CN117088952B

CN117088952B - A Masson pine PmSND4 gene and its expression protein and application

Info

Publication number: CN117088952B
Application number: CN202310925452.XA
Authority: CN
Inventors: 姚圣; 季孔庶; 娄轩; 王登宝; 程翔; 张景景
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2023-07-26
Filing date: 2023-07-26
Publication date: 2025-03-25
Anticipated expiration: 2043-07-26
Also published as: CN117088952A

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

Pinus massoniana PmSND gene and expression protein and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and in particular relates to a Pinus massoniana PmSND gene, an expression protein and application thereof.

Background

Pinus massoniana (Pinus massoniana lamb.) is an important strategic tree species for the wood industry in south China. The pinus massoniana wood is excellent and widely used for building, road and bridge engineering and other materials, has high cellulose content, is a high-quality pulping and papermaking raw material, and is also an important raw material of artificial fiber boards. The pinus massoniana wood has wide application, so that the advanced technology is utilized to cultivate and screen excellent germplasm materials, and the method for improving the yield of pinus massoniana wood and improving the lumber property of pinus massoniana has great social benefit and economic benefit.

The formation of wood is mainly realized by the deposition of plant secondary wall substances, and the deposition comprises lignin, hemicellulose and cellulose which are mutually crosslinked and combined to form a basic skeleton of plant cells and bear the function of mechanical support of the plant cells. Prior studies have demonstrated that plant secondary wall formation and deposition is controlled by a very complex cell regulatory network consisting of a number of different families of transcription factors. The transcription factor of the regulation network has three layers, the top layer regulation factor is mainly NAC transcription factor, can regulate and control the downstream transcription factor, the secondary transcription factor can be directly activated or inhibited by the top layer transcription factor, can regulate and control the downstream transcription factor or be directly combined with cis-acting elements of the structural gene to promote or inhibit gene expression, and the three layers of transcription factors are regulated and controlled by the upstream top layer and the secondary transcription factor to be combined with cis-elements of the structural gene. The secondary transcription factor and the third layer transcription factor are mainly MYB transcription factors, and can be combined with a promoter cis-acting element of the SCW synthetic gene. NAC transcription factors and MYB transcription factors play different roles, respectively, and together regulate the deposition of lignin, cellulose and hemicellulose.

NAC transcription factor family members are top-level regulators of xylem cell development and deposition of plant secondary cell wall components, and play an important role in the transcriptional regulation network of wood formation. The invention discloses a sequence and function research of Pinus massoniana PmSND, which is helpful for the deep research of the function of Pinus massoniana PmSND gene and provides a molecular means and basis for the directional breeding work of improving the yield of Pinus massoniana wood and improving the lumber property of Pinus massoniana.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a Pinus massoniana PmSND gene which meets the use requirements. The invention aims to provide a coding protein of a Pinus massoniana PmSND gene. The invention also solves the technical problem of providing an application of a Pinus massoniana PmSND gene for plant improvement breeding.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a Pinus massoniana PmSND gene has a nucleotide sequence shown in SEQ ID NO. 1.

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

The application of the Pinus massoniana PmSND gene in improving the growth rate of mountain new poplar comprises the following steps:

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

(2) Transforming the constructed vector of the Pinus massoniana PmSND gene into leaves of the mountain new poplar;

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

The application of the Pinus massoniana PmSND 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 PmSND gene is shown as SEQ ID NO. 1.

The application of the Pinus massoniana PmSND 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 PmSND gene is shown as SEQ ID NO. 1.

The application of the Pinus massoniana PmSND gene in thickening the cell wall between Yang Di eight internodes of Pinus massoniana PmSND gene is shown in SEQ ID NO. 1.

The application of the Pinus massoniana PmSND gene in increasing the content of aspen lignin is provided, and the nucleotide sequence of the Pinus massoniana PmSND gene is shown as SEQ ID NO. 1.

The application of the Pinus massoniana PmSND gene in increasing the cellulose content of Pinus massoniana PmSND, wherein the nucleotide sequence of the Pinus massoniana PmSND gene is shown as SEQ ID NO. 1.

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

Compared with the prior art, the invention has the beneficial effects that:

The invention takes 2-year-old masson pine leaves as materials, obtains the masson pine PmSND gene by cloning, constructs an over-expression vector pCAMBIA1301-PmSND on the basis, and transfers the over-expression vector pCAMBIA1301-PmSND into mountain new poplar to obtain transgenic plants. Compared with the wild mountain fresh poplar, the transgenic mountain fresh Yang Zhu line grows faster, the leaf areas of the 3 rd, the 6 th and the 9 th leaves are larger, the plant line stem height and the ninth internode stem diameter are remarkably increased, the cell wall is remarkably thickened, the thicknesses of the stem xylem and phloem are increased, and the contents of lignin, cellulose and hemicellulose are remarkably increased.

Drawings

FIG. 1 is a diagram showing tissue-specific expression of PmSND gene in Pinus massoniana;

FIG. 2 is a graph of GUS staining analysis of PmSND4 promoter transgenic Arabidopsis;

FIG. 3 is a diagram of PmSND4 transcriptional self-activation assay;

FIG. 4 is a genomic DNA electrophoresis detection diagram of PmSND4 transgenic aspen;

FIG. 5 is an analysis of the expression of PmSND4 transgenic aspen;

FIG. 6 is a plant phenotyping diagram of PmSND4 transgenic aspen;

FIG. 7 is an electron microscope scan of a stem cross section of PmSND4 transgenic aspen;

FIG. 8 is a map of PmSND4 transgenic aspen versus wild plant tissue staining;

FIG. 9 is a diagram showing PmSND4 transgenic aspen lignin, cellulose and hemicellulose content assays;

FIG. 10 is a yeast single hybrid diagram of PmSND and PmMYB4 promoters;

FIG. 11 is a dual luciferase fluorescence intensity analysis of PmSND and PmMYB4 promoters.

Detailed Description

The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.

The 2-year old masson pine used in the experiment was obtained from a tree garden in the university of Nanjing forestry in the Xuanwu area of Nanjing, jiangsu province.

Example 1

1. Screening according to Pinus massoniana transcriptome data (PRJNA 655997) to obtain a coding reading frame sequence of PmSND gene, and amplifying PmSND gene by using Oligo 6 design specific primers, wherein the sequences of the primers are as follows:

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

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

2. Amplification of target Gene

Total RNA from 2-year-old masson pine was extracted using the Aidlab's EASYspinPlus plant RNA flash extraction kit. Using gold in its entiretyThe One-StepRT-PCRSuperMix reverse transcription kit reverse transcribes the extracted RNA into cDNA.

The cDNA after reverse transcription of RNA extracted from 2-year-old masson pine leaves is used as a template and a specific primer of PmSND gene to carry out PCR, and the PmSND4 gene open reading frame cloning reaction system is as follows:

The reaction procedure is:

The PCR product was purified to give PmSND gene and the fragment of interest was ligated to Blunt vector using pClone007 Blunt Simple Vector Kit kit. Transferring the vector into escherichia coli, screening positive monoclonal colony for sequencing, and measuring the nucleotide sequence of PmSND gene as shown in SEQ ID NO.1 and the amino acid sequence as shown in SEQ ID NO. 2.

Example 2

1. The specificity expression condition of PmSND genes in 8 tissues of 2-year-old masson pine root (R), old Stem (OS), young Stem (YS), old needle leaf (ON), young leaf (YN), terminal Bud (TB), xylem (X) and phloem (P) is analyzed by using qRT-PCR technology, and the xylem (X) is a control group. Adopting Pinus massoniana PmTUA (KM 496535.1) as an internal reference gene, and adopting Primer 5.0 software to design a specific Primer of PmSND gene, wherein the Primer sequence is as follows:

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

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

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

PmSND4-Q-R:5'-TGCCTGTATTTTCCTTCGT-3'。

The following reaction system (on ice) was added to a sterile, enzyme-free 96-well PCR plate:

after the sample is added, the 96-well plate is sealed by using a sealing film, and is centrifuged at 3000rpm for 5min at 4 ℃, and then the sample is put into a real-time fluorescence quantitative PCR instrument.

The reaction procedure was a standard procedure, i.e., 95℃pre-denaturation for 2min,95℃denaturation for 10s,60℃annealing/extension for 30s,40 cycles, total volume of 10. Mu.L, rest of the settings were referred to HieffUniversal Blue qPCR SYBR GREEN MASTER Mix specification. After the completion of the reaction, the relative expression level was calculated by the method of 2 ^-△△CT, and data analysis was performed using Excel and GRAPHPAD PRISM 9.0.0.

As shown in FIG. 1, pmSND4 gene was expressed in Terminal Bud (TB), root (R), old Stem (OS), young Stem (YS), old leaf (ON), young leaf (YN), xylem (X), phloem (P), which indicated that the gene was expressed in general in each tissue, but the expression level was slightly different, and the expression level was slightly higher in the old leaf than in other parts, and then in the stem.

Example 3

1. The promoter sequence of the SND4 gene was obtained by screening with reference to the genomic data of Pinus koraiensis (doi: https:// doi. Org/10.1016/j. Cell. 2021.12.006), and the PmSND gene was amplified using Oligo 6-designed specific primers, the primer sequences were:

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

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

2. Cloning of PmSND Gene promoter

Genomic DNA (gDNA) from 2-year old Pinus massoniana seedlings was extracted using FastPure Plant DNA Isolation Mini Kit kit, for specific steps reference FastPure Plant DNA Isolation Mini Kit.

Taking Pinus massoniana gDNA as a template, and carrying out PCR by using a PmSND gene promoter specific primer, wherein a cloning reaction system of the PmSND4 gene promoter is as follows:

PmSND4-F	2.5μL
		PmSND4-R	2.5μL
gDNA template	2.5μL
		2×Taq PCR Master Mix	25μL
ddH₂O	Up to 50μL

The reaction procedure is:

The PCR product was purified to give PmSND gene promoter fragment, and the target fragment was ligated to Blunt vector. Transferring the vector connected with the target fragment into escherichia coli, screening positive monoclonal colony for sequencing, and measuring the nucleotide sequence of PmSND gene promoter as shown in SEQ ID NO. 3.

3. GUS staining analysis of PmSND4 promoter

GUS vector is transformed into agrobacterium tumefaciens EHA105, the bacterial liquid with correct sequence is recovered and purified, and then the inflorescence of the arabidopsis thaliana is infected by using an inflorescence infection method, so that the transformation efficiency is improved, and each plant can be repeatedly infected for 2-3 times.

Collecting the T0 generation Arabidopsis seeds, sterilizing, planting in MS culture medium containing Hyg, transferring to soil after growing for two weeks to obtain T1 generation Arabidopsis, collecting T1 generation Arabidopsis seeds, planting by the same method to obtain T2 generation Arabidopsis, fully soaking T2 generation Arabidopsis seedlings in culture solution containing GUS dye solution, culturing overnight at 37 ℃ under the condition of avoiding light, treating plants for 10min by using 70% ethanol and 30% glacial acetic acid mixed solution, decolorizing the plants by using 75% ethanol, and making slices for observation after decolorizing.

As a result, as shown in FIG. 2, there was no staining of the wild type Arabidopsis seedlings (WT), whereas the transgenic Arabidopsis seedlings were stained blue, with a darker degree of staining at the stems and veins. This indicates that PmSND gene promoter is ubiquitously expressed in various tissues of plants and higher in leaves and stems.

4. PmSND4 transcriptional activation Activity of 4

Constructing pGBKT7-PmSND4 vector by using a homologous recombination method, wherein the sequence of a homologous recombination primer is as follows:

pGBKT7-PmSND4-F:

5'-aggccgaattcccggggatccATGGAGTGGTTTATCCATGGAAA-3',

pGBKT7-PmSND4-R:

5'-ggttatgctagttatgcggccgcTCAAATCAGTCCTCCATAGTGGG-3'。

The homologous recombinant vector pGBKT7-PmSND4 was transformed into AH109 yeast competence (Shanghai Biotechnology Co., ltd.). Cloning plates were randomly selected from SD/-Trp medium onto SD/-Trp/-Leu/-His medium using pGBKT7 empty plasmid as negative control. Culturing at 28 ℃ for 72 hours, and observing whether the bacterial plaques of the experimental group and the control group grow.

As shown in FIG. 3, the recombinant plasmid of pGBKT7-PmSND4 can only grow on SD/-Leu medium, the growth state on SD/-Leu/-Trp/-His medium is consistent with that of the negative control pGBKT7 vector, and the color of bacterial plaque is not changed after the SD/-Leu/-Trp/-His is added to X-alpha-gal, so that the experiment proves that PmSND has no self-excitation activity.

Example 4

1. Construction of PmSND Gene overexpression vector

Constructing pCAMBIA1301-PmSND4 plant over-expression vector by using a homologous recombination method, transforming the successfully constructed recombinant vector into agrobacterium tumefaciens EHA105, and screening to obtain positive monoclonal colonies for subsequent transformation experiments. The sequences of the homologous recombination primers are as follows:

pCAMBIA1301-PmSND4-F:

5'-ggtacccggggatcctctagaATGGAGTGGTTTATCCATGGAAA-3',

pCAMBIA1301-PmSND4-R:

5'-ttaccctcagatctaccatggTCAAATCAGTCCTCCATAGTGGG-3'。

2. leaf disc method for stably transforming mountain new poplar

Inoculating a positive monoclonal colony containing pCAMBIA1301-PmSND4 vectors into a 15mL sterile enzyme-free centrifuge tube, adding 5mL of liquid LB culture medium containing Kan and Rif antibiotics, and culturing in a shaking table at 28 ℃ at 220rpm overnight to obtain bacterial liquid; transferring 3mL of bacterial liquid into a 250mL conical flask, adding 50mL of liquid LB culture medium containing Kan and Rif antibiotics, centrifuging at 5000rpm at room temperature for 30min when the OD value is 0.4, and discarding the supernatant; re-suspending agrobacterium precipitation by using an equal volume of MS liquid culture medium containing AS to obtain re-suspended agrobacterium liquid, taking mountain fresh poplar tissue culture seedlings which grow for about 30d and have good growth state, shearing off the edges of leaves of mountain fresh poplar tissue culture seedlings by using scissors to manufacture wounds, shearing the leaves into square leaves with the length of about 1cm multiplied by 1cm, placing the square leaves into the re-suspended agrobacterium liquid culture medium, sealing the square leaves, standing for 20min at room temperature, taking out the leaves in the agrobacterium liquid culture medium by using sterilized tweezers, sucking the surface bacterial liquid of the leaves by using filter paper, placing the front of the leaves in a dark culture medium (differentiation medium+AS) upwards for 2d, washing the leaves in an ultra-clean workbench twice by using sterile water after the dark culture is finished, washing the third time by using sterile water containing Tim, sucking water by using sterilized water paper after the washing, culturing for 7d by using a light culture medium (differentiation medium+Tim), washing the leaves in a sterile water-absorbing medium for two times (sterile water-absorbing medium of 53 mg/25 mg after the end of light culture medium) continuously washing the leaves until the green tissue culture medium is washed for 150mg (medium) after the time of drying the culture medium is continuously washed, culturing the buds in strong seedling culture medium (elongation culture medium+Tim (150 mg/L) +Kan (25 mg/mL)) for 20-25 days, if agrobacterium colony appears in the culture medium, cleaning plant materials in time and replacing new strong seedling culture medium, culturing strong seedlings, transferring adventitious buds into rooting culture medium (rooting culture medium+Tim (150 mg/L) +Kan (25 mg/mL)) containing Kan resistance to induce rooting, taking seedlings of wild type and over-expressed strain cultured in rooting culture medium for about 40 days, taking out the plants after opening cover and culturing for 3 days, washing plant root agar with clear water, transplanting into flower pot, and culturing in greenhouse.

3. Transgenic mountain new Yang Yangxing detection

Extracting gDNA of transgenic aspen (I, II and III) and wild aspen (negative control, WT) by using FastPure Plant DNA Isolation Mini Kit kit, and identifying the over-expression condition of PmSND gene by PCR method by using gDNA of wild aspen, transgenic aspen and over-expression vector plasmid (constructed pCAMBIA1301 recombinant plasmid, CK) as templates.

The results are shown in FIG. 4, and all three transgenic mountain new Yang Zhu lines (I, II and III) are positive plants. And transferring the cultured domesticated seedlings to soil for continuous greenhouse growth after 3d of domestication and seedling hardening culture.

The Trizol method is used for extracting RNA of transgenic aspen and reversely transcribing the RNA into cDNA, and diluting the cDNA obtained by reverse transcription by 20 times for analysis of the expression quantity of the gene PmSND of the transgenic aspen.

As a result, as shown in FIG. 5, the expression level of the transgenic mountain fresh Yang Zhu lines (I, II, III) was higher than that of the wild-type mountain fresh poplar (WT), and the highest line expression level (II) was 60 times that of the wild-type.

4. Phenotypic analysis of transgenic mountain poplar

Three transgenic shanxin Yang Zhu lines (I, II, III) were selected as test groups and untransformed wild shanxin poplar (WT) as controls, and after 90d growth, statistical growth rates, leaf area (3 rd, 6 th, 9 th leaves), plant height and ninth internode diameter were observed.

As shown in FIG. 6, transgenic mountain fresh Yang Zhu lines (I, II and III) grew faster than wild mountain fresh poplar (WT) (FIG. 6A), and the observation of 3 rd, 6 th and 9 th leaves showed that the leaf area of transgenic mountain fresh Yang Zhu lines was larger (FIG. 6B), and the statistical analysis of plant height and ninth internode diameter showed that both the stem height (FIG. 6C) and stem diameter (FIG. 6D) of transgenic mountain fresh Yang Zhu lines were significantly increased.

Taking 90d wild type and transgenic shanxin Yang Zhu lines, taking down 8 th internode, rapidly placing into FAA fixing solution (taking 5mL of 38% formaldehyde solution and glacial acetic acid solution respectively, adding into 90mL of 70% alcohol solution, mixing uniformly upside down), fixing for more than 24 hours, drying, slicing, and quantitatively analyzing the cell morphology of xylem by using a scanning electron microscope (Scanning electron microscopy, SEM) and image analysis software IMAGEJ (https:// imagej.

The results are shown in fig. 7, in which the cell walls of the transgenic aspen were significantly thickened.

5. Toluidine blue tissue staining

The stem section of transgenic mountain new poplar is used as material to dye toluidine blue tissue, and the tissue is observed under microscope after being washed with water. For specific methods reference is made to the plant tissue staining solution (toluidine blue method) instructions of Solarbio.

The results are shown in fig. 8, where both the stem xylem and phloem thickness of the transgenic aspen were increased compared to the wild type.

6. Determination of secondary wall component content of positive transgenic mountain new Yang Zhu line

The lignin content is determined by a sulfuric acid method, and the method comprises the following specific steps:

Taking the whole stem trunk of the wild-type and positive transgenic mountain new Yang Zhu line growing for 90d as a material, drying the stem trunk to constant weight by using an oven, grinding the stem trunk into powder, and placing 0.1g into a centrifuge tube. Three repetitions of each sample are arranged, 10mL of acetic acid (CH ₃ COOH) solution (1%) is added into the centrifuge tube, the mixture is stirred and mixed uniformly, the supernatant is discarded, 5mL of CH ₃ COOH solution is used for washing and precipitating once, 3.5mL of mixed solution of ethanol (CH ₃CH₂ OH) and diethyl ether (C ₂H₅OC₂H₅) is added, the mixture is kept stand for 3min, the supernatant is repeated for three times, the centrifuge tube is dried and precipitated by boiling water bath, 72 percent sulfuric acid (H ₂SO₄) solution is added into the centrifuge tube after drying, the mixture is stirred and mixed uniformly by using a glass rod, the mixture is kept stand for 16H at room temperature, the cellulose is completely dissolved, 10mL of sterile water is added, the mixture is stirred and mixed uniformly by using a glass rod, the mixture is cooled to room temperature, 5mL of sterile water and 0.5mL of barium chloride (BaCl ₂) solution (10%), the mixture is stirred and centrifuged for 5min at 3000rpm after shaking and precipitating twice, 10mL of 10 percent sulfuric acid solution and 0.1mol/L potassium dichromate (K ₂Cr₂O₇) solution are respectively added, boiling water is dried and added into the centrifuge tube, the mixture is cooled and titrated by using a solution of 10mL of sodium iodide and 0.2 mol/6 mol solution (0.42 mol) of sodium sulfate solution after cooling and 0.42 mol percent sulfuric acid solution are added into the mixture, and the mixture is cooled and cooled by using a glass rod for titration.

The lignin content (%) was calculated as:

In the calculation formula, k represents the concentration (mol/L) of Na ₂S₂O₃, a represents the volume (mL) of Na ₂S₂O₃ consumed by blank titration, b represents the volume (mL) of Na ₂S₂O₃ consumed by titration, 48 represents the titer of 1mol lignin (represented by C ₁₁H₁₂O₄) corresponding to Na ₂S₂O₃, and n is the mass (g) of the sample powder.

Cellulose content was determined using the anthrone sulfate colorimetric method and hemicellulose content was determined using the hydrochloric acid hydrolysis method.

In the above assay, three technical replicates and three biological replicates were taken for each positive transgenic line and data analyzed using graphpadprism9.0, resulting in the detection of their significance using T-test.

As shown in FIG. 9, compared with the wild type mountain fresh Yang Zhu line, the positive transgenic mountain fresh Yang Zhu line has the advantages that the lignin and hemicellulose content is remarkably increased, and the cellulose content is increased, so that the PmSND gene can promote the synthesis of lignin, cellulose and hemicellulose.

7. Yeast single hybrid interaction verification

The recombinant vectors which were successfully constructed were transformed into yeast competent Y187 by homologous recombination method to construct PGADT-PmSND and pHis2-ProPmMYB vectors, positive clone colonies were selected and isolated from the plates, and were spotted on 3-AT-deficient medium corresponding to SD/-His but with different concentrations (10, 20, 30, 40, 50, 60, 70 mM) after re-suspension with 1mL of sterile water, respectively, and cultured for 3d AT 28 ℃. The single colony which was confirmed to be successful was resuspended by the same method, 5. Mu.L of the single colony was spotted on SD/-Trp/-Leu/-His and 3-AT medium of the corresponding concentration, and cultured AT 28℃for 4d, and the growth was observed. PGADT7-PmSND4+ pHis2 was used as negative control.

The results are shown in FIG. 10, in which the growth status of negative control PGADT-PmSND 4+ pHis2 on SD/-Trp/-Leu/-His medium was completely inhibited, while PGADT-PmSND 4+ pHis2-Pro-PmMYB4 could normally grow on SD/-Trp/-Leu/-His medium, indicating that PmSND4 could bind to the promoter of PmMYB 4.

8. Dual luciferase assay

Constructing pGreenII-0800-LUC-ProPmMYB and pGreenII-62-SK-PmSND4 vectors by using a homologous recombination method, transforming the successfully constructed recombinant vectors into agrobacterium tumefaciens EHA105, screening to obtain positive clone single colony, and referring to a holy Dual Luciferase Reporter GENE ASSAY KIT kit by transient expression of a double luciferase test. The control group of ProPmMYB4-LUC+SK was used as a standard and was set to 1.

As a result, as shown in FIG. 11, the promoter of PmMYB.sup.4 gene was able to bind to PmSND.sup.24, and the fluorescence intensity of ProPmMYB.sup.4-LUC+ PmSND.sup.4-SK was 6 times that of the control group ProPmMYB.sup.4-LUC+SK.

Claims

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

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

3. Application of the Masson pine PmSND4 gene in improving the growth rate of Populus davidianus, the Masson pine PmSND4 gene nucleotide sequence is shown in SEQ ID NO.1.

4. The use of the Masson pine PmSND4 gene in improving the growth rate of Populus davidianus according to claim 3, characterized in that it comprises the following steps:

(1) Construction of a vector for the Masson pine PmSND4 gene;

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

(3) Cultivate and screen transgenic Populus dasyphylla with accelerated growth rate.

5. Application of the Masson pine PmSND4 gene in increasing the thickness of xylem and phloem in Populus dasyphylla stems, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

6. Application of the Masson pine PmSND4 gene in increasing the height of Populus davidianus plants, 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.

7. 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.

8. 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.

9. Application of the Masson pine PmSND4 gene in increasing the cellulose content of Populus davidianus, wherein the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.

10. Application of the Masson pine PmSND4 gene in increasing the hemicellulose content of Populus davidianus, the nucleotide sequence of the Masson pine PmSND4 gene is shown in SEQ ID NO.1.