Gene PbrSND1 for regulating formation of stone cells of pear fruits

Abstract

The invention discloses a gene PbrSND1 for regulating and controlling synthesis of pear fruit lignin, the CDS sequence of the gene is as shown in SEQ ID NO.1, and the sequence of a coded protein is as shown in SEQ ID NO.2. Fluorescent quantitative analysis shows that the gene is mainly expressed in a key stage of development of stone cells of pear fruits. When an overexpression vector constructed by the gene is injected into a Dangshan pear fruit, the contents of stone cells, lignin and cellulose are obviously increased compared with those of injection of a no-load control part; an overexpression vector constructed by the gene is introduced into arabidopsis thaliana, secondary cell walls of conduit cells and intervascular bundle fiber cells of the obtained PbrSND1 transgenic arabidopsis thaliana are remarkably thickened, and accumulation of lignin and cellulose in stems is remarkably increased. The discovery of the gene provides a new gene resource for fruit quality breeding, and the gene is an important candidate gene for improving fruit quality breeding in future genetic engineering.

Description

Technical Field

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

[0002] Pear (Pyrus spp.) is one of the important economic fruit trees, and the quality of its fruits directly affects the market value. Stone cells are a special type of cells in pear fruits, and their content and distribution directly affect the texture and taste of the fruits. Excessive stone cells will cause the fruits to be rough and reduce the edible quality. At present, the molecular mechanism of stone cell formation in pear fruits is not clear, and there is a lack of effective gene resources for regulating the stone cell content and improving the fruit quality. As early as 1935, Smith found that the lignin content in the cell walls of stone cells in pear fruits exceeded 30%; in 2009, Tao et al. further clarified the process of lignification of parenchyma cells in pear fruits leading to the deposition of lignin in the cell walls to form stone cells by comprehensively using a variety of microscopy techniques, chemical methods, spectroscopy methods, etc. (Tao et al., 2009). However, due to the complexity of the stone cell formation mechanism, the regulatory network behind it has not been fully analyzed. Therefore, further exploring the key factors of stone cell formation is of great significance for accelerating the improvement of pear fruit quality. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a key gene that regulates the formation of stone cells in pear fruits. This gene was isolated and cloned from the cultivar 'Dangshansuli' (Pyrus bretschneideri) with a high stone cell content. The applicant 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 Sequence Listing SEQ ID NO.2. The discovery of this gene may provide new insights into the improvement of pear fruit quality.

[0004] Another purpose of the present invention is to provide an application of the aforementioned gene PbrSND1. An overexpression vector was constructed with this gene and introduced into Arabidopsis thaliana through Agrobacterium-mediated genetic transformation. The obtained transgenic materials were verified for biological functions, indicating that the cloned PbrSND1 gene of the present invention has the functions of promoting lignin accumulation and secondary cell wall thickening.

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

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

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

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

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

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

[0011] In the second aspect, the present invention also provides the application of the protein encoded by the gene PbrSND1 in the following (A1)-(A3):

[0012] (A1) Use in increasing the lignin and cellulose content in plants;

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

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

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

[0016] Where * represents the stop codon. The secondary structure of this protein is mainly random coil and α-helix, and it is a stable protein. The number of amino acids of this protein is 422.

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

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

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

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

[0021] The recombinant expression vector containing the gene PbrSND1 can be constructed by using the existing plant expression vectors in the present invention.

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

[0023] For the convenience of identifying and screening transgenic plants, the used plant expression vector is processed by adding a gene encoding a luminescent compound (luciferase gene) that can be expressed in plants and an antibiotic marker with resistance (kanamycin marker). Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened with hygromycin.

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

[0025] Fourthly, the present invention also protects the application of the recombinant bacterium containing the aforementioned gene PbrSND1 in the following (A1)-(A3):

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

[0027] (A2) Application 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 above application is achieved by overexpressing the gene PbrSND1 in the target plant.

[0030] In an embodiment of the present invention, using the vector pSAK778 that guides the expression of foreign genes in plants, the gene encoding the protein is introduced into Arabidopsis thaliana, and transgenic Arabidopsis thaliana plants can be obtained. The expression vector carrying the gene can be transformed into Arabidopsis thaliana by using the Agrobacterium-mediated method (floral dip method), and the transformed Arabidopsis thaliana seeds can be collected.

[0031] In an embodiment of the present invention, using the vector pSAK778 that guides the expression of foreign genes in plants, the gene encoding the protein is transiently introduced into the young fruits of 'Dangshansuli' 35 days after flowering, and the transiently injected fruits are obtained, and their related indexes are measured.

[0032] The plants described in the present invention can be either monocotyledonous plants or dicotyledonous plants, such as Arabidopsis thaliana, pears, 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 lines were verified for biological functions. The lignin content was significantly increased, the secondary cell wall of the stem vascular cells was significantly thickened, the expression levels of genes related to lignin synthesis were also significantly increased, and the cellulose content was also significantly increased.

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

[0035] In a specific embodiment, the method is achieved by overexpressing the gene PbrSND1 in a target plant.

[0036] Sixthly, the present invention protects a method for increasing the lignin and / or cellulose content 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 overexpressing the gene PbrSND1 in a target plant.

[0038] Beneficial effects

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

[0040] (1) The present invention discovers for the first time that PbrSND1 can positively regulate the lignin and cellulose contents in pear fruits.

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

[0042] (3) The discovery of the gene PbrSND1 of the present invention provides a new gene resource for fruit quality breeding and is an important candidate gene for future genetic engineering improvement of fruit quality breeding. Description of the drawings

[0043] Figure 1 It is the analysis of the relative expression levels of PbrSND1 in different parts of pears and at different stages of pear fruits.

[0044] Figures 2-5 It is the transient overexpression analysis of PbrSND1 in pear fruits, wherein,

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

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

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

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

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

[0050] Figure 7 It is the toluidine blue and phloroglucinol staining analysis of the stem sections of PbrSND1 overexpressing plants.

[0051] Figure 8 It is the cellulose content of the stems of PbrSND1 overexpressing plants. Detailed implementation manners

[0052] The following describes the present invention in detail with reference to specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention to make it applicable to various uses and conditions.

[0053] For the experimental methods without specific conditions in the following examples, they are usually carried out according to the well-known means in the art. For the test materials used in the following examples, unless otherwise specified, they are all purchased from regular biochemical reagent stores.

[0054] Example 1 Obtaining of the pear PbrSND1 gene

[0055] According to the PbrSND1 gene sequence, specific primer pairs for amplifying this sequence were designed using Primer Premier 5.0.

[0056] The specific steps are as follows:

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

[0058] PbrSND1-F: acgctcgacactagtggatccATGGCGCCTGAAAACATGAG;

[0059] PbrSND1-R: tcattaaagcaggactctagaTTATATAGGACCGTTCGACACGAG.

[0060]

[0061]

[0062] The amplified products were 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 reacting 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 to construct the expression vector 35S-PbrSND1 using the ClonExpress II One Step Cloning Kit (Vazyme, China), a non-ligation enzyme-dependent single-fragment rapid cloning kit. The ligation system is shown in Table 4. After incubating at 37 °C for 30 min, it was transformed into Escherichia coli competent DH5α (Tsingke, China). The method for Escherichia coli transformation is as follows:

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

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

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

[0066] (4) After centrifuging at 4000 rpm for 3 min, discard 500 μL of the supernatant. After resuspension, take 100 μL of the resuscitated competent cells and evenly spread them on the LB solid medium containing the corresponding antibiotics. The culture dish was inverted and placed in a 37 °C constant temperature incubator and cultured overnight.

[0067]

[0068]

[0069]

[0070] After 12 - 16 h of transformation, pick monoclonal colonies on the plate into a 1 mL centrifuge tube, add LB liquid medium containing the corresponding antibiotics, and culture with shaking at 37 °C until the bacterial liquid becomes turbid, and then perform positive identification. The reagent used is 2 × Rapid Taq MasterMix (Vazyme, China). The reaction system is shown in Table 5, and the PCR program is shown in Table 6. After obtaining positive clones, send the positive clones to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. According to the sequencing results, obtain the gene sequence of PbrSND1.

[0071]

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

[0073] Amplify the correctly sequenced bacterial liquid for use in preserving the bacterial strain and plasmid extraction. Preserve the bacterial liquid according to the ratio of bacterial liquid: glycerol = 7:3 (V:V), mix well, quickly freeze in liquid nitrogen, and store at - 80 °C for later use. Use the FastPure Plasmid Mini Kit (Vazyme, China) kit to extract the plasmid.

[0074] Thaw the Agrobacterium tumefaciens competent cells GV3101 (Vidi Biotechnology Co., Ltd., Shanghai, China) on ice, then add the recombinant vector plasmid containing the target gene, let it stand on ice for 5 min, quickly freeze in liquid nitrogen for 5 min, then perform heat shock at 37 °C for 5 min, place on ice for 5 min, and then add 700 μL of antibiotic - free liquid LB, and culture with shaking for 3 h (set the shaker at 28 °C, 250 rpm / min). Centrifuge at low speed for 5 min, discard part of the supernatant, leave 100 μL to resuspend, and evenly coat it on the LB solid medium containing antibiotics (kana 50 μg / mL; rifampicin 50 μg / mL) with a spreading rod. Invert and place it in an incubator at 28 °C. After 48 h, pick monoclonal colonies with a sterile toothpick for PCR verification. The PCR amplification system is shown in Table 6. Preserve the Agrobacterium tumefaciens bacterial liquid according to the ratio of bacterial liquid: glycerol = 7:3 (V:V), mix well, quickly freeze in liquid nitrogen, and store at - 80 °C for later use.

[0075] Example 2 Analysis of the contents of stone cells, lignin, and cellulose

[0076] The content of stone cells in the pulp was determined by the freezing separation method. Three fruits of the same size (more young fruits were taken) were selected, the peel was removed, and the edible part of the fruit was taken according to the quartering method. 100 g was weighed and placed in a refrigerator at -20 °C for 24 h. Then it was taken out and thawed at room temperature. 200 ml of distilled water was added, and it was mashed with a tissue homogenizer (1000 - 1500 r·min -1 ) for 5 min. Then the homogenate was transferred to a 1000 ml beaker, stirred with a glass rod for 1 min, and left to stand for 5 min to allow the stone cells to fully precipitate at the bottom of the beaker. The upper suspension was poured out, and the precipitate was suspended in 0.5 M hydrochloric acid solution for 30 min, with stirring every 5 min during this period. The floating substances were removed, and it was rinsed 5 - 6 times with distilled water. The suspensions from the first few rinses were collected and rinsed. The obtained stone cells were combined, filtered through a coarse filter paper, and finally pure stone cells were separated, dried to a constant weight, and weighed.

[0077] Accurately weigh 0.01 g of the pulp powder sample using an analytical balance. Grind it to a homogenate with 95% ethanol, make the volume up to 5 ml, centrifuge at 12000 g for 2 min, discard the supernatant, wash it 3 times with 95% ethanol, and then wash it 3 times with alcohol:n - hexane = 1:2 (V / V). Place it in a fume hood to dry. Then add 2 ml of 25% bromoacetyl acetic acid solution, warm it in a water bath at 70 °C for 30 min, add 0.9 ml of 2 M NaOH solution to terminate the reaction, then add 5 ml of acetic acid and 0.1 ml of 7.5 M hydroxylamine chloride solution, make the volume up to 10 ml with glacial acetic acid, measure the absorbance at 280 nm, and finally determine the lignin content by referring to the curve of the lignin standard sample (Sigma - Aldrich, USA) (Syros et al., 2004).

[0078] After drying to a constant weight, it was ground into powder with a grinding machine. Weigh 0.2 g of the powder in a beaker, place the beaker in a cold water bath, add 60 ml of 60% H2SO4, and digest for 30 minutes. Then transfer the digested solution to a 100 ml volumetric flask, and adjust it to the etching line with 60% H2SO4, shake it well, and filter it into a separate beaker using a Buchner funnel. Add 5 ml of the filtrate to a 100 ml volumetric flask, dilute it with distilled water on a cold water bath, and shake it well. 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 to it. Shake the mixture well and let it stand for 12 minutes. Finally, measure the absorbance at 620 nm, and determine the cellulose content in the stone cells by referring to the standard curve calculated for microcrystalline cellulose (Macklin). Each sample was subjected to 3 independent biological replicates.

[0079] Example 3 Transient transformation of pear fruits

[0080] The culture conditions of the Agrobacterium strain were the same as those in Example 1. After discarding the supernatant, the fungal precipitate was resuspended in the osmotic medium (10 mM MgCl2, 10 mM MES, 200 μM AS, pH 5.6, OD 600 = 1.0). At room temperature, after 2 - 4 h of induction in the dark, infiltration was performed by injection at the equatorial part of the young fruit of 'Dangshansuli' at 35 DAF.

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

[0082] Example 4 Arabidopsis thaliana genetic transformation

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

[0084] (2) After the bacterial cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in the same 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 SILWET L-77 was added to a final concentration of 0.025%;

[0085] (3) The wild-type Arabidopsis thaliana to be transformed (bolting 10 - 15 cm) was trimmed of siliques and open flowers;

[0086] (4) The remaining flowers of Arabidopsis thaliana were immersed in the bacterial solution and vacuumed to 0.6 - 0.8 KPa, and immersed for 5 min;

[0087] (5) The plants were cultured in the dark at 22 °C for 24 h, and then the plants were taken out for normal culture, and the seeds were harvested for screening.

[0088] The harvested T0 generation seeds were planted in a screening medium (containing MS medium, 30 g / L sucrose, 0.75% agar, 20 mg / L hygromycin, 100 mg / L ticarcillin and 100 mg / L carbenicillin) for screening. The germinated seedlings were transplanted 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 harvested after they matured.

[0089] Example 5 Microscopic Observation of Transgenic Arabidopsis

[0090] The T2 generation seeds of Example 4 and wild-type seeds were planted in MS medium to form T3 generation transgenic lines for physiological measurement. They were cultured in a greenhouse with a photoperiod of 16 h light / 8 h dark and a relative humidity of 40%. The primary inflorescence stems were dried to a constant weight and then ground into powder using a grinding machine to measure the lignin and cellulose contents.

[0091] After 8 weeks of Arabidopsis cultivation, 3 T3 generation transgenic lines and wild-type Arabidopsis were randomly selected. The stems of Arabidopsis (about 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 dimethoxy acid buffer (pH = 7.4) and then with 1% OsO4 at 4 °C. Dehydration of the sections was performed using different concentrations of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%), and they were embedded in epoxy resin. After 2 days, they were cut using an ultramicrotome (LKB-8800, Sweden).

[0092] Paraffin sections were prepared and stained with toluidine blue and phloroglucinol according to the following steps:

[0093] Ethanol dehydration: Dehydration was carried out successively with 75% - 100% ethanol in 5 grades for 2 h each.

[0094] Clearing: Ethanol and xylene were used for stepwise clearing according to the ratio. Ethanol:xylene = 3:1 for 40 min;

[0095] Ethanol:xylene = 1:1 for 40 min; Ethanol:xylene = 1:3 for 4 min; Immersion in pure xylene for 1 h, repeated once.

[0096] Wax infiltration: Half the volume of xylene was added, and then half the volume of paraffin was added. The temperature was raised to 75 °C in an oven, and the melted paraffin was allowed to infiltrate for 2 h, and this was repeated once.

[0097] Embedding: After wax infiltration, the materials were picked up with forceps and placed in a paper box, and then embedded with melted pure wax solution.

[0098] Trimming the sections: Trim the embedded materials into a trapezoid according to their positions, and use a Leica RM 2015 manual microtome to cut sections with a thickness of about 6 μm.

[0099] Spreading and adhering the sections: Gently pick up the cut samples with small forceps, place them in a water bath at 35 - 45 °C to spread the sections, pick them up with a glass slide after the wax sections are spread, and dry them in an oven at 40 °C.

[0100] Deparaffinization: Insert the glass slides with samples into xylene for 15 min of deparaffinization, wash with xylene: absolute ethanol = 1:1 for 2 min, and rinse with absolute ethanol of different grades (100%, 95%, 90%, 85%, 80%, 75%, 70%) in sequence from high concentration to low concentration for 2 min each.

[0101] Staining: Stain with toluidine blue staining solution for 24 h, wash twice with 95% ethanol, absolute ethanol, xylene: absolute ethanol = 1:1, and pure xylene for 30 s each, cover with neutral balsam for mounting, and dry in an oven at 40 °C.

[0102] Observe and collect pictures with an upright fluorescence microscope.

[0103]

[0104] Determination of lignin content showed that the contents of G-type lignin and H-type lignin in the inflorescence stems increased significantly (Table 7). In addition, toluidine blue and phloroglucinol-hydrochloric acid staining were performed on paraffin sections of the stems of wild-type and PbrSND1 transgenic plants. It was found that the staining of lignified tissues in transgenic plants was stronger than that in wild-type plants, and the secondary cell wall thickness of vessel cells and interfascicular fiber cells was higher than that in wild-type plants ( Figure 6 ), further confirming that PbrSND1 positively regulates lignin deposition. Generally speaking, these findings indicate that PbrSND1 promotes lignin deposition and SCW thickening during sclereid development. Figure 8 It is shown that the cellulose content in the stems of Arabidopsis thaliana overexpressing PbrSND1 also increases significantly.

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

Claims

1. Gene PbrSND1 Use in the following (A1)-(A3): (A1) Use for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A3) Use in breeding for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; The gene PbrSND1 has a CDS sequence as shown in SEQ ID NO.

1.

2. The gene according to claim 1 PbrSND1 Use of the encoded protein in the following (A1)-(A3): (A1) Use for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A3) Use in breeding for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; The amino acid sequence of the said protein is shown as SEQ ID NO.

2.

3. A recombinant expression vector and / or transient expression vector containing the gene recited in claim 1 is used in the following (A1)-(A3): PbrSND1 ​ (A1) Use for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A3) Use in breeding for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; The gene PbrSND1 has a CDS sequence as shown in SEQ ID NO.

1.

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

5. Use of the recombinant bacterium containing the gene recited in claim 1 PbrSND1 in the following (A1)-(A3): (A1) Use for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A2) Use in the preparation of products for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; (A3) Use in breeding for increasing the lignin and / or cellulose content in pear fruits or Arabidopsis thaliana; The gene PbrSND1 has a CDS sequence as shown in SEQ ID NO.

1.

6. The application according to any one of claims 1-5, characterized in that The application is achieved by overexpressing the gene PbrSND1 transferred into the target plant.

7. A method for increasing the lignin and / or cellulose content of pear fruits, characterized in that, The method is achieved by increasing the expression level of a gene in pears PbrSND1 The CDS sequence of the gene PbrSND1 is shown in SEQ ID NO.1 。 8. The method according to claim 7, characterized in that, The method is achieved by overexpressing the gene PbrSND1 by transferring it into pears.

9. A method for increasing the lignin and / or cellulose content in Arabidopsis thaliana, characterized in that, The method is achieved by increasing the expression level of a gene in Arabidopsis thaliana PbrSND1 and the CDS sequence of the gene PbrSND1 is shown in SEQ ID NO.1 。 10. The method according to claim 9, wherein The method is achieved by overexpressing the gene PbrSND1 by transferring it into Arabidopsis thaliana.

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