A gene pbtcp13 for regulating stone cells of pear fruit

Abstract

The application discloses a gene for regulating stone cells of pear fruits PbTCP 13 The CDS sequence of the gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the gene is shown as SEQ ID NO. 2. The gene is constructed into an overexpression vector, and the overexpression vector is injected into 'Dangshan Quli' pear fruits, so that the stone cell content and the lignin content of the injection part are significantly lower than those of a part injected with an empty control; the gene is introduced into Arabidopsis thaliana to obtain PbTCP13 transgenic Arabidopsis thaliana, and the secondary cell wall of the vessel cell of the transgenic Arabidopsis thaliana is significantly thinned, and the lignin accumulation in the stem is significantly reduced. The gene provides a new gene resource for fruit quality breeding, and is an important candidate gene for future gene engineering improvement of fruit quality breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and fruit tree genetics and breeding, specifically to a key gene, PbTCP13, that regulates the formation of stone cells in pear fruit and its application in improving the quality of pear fruit. Background Technology

[0002] Pear is one of the important fruit tree species in Jiangsu Province. Due to varietal characteristics, pear fruits often develop stone cells due to cell lignification. These unique lignified cells in pears, which significantly affect quality, involve the gradual thickening of the secondary cell wall of parenchyma cells to form a hard cell wall. Lignin is the main component of the secondary cell wall, so lignin metabolism is inextricably linked to stone cell formation (Tao et al., 2009). However, due to the complexity of the stone cell formation mechanism, the underlying regulatory network has not yet been fully elucidated.

[0003] Therefore, further exploration of the key factors in stone cell formation is of great significance for accelerating the improvement of pear fruit quality. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a key gene regulating stone cell formation in pear fruits. This gene was isolated and cloned from the high-stone-cell content variety 'Dangshan Crisp Pear' (Pyrus bretschneideri), and named PbTCP13 by the applicant. Its CDS sequence is shown in SEQ ID NO.1, and its corresponding protein sequence is shown in the sequence listing SEQ ID NO.2. The discovery of this gene may provide new insights into improving the quality of pear fruits.

[0005] Another objective of this invention is to provide an application of the aforementioned gene PbTCP13 and related biological materials. An overexpression vector of this gene was constructed and introduced into Arabidopsis thaliana via Agrobacterium-mediated genetic transformation. The resulting transgenic material underwent biological function verification, demonstrating that the PbTCP13 gene cloned in this invention has the function of reducing lignin accumulation and secondary cell wall thickening.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides the application of the gene PbTCP13 in the following (A1)-(A3):

[0008] (A1) Applications that reduce the lignin content in plants;

[0009] (A2) Application in the preparation of products that reduce the lignin content in plants;

[0010] (A3) Application in breeding to reduce lignin content in plants;

[0011] The CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.1.

[0012] Secondly, the present invention also provides the use of the protein encoded by the gene PbTCP13 in the following (A1)-(A3):

[0013] (A1) Applications that reduce the lignin content in plants;

[0014] (A2) Application in the preparation of products that reduce the lignin content in plants;

[0015] (A3) Application in breeding to reduce lignin content in plants;

[0016] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0017] The protein's secondary structure is mainly composed of irregular coils and α-helices, making it a stable protein; the protein has 601 amino acids.

[0018] Thirdly, the present invention also provides the application of recombinant expression vectors and transient expression vectors containing the gene PbTCP13 in the following (A1)-(A3):

[0019] (A1) Applications that reduce the lignin content in plants;

[0020] (A2) Application in the preparation of products that reduce the lignin content in plants;

[0021] (A3) Application in breeding to reduce lignin content in plants.

[0022] The present invention can construct a recombinant expression vector containing the gene PbTCP13 using existing plant expression vectors.

[0023] When constructing a recombinant plant overexpression vector using the PbTCP13 gene, a strong cauliflower mosaic virus (CAMV) 35S promoter can be added before its transcription initiation nucleotide. When constructing a plant expression vector using the gene of this invention, ATG can be used as the start codon, but it must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0024] To facilitate the identification and screening of transgenic plants, the plant expression vectors used are processed to incorporate genes that encode luminescent compounds (luciferase genes) and antibiotic resistance markers (kanamycin markers). For safety reasons, no selective marker genes may be added, and transformed plants can be screened directly with hygromycin.

[0025] In a specific implementation, the backbone vector of the recombinant expression vector is pCAMBIA1300-GFP.

[0026] Fourthly, the present invention also protects the use of recombinant bacteria containing the aforementioned gene PbTCP13 in the following (A1)-(A3):

[0027] (A1) Applications that reduce the lignin content in plants;

[0028] (A2) Application in the preparation of products that reduce the lignin content in plants;

[0029] (A3) Application in breeding to reduce lignin content in plants.

[0030] In a specific implementation, the application is achieved by overexpressing the gene PbTCP13 in the target plant.

[0031] In one embodiment of the present invention, a vector carrying the pCAMBIA1300-GFP to guide the expression of a foreign gene in plants is used to introduce the gene encoding the protein into Arabidopsis thaliana, thereby obtaining transgenic Arabidopsis plants. The expression vector carrying the gene can be transformed into Arabidopsis thaliana using an Agrobacterium-mediated transformation method (floret infection method), and the transformed Arabidopsis seeds can be harvested.

[0032] In one embodiment of the present invention, a vector using pCAMBIA1300-GFP to guide the expression of a foreign gene in plants was used to transiently introduce the gene encoding the protein into young fruit of 'Dangshan Crisp Pear' 35 days after flowering. The transiently injected fruit was obtained, and relevant indicators were measured, revealing overexpression. PbTCP13 It significantly reduced the content of stone cells and lignin in pear fruits.

[0033] The existing technology (Zhao Y, Su X, Wang X, Wang M, Chi X, Aamir Manzoor M, Li G, Cai Y. Comparative Genomic Analysis of TCP Genes in Six Rosaceae Species and Expression Pattern Analysis in Pyrus bretschneideri. Front Genet. 2021 May17;12:669959. doi: 10.3389 / fgene.2021.669959. PMID: 34079584; PMCID:PMC8165447) shows that, PbTCP14 and PbTCP15 Genes may be involved in the thickening of the secondary cell wall during pear stone cell formation, which is consistent with the present application. PbTCP13 Negative regulation of sclereid content and lignin content in pear fruit produces opposite effects.

[0034] The plants described in this invention can be either monocotyledonous or dicotyledonous, such as Arabidopsis thaliana and pear.

[0035] This 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 resulting transgenic lines were verified by biological function. The lignin content was significantly reduced, the secondary cell walls of the stem vascular cells were significantly thinned, and the expression levels of lignin synthesis-related genes were also significantly reduced.

[0036] Fifthly, the present invention protects a method for reducing the lignin content of pear fruit, said method being achieved by increasing the expression level of the gene PbTCP13 in pear.

[0037] In a specific implementation, the method is achieved by overexpressing the gene PbTCP13 in the target plant.

[0038] Sixthly, the present invention protects a method for reducing the lignin content in Arabidopsis thaliana, said method being achieved by increasing the expression level of the gene PbTCP13 in Arabidopsis thaliana.

[0039] In a specific implementation, the method is achieved by overexpressing the gene PbTCP13 in the target plant.

[0040] Beneficial effects

[0041] The present invention provides a gene, PbTCP13, that regulates the formation of stone cells in pear fruit, which has the following advantages compared with the prior art:

[0042] (1) This invention is the first to discover that PbTCP13 can negatively regulate the lignin content in pear fruit.

[0043] (2) The gene PbTCP13 provided by this invention was overexpressed in Arabidopsis thaliana. The transgenic lines obtained were verified by biological function, showing a significant reduction in lignin content and a significant thinning of the secondary cell wall of stem vascular cells.

[0044] (3) The discovery of the gene PbTCP13 in this invention provides new gene resources for fruit quality breeding and is an important candidate gene for future genetic engineering to improve fruit quality breeding. Attached Figure Description

[0045] Figure 1 This is an analysis of transient overexpression of PbTCP13 in pear fruit; among which, Figure 1Figure A in the image shows the phloroglucinol-hydrochloric acid staining image of 'Dangshan Crisp Pear' 7 days after PbTCP13 overexpression; 35S represents the empty vector control; 35S-PbTCP13 represents PbTCP13 overexpression mediated by the 35S strong promoter; scale bar = 1cm. Figure 1 Figure B in the figure shows the lignin content in pear fruit that overexpresses PbTCP13; Figure 1 Figure C in the figure represents the content of stone cells overexpressing PbTCP13 in pear fruit; Figure 1 Figure D in the figure shows the expression level of lignin-related genes and PbTCP13 in pear fruits overexpressing PbTCP13; (*p < 0.05, **p < 0.01, ***p < 0.001).

[0046] Figure 2 This is an analysis of the overexpression of PbTCP13 in Arabidopsis thaliana; among which, Figure 2 Figure A shows the phenotypes of wild-type and PbTCP13-overexpressing plants after 55 days of growth; scale bar = 5cm. Figure 2 Figure B in the diagram shows the qRT-PCR analysis, which indicates that PbTCP13 was successfully overexpressed in the transgenic lines. Figure 2 Figure C in the figure represents the lignin content analysis of wild-type and PbTCP13-overexpressing plants; Figure 2 Figure D in the figure shows the observation of toluidine blue and safranin-fast green staining of paraffin sections of stems from wild-type and PbTCP13-overexpressing Arabidopsis thaliana plants. Scale bar = 25 μm. Figure 2 Figure E in the figure is a statistical analysis of the thickness of the secondary cell wall (SCW) of duct cells in wild-type and PbTCP13 transgenic lines; Figure 2 The F-plot in the figure represents the expression level of lignin-related genes in Arabidopsis thaliana overexpressing PbTCP13; (*p < 0.05, **p < 0.01, ***p < 0.001). Detailed Implementation

[0047] The present invention will now be described 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 its spirit and scope to make it suitable for various uses and conditions.

[0048] Experimental methods in the following examples, unless otherwise specified, are generally performed using methods known in the art. Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0049] Example 1: Obtaining the PbTCP13 gene from pear

[0050] Based on the PbTCP13 gene sequence, specific primer pairs for amplifying this sequence were designed using Primer Premier 5.0.

[0051] The specific steps are as follows:

[0052] Using cDNA from Dangshan pear 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 primer sequences are as follows:

[0053] PbTCP13-F:

[0054] gagaacacgggggactctagaATGGACTTATCAAATTTCCAACCC, as shown in SEQ ID NO.3;

[0055] PbTCP13-R:

[0056] gcccttgctcaccatggatccTTGAGAACTGTTTGGGGCTTCA, as shown in SEQ ID NO.4.

[0057] Table 1 Gene amplification system

[0058]

[0059] Table 2 Gene Amplification PCR Procedure

[0060]

[0061] The amplified products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The pCAMBIA1300-GFP vector (TransGen, China) 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℃ 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-PbTCP13. The ligation system is shown in Table 4. After incubation at 37℃ for 30 min, the vector was transformed into competent E. coli DH5α cells (Tsingke, China). The E. coli transformation method is as follows:

[0062] (1) Add 20 μL of ligation product to 50 μL of Escherichia coli competent DH5α (Tsingke, China) cells thawed in an ice bath, mix gently, and place on ice for 30 min;

[0063] (2) After heat shock in a 42℃ water bath for 45 seconds, place it in ice for 2 minutes. During this process, the centrifuge tube should be kept still.

[0064] (3) Add 600 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 200 rpm for 1-2 h to allow the bacteria to recover;

[0065] (4) After centrifuging at 4000 rpm for 3 min, discard 500 μL of supernatant, resuspend, take 100 μL of recovered competent cells and spread them evenly on LB solid medium containing the corresponding antibiotics. Place the culture dish upside down in a 37℃ constant temperature incubator and culture overnight.

[0066] Table 3 GFP vector double enzyme digestion lines

[0067]

[0068] Table 4 Overexpression vector linkage system

[0069]

[0070] Table 5 Positive clone identification system

[0071]

[0072] 12-16 hours after transformation, single clones from the plate were picked and transferred to 1 mL centrifuge tubes. LB broth containing the appropriate antibiotic was added, and the mixture was incubated at 37°C with shaking until the culture became turbid. Positive identification was then performed. The reagents used were 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, they were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Based on the sequencing results, the gene sequence of PbTCP13 was obtained.

[0073] Table 6 Gene Amplification PCR Procedure

[0074]

[0075] The gene sequence of this gene in pear was obtained by cloning the expression vector PbTCP13-F. After sequencing, it was found that a 1806bp CDS sequence was isolated from the GFP vector, which is SEQ ID NO.1 and has a length of 1806bp. The gene encodes a protein of 601 amino acids, which is SEQ ID NO.2.

[0076] The correctly sequenced bacterial cultures were amplified and used for bacterial strain and plasmid extraction. The bacterial culture was preserved by mixing the bacterial culture and glycerol in a ratio of 7:3 (V:V), then flash-frozen in liquid nitrogen and stored at -80°C for later use. Plasmid extraction was performed using the FastPure Plasmid Mini Kit (Vazyme, China).

[0077] Thaw Agrobacterium competent cells GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China) on ice, then add the recombinant vector plasmid containing the target gene. Incubate on ice for 5 min, then flash-freeze in liquid nitrogen for 5 min, followed by heat shock at 37°C for 5 min, then on ice for 5 min. Add 700 μL of antibiotic-free LB medium and incubate on a shaker at 28°C and 250 rpm for 3 h. Centrifuge at low speed for 5 min, discard some of the supernatant, and resuspend 100 μL. Spread evenly on LB solid medium containing antibiotics (kana 50 μg / mL; rifampin 50 μg / mL). Invert the container at 28°C. After 48 h, use a sterile toothpick to pick single cloning sites for PCR verification. The PCR amplification system is shown in Table 6. Agrobacterium culture was preserved by mixing the culture with glycerol at a ratio of 7:3 (V:V), flash-freezing in liquid nitrogen, and storing at -80°C for later use.

[0078] Example 2: Analysis of stone cell and lignin content

[0079] The content of stone cells in the pulp was determined using a cryo-separation method. Three fruits of similar size (more from young fruits) were taken, the peel removed, and the edible portion was quartered. 100 g of each portion was weighed and placed in a -20℃ freezer for 24 hours. After thawing at room temperature, 200 ml of distilled water was added, and the mixture was homogenized using a tissue homogenizer (1000-1500 r·min). -1 Crush the mixture for 5 minutes. Then transfer the homogenate to a 1000 ml beaker, stir with a glass rod for 1 minute, and let it stand for 5 minutes to allow the stone cells to fully precipitate 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 to remove floating matter. Rinse with distilled water 5-6 times, collecting the first few suspensions and rinsing. Combine the obtained stone cells, filter through coarse filter paper, and finally separate the pure stone cells. Dry to constant weight and weigh.

[0080] Accurately weigh 0.01 g of fruit pulp powder sample using a 0.01 g balance, grind it into a homogenate with 95% ethanol, bring the volume to 5 ml, centrifuge at 12000 g for 2 min and discard the supernatant, wash three times with 95% ethanol, then wash three times with ethanol:n-hexane = 1:2 (V / V), and dry in a fume hood. Then add 2 ml of 25% bromoacetate solution, incubate in a 70°C water bath for 30 min, add 0.9 ml of 2M NaOH solution to terminate the reaction, then add 5 ml of acetic acid and 0.1 ml of 7.5M hydroxylamine chloride solution, bring the volume to 10 ml with glacial acetic acid, and measure the absorbance at 280 nm. Finally, determine the lignin content using a lignin standard sample (Sigma-Aldrich, USA) curve (Syros et al., 2004).

[0081] This experiment investigated the content of stone cells and lignin in pear fruits transiently overexpressed with PbTCP13, and found that overexpression of PbTCP13 significantly reduced the content of stone cells and lignin in pear fruits.

[0082] Example 3 Instantaneous transformation of pear fruit

[0083] The Agrobacterium strain was cultured under the same conditions as in Example 1. After discarding the supernatant, the bacterial precipitate was resuspended in osmotic medium (10 mM MgCl2, 10 mM MES, 200 μM AS, pH 5.6, OD 5.5). 600 =1.0). At room temperature, after induction in the dark for 2-4 hours, the young fruit of 'Dangshan Crisp Pear' at 35 DAF was injected into the equatorial region.

[0084] The results showed that lignin staining was reduced at the PbTCP13 injection site 7 days after injection, compared with the corresponding non-injection site and GFP. Figure 1 Figure A in the diagram shows a significant decrease in lignin content. Figure 1 (Figure B in the diagram) The content of stone cells is also significantly reduced ( Figure 1 (See Figure C in the diagram). After overexpression, the gene expression level of PbTCP13 at the injection site significantly increased, indicating successful overexpression of PbTCP13. Furthermore, the expression of lignin synthesis-related genes at the injection site significantly decreased (…). Figure 1 (See Figure D in the diagram). Therefore, PbTCP13 negatively regulates the lignin content in pear fruits.

[0085] Example 4: Genetic transformation of Arabidopsis thaliana

[0086] (1) The Agrobacterium strain containing the expression vector 35S-PbTCP13 (hereinafter referred to as PbTCP13 Agrobacterium strain) that was verified by PCR in Example 1 was added to 20 mL LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) with 100 μL of PbTCP13 Agrobacterium strain and cultured in a shaker at 28°C for 16 h.

[0087] (2) After centrifuging the bacterial cells at 4000 rpm for 10 min, discard the supernatant and resuspend them 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 add SILWETL-77 to make the final concentration 0.025%.

[0088] (3) Cut off the siliques and open flowers from the wild-type Arabidopsis thaliana (10-15 cm bolting) to be transformed;

[0089] (4) Soak the preserved flowers of Arabidopsis thaliana in the bacterial solution and vacuum them to 0.6-0.8 kPa for 5 min;

[0090] (5) Cultivate in the dark at 22℃ for 24 hours, then take out the plants for normal cultivation, harvest the seeds and wait for screening.

[0091] The harvested T0 generation seeds were screened on a selection medium (containing MS medium, 30 g / L sucrose, 0.75% agar, 20 mg / L hygromycin, 100 mg / L termethin and 100 mg / L carboxylation). The resulting seedlings were then transferred to plastic containers containing 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.

[0092] Example 5 Transgenic Arabidopsis thaliana

[0093] T2 generation seeds and wild-type seeds from Example 4 were planted in MS medium to form T3 generation transgenic lines for physiological assays. The plants were cultured in a greenhouse with a photoperiod of 16 h light / 8 h dark and a relative humidity of 40%. Primary inflorescence stems were dried to constant weight and then ground into powder using a sample grinder to determine the lignin content.

[0094] After 8 weeks of Arabidopsis thaliana culture, three T3 transgenic lines and wild-type Arabidopsis thaliana were randomly selected. Paraffin sections were prepared and stained with toluidine blue and safranin-fast green according to the following steps:

[0095] Ethanol dehydration: Use 75% to 100% ethanol to dehydrate in 5 grades for 2 hours each.

[0096] Transparency: Ethanol and xylene were gradually diluted in a specific ratio to achieve transparency. Anhydrous ethanol:xylene = 3:1 was used for elution for 40 minutes.

[0097] Treatment with anhydrous ethanol:xylene = 1:1 for 40 min; treatment with anhydrous ethanol:xylene = 1:3 for 4 min; soaking in pure xylene for 1 h, repeated once.

[0098] Wax impregnation: Add half the volume of xylene and half the volume of paraffin wax, heat to 75°C in an oven, and impregnate with the molten paraffin wax for 2 hours. Repeat once.

[0099] Embedding: After the wax impregnation is completed, the material is picked up with tweezers and placed in a cardboard box, and then embedded with a pure wax solution that has been melted into liquid.

[0100] Trimming and sectioning: The embedded material is trimmed into a trapezoidal shape according to its position and sectioned using a Leica RM 2015 hand-cranked microtome to a thickness of approximately 6 μm.

[0101] Spreading and mounting: Gently pick up the cut sample with small tweezers and place it in a water bath at 35-45℃ to spread. After the wax slide has spread, remove it with a glass slide and place it in a 40℃ oven to dry.

[0102] Dewaxing: Insert the glass slide with the sample attached into xylene and dewax for 15 minutes. Wash with xylene: anhydrous ethanol = 1:1 for 2 minutes. Rinse with anhydrous ethanol in different grades (100%, 95%, 90%, 85%, 80%, 75%, 70%), washing for 2 minutes in sequence from high concentration to low concentration.

[0103] Staining: Stain with toluidine blue staining solution for 24 hours, then wash twice for 30 seconds each with 95% ethanol, anhydrous ethanol, xylene:anhydrous ethanol = 1:1, and pure xylene. Cover and mount with neutral resin and dry in an oven at 40℃.

[0104] Images were captured and observed using an upright fluorescence microscope.

[0105] The results showed that plants overexpressing PbTCP13 had significantly higher yields than those expressing WT ( Figure 2 Figure A in the diagram shows that qRT-PCR confirmed the successful overexpression of PbTCP13 in the transgenic lines. Figure 2 Figure B in the diagram shows that the lignin content in the inflorescence stem was significantly reduced. Figure 2 (Figure C in the diagram). Furthermore, observation of paraffin sections of stems from wild-type and PbTCP13 transgenic plants stained with toluidine blue and safranin-fast green revealed that the staining of ligninized tissue in transgenic plants was weaker than that in wild-type plants (Figure C in the diagram). Figure 2 (Figure D in the diagram). The SCW thickness of ductal cells was significantly lower than that of wild-type cells (Figure D in the diagram). Figure 2 Figure E in the diagram shows that the expression levels of lignin-related genes are downregulated. Figure 2 (See Figure F in the diagram). This further confirms that PbTCP13 negatively regulates lignin deposition. In summary, these findings suggest that PbTCP13 reduces lignin deposition and thins the SCW during sclereid development.

[0106] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. Application of gene PbTCP13 in any of the following (A1)-(A3): (A1) Application in reducing lignin content in pear fruit or Arabidopsis thaliana; (A2) Application in the preparation of products that reduce the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding to reduce lignin content in pear fruits or Arabidopsis thaliana; The CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.1; The application is achieved by overexpressing the gene PbTCP13 in pear fruit or Arabidopsis thaliana.

2. The use of the protein encoded by the gene PbTCP13 according to claim 1 in any one of the following (A1)-(A3): (A1) Application in reducing lignin content in pear fruit or Arabidopsis thaliana; (A2) Application in the preparation of products that reduce the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding to reduce lignin content in pear fruits or Arabidopsis thaliana; The amino acid sequence of the protein is shown in SEQ ID NO.2; The application is achieved by overexpressing the gene PbTCP13 in pear fruit or Arabidopsis thaliana.

3. The use of the recombinant expression vector and / or transient expression vector containing the gene PbTCP13 as described in claim 1 in any of the following (A1)-(A3): (A1) Application in reducing lignin content in pear fruit or Arabidopsis thaliana; (A2) Application in the preparation of products that reduce the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding to reduce lignin content in pear fruits or Arabidopsis thaliana; The CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.1; The application is achieved by overexpressing the gene PbTCP13 in pear fruit or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The backbone vector of the recombinant expression vector is pCAMBIA1300-GFP.

5. The use of the recombinant bacteria containing the PbTCP13 gene as described in claim 1 in any one of the following (A1)-(A3): (A1) Application in reducing lignin content in pear fruit or Arabidopsis thaliana; (A2) Application in the preparation of products that reduce the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding to reduce lignin content in pear fruits or Arabidopsis thaliana; The CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.1; The application is achieved by overexpressing the gene PbTCP13 in pear fruit or Arabidopsis thaliana.

6. A method for reducing the lignin content of pear fruit, characterized in that, The method is achieved by overexpressing the gene PbTCP13 in pear fruit, and the CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.

1.

7. A method for reducing the lignin content in Arabidopsis thaliana, characterized in that, The method is achieved by overexpressing the gene PbTCP13 in Arabidopsis thaliana, and the CDS sequence of the gene PbTCP13 is shown in SEQ ID NO.1.

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