Pear transcription factor PbrMYB24 and its application in promoting lignin and cellulose synthesis in sclereids

Abstract

This invention discloses the pear transcription factor PbrMYB24 and its application in promoting lignin and cellulose synthesis in pear stone cells. The PbrMYB24 gene, isolated from 'Dangshan Crisp Pear', has the nucleotide sequence shown in SEQ ID No. 1. Transient overexpression in pear fruit confirmed that PbrMYB24 can promote the accumulation of lignin and cellulose in fruit stone cells. In transgenic pear pulp callus tissue and Arabidopsis inflorescence stems overexpressing PbrMYB24, lignin and cellulose contents were significantly increased, and the signals of phloroglucinol hydrochloride staining, Fast Green staining, and Karl von Willebrand fluorescence staining were significantly enhanced at the same time. This demonstrates that the PbrMYB24 gene participates in regulating the formation of lignin and cellulose in pear fruit stone cells.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the pear transcription factor PbrMYB24 and its application. Specifically, it relates to the isolation and cloning of the PbrMYB24 gene, a member of the MYB family that regulates the development of stone cells in pear fruit, from 'Dangshan Crisp Pear'. Background Technology

[0002] Pear is a perennial woody plant belonging to the genus *Pyrus* of the subfamily Amygdaloideae in the family Rosaceae. It is the third largest fruit tree species in my country, with a cultivation history of over 3,000 years (Teng Yuanwen, 2017). The Asian pear, including white pear, sand pear, autumn pear, and Xinjiang pear, is widely cultivated in my country; while Western countries such as Europe and America mainly cultivate European pears. As the world's largest pear producer, my country has a cultivation area of ​​13.05 million mu (approximately 933,333 hectares) and a total output of 16.1 million tons, accounting for 67.3% and 69.7% of the world's total respectively (FAO, 2020), holding a pivotal position in international pear production. However, some traditional main varieties in my country, such as the Dangshan crisp pear, have high stone cell content, resulting in poor fruit quality and weak market competitiveness. Stone cells are a unique characteristic of pear flesh; a large number of stone cells can affect the taste and processing quality. Therefore, clarifying the differences in stone cell content and elucidating the stone cell formation mechanism are particularly important for improving the quality of pear fruits.

[0003] Stone cells are actually thick-walled tissue cells that develop through the gradual thickening of secondary cell walls. What are commonly referred to as fruit stone cells are actually "stone cell clusters" composed of multiple stone cells. The size, number, and density of these clusters directly affect the texture and taste of pear fruit (Xue et al., 2020). Pear fruit stone cells are mainly composed of lignin and cellulose. The average lignin content (29.73%) is higher than the average cellulose content (18.03%), indicating that lignin is the main component of pear fruit stone cells (Zhang et al., 2020). Current research on pear stone cells mainly focuses on physiology and anatomy. The formation of pear pulp stone cells is highly correlated with the biosynthesis, transport, and accumulation of lignin and cellulose. Further analysis is needed to elucidate the key genes and molecular mechanisms underlying pear fruit stone cell formation.

[0004] Lignin biosynthesis begins with the deamination of the aromatic amino acid phenylalanine, and proceeds through a series of hydroxylation, methylation, and reduction reactions to ultimately produce the three most common basic monomers in lignin: coniferyl alcohol, sinigrin, and p-coumaryl alcohol. These monomers, during polymerization into lignin, produce guaiacyl lignin monomers (G-type monomers), syringyl lignin monomers (S-type monomers), and p-hydroxyphenyl lignin monomers (H-type monomers), respectively. Finally, these lignin monomers polymerize under the action of LAC and POD to form lignin (Vanholme et al., 2010, Vanholme et al., 2013). Cellulose synthesis can be summarized as follows: the rose cyclization complex located on the cell membrane utilizes UDP-glucose in the cytoplasm as a direct substrate for cellulose synthesis, catalyzing the elongation of dextran chains. Then, glucose is further polymerized to form microfibrils, which are then deposited orderly on the cell wall (Zhong et al., 2019). Currently, there are no reports on the pear PbrMYB24 gene promoting lignin and cellulose synthesis in sclereids. Summary of the Invention

[0005] The purpose of this invention is to provide a PbrMYB24 gene that regulates the development of stone cells in pear fruits.

[0006] Another object of the present invention is to provide the application of this gene.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A transcription factor PbrMYB24 gene, isolated from 'Dangshan Crisp Pear', is a member of the MYB family and promotes the synthesis of lignin and cellulose in sclereids. Its nucleotide sequence is shown in SEQ ID No. 1, containing a 1044bp open reading frame; it encodes 347 amino acids, the amino acid sequence of which is shown in SEQ ID No. 2 of the sequence listing.

[0009] A recombinant expression vector containing the PbrMYB24 gene described in this invention.

[0010] The recombinant expression vector uses pCAMBIA1301 as the starting vector, and the insertion site of the PbrMYB24 gene is between Xba I and BamHI.

[0011] Genetically engineered bacteria containing the PbrMYB24 gene described in this invention.

[0012] Primer pairs for cloning the cDNA sequence of the PbrMYB24 gene described in this invention are shown in SEQ ID No. 3 for the upstream primer PbrMYB24-F1 and in SEQ ID No. 4 for the downstream primer PbrMYB24-R1.

[0013] The application of the PbrMYB24 gene described in this invention in promoting lignin and cellulose synthesis in sclereids.

[0014] The application of the recombinant expression vector described in this invention in promoting the synthesis of lignin and cellulose in sclereids.

[0015] Beneficial effects

[0016] Combining co-expression network analysis and transcriptome analysis, the applicant identified an R2R3-MYB transcription factor, PbrMYB24, in pear varieties with varying levels of stone cell content. The expression level of this gene in different varieties was significantly positively correlated with the stone cell content. Transient overexpression in pear fruit confirmed that PbrMYB24 promotes the accumulation of stone cells, lignin, and cellulose. Transgenic pear pulp callus tissue and Arabidopsis inflorescence stems overexpressing PbrMYB24 showed significantly increased lignin and cellulose content, with significantly enhanced signals in pyrogallol hydrochloride staining, Fast Green staining, and Karl von Willebrand fluorescence staining. This demonstrates that the PbrMYB24 gene participates in regulating the formation of lignin and cellulose in pear fruit stone cells. The discovery of this gene supplements and refines the MYB transcriptional regulation mechanism of lignin metabolism in pear, providing a theoretical basis for improving pear fruit stone cell content and offering genetic resources for the paper and bioenergy industries to reduce lignin content through gene editing.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] 1. The discovery of the PbrMYB24 gene provides a theoretical basis for improving the synthesis of stone cells in pear fruits. At the same time, it provides genetic resources for the paper industry and the bioenergy industry to reduce lignin content through gene editing. The development and utilization of this genetic resource is conducive to reducing agricultural costs and achieving environmental friendliness.

[0019] 2. The PbrMYB24 gene was functionally verified in pear juvenile fruit, pear flesh callus, and Arabidopsis thaliana using Agrobacterium-mediated genetic transformation. The results showed that the PbrMYB24 gene cloned in this invention has the advantage of simultaneously regulating enzymes in the lignin and cellulose synthesis pathways encoded by multiple genes, providing a more efficient approach for molecular breeding. Attached Figure Description

[0020] Figure 1 This invention analyzes the spatiotemporal expression patterns of the PbrMYB24 gene in different varieties and tissues.

[0021] The study included: A) Correlation analysis of PbrMYB24 expression and stone cell content in the pulp of 20 varieties 35 days after flowering. The bar chart represents PbrMYB24 expression, and the dots represent the stone cell content in the pulp. The PbrMYB24 expression level in the pulp of 'Zaosu Pear' 35 days after flowering was set to 1. B) Correlation analysis of PbrMYB24 expression and lignin content in the pulp of 20 varieties 35 days after flowering. The bar chart represents PbrMYB24 expression, and the dots represent the lignin content in the pulp. C) Correlation analysis of PbrMYB24 expression and cellulose content in the pulp of 20 varieties 35 days after flowering. The bar chart represents PbrMYB24 expression, and the dots represent the cellulose content in the pulp. D) Correlation analysis of the stone cell content and the relative expression level of PbrMYB24 in the fruit of 'Dangshan Crisp Pear' at different developmental stages. The bar chart represents the expression level of PbrMYB24, and the dots represent the content of stone cells in the pulp. DAFB indicates the number of days of fruit development after full bloom.

[0022] Figure 2 This is a schematic diagram of the carrier in Embodiment 2 of the present invention.

[0023] Figure 3 Functional analysis of transient injection of the PbrMYB24 gene into young pear fruits according to this invention.

[0024] Where: A, phloroglucinol hydrochloride staining of pear fruit sections transiently expressing the PbrMYB24 gene; EV represents the site of transformation into the empty pCAMBIA1301 vector; EV-OE represents the site of transformation into the 35S-PbrMYB24-GFP recombinant vector. B and C, lignin (B) and cellulose (C) content of the injected portion of the pulp. D, expression levels of genes related to lignin and cellulose synthesis in the injected pear pulp. * indicates significant difference.

[0025] Figure 4 Functional analysis of the PbrMYB24 gene in transgenic pear flesh callus tissue for the present invention.

[0026] Wherein: A, relative expression level of PbrMYB24 in transgenic pear callus. EV represents pear callus transformed with empty pCAMBIA1301 plasmid; the rest are transgenic pear callus. B and C, lignin (B) and cellulose (C) content in transgenic pear callus. D and E, phloroglucinol hydrochloride staining of pear callus showing lignin synthesis. F and G, paraffin sections of pear callus stained with Karl von Fluor fluorescence showing cellulose synthesis. H, expression levels of genes related to lignin and cellulose synthesis in transgenic pear callus. * indicates significant difference.

[0027] Figure 5 Functional analysis of the PbrMYB24 gene in transgenic Arabidopsis thaliana plants for the present invention.

[0028] Wherein: A and B represent the lignin (A) and cellulose (B) contents in wild-type and transgenic Arabidopsis thaliana plants. WT: Wild-type; the rest are transgenic lines. C: Expression levels of genes related to lignin and cellulose synthesis in wild-type and transgenic Arabidopsis thaliana plants. DK: Histological analysis of inflorescence stems of wild-type and transgenic Arabidopsis thaliana plants: DE, toluidine blue staining to indicate cell morphology; FI, Wiesner and UV light detection of lignin deposition spatial distribution; JK, Karl von Fluor fluorescence staining to detect cellulose deposition spatial distribution. LQ: Transmission electron microscopy to detect secondary cell wall thickness and statistical analysis. * indicates significant differences. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these 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 to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0030] Example 1: Spatiotemporal Expression Pattern Analysis of the PbrMYB24 Gene

[0031] Different tissue samples of 'Dangshan Crisp Pear' were collected from orchards in Suining County, Xuzhou City, Jiangsu Province. Pear varieties with high and low stone cell content were collected from the Jiangsu Academy of Agricultural Sciences. Total RNA was extracted using the CTAB method (Porebski et al., 1997), and the quality of the extracted samples was determined by spectrophotometry and agarose gel electrophoresis. 3 μg of extracted total RNA was used for reverse transcription using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. The primers used for real-time PCR were gene-specific primer pairs: SEQ ID No. 5 and SEQ ID No. 6; GAPDH was used as an internal reference gene. The real-time PCR kit was purchased from Roche. The instrument used for real-time PCR was a Roche 480 quantitative PCR instrument. The reaction system consisted of: 10 μL of 2×SYBR Green I Master Mix, 0.4 μL of forward and reverse primers (10 μM), 2 μL of cDNA, and 7.2 μL of PCR-grade water. The reaction conditions were: denaturation at 95℃ for 5 min; pre-denaturation at 95℃ for 5 s, annealing at 60℃ for 5 s, and extension at 72℃ for 10 s, repeated for 45 cycles; melting curve analysis was performed at 65℃ to 95℃, with an increase of 1℃ every 5 s.

[0032] We selected 20 varieties each from those with high and low pulp stone cell content. After detecting the stone cell content, lignin content, cellulose content, and relative expression level of PbrMYB24 in the pulp 35 days after flowering, we found that the pulp stone cell content, lignin content, cellulose content, and PbrMYB24 expression level were significantly positively correlated, with correlations of 0.86, 0.68, and 0.63, respectively. Figure 1 AC). The expression level of the PbrMYB24 gene in the pulp tissue 21-49 days after flowering was significantly higher than that in the pulp tissue after 63 days after flowering. Furthermore, the expression level of PbrMYB24 was significantly correlated with the content of stone cells in fruit development dynamics (correlation rate 0.74). Figure 1 D). Based on the above results, we speculate that the significant differences in PbrMYB24 expression levels may cause differences in the content of stone cells in the pulp of different varieties.

[0033] Example 2: Isolation, cloning, and construction of the overexpression vector for the PbrMYB24 gene

[0034] 3 μg of RNA from the early pulp of 'Dangshan Crisp Pear' was reverse transcribed using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Based on the multiple cloning site of the pCAMBIA-1301 vector and the restriction enzyme sites in the coding region of the PbrMYB24 gene, Xba I and BamHI were selected as restriction enzymes. Primers with restriction sites (SEQ ID NO. 3 and SEQ ID NO. 4) were designed using Snapgene software according to general primer design principles. The 50 μL reaction mixture included 200 ng cDNA, 1× buffer (TransStart FastPfu Buffer), 10 mM dNTPs, 1 U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500 nM of the primers. The PCR reaction was performed on an Eppendorf amplification instrument according to the following program: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 20 seconds, 60°C annealing for 20 seconds, 72°C extension for 1 minute, 35 thermal cycles, 72°C extension for 10 minutes, and storage at 4°C. A single PCR band was produced.

[0035] After PCR products were detected by 1% agarose gel electrophoresis, DNA fragments were recovered using a small-volume gel recovery kit (purchased from Kangwei Century, and the operation was performed according to the kit's instructions). The total volume of the double digestion system for the pCAMBIA1301 vector was 50 μL, containing 10 μL of the pCAMBIA1301 vector plasmid obtained from plasmid extraction, 5 μL of 10×Buffer (purchased from NEB), 1 μL of Xba I, 1 μL of BamHI, and 33 μL of water. Digestion was performed at 37°C for 3 hours, followed by recovery. The expression vector pCAMBIA1301, digested with restriction endonucleases, was ligated to the PbrMYB24 gene using recombinase Exnase II (purchased from Vazyme) at 37°C for 30 minutes. The total reaction volume was 20 μL, containing 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 2 μL of the PCR product of the PbrMYB24 gene, 6 μL of the double digestion product of the pCAMBIA1301 vector, and 6 μL of water. 10 μL of the ligation product was transformed into competent E. coli DH5α cells. Positive clones were screened on LB agar plates containing 50 mg / L kanamycin. Plasmids were extracted, digested, and identified by PCR. The recombinant plasmid samples were sent to a biotechnology company for sequencing. Sequencing results showed that the full-length PbrMYB24 gene is 1044 bp, and its nucleotide sequence is shown in SEQ ID NO.1. It encodes a protein with 348 amino acid residues, the sequence of which is shown in SEQ ID NO.2. We named the recombinant vector 35S-PbrMYB24-GFP. The constructed vector map is shown below. Figure 2 As shown, the recombinant vector was introduced into Agrobacterium GV3101 using the freeze-thaw method.

[0036] Example 3: Transient transformation and functional analysis of pear fruit

[0037] (1) Instantaneous transformation of pear fruit

[0038] Agrobacterium containing the PbrMYB24 overexpression vector was injected into 'Dangshan Crisp Pear' approximately 35 days after flowering using Agrobacterium-mediated transformation. The method is as follows:

[0039] 1. Activate Agrobacterium containing the correct plasmid on a solid culture medium and grow it in an incubator at 28°C for 48 hours;

[0040] 2. Add 30 mL of solution containing R to a 100 mL Erlenmeyer flask. + With K + Activated Agrobacterium was picked up with a pipette tip from the liquid LB medium and grown in a shaker at 28°C and 200 rpm for 12 hours.

[0041] 3. Pour the bacterial culture into a 50mL centrifuge tube and centrifuge at 6000rpm for 15 minutes to collect all bacterial cells;

[0042] 4. Resuspend the precipitate with an appropriate amount of induction medium (10mM MgCl2, 10mM MES, 200mM acetylsalicylic acid, pH 5.6), adjust the OD value to 0.8-1.2, and induce it for 4 hours at room temperature using a decolorizing shaker.

[0043] 5. Inject the infection solution into pear fruits about 35 days after flowering, injecting at least 10 fruits each time, and conduct three biological replicates of the experiment;

[0044] 6. Incubate in the dark for 24 hours, then place in an incubator with a photoperiod of 16 hours of light / 8 hours of darkness, maintaining a temperature of 22℃, and incubate for 7 days;

[0045] (2) Histological analysis

[0046] Cut the fruit open with a blade, and stain it with phloroglucinol-hydrochloric acid. The specific steps are as follows:

[0047] 1. First, treat with 30% hydrochloric acid solution (V / V) for 1 minute;

[0048] 2. 10% phloroglucinol is soluble in 80% ethanol (w / v);

[0049] 3. Then drip it onto the slices that have been treated with hydrochloric acid and stain for 5 minutes;

[0050] 4. Finally, rinse with plenty of water to stop the reaction.

[0051] Phloroglucinol-hydrochloric acid staining revealed significantly enhanced lignin staining in sites overexpressing PbrMYB24, while there was no significant difference between sites injected with pCAMBIA1301 vector and uninjected sites. Figure 3 A).

[0052] (3) Detection of lignin and cellulose content at the injection site

[0053] The lignin content of pear pulp callus was analyzed using the bromoacetylation method. After acetylation, the phenolic hydroxyl groups in lignin exhibit a characteristic absorption peak at 280 nm, and the absorbance at 280 nm is positively correlated with lignin content. The specific method is as follows:

[0054] 1. Weigh approximately 0.01 g of the dried sample into a mortar, with three replicates per sample. Add 1 mL of pH 7.0 PBS buffer, grind into a homogenate, and wash the mortar and pestle with phosphate buffer. Transfer the homogenate to a 15 mL centrifuge tube and centrifuge at 3000 g for 5 min (the centrifugation speed and time are the same throughout this method). Slowly discard the supernatant. Wash the precipitate twice with 5 mL of PBS buffer and twice with 5 mL of distilled water to remove as much soluble sugar as possible.

[0055] 2. Add 5 mL of chloroform-methanol (1:1, v / v) to the precipitate using a pipette, incubate at room temperature (approximately 25°C), shake at 150 rpm for 1 hour, centrifuge, and discard the supernatant. Wash the precipitate once with 5 mL of methanol, then once with 5 mL of acetone, and finally once with 5 mL of distilled water, discarding the supernatant.

[0056] 3. Add 5 mL of DMSO-H2O (9:1, v / v) to the precipitate, shake overnight at room temperature (12 h), centrifuge, wash the precipitate twice with 5 mL of DMSO-H2O, and then wash three times with 5 mL of distilled water. The precipitate at this point is coarse cell wall material.

[0057] 4. Add 2 mL of 25% bromoacetate solution (bromoacetyl:acetic acid = 1:3) and 4 μL of 60% perchloric acid. After incubating at 70°C for 30 min, add 0.9 mL of 2 mol / L NaOH to stop the reaction.

[0058] 5. Add 5 ml of acetic acid and 0.1 ml of 7.5 mol / L hydroxylamine chloride, and dilute to 10 ml with acetic acid. After standing for 10-20 min, measure the absorbance at 280 nm. The lignin content is expressed as the percentage of the measured value / the weight of the sample used.

[0059] Cellulose is a polysaccharide composed of β-glucose residues, and its measurement method follows the anthrone-sulfuric acid method. The specific method is as follows:

[0060] 1. The extraction of coarse cell wall material shall be performed in accordance with the method described above.

[0061] 2. Add 5 ml of 4 mol / L KOH to the coarse cell wall material and shake at room temperature for 1 h. Centrifuge to remove the supernatant. Wash the precipitate once with 5 ml of 4 mol / L KOH and twice with 5 ml of water.

[0062] 3. Add 5 ml of 60% H2SO4 to the above precipitate and shake at room temperature for 2 h. Then, dilute to 10 ml with water and centrifuge to collect all the supernatant.

[0063] 4. Take 1 ml of supernatant and add it to a glass test tube. Add 2 ml of 0.2% anthrone sulfuric acid reagent under cold water bath conditions. Then transfer it to boiling water and heat for 5 min. Cool it in water and measure the absorbance at 620 nm.

[0064] Compared with the pulp portion injected with pCAMBIA1301 vector, the lignin and cellulose content of the portion overexpressing PbrMYB24 was significantly increased. Figure 3 BC).

[0065] (4) Detection of relative expression levels of genes related to lignin and cellulose synthesis at the injection site

[0066] RNA extraction, cDNA synthesis, and the system and procedures for quantitative real-time PCR were performed according to Example 1. Overexpression of PbrMYB24 in the pulp significantly increased the expression levels of Pbr4CL1, Pbr4CL4, PbrCSE1, PbrCCOAMT6, PbrCCOAMT1, PbrF5H2, PbrLAC4, PbrSTONE, PbrCESA8a, PbrCESA8b, PbrMYB169, and PbrNSC. Figure 3 D). In summary, these results indicate that overexpression of PbrMYB24 promotes lignin and cellulose synthesis in pear fruit stone cells.

[0067] Table 1 Primers for real-time fluorescence quantitative PCR of pear

[0068]

[0069]

[0070] Example 4: Stable transformation and functional analysis of pear pulp callus

[0071] (1) Stable transformation of pear pulp callus tissue

[0072] Agrobacterium containing the PbrMYB24 overexpression vector was used to infect pear pulp callus tissue via Agrobacterium-mediated infection, as follows:

[0073] 1. Activate Agrobacterium containing the correct plasmid on a solid culture medium and grow it in an incubator at 28°C for 48 hours;

[0074] 2. Add 30 mL of solution containing R to a 100 mL Erlenmeyer flask. + With K + Activated Agrobacterium was picked up with a pipette tip from the liquid LB medium and grown in a shaker at 28°C and 200 rpm for 12 hours.

[0075] 3. Pour the bacterial culture into a 50mL centrifuge tube and centrifuge at 6000rpm for 15 minutes to collect all bacterial cells;

[0076] 4. Resuspend the precipitate in an appropriate amount of MS liquid medium (100mM acetylsalicylic acid, pH 5.8), adjust the OD value to 0.8-1.2, and induce at 28℃ for 1h;

[0077] 5. Add the pulp callus to MS liquid medium and induce at 28℃ for 15 min;

[0078] 6. Filter the fruit pulp callus tissue through gauze, place it on MS medium and grow for 2 days, cultured in the dark at 25℃.

[0079] 7. The fruit pulp callus was grown on MS medium containing hygromycin, and transgenic callus was screened.

[0080] (2) Identification of callus in transgenic pear pulp

[0081] RNA extraction, cDNA synthesis, and the system and steps for quantitative real-time PCR were as described in Example 1. We observed that nine fruit pulp callus tissues could grow on hygromycin-containing medium. These callus tissues were then transferred to new hygromycin-containing medium in a clean bench for continued growth. Seven of these fruit pulp callus tissues grew normally. Further analysis of the relative expression level of the PbrMYB24 gene in these seven callus tissues using quantitative real-time PCR showed that the expression level of the PbrMYB24 gene was higher in all seven transgenic callus tissues than in callus tissues transformed with the pCAMBIA1301 empty vector (EV). OE-2, OE-4, and OE-6 showed good growth and significantly higher expression levels than other positive callus tissues. Figure 4 A), therefore we selected these three transgenic lines for subsequent research.

[0082] (3) Histological analysis

[0083] After subculturing the above-mentioned transgenic calluses (EV, OE-2, OE-4, OE-6) for 15 days, they were transferred to a treatment medium containing 10 μM EBR for further culture. After 20 days of growth, pear pulp calluses were harvested for phloroglucinol-hydrochloric acid staining, following the specific steps outlined in Example 3-2. Phloroglucinol-hydrochloric acid staining revealed that pear pulp calluses overexpressing PbrMYB24 exhibited significantly enhanced lignin staining effects. Figure 4 DE), indicating that PbrMYB24 promotes lignin synthesis in pear callus tissue.

[0084] A small amount of transgenic pear callus tissue was further taken and fixed with FAA fixative at 4°C for more than one week. Paraffin sections were then prepared by following these steps:

[0085] 1. Dehydration: Dehydrate with 85%, 95%, 100%, and 100% ethanol sequentially for 2 hours, then dehydrate with anhydrous ethanol and xylene in a volume ratio of 1:1 for 2 hours.

[0086] 2. To make it transparent: Soak in xylene for 2 hours, then repeat once.

[0087] 3. Wax infiltration: Add 1 / 2 volume of xylene to the fixed bottle, then add 1 / 2 volume of molten wax. Heat in a drying oven to about 3°C ​​above the melting point of paraffin. After the paraffin melts, remove the cap. After 2 hours, infiltrate with molten pure wax for 2 hours. Repeat once.

[0088] 4. Embedding: The wax-impregnated material is embedded in a folded cardboard box using a pure wax solution with a temperature approximately 3°C higher than its melting point.

[0089] 5. Sectioning: Use a Leica RM 2015 microtome (Leica Mikrosysteme, Germany) to section the sample into 5μm thick sections;

[0090] 6. Spreading slides: Gently pick up the cut wax strip with a brush and place it in a 35℃ water bath to spread the slide. After it is fully spread, pick up the slide with a glass slide and place it in a 37℃ incubator to dry.

[0091] 7. Dewaxing: Place the dried material in pure xylene for 10 minutes (repeat once), anhydrous ethanol for 4 minutes (repeat once), 95% ethanol for 4 minutes, 85% ethanol for 4 minutes, and 70% ethanol for 2 minutes in sequence.

[0092] 8. Calcofluor staining: Stain the sections with 10ul Calcofluor White Stain for 5 minutes, then blot off the excess Calcofluor with filter paper and wash with water three times.

[0093] Paraffin sections were observed under UV excitation using an upright fluorescence microscope. The results showed that the fluorescence signal of pear pulp callus overexpressing PbrMYB24 was significantly enhanced. Figure 4 FG) indicates that PbrMYB24 promotes cellulose synthesis in pear callus tissue.

[0094] (4) Analysis of lignin and cellulose content in transgenic callus

[0095] The lignin content of transgenic callus was measured using the bromoacetyl method, with specific steps as described in Examples 3-3. The results showed that the lignin and cellulose contents of pear pulp callus overexpressing PbrMYB24 were significantly higher than those of pear pulp callus transformed without vector. Figure 4 BC).

[0096] (5) Detection of relative expression levels of genes related to lignin synthesis in transgenic callus

[0097] RNA extraction, cDNA synthesis, and the system and steps for quantitative real-time PCR were as described in Example 1. The expression levels of endogenous genes related to lignin synthesis in transgenic callus tissue were analyzed using quantitative real-time PCR. The results showed that, compared with pear pulp callus transformed with an empty vector, the expression levels of endogenous genes related to lignin synthesis, including Pbr4CL1, PbrCSE1, PbrCCr21, PbrCCOAMT1, PbrCAD24, PbrLAC4, PbrLAC19, PbrSTONE, PbrCESA8b, PbrCESA4b, PbrCESA7a, PbrCESA4a, PbrNSC, and PbrMYB169, were significantly increased in pear pulp callus overexpressing PbrMYB24. Figure 4 H). In summary, these results indicate that overexpression of PbMYB24 promotes lignin and cellulose synthesis in pear pulp callus. Example 5: Determination of lignin and cellulose-related physiological indicators in transgenic Arabidopsis plants.

[0098] (1) Lignin and cellulose content and determination in transgenic Arabidopsis thaliana

[0099] Six wild-type and six T3 transgenic lines were collected, and the lower 10cm stems of the inflorescence were cut into 2mm lengths. The samples were then mixed and dried to constant weight. 10mg of the dried sample was used to extract the coarse cell wall for lignin and cellulose content analysis. The specific steps are as described in Example 3-3.

[0100] The differences in lignin content in coarse cell walls were compared using the bromoacetyl method, and the lignin content in the transgenic lines was significantly increased. Figure 5 A). The difference in cellulose content in coarse cell walls was compared using the anthrone sulfate method. The cellulose content of the transgenic line was significantly increased compared to the wild type. Figure 5 B)

[0101] (2) Detection of relative expression levels of genes related to lignin synthesis in transgenic Arabidopsis thaliana inflorescence stems

[0102] The primary inflorescence stems of wild-type and transgenic plants at 4 weeks old were taken. The RNA extraction, cDNA synthesis, and the system and steps of real-time PCR were as described in Example 1.

[0103] Analysis of the expression levels of endogenous lignin and cellulose synthesis metabolism genes in Arabidopsis plants overexpressing PbrMYB24 revealed a highly significant increase in the expression levels of both lignin and cellulose synthesis-related genes. Figure 5 C) indicates that PbrMYB24 causes excessive accumulation of lignin and cellulose in the stem by activating the expression of genes related to lignin and cellulose synthesis.

[0104] (3) Observation of tissue structure of transgenic Arabidopsis thaliana inflorescence stem

[0105] 1 Paraffin section

[0106] Take the base of the primary inflorescence stem of 8-week-old wild-type and transgenic plants and fix it with FAA fixative at 4°C for more than one week. Refer to 4-3 for the preparation steps of paraffin sections.

[0107] Toluidine blue staining: Stain the sections with 1% toluidine blue boric acid solution (W / V) for 5 minutes, then with 95% ethanol for 2 minutes, 100% ethanol for 3 minutes (repeat once), xylene for 10 minutes, xylene for 5 minutes, cover with neutral resin, and dry in an incubator at 37°C.

[0108] Safranin-Fix-Green staining: Immerse the sections in safranin staining solution for 1-2 hours, rinse briefly with tap water to remove excess dye, then immerse the sections in Fast Green staining solution for 30-60 seconds, and dehydrate with anhydrous ethanol in three tanks.

[0109] Calcofluor staining: Stain the sections with 10ul Calcofluor White Stain for 5 minutes, then blot off excess Calcofluor with filter paper and wash three times with water.

[0110] The stained sections were observed and photographed using a Leica TCs SP2 spectral confocal microscope.

[0111] 2. Observation of autofluorescence in lignin

[0112] Unstained sections were observed using a Leica TCs SP2 confocal microscope with a diode laser emitting 405nm laser light. The laser intensity, magnification, and photomultiplier tube gain settings were kept consistent across different samples.

[0113] 3 Transmission electron microscopy

[0114] Samples identical to paraffin sections were fixed in 2.5% glutaraldehyde fixative at 4°C for 12 hours. After collection, the samples were immediately immersed in fresh 2.5% glutaraldehyde fixative. Collection should be performed at a low temperature of 0-4°C, and the instruments and fixative used should also be cooled. Due to the poor permeability of the fixatives (glutaraldehyde penetration depth is 0.5 mm, and osmium tetroxide penetration depth is 0.25 mm), the sample size was approximately 1.5 mm × 3 mm, with a thickness not exceeding 2 mm. For materials floating on the fixative, air was aspirated to ensure complete immersion in the fixative for thorough fixation. The method was referenced (Whitehill et al., 2016). The thickness of the secondary cell wall of xylem cells was measured using Image-Pro Plus software.

[0115] Paraffin sectioning, lignin autofluorescence detection, and transmission electron microscopy revealed that the xylem cells of the transgenic Arabidopsis stems had normal morphology, but significant lignin accumulation, strong autofluorescence, and thicker secondary cell walls. Figure 5 DQ).

[0116] Table 2 Primers for real-time fluorescence quantitative analysis of Arabidopsis thaliana

[0117]

[0118] Main References

[0119] Porebski S,Bailey LG,Baum BR(1997)Modification of a CTAB DNAextraction protocol for plants containing high polysaccharide and polyphenolcomponents.Plant molecular biology reporter15:8-15

[0120] Vanholme R,Cesarino I,Rataj K,Xiao Y,Sundin L,Goeminne G,Kim H,CrossJ,Morreel K,Araujo P(2013)Caffeoyl shikimate esterase(CSE)is an enzyme in thelignin biosynthetic pathway inArabidopsis.Science 341:1103-1106

[0121] Vanholme R,Demedts B,Morreel K,Ralph J,Boerjan W(2010)Ligninbiosynthesis and structure.Plant physiology 153:895-905

[0122] Whitehill JGA,Henderson H,Schuetz M,Skyba O,Yuen MMS,King J,SamuelsAL,Mansfield SD,Bohlmann J(2016)Histology and cell wall biochemistry of stonecells in the physical defence ofconifers against insects.Plant,cell&environment 39:1646-1661

[0123] XueYS,Shao-Zhuo XU,Xue C,Wang RZ,Zhang MY,Jia-Ming LI,Zhang SL,Jun WU(2020)Pearprocess:A new phenotypic tool for stone cell trait evaluation inpear fruit.Journal of Integrative Agriculture 19:1625-1634

[0124] Zhang J,Li J,Xue C,Wang R,Wu J(2020)The Variation of Stone CellContent in 236Germplasms of Sand Pear(Pyrus pyrifolia)and Identification ofRelated Candidate Genes.Horticultural Plant Journal:

[0125] Zhong R, Cui D, Ye ZH (2019) Secondary cell wall biosynthesis. New Phytol 221:1703-1723. Teng Yuanwen (2017) Research progress on phylogeny of Pyrus species and origin of Oriental pear varieties. Journal of Fruit Science 34:370-378.

Claims

1. Overexpression PbrMYB24 The application of the gene in promoting lignin and cellulose synthesis in pear pulp callus tissue or Arabidopsis inflorescence stem, as described PbrMYB24 The gene, whose CDS sequence is shown in SEQ ID No.

1.

2. Contains PbrMYB24 The application of recombinant gene expression vectors in promoting lignin and cellulose synthesis in pear pulp callus or Arabidopsis inflorescence stems, as described above. PbrMYB24 The gene, whose CDS sequence is shown in SEQ ID No. 1.

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