA1301-PmSND4-R:

5'-ttaccctcagatctaccatggTCAAATCAGTCCTCCATAGTGGG-3'.

2. Stable transformation of Populus elata by leaf disk method

Inoculate the positive single clone colony containing the pCAMBIA1301-PmSND4 vector into a 15 mL sterile enzyme-free centrifuge tube, add 5 mL of liquid LB medium containing Kan and Rif antibiotics, and culture it in a 28°C shaker at 220 rpm overnight to obtain a bacterial liquid; Transfer 3 mL of bacterial solution into a 250 mL Erlenmeyer flask and add 50 mL of liquid LB medium containing Kan and Rif antibiotics. When the OD value reaches 0.4, centrifuge at 5000 rpm for 30 min at room temperature and discard the supernatant; use an equal volume of MS liquid containing AS. Resuspend the Agrobacterium pellet in the culture medium to obtain the resuspended Agrobacterium bacteria liquid; take the tissue culture seedlings of Populus albaensis that have been grown for about 30 days and are in good growth condition, use scissors to cut off the edge of the leaves, create wounds, and cut them into Place a square leaf of about 1cm x 1cm into the resuspended Agrobacterium bacteria liquid, seal it, and leave it at room temperature for 20 minutes; use sterilized tweezers to take out the leaves from the Agrobacterium bacteria liquid, and use filter paper to absorb the bacteria liquid on the surface of the leaves. Place the leaves face up in the dark medium (differentiation medium + AS) and cultivate in the dark for 2 days; after the dark culture, wash the leaves twice with sterile water in a clean workbench, and wash them with Tim-containing solution for the third time. Wash with sterile water. 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 is completed, clean the leaves with sterile water in a clean workbench. Twice, the third time was washed with sterile water containing Tim. After cleaning, use sterilized absorbent paper to absorb the water, and use screening medium (leaf differentiation medium + Tim (150mg/L) + Kan (25mg/ L)) Continue to culture until callus grows on the leaves, cleaning the leaves once every 7 days; when the leaves grow small green buds, transfer the buds to the seedling growth medium (elongation medium + Tim (150mg/L) + Kan (25 mg/mL)) for 20-25 days. If Agrobacterium colonies appear in the medium, clean the plant materials in time and replace them with new seedling strengthening medium; after the seedlings are cultured, the adventitious shoots are transferred to rooting medium containing Kan resistance. culture medium (rooting medium + Tim (150mg/L) + Kan (25mg/mL)) to induce rooting; take the seedlings of wild type and overexpression lines that have been cultured in the rooting medium for about 40 days, each strain Three seedlings were selected, opened and cultured for 3 days. The plants were taken out, washed with water to remove agar from the roots of the plants, transplanted into flower pots, and placed in a greenhouse for cultivation.

3. Positive test of transgenic mountain poplar

Use the FastPure Plant DNA Isolation Mini Kit to extract the gDNA of transgenic Populus elata (Ⅰ, Ⅱ, Ⅲ) and wild-type Populus elata (negative control, WT). The overexpression vector plasmid (the successfully constructed pCAMBIA1301 recombinant plasmid, CK) was used as a template to identify the overexpression of the PmSND4 gene by PCR.

The results are shown in Figure 4. The three transgenic poplar lines (Ⅰ, Ⅱ, Ⅲ) are all positive plants. After 3 days of domestication and hardening, the seedlings were transferred to soil and continued to grow in the greenhouse.

The RNA of transgenic Populus aspinus was extracted using the Trizol method and reverse transcribed into cDNA. The cDNA obtained by reverse transcription was diluted 20 times and used to analyze the expression level of the PmSND4 gene of transgenic Populus aspinum.

The results are shown in Figure 5. The expression levels of the transgenic Populus elata strains (Ⅰ, Ⅱ, and Ⅲ) are all higher than those of the wild-type Populus elegans (WT), and the highest expression level (II) is 60% of the wild type. times.

4. Phenotypic analysis of transgenic poplar

Three transgenic Populus elata strains (Ⅰ, Ⅱ, Ⅲ) were selected as the test group and the untransformed wild-type Populus elegans (WT) was used as the control. After 90 days of growth, the growth rate and leaf area (3rd and 6th) were observed. , 9 leaves), plant height and the growth status of the ninth internode diameter.

The results are shown in Figure 6. Compared with the wild-type Populus elegans (WT), the transgenic Populus elata strains (Ⅰ, Ⅱ, Ⅲ) grew faster (Figure 6A); the 3rd, 6th, and 9th leaves were tested respectively. Observation, the results also showed that the leaf area of the transgenic Populus elegans line was larger (Figure 6B); statistical analysis of the plant height and the diameter of the ninth internode also showed that the stem height of the transgenic Populus elegans strain (Figure 6C) and Stem diameter (Figure 6D) showed a significant increase.

Take the 90-d wild-type and transgenic Populus aspinis strains, remove the 8th internode and quickly put them into FAA fixative (take 5 mL each of 38% formaldehyde solution and glacial acetic acid solution, add 90 mL of 70% alcohol solution, up and down Mix by inversion), fixed for more than 24 hours, dried and sliced, and the morphology of each xylem cell was analyzed using a scanning electron microscope (Scanning electron microscopy, SEM) and image analysis software IMAGEJ (https://imagej.nih.gov/ij/) Perform quantitative analysis.

The results are shown in Figure 7. The cell walls of the transgenic poplars were significantly thickened.

5. Toluidine blue tissue staining

Using transgenic poplar stem segments as materials, tissue staining with toluidine blue was performed, washed with water, and observed under a microscope. For specific methods, refer to Solarbio's plant tissue staining solution (toluidine blue method) instructions.

The results are shown in Figure 8. Compared with the wild type, the stem xylem and phloem thickness of the transgenic Aspen poplar increased.

6. Determination of the content of secondary wall components of positive transgenic poplar lines

Use the sulfuric acid method to determine lignin content. The specific steps are as follows:

Take the complete stem trunks of wild-type and positive transgenic Populus aspermifera strains grown for 90 days as materials, dry them in an oven to constant weight, grind them into powder, and place 0.1g in a centrifuge tube. Set three replicates for each sample; add 10 mL of acetic acid (CH ₃ COOH) solution (1%) to the above-mentioned centrifuge tube, shake and mix well, then centrifuge, discard the supernatant, and wash the precipitate once with 5 mL of CH ₃ COOH solution; add 3.5 mL of Mix ethanol (CH ₃ CH ₂ OH) and diethyl ether (C ₂ H ₅ OC ₂ H ₅ ) in equal volumes, let it stand for 3 minutes, discard the supernatant, and repeat this step three times; dry the precipitate in the centrifuge tube in a boiling water bath, dry and centrifuge Add 72% sulfuric acid (H ₂ SO ₄ ) solution to the tube, use a glass rod to stir and mix, and let stand at room temperature for 16 hours, waiting for the cellulose to completely dissolve; add 10 mL of sterile water, mix with a glass rod, and then place in a boiling water bath for 5 minutes and cool to At room temperature, add 5 mL of sterile water and 0.5 mL of barium chloride (BaCl ₂ ) solution (10%), shake and mix, and centrifuge at 3000 rpm for 5 min. After centrifugation, wash the precipitate twice with sterile water; add 10 mL of 10% sulfuric acid respectively. solution and 0.1 mol/L potassium dichromate (K ₂ Cr ₂ O ₇ ) solution, boiling water bath for 15 minutes, use a glass rod to stir continuously during the boiling water bath, cool and set aside; transfer the cooled sample to a beaker and add 20% 5 mL of potassium iodide solution (KI) and 1 mL of 0.5% starch solution were titrated using 0.2 mol/L sodium thiosulfate (Na ₂ S ₂ O ₃ ). The control group used 10 mL of 10% sulfuric acid solution and 0.1 mol/L Titrate the potassium dichromate solution separately.

The calculation formula of lignin content (%) is:

In this calculation formula, k represents the concentration of Na ₂ S ₂ O ₃ (mol/L); a represents the volume of Na ₂ S ₂ O ₃ consumed in the blank titration (mL); b represents the Na ₂ S consumed in the titration. The volume of ₂ O ₃ (mL); 48 indicates that 1 mol of lignin (represented by C ₁₁ H ₁₂ O ₄ ) is equivalent to the titer of Na ₂ S ₂ O ₃ ; n is the mass of the sample powder (g).

The cellulose content was determined using the anthrone sulfate colorimetric method, and the hemicellulose content was determined using the hydrochloric acid hydrolysis method.

In the above content determination, three technical repeats and three biological repeats were taken from each positive transgenic line, GraphPad Prism9.0 was used for data analysis, and the significance of the results was tested using T-test.

The results are shown in Figure 9. Compared with the wild-type Populus elegans strain, the content of lignin and hemicellulose in the positive transgenic Populus elegans strain was significantly increased, and the content of cellulose was also increased. The results show that The PmSND4 gene can promote the synthesis of lignin, cellulose and hemicellulose.

7. Verification of yeast single-hybrid interaction

Use the homologous recombination method to construct the PGADT7-PmSND4 and pHis2-ProPmMYB4 vectors, transform the successfully constructed recombinant vectors into competent yeast Y187, and screen to obtain positive clone colonies; pick the positive clone colonies from the plates and use them 1mL of sterile water was resuspended and then spotted on the corresponding SD/–His 3-AT-deficient medium with different concentrations (10, 20, 30, 40, 50, 60, 70mM), and cultured at 28°C for 3 days. Observe its growth status; resuspend the single colony that was successfully verified above using the same method, pipet 5 μL and spot it in the culture medium of SD/-Trp/-Leu/-His and the corresponding concentration of 3-AT, 28°C Cultivate for 4 days and observe its growth status. PGADT7-PmSND4+pHis2 was used as a negative control.

The results are shown in Figure 10. The growth status of negative control PGADT7-PmSND4+pHis2 on SD/-Trp/-Leu/-His medium was completely inhibited, while PGADT7-PmSND4+pHis2-Pro-PmMYB4 could be grown on SD/- It grew normally on Trp/-Leu/-His medium, and the results showed that PmSND4 could bind to the promoter of PmMYB4.

8. Dual luciferase test

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

The results are shown in Figure 11. 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 (10)

1. A nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

2. The expressed protein of the Pinus massoniana PmSND4 gene as set forth in claim 1, wherein the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2.

3. The application of the Pinus massoniana PmSND4 gene in improving the growth rate of the Pinus massoniana, wherein the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

4. The use of the PmSND4 gene of pinus massoniana in improving the growth rate of pinus massoniana according to claim 3, comprising the steps of:

(1) Constructing a vector of a Pinus massoniana PmSND4 gene;

(2) Transforming the constructed vector of the Pinus massoniana PmSND4 gene into leaves of the Pinus massoniana;

(3) And (5) culturing and screening to obtain the transgenic mountain new poplar with accelerated growth speed.

5. The application of the Pinus massoniana PmSND4 gene in increasing the thickness of the xylem and phloem of Pinus massoniana Yang Jing is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

6. The application of the Pinus massoniana PmSND4 gene in increasing the plant height of Pinus massoniana or increasing the diameter of the ninth internode or increasing the leaf area is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

7. The application of the Pinus massoniana PmSND4 gene in thickening the cell wall between the eight nodes of Pinus massoniana Yang Di is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

8. The application of the Pinus massoniana PmSND4 gene in increasing the woody substance content of the Pinus massoniana is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

9. The application of the Pinus massoniana PmSND4 gene in increasing the cellulose content of Pinus massoniana is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.

10. The application of the Pinus massoniana PmSND4 gene in increasing the cellulose content of Pinus massoniana Yang Ban is provided, and the nucleotide sequence of the Pinus massoniana PmSND4 gene is shown as SEQ ID NO. 1.