A gene PbrSND1 regulating stone cell formation in pear fruit

Abstract

The present invention discloses a gene for regulating lignin synthesis in pear fruit PbrSND1, The CDS sequence of this gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. Fluorescence quantitative analysis showed that this gene is mainly expressed in the key stage of stone cell development in pear fruit. When the gene was overexpressed in the fruit of Dangshan Pear, the stone cell, lignin and cellulose contents were significantly increased compared with the empty control site. When the gene was overexpressed in Arabidopsis, the stone cell, lignin and cellulose contents were significantly increased. PbrSND1 The secondary cell walls of the vascular cells and intervascular fiber cells of transgenic Arabidopsis thaliana were significantly thickened, and the accumulation of lignin and cellulose in the stems was significantly increased. The discovery of this gene provides a new genetic resource for fruit quality breeding and is an important candidate gene for future genetic engineering to improve fruit quality breeding.

Description

Technical Field

[0001] The present invention relates to the fields of plant molecular biology and fruit tree genetic breeding, and in particular to a key gene PbrSND1 regulating the formation of stone cells in pear fruits and application of the gene in improving the quality of pear fruits. Background Art

[0002] Pear (Pyrus spp.) is an important economic fruit tree, and its fruit quality directly affects its market value. Stone cells are a specialized cell type in pear fruit, and their abundance and distribution directly influence the fruit's texture and taste. Excessive stone cells can lead to rough fruit and reduced edible quality. Currently, the molecular mechanisms underlying stone cell formation in pear fruit remain unclear, and effective genetic resources are lacking for regulating stone cell abundance and improving fruit quality. As early as 1935, Smith discovered that the lignin content of the cell walls of pear stone cells exceeded 30%. In 2009, Tao et al., using a combination of microscopy, chemical, and spectroscopic techniques, further clarified the process by which pear parenchyma cells lignify, leading to lignin deposition in the cell walls and forming stone cells (Tao et al., 2009). However, due to the complexity of the stone cell formation mechanism, the underlying regulatory network remains largely unresolved. Therefore, further understanding the key factors underlying stone cell formation is crucial for accelerating pear fruit quality improvement. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a key gene that regulates stone cell formation in pear fruit. This gene was isolated and cloned from the cultivar 'Dangshan Suli Pear' (Pyrus bretschneideri), which has a high stone cell content. The applicant has named it Stone cell-associated NAC domain protein 1 (PbrSND1). Its CDS sequence is shown in SEQ ID NO. 1, and its corresponding protein sequence is shown in SEQ ID NO. 2 in the sequence listing. The discovery of this gene may provide new insights into improving the quality of pear fruit.

[0004] Another object of the present invention is to provide a use of the aforementioned gene, PbrSND1. An overexpression vector was constructed for this gene and introduced into Arabidopsis thaliana via Agrobacterium-mediated genetic transformation. The resulting transgenic material was biologically validated, demonstrating that the cloned PbrSND1 gene promotes lignin accumulation and secondary cell wall thickening.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides the use of the gene PbrSND1 in the following (A1)-(A3):

[0007] (A1) Applications for increasing the content of lignin and / or cellulose in plants;

[0008] (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in plants;

[0009] (A3) Application in breeding for increasing the lignin and / or cellulose content in plants;

[0010] The CDS sequence of the gene PbrSND1 is shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention further provides the use of the protein encoded by the gene PbrSND1 in the following (A1) to (A3):

[0012] (A1) Applications for increasing the content of lignin and cellulose in plants;

[0013] (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in plants;

[0014] (A3) Application in breeding for increasing the lignin and / or cellulose content in plants;

[0015] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0016] The * indicates a stop codon. The secondary structure of this protein is mainly irregular coils and α-helices, making it a stable protein. The protein has 422 amino acids.

[0017] In a third aspect, the present invention further provides the use of a recombinant expression vector and / or transient expression vector containing the gene PbrSND1 in the following (A1) to (A3):

[0018] (A1) Applications for increasing the content of lignin and / or cellulose in plants;

[0019] (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in plants;

[0020] (A3) Application in breeding for increasing the lignin and / or cellulose content in plants.

[0021] The present invention can use existing plant expression vectors to construct a recombinant expression vector containing the gene PbrSND1.

[0022] When using the gene PbrSND1 to construct a recombinant plant overexpression vector, the cauliflower mosaic virus (CAMV) 35S strong promoter can be added before its transcription start nucleotide; when using the gene of the present invention to construct a plant expression vector, ATG can be used as the start codon, but it must be consistent with the reading frame of the coding sequence to ensure correct translation of the entire sequence.

[0023] To facilitate identification and screening of transgenic plants, the plant expression vectors used are modified to incorporate a gene encoding a luminescent compound (luciferase) expressed in plants and an antibiotic resistance marker (kanamycin marker). For transgenic plant safety, it is possible to omit any selectable marker genes and directly screen transformed plants using hygromycin.

[0024] In a specific embodiment, the backbone vector of the recombinant expression vector is pSAK778.

[0025] In a fourth aspect, the present invention also protects the use of a recombinant bacterium containing the gene PbrSND1 described above in the following (A1) to (A3):

[0026] (A1) Applications for increasing the content of lignin and / or cellulose in plants;

[0027] (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in plants;

[0028] (A3) Application in breeding for increasing the lignin and / or cellulose content in plants.

[0029] In a specific embodiment, the application is achieved by transferring the gene PbrSND1 into the target plant for overexpression.

[0030] In one embodiment of the present invention, the gene encoding the protein is introduced into Arabidopsis thaliana using the pSAK778 vector, which directs exogenous gene expression in plants, to obtain transgenic Arabidopsis plants. The expression vector carrying the gene can be transformed into Arabidopsis thaliana using Agrobacterium-mediated transformation (floral infection), and the transformed Arabidopsis seeds can be harvested.

[0031] In one embodiment of the present invention, the gene encoding the protein was transiently introduced into the young fruit of 'Dangshan Pear' 35 days after flowering using the vector pSAK778 for directing the expression of exogenous genes in plants, and the transiently injected fruits were obtained and their related indicators were measured.

[0032] The plant described in the present invention can be either a monocot or a dicot, such as Arabidopsis thaliana, pear, etc.

[0033] The present invention also relates to the application of this gene in the genetic improvement of fruit quality. The gene was overexpressed in Arabidopsis thaliana, and the obtained transgenic strains were verified by biological functions. The lignin content was significantly increased, the secondary cell walls of stem vascular cells were significantly thickened, the expression levels of lignin synthesis-related genes were also significantly increased, and the cellulose content was also significantly increased.

[0034] In a fifth aspect, the present invention protects a method for increasing the lignin and / or cellulose content of pear fruit, which is achieved by increasing the expression level of the gene PbrSND1 in pear.

[0035] In a specific embodiment, the method is achieved by transferring the gene PbrSND1 into the target plant for overexpression.

[0036] In a sixth aspect, the present invention protects a method for increasing the content of lignin and / or cellulose in Arabidopsis thaliana, which is achieved by increasing the expression level of the gene PbrSND1 in Arabidopsis thaliana.

[0037] In a specific embodiment, the method is achieved by transferring the gene PbrSND1 into the target plant for overexpression.

[0038] Beneficial effects

[0039] The gene PbrSND1 for regulating stone cell formation in pear fruit provided by the present invention has the following beneficial effects compared with the prior art:

[0040] (1) This study first discovered that PbrSND1 can positively regulate the content of lignin and cellulose in pear fruit.

[0041] (2) The gene PbrSND1 provided by the present invention was overexpressed in Arabidopsis thaliana, and the obtained transgenic strains were verified by biological functions. The lignin and cellulose contents were significantly increased, and the secondary cell walls of the stem vascular cells were significantly thickened.

[0042] (3) The discovery of the gene PbrSND1 in the present invention provides a new gene resource for fruit quality breeding and is an important candidate gene for future genetic engineering to improve fruit quality breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is an analysis of the relative expression levels of PbrSND1 in different parts of pear and at different stages of pear fruit.

[0044] Figure 2-5 is the transient overexpression analysis of PbrSND1 in pear fruit, in which,

[0045] Figure 2The phloroglucinol-hydrochloric acid staining image of 'Dangshan Pear' after 7 days of PbrSND1 overexpression. 35S represents the empty control, and 35S-PbrSND1 represents the overexpression of PbrSND1 mediated by the 35S strong promoter.

[0046] Figure 3 is the expression level of overexpressed PbrSND1 in pear fruit.

[0047] Figure 4 is the stone cell content in pear fruit after overexpression of PbrSND1.

[0048] Figure 5 is the lignin content in pear fruit after overexpression of PbrSND1.

[0049] Figure 6 is the cellulose content in pear fruit after overexpression of PbrSND1.

[0050] Figure 7 Toluidine blue and phloroglucinol staining analysis of stem sections of PbrSND1-overexpressing plants.

[0051] Figure 8 is the cellulose content in the stems of PbrSND1-overexpressing plants. DETAILED DESCRIPTION

[0052] The present invention is described in detail below with reference to specific embodiments. Based on the following description and examples, those skilled in the art can ascertain the essential features of the present invention and, without departing from the spirit and scope of the present invention, can make various changes and modifications to the present invention to adapt it to various uses and conditions.

[0053] The experimental methods in the following examples, where specific conditions are not specified, are generally based on well-known methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0054] Example 1 Obtaining the Pear PbrSND1 Gene

[0055] According to the PbrSND1 gene sequence, a specific primer pair for amplifying the sequence was designed using Primer Premier 5.0.

[0056] The specific steps are as follows:

[0057] Using Dangshan pear cDNA as a template, amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). The amplification system is shown in Table 1, the amplification program is shown in Table 2, and the amplification primer sequences are:

[0058] PbrSND1-F: acgctcgacactagtggatccATGGCGCCTGAAAACATGAG;

[0059] PbrSND1-R:tcattaaagcaggactctagaTTATATAGGACCGTTCGACACGAG.

[0060]

[0061]

[0062] The amplified product was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The pSAK778 overexpression vector was digested with Xba I and BamH I restriction endonucleases (Themo Scientific, China). The digestion system is shown in Table 3. After incubation at 37°C for 2 h, the digested vector was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The purified product and the double-digested vector were ligated using the ClonExpress II One Step Cloning Kit (Vazyme, China) to construct the expression vector 35S-PbrSND1. The ligation system is shown in Table 4. After incubation at 37°C for 30 min, the vector was transformed into competent Escherichia coli DH5α (Tsingke, China). The E. coli transformation method is as follows:

[0063] (1) Add 20 μL of ligation product to 50 μL of E. coli competent DH5α (Tsingke, China) cells melted in an ice bath, mix gently, and place on ice for 30 min;

[0064] (2) After heat shock in a 42°C water bath for 45 seconds, place in ice for 2 minutes. During this process, the centrifuge tube should not be shaken;

[0065] (3) Add 600 μL of LB liquid medium without antibiotics and culture at 37°C in a shaker at 200 rpm for 1-2 h to allow the bacteria to recover;

[0066] (4) After centrifugation at 4000 rpm for 3 minutes, discard 500 μL of the supernatant, resuspend and take 100 μL of the revived competent cells and evenly spread them on LB solid culture medium containing the corresponding antibiotics. The culture dish is placed upside down in a 37°C constant temperature incubator and cultured overnight.

[0067]

[0068]

[0069]

[0070] 12-16 hours after transformation, single colonies were picked from the plates and placed in 1 mL centrifuge tubes. LB liquid medium containing the appropriate antibiotics was added and cultured on a shaker at 37°C until the culture became turbid. Positive identification was then performed. 2× Rapid Taq MasterMix (Vazyme, China) was used. The reaction system is shown in Table 5, and the PCR procedure is shown in Table 6. After obtaining positive clones, they were sent to Shanghai Sangon for sequencing. The PbrSND1 gene sequence was determined based on the sequencing results.

[0071]

[0072] The gene sequence of this gene in pear was cloned using the expression vector PbrSND1-F. After sequencing, it was found that the pSAK778 vector isolated a 1266bp CDS sequence, whose sequence is SEQ ID NO.1, with a length of 1266bp; this gene encodes a protein of 422 amino acids, whose sequence is SEQ ID NO.2.

[0073] Correctly sequenced bacterial cultures were expanded and used for bacterial culture and plasmid extraction. The culture was maintained at a ratio of 7:3 (v:v) of bacterial culture to glycerol. The culture was mixed, quickly frozen in liquid nitrogen, and stored at -80°C until further use. Plasmids were extracted using the FastPurePlasmid Mini Kit (Vazyme, China).

[0074] Competent Agrobacterium tumefaciens GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China) was thawed on ice, and the recombinant vector plasmid containing the target gene was added. After 5 minutes of ice-rest, the cells were snap-frozen in liquid nitrogen for 5 minutes. The cells were then heat-shocked at 37°C for 5 minutes and kept on ice for 5 minutes. Subsequently, 700 μL of antibiotic-free liquid LB was added and the cells were shaken for 3 hours (28°C, 250 rpm / min). The cells were centrifuged at low speed for 5 minutes, and the supernatant was discarded. 100 μL of the suspension was resuspended and evenly spread using a spreader on LB solid medium supplemented with antibiotics (50 μg / mL kana and 50 μg / mL rifampicin). The cells were placed upside down and incubated in a 28°C incubator. After 48 hours, single colonies were picked with a sterile toothpick and verified by PCR. The PCR amplification system is shown in Table 6. For stock preparation, the Agrobacterium culture solution was prepared at a ratio of 7:3 (v:v) of culture solution to glycerol. The mixture was then snap-frozen in liquid nitrogen and stored at -80°C until further use.

[0075] Example 2 Analysis of Stone Cell, Lignin and Cellulose Content

[0076] The stone cell content in the fruit pulp was determined using the freeze fractionation method. Three fruits of equal size (young fruits were preferred), the peels removed, and the edible portion of the fruit was quartered. 100 g of the fruit was weighed and placed in a -20°C refrigerator for 24 h. Thawed at room temperature, 200 ml of distilled water was added, and the fruit was thawed in a tissue crusher (1000-1500 rpm). -1 ) and mash for 5 minutes. Then transfer the homogenate to a 1000ml beaker and stir with a glass rod for 1 minute. Let it sit for 5 minutes to allow the stone cells to fully settle at the bottom of the beaker. Pour off the upper suspension and suspend the precipitate in 0.5 M hydrochloric acid solution for 30 minutes, stirring every 5 minutes. Remove floating matter and rinse with distilled water 5-6 times. Collect the suspension from the first few times and rinse again. Combine the resulting stone cells and filter through coarse filter paper to isolate the pure stone cells. Dry to a constant weight and weigh.

[0077] 0.01 g of pulp powder was accurately weighed using a microbalance and ground with 95% ethanol to a homogenate. The volume was then adjusted to 5 ml and centrifuged at 12,000 g for 2 min. The supernatant was discarded and the mixture was washed three times with 95% ethanol and three times with ethanol:n-hexane (1:2, v / v). The mixture was then air-dried in a fume hood. Then, 2 ml of 25% bromoacetoacetic acid solution was added and the mixture was incubated in a 70°C water bath for 30 min. The reaction was terminated by adding 0.9 ml of 2 M NaOH solution. 5 ml of acetic acid and 0.1 ml of 7.5 M hydroxylamine chloride solution were then added and the volume was adjusted to 10 ml with glacial acetic acid. The absorbance was measured at 280 nm. The lignin content was determined by comparing the absorbance of a lignin standard (Sigma-Aldrich, USA) to the absorbance curve (Syros et al., 2004).

[0078] After drying to constant weight, grind the sample into a powder using a sample grinder. Weigh 0.2 g of the powder into a beaker, place the beaker in a cold water bath, add 60 ml of 60% H₂SO₄, and digest for 30 minutes. The digested solution is then transferred to a 100 ml volumetric flask, adjusted to the etch line with 60% H₂SO₄, shaken thoroughly, and filtered using a Büchner funnel into a separate beaker. Add 5 ml of the filtrate to a 100 ml volumetric flask, dilute with distilled water on a cold water bath, and shake thoroughly. Then, transfer 2 ml of the solution to a test tube, add 0.5 ml of 2% anthrone reagent and 5 ml of concentrated H₂SO₄, shake the mixture thoroughly, and let it stand for 12 minutes. Finally, the absorbance is measured at 620 nm, and the cellulose content in the stone cells is determined by reference to a standard curve calculated for microcrystalline cellulose (Macklin). Three independent biological replicates were performed for each sample.

[0079] Example 3 Transient transformation of pear fruit

[0080] The culture conditions of Agrobacterium strain were the same as those in Example 1. After discarding the supernatant, the bacterial pellet was resuspended in an infiltration medium (10 mM MgCl2, 10 mM MES, 200 μM AS, pH 5.6, OD 600 =1.0). After 2-4 h of induction in the dark at room temperature, the equator of 35-day-old Dangshan Pear fruit was injected and infiltrated.

[0081] The results showed that 7 days after injection, increased lignin staining was observed at the injection sites of PbrSND1 compared with the corresponding non-injection sites and empty injection ( Figure 2 ). PbrSND1 was successfully overexpressed at the injection site ( Figure 3 ). Stone cells ( Figure 4 ), lignin ( Figure 5 ) and cellulose ( Figure 6 ) content was significantly increased. Therefore, PbrSND1 positively regulates the lignin and cellulose contents in pear fruit.

[0082] Example 4 Arabidopsis genetic transformation

[0083] (1) The Agrobacterium strain containing the expression vector 35S-PbrSND1 (hereinafter referred to as PbrSND1 Agrobacterium strain) verified to be correct by PCR in Example 1 was added with 100 μL of PbrSND1 Agrobacterium strain to 20 mL of LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampicin), and cultured in a shaking incubator at 28°C for 16 h;

[0084] (2) The cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in an equal volume of transformation medium (2.25 g / L MS medium, 5 g / L sucrose, 10 μg / L 6-BA, pH adjusted to 5.7 with KOH), and SILWETL-77 was added to a final concentration of 0.025%.

[0085] (3) Cut off the siliques and opened flowers of the wild-type Arabidopsis thaliana to be transformed (bolting 10-15 cm);

[0086] (4) Soak the preserved Arabidopsis flowers in the bacterial solution and evacuate to 0.6-0.8 kPa for 5 minutes;

[0087] (5) Culture in the dark at 22°C for 24 hours, then remove the plants and culture them normally, and harvest the seeds for screening.

[0088] The harvested T0 generation seeds were planted in screening medium (containing MS medium, 30 g / L sucrose, 0.75% agar, 20 mg / L hygromycin, 100 mg / L timentin and 100 mg / L carboxybarbital) for screening, and the grown seedlings were moved into plastic pots with a mixture of vermiculite and soil (1:2) and cultured in a greenhouse with a photoperiod of 16 h light / 8 h dark and a relative humidity of 40%. The seeds were collected after they matured.

[0089] Example 5 Microscopic observation of transgenic Arabidopsis

[0090] T2 seeds from Example 4 and wild-type seeds were seeded in MS medium to generate T3 transgenic plants for physiological measurements. Plants were cultured in a greenhouse with a photoperiod of 16 hours light / 8 hours dark and a relative humidity of 40%. Primary inflorescence stems were dried to constant weight and ground into powder using a grinder. Lignin and cellulose contents were then measured.

[0091] After 8 weeks of Arabidopsis cultivation, three T3 transgenic lines and wild-type Arabidopsis were randomly selected. Arabidopsis stems (approximately 5 cm) were cut and fixed in 0.1 M sodium bicarbonate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde at 4°C (Kim et al., 2017). The stems were rinsed with 0.1 M dimethicone buffer (pH 7.4) and then incubated with 1% O2O4 at 4°C. The sections were dehydrated using various concentrations of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) and embedded in butyrrolidone. After 2 days, sections were cut using an ultramicrotome (LKB-8800, Sweden).

[0092] Prepare paraffin sections and stain with toluidine blue and phloroglucinol as follows:

[0093] Ethanol dehydration: Use 75% to 100% ethanol and dehydrate in 5 levels for 2 hours.

[0094] Transparency: ethanol and xylene are gradually transparent according to the proportion, anhydrous ethanol: xylene = 3:1 elution for 40 minutes;

[0095] Treat with anhydrous ethanol: xylene = 1:1 for 40 minutes; treat with anhydrous ethanol: xylene = 1:3 for 4 minutes; soak in pure xylene for 1 hour, repeat once.

[0096] Wax dipping: add half volume of xylene and half volume of paraffin wax, heat to 75°C in an oven, infiltrate with melted paraffin wax for 2h, and repeat once.

[0097] Embedding: After the wax immersion is completed, use tweezers to pick up the material and place it in a paper box and embed the material with pure wax solution that has melted into liquid.

[0098] Trimming and sectioning: The embedded material was trimmed into a trapezoidal shape according to the position of the material and sectioned using a Leica RM 2015 hand-cranked microtome with a thickness of approximately 6 μm.

[0099] Spreading and sticking: Pick up the cut sample gently with tweezers, place it in a 35-45℃ water bath for spreading, pick it up with a glass slide after the wax sheet is spread, and put it in a 40℃ oven to dry.

[0100] Dewaxing: Place the slide with the sample in xylene and dewax for 15 minutes. Wash with xylene and anhydrous ethanol in a ratio of 1:1 for 2 minutes. Rinse with anhydrous ethanol at different concentrations (100%, 95%, 90%, 85%, 80%, 75%, 70%), washing for 2 minutes from high to low concentration.

[0101] Staining: Stain with toluidine blue staining solution for 24 hours, wash twice with 95% ethanol, anhydrous ethanol, xylene: anhydrous ethanol = 1:1, and pure xylene for 30 seconds respectively, cover with neutral gum, and dry in a 40℃ oven.

[0102] The images were observed and collected using an upright fluorescence microscope.

[0103]

[0104] Lignin content determination showed that the content of G-type lignin and H-type lignin in the inflorescence stems was significantly increased (Table 7). In addition, paraffin sections of the stems of wild-type and PbrSND1 transgenic plants were stained with toluidine blue and phloroglucinol-hydrochloric acid. It was found that the staining of lignin tissue in transgenic plants was stronger than that in wild-type plants, and the thickness of the secondary cell walls of vascular cells and interfascicular fiber cells was higher than that in wild-type plants ( Figure 6 ), further confirming that PbrSND1 positively regulates lignin deposition. Collectively, these findings suggest that PbrSND1 promotes lignin deposition and SCW thickening during stone cell development. Figure 8 It was shown that the cellulose content in the stems of PbrSND1-overexpressing Arabidopsis was also significantly increased.

[0105] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. Genes PbrSND1 Application in the following (A1)-(A3): (A1) Application of increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; The gene PbrSND1 The CDS sequence is shown in SEQ ID NO. 1; The application is to PbrSND1 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

2. The gene according to claim 1 PbrSND1 Application of the encoded protein in the following (A1)-(A3): (A1) Application of increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; The amino acid sequence of the protein is shown in SEQ ID NO.2; The application is to PbrSND1 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

3. Containing the gene according to claim 1 PbrSND1 Use of the recombinant expression vector and / or transient expression vector in the following (A1)-(A3): (A1) Application of increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; The gene PbrSND1 The CDS sequence is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that The backbone vector of the recombinant expression vector is pSAK778.

5. Containing the gene according to claim 1 PbrSND1 Application of the recombinant bacteria in the following (A1)-(A3): (A1) Application of increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin and / or cellulose content in pear fruit or Arabidopsis thaliana; The gene PbrSND1 The CDS sequence is shown in SEQ ID NO.

1.

6. A method for increasing the lignin and / or cellulose content of pear fruit, characterized in that: The method improves the pear gene PbrSND1 The expression level of the gene is achieved PbrSND1 The CDS sequence is shown in SEQ ID NO.1 。 7. The method according to claim 6, characterized in that The method is to PbrSND1 This was achieved by overexpression in pear.

8. A method for increasing the content of lignin and / or cellulose in Arabidopsis thaliana, characterized in that: The method improves the expression of genes in Arabidopsis thaliana PbrSND1 The expression level of the gene is achieved PbrSND1 The CDS sequence is shown in SEQ ID NO.1 。 9. The method according to claim 8, characterized in that The method is to PbrSND1 This was achieved by overexpression in Arabidopsis thaliana.

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