Gene PbMYB20 for regulating formation of stone cells of pear fruits

Abstract

The invention discloses a gene PbMYB20 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. When an overexpression vector constructed by the gene is injected into a Dangshan pear fruit, the content of stone cells and lignin is remarkably increased compared with that of injection of a no-load control part; when a silent vector constructed by the gene is injected into a Dangshan pear fruit, the stone cell content and the lignin content are obviously reduced compared with those of a no-load control part; an overexpression vector constructed by the gene is introduced into arabidopsis thaliana, the secondary cell wall of conduit cells of the obtained PbMYB20 transgenic arabidopsis thaliana is obviously thickened, and the accumulation of lignin in stems is obviously 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] This invention relates to the fields of plant molecular biology and fruit tree genetics and breeding, specifically to a key gene, PbMYB20, 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 an important fruit tree species in my country. Pears are highly favored by consumers for their crisp, juicy, and sweet-and-sour flavor. The quality of pears is mainly determined by factors such as peel characteristics, fruit shape, flesh texture, and aroma. Among these factors, stone cells, thick-walled cells unique to pears, are a core bottleneck restricting flesh texture and commercial value (Jin et al., 2013; Kolniak-Ostek, 2016). Studies have shown that the formation of stone cells mainly depends on the thickening of secondary cell walls and the large accumulation of lignin (Tao et al., 2009). When the stone cell content is too high, it leads to coarse pear flesh, insufficient juice, and increased hardness, which not only severely reduces the fruit's palatability but also affects the accumulation of nutrients such as sugars, organic acids, and vitamins, thereby lowering the market price and commercial competitiveness of the fruit (Choi et al., 2007; Zhang et al., 2024). In recent years, researchers have identified some key genes involved in the formation of stone cells in pear fruits. However, since stone cell formation is a complex regulatory process, its complete regulatory mechanism has not yet been fully elucidated. Therefore, in-depth exploration of the key factors regulating stone cell formation in pear fruits has important theoretical support and practical production application value for improving pear fruit quality, promoting the quality and efficiency of the pear industry, and accelerating the breeding of high-quality pear varieties. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide a key gene regulating the formation of stone cells in pear fruits. This gene was isolated and cloned from the high-stone-cell content variety 'Dangshan Crisp Pear' (Pyrus bretschneideri), and named PbMYB20. 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 provides new insights for improving the quality of pear fruits.

[0004] Another objective of this invention is to provide an application of the aforementioned gene PbMYB20 and related biological materials; an overexpression vector of the gene was constructed and introduced into Arabidopsis thaliana through Agrobacterium-mediated genetic transformation. The resulting transgenic material was verified by biological function, showing that the PbMYB20 gene cloned in this invention has the function of promoting lignin accumulation and secondary cell wall thickening; when the gene was silenced and transferred into pear fruit, the lignin content was significantly reduced.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides the application of a substance that reduces the content or activity of PbMYB20 protein, or reduces / silences the expression level of the PbMYB20 gene, in reducing the lignin content in pear fruit or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following:

[0007] a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20;

[0008] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0009] a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2);

[0010] a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3);

[0011] The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.1.

[0012] Secondly, the present invention provides the use of a substance that reduces the content or activity of PbMYB20 protein, or reduces / silences the expression level of the PbMYB20 gene, in the preparation of a product that reduces the lignin content in pear fruit or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following:

[0013] a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20;

[0014] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0015] a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2);

[0016] a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3);

[0017] The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.1.

[0018] Thirdly, the present invention provides the application of substances that reduce the content or activity of PbMYB20 protein, or reduce / silence the expression level of the PbMYB20 gene, in breeding to reduce the lignin content in pear fruits or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following:

[0019] a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20;

[0020] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0021] a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2);

[0022] a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3);

[0023] The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.1.

[0024] In specific implementation plans, reducing the content or activity of PbMYB20 protein can be achieved by reducing / silencing the expression level of the PbMYB20 gene.

[0025] Any methods known in the art for reducing the content or activity of PbMYB20 protein, such as using small molecule inhibitors or CRISPR-Cas9-mediated gene knockout, are within the scope of protection of this invention as long as they can reduce the content or activity of PbMYB20 protein.

[0026] In specific implementation schemes, the nucleic acid molecules described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules may also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0027] In a specific implementation, the nucleic acid molecule sequence in a1) is shown in SEQ ID NO.3.

[0028] In a specific implementation scheme, in a3), the backbone vector of the recombinant vector is TRV2.

[0029] Fourthly, the present invention provides a method for reducing the lignin content of pear fruit, the method being achieved by reducing the expression level of the gene PbMYB20 in pear, the CDS sequence of the gene PbMYB20 being shown in SEQ ID NO.1.

[0030] Fifthly, the present invention provides a method for reducing the lignin content in Arabidopsis thaliana, the method being achieved by reducing the expression level of the gene PbMYB20 in Arabidopsis thaliana, the CDS sequence of the gene PbMYB20 being shown in SEQ ID NO.1.

[0031] In a sixth aspect, the present invention provides a breeding method for reducing the lignin content of pear fruit, wherein the method is achieved by reducing the expression level of gene PbMYB20 in pear fruit, and the CDS sequence of gene PbMYB20 is shown in SEQ ID NO.1.

[0032] In a seventh aspect, the present invention provides a breeding method for reducing the lignin content in Arabidopsis thaliana, the method being achieved by reducing the expression level of the gene PbMYB20 in Arabidopsis thaliana, the CDS sequence of the gene PbMYB20 being shown in SEQ ID NO.1.

[0033] In specific implementation schemes, the method can be achieved through RNA interference technology, antisense oligonucleotide technology, or CRISPR / Cas9 gene editing methods.

[0034] In a specific implementation plan, the method includes the following steps:

[0035] (1) Construct a recombinant expression vector to silence the gene PbMYB20 mentioned above;

[0036] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant.

[0037] In a specific implementation scheme, the backbone vector of the recombinant expression vector is TRV2.

[0038] Lignin is the main component of stone cells, and changes in lignin content are a key indicator of stone cell formation and accumulation. As those skilled in the art can understand, regulating lignin content can effectively regulate stone cell formation.

[0039] Eighthly, the present invention also protects the use of the gene PbMYB20, the protein encoded by the gene PbMYB20, recombinant expression vectors and / or transient expression vectors containing the gene PbMYB20, and recombinant bacteria containing the gene PbMYB20 in the following (A1)-(A3):

[0040] (A1) Applications to increase lignin content in pear fruits or Arabidopsis thaliana;

[0041] (A2) Application in the preparation of products that increase the lignin content in pear fruit or Arabidopsis thaliana;

[0042] (A3) Application in breeding to increase lignin content in pear fruits or Arabidopsis thaliana;

[0043] The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.1.

[0044] In a specific implementation, the amino acid sequence of the protein encoded by the gene PbMYB20 is shown in SEQ ID NO.2.

[0045] In a specific implementation, the application is achieved by overexpressing the gene PbMYB20 in the target plant.

[0046] In a specific implementation, the backbone vector of the recombinant expression vector is pCAMBIA1300-GFP.

[0047] In a specific implementation, the recombinant expression vector can be constructed using methods disclosed in the prior art. This application provides an example method: inserting the gene PbMYB20 between the XbaI and BamHI sites of pCAMBIA1300 to obtain the recombinant expression vector 35S-PbMYB20-GFP.

[0048] 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.

[0049] 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 are harvested. Overexpression of this gene in Arabidopsis thaliana yields transgenic lines that, upon biological function verification, show a significant increase in lignin content, a significant thickening of the secondary cell wall of stem vascular cells, and a significant increase in the expression levels of lignin synthesis-related genes.

[0050] In one embodiment of the present invention, a vector using pCAMBIA1300-GFP and TRV2 to guide the expression of exogenous genes in plants was used to transiently introduce the gene encoding the protein into the young fruit of 'Dangshan Crisp Pear' 35 days after flowering, and the transiently injected fruit was obtained and its relevant indicators were measured.

[0051] Ninthly, the present invention provides a method for increasing the lignin content of pear fruit or Arabidopsis thaliana, characterized in that the method is achieved by increasing the expression level of gene PbMYB20 in pear or Arabidopsis thaliana, the CDS sequence of gene PbMYB20 being shown in SEQ ID NO.1.

[0052] In a tenth aspect, the present invention provides a breeding method for increasing the lignin content in pear fruit or Arabidopsis thaliana, characterized in that the method is achieved by increasing the expression level of gene PbMYB20 in pear or Arabidopsis thaliana, the CDS sequence of gene PbMYB20 being shown in SEQ ID NO.1.

[0053] In a specific implementation, the method is achieved by overexpressing the gene PbMYB20 in Arabidopsis thaliana.

[0054] Beneficial effects

[0055] The present invention provides a gene, PbMYB20, that regulates the formation of stone cells in pear fruit, which has the following advantages compared with the prior art:

[0056] (1) This invention is the first to discover that PbMYB20 can positively regulate the lignin content in pear fruit. Overexpression of PbMYB20 significantly increases the lignin content, while silencing PbMYB20 significantly reduces the lignin content.

[0057] (2) The gene PbMYB20 provided by this invention was overexpressed in Arabidopsis thaliana. The transgenic lines obtained were verified by biological function, showing a significant increase in lignin content and a significant thickening of the secondary cell wall of stem vascular cells.

[0058] (3) The discovery of the gene PbMYB20 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

[0059] Figure 1 This is an analysis of the transient overexpression of PbMYB20 in pear fruit; among which, Figure 1 Figure A in the image shows the phloroglucinol-hydrochloric acid staining image of 'Dangshan Crisp Pear' 7 days after PbMYB20 overexpression; EV-OE and EV-VIGS represent the empty vector control in the overexpression and silencing experiments, respectively; PbMYB20-OE and PbMYB20-VIGS represent PbMYB20 overexpression mediated by the 35S strong promoter and TRV2, respectively; scale bar = 1 cm; Figure 1 Figure B in the figure shows the lignin content of PbMYB20 overexpressed and silenced in pear fruit; Figure 1 Figure C in the figure represents the sclereid content of PbMYB20 overexpression and silencing in pear fruit; Figure 1 Figure D in the figure shows the expression levels of lignin-related genes and PbMYB20 in pear fruits overexpressing PbMYB20. Figure 1 Figure E in the figure shows the expression levels of lignin-related genes and PbMYB20 in pear fruit with silenced PbMYB20 (*p < 0.05, **p < 0.01, ***p < 0.001).

[0060] Figure 2 This is an analysis of the overexpression of PbMYB20 in Arabidopsis thaliana; among which, Figure 2 Figure A shows the phenotypes of wild-type and PbMYB20-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 PbMYB20 was successfully overexpressed in the transgenic lines. Figure 2 Figure C in the figure shows the lignin content analysis of wild-type and PbMYB20 overexpressing plants; Figure 2 Figure D in the figure shows the observation of toluidine blue staining on paraffin sections of stems from wild-type and PbMYB20-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 PbMYB20 transgenic lines; Figure 2 The F-plot in the figure represents the expression level of lignin-related genes in Arabidopsis thaliana overexpressing PbMYB20; (*p < 0.05, **p < 0.01, ***p < 0.001). Detailed Implementation

[0061] 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.

[0062] 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.

[0063] Example 1: Obtaining the PbMYB20 gene from pear

[0064] Based on the PbMYB20 gene sequence, specific primer pairs for amplifying this sequence were designed using Primer Premier 5.0.

[0065] The specific steps are as follows:

[0066] 1. Cloning of the PbMYB20 gene in pear

[0067] Using cDNA from Dangshan pear as a template, amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). The primer sequences were as follows:

[0068] PbMYB20-GFP-F:

[0069] gagaacacgggggactctagaATGGCTGCTCCTACAACCCC, as shown in SEQ ID NO.5;

[0070] PbMYB20-GFP-R:

[0071] gcccttgctcaccatggatccCGGCCCATCCATGTTCCA, as shown in SEQ ID NO.6.

[0072] The 50 μL PCR reaction system was as follows: 25 μL PCR Buffer for KOD FX Neo (2×), 1 μL forward primer F1 (10 µM), 1 μL reverse primer R1 (10 µM), 1 μL dNTPs (2.0 mM), 1 μL KOD FX Neo (1.0 U / μL), 200 ng cDNA, and sterile distilled water to bring the total volume to 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 35 amplification cycles, including 95℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, followed by a 72℃ extension for 10 min, and then incubation at 10℃. After amplification, a single target band of PCR product was detected by 1.5% agarose gel electrophoresis. The amplified product was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China).

[0073] 2. Construction of a plant overexpression vector for the pear PbMYB20 gene

[0074] The purified PCR product was ligated into the pCAMBIA1300-GFP vector and transformed into *E. coli* DH5α using a heat shock method. PCR detection was performed using gene-specific primers, and positive bacterial cultures were sequenced (Shanghai Sangon Biotech Co., Ltd.). Sequencing results showed that the target fragment amplified in this invention was 897 bp in length, with the sequence SEQ ID NO.1, and the gene encodes a 298-amino acid protein, with the sequence SEQ ID NO.2. The correctly obtained recombinant target vector was named 35S-PbMYB20-GFP. The recombinant vector 35S-PbMYB20-GFP and the control empty vector 35S-GFP were transformed into *Agrobacterium* GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China) using a heat shock method.

[0075] 3. Constructing a plant silencing vector for the pear PbMYB20 gene

[0076] The PbMYB20 gene was amplified using the one-stranded cDNA obtained in Part 1 of Example 1. The nucleotide sequences of the primer pairs are shown below:

[0077] PbMYB20-TRV2-F:

[0078] aaggttaccgaattctctagaGGAAAAAGCTGCAGATTGAGATG, as shown in SEQ ID NO.7;

[0079] PbMYB20-TRV2-R:

[0080] ggcctcgagacgcgtgagctcAGAAGAAGATTGCTCCATTTTTTGC, as shown in SEQ ID NO.8.

[0081] The target product was amplified using the PCR reaction system described in Part 1 of Example 1 by adding PbMYB20-TRV2-F and PbMYB20-TRV2-R, and the specific target band was recovered. The recovered and purified PCR product was ligated into the TRV vector and transformed into *E. coli* DH5α using a heat shock method. PCR detection was performed using gene-specific primers, and the positive bacterial cultures were sequenced (Shanghai Sangon Biotech Co., Ltd.). Sequencing results showed that a 252 bp CDS sequence was isolated from the TRV2 vector, which is SEQ ID NO. 3 and has a length of 252 bp; the theoretical protein sequence encoded by this gene is SEQ ID NO. 4. The correct recombinant target vector was named TRV-PbMYB20. The recombinant vector TRV-PbMYB20 and the control empty TRV vector were transformed into *Agrobacterium* GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China) using a heat shock method.

[0082] Example 2: Analysis of stone cell and lignin content

[0083] 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). -1Crush 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.

[0084] 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).

[0085] This experiment investigated the content of stone cells and lignin in pear fruits transiently overexpressed with PbMYB20, and found that overexpression of PbMYB20 significantly increased the content of stone cells and lignin in pear fruits.

[0086] Example 3 Instantaneous transformation of pear fruit

[0087] 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). After induction at room temperature in the dark for 2–4 hours, the lignin was injected into the equatorial region of young 'Dangshan Crisp Pear' fruits at 35 DAF. Results showed that 7 days after injection, the lignin staining effect at the site injected with the PbMYB20 overexpression vector was significantly stronger than that at the site injected with the empty control vector; conversely, the lignin staining effect at the site injected with the PbMYB20 silencing vector was weaker than that at the site injected with the empty control vector. Figure 1 (Figure A in the text).

[0088] Overexpression of PbMYB20 significantly increased lignin content, while silencing PbMYB20 had the opposite effect, significantly decreasing lignin content. Figure 1(Figure B in the diagram). Simultaneously, overexpression of PbMYB20 significantly increased sclereocyte content, while silencing of PbMYB20 significantly decreased sclereocyte content. Figure 1 (See Figure C in the diagram). After overexpression, the gene expression level of PbMYB20 at the injection site significantly increased, indicating successful overexpression of PbMYB20. Furthermore, the expression of lignin synthesis-related genes at the injection site significantly increased (…). Figure 1 (See Figure D in the diagram). After silencing, the gene expression level of PbMYB20 at the injection site was significantly reduced, indicating successful silencing of PbMYB20. The expression of lignin synthesis-related genes at the injection site was also significantly reduced (…). Figure 1 (See Figure E in the diagram). Therefore, PbMYB20 positively regulates the lignin content in pear fruits.

[0089] Example 4: Genetic transformation of Arabidopsis thaliana

[0090] (1) The Agrobacterium strain containing the expression vector 35S-PbMYB20 (hereinafter referred to as PbMYB20 Agrobacterium strain) that was verified by PCR in Example 1 was added to 20 mL of LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and cultured in a shaker at 28°C for 16 h.

[0091] (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%.

[0092] (3) Cut off the siliques and open flowers from the wild-type Arabidopsis thaliana (10-15 cm bolting) to be transformed;

[0093] (4) Soak the preserved flowers of Arabidopsis thaliana in the bacterial solution and vacuum them to 0.6-0.8 kPa for 5 min;

[0094] (5) Incubate in the dark at 22℃ for 24 hours, then remove the plants and culture them normally, and harvest the seeds for screening.

[0095] 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.

[0096] Example 5 Transgenic Arabidopsis thaliana

[0097] 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.

[0098] After 8 weeks of Arabidopsis thaliana culture, three T3 transgenic lines and wild-type Arabidopsis thaliana were randomly selected, and paraffin sections were prepared and stained with toluidine blue according to the following steps:

[0099] Ethanol dehydration: Use 75% to 100% ethanol to dehydrate in 5 grades for 2 hours each.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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℃.

[0108] Images were captured and observed using an upright fluorescence microscope.

[0109] The results showed that plants overexpressing PbMYB20 were significantly lower than WT ( Figure 2 Figure A in the diagram shows that PbMYB20 was successfully overexpressed in the transgenic lines by qRT-PCR. Figure 2 Figure B in the diagram shows that the lignin content in the inflorescence stem was significantly increased. Figure 2 (See Figure C in the diagram). Furthermore, toluidine blue staining of paraffin sections of stems from wild-type and PbMYB20 transgenic plants revealed that the staining of ligninized tissue in transgenic plants was stronger than that in wild-type plants (see Figure C in the diagram). Figure 2 (Figure D in the diagram). The SCW thickness of ductal cells was significantly higher 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 upregulated. Figure 2 (See Figure F in the diagram). This further confirms that PbMYB20 positively regulates lignin deposition. In summary, these findings suggest that PbMYB20 promotes lignin deposition and thickens the SCW during sclereid development.

[0110] 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. The application of substances that reduce the content or activity of PbMYB20 protein, or reduce / silence the expression level of the PbMYB20 gene, in reducing the lignin content in pear fruit or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following: a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2); a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3); in, The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.

1.

2. The application of substances that reduce the content or activity of PbMYB20 protein, or reduce / silence the expression level of the PbMYB20 gene, in the preparation of products that reduce the lignin content in pear fruits or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following: a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2); a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3); in, The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.

1.

3. The application of substances that reduce the content or activity of PbMYB20 protein, or reduce / silence the expression level of the PbMYB20 gene, in breeding to reduce the lignin content in pear fruits or Arabidopsis thaliana, wherein the substance that reduces / silences the expression level of the PbMYB20 gene is any one of the following: a1) Nucleic acid molecules representing the expression level of the silenced gene PbMYB20; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecule described in a1), or a recombinant vector containing the expression cassette described in a2); a4) Recombinant microorganisms containing the nucleic acid molecules described in a1), or recombinant microorganisms containing the expression cassette described in a2), or recombinant microorganisms containing the recombinant vector described in a3); in, The CDS sequence of the gene PbMYB20 is shown in SEQ ID NO.

1.

4. The application according to any one of claims 1-3, characterized in that, The scaffold vector of the recombinant vector is TRV2.

5. A method for reducing the lignin content of pear fruit, characterized in that, The method is achieved by reducing the expression level of the gene PbMYB20 in pear, the CDS sequence of which is shown in SEQ ID NO.

1.

6. A method for reducing the lignin content in Arabidopsis thaliana, characterized in that, The method is achieved by reducing the expression level of the gene PbMYB20 in Arabidopsis thaliana, the CDS sequence of which is shown in SEQ ID NO.

1.

7. A breeding method for reducing the lignin content of pear fruits, characterized in that, The method achieves this by reducing the expression level of the gene PbMYB20 in pear fruit, the CDS sequence of which is shown in SEQ ID NO.

1.

8. A breeding method for reducing the lignin content in Arabidopsis thaliana, characterized in that, The method is achieved by reducing the expression level of the gene PbMYB20 in Arabidopsis thaliana, the CDS sequence of which is shown in SEQ ID NO.

1.

9. The method according to any one of claims 5-8, characterized in that, The method includes the following steps: (1) Construct a recombinant expression vector to silence the gene PbMYB20 described in claim 1; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant.

10. The method according to claim 9, characterized in that, The backbone vector of the recombinant expression vector is TRV2.

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