PtoPASPA3A gene for regulating tree cell wall lignin synthesis and application of PtoPASPA3A gene

Abstract

The invention relates to a PtoPASPA3A gene for regulating and controlling tree cell wall lignin synthesis and application of the PtoPASPA3A gene, and belongs to the technical field of gene engineering. The invention provides a PtoPASPA3A gene for regulating and controlling synthesis of lignin in a tree cell wall. The nucleotide sequence of the PtoPASPA3A gene is as shown in SEQ ID NO. 1. A populus tomentosa plant with an overexpressed PtoPASPA3A gene is obtained through an agrobacterium-mediated populus tomentosa genetic transformation system, and analysis finds that the overexpressed PtoPASPA3A gene does not influence the external growth phenotype of the plant, so that the xylem fiber cell secondary wall of the plant becomes thin, finally, the lignin content is reduced, the cell wall saccharification ability is improved, and the xylem fiber cell secondary wall becomes thin. It is shown that the PtoPASPA3A gene has the effect of regulating and controlling lignin on trees and has important theoretical guiding significance on cultivation of new varieties of paper pulp.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a PtoPASPA3A gene that regulates lignin synthesis in tree cell walls and its applications. Background Technology

[0002] Wood is an essential raw material for energy, papermaking, and construction, and is also an important renewable resource worldwide. Wood formation is a highly ordered developmental process, with its structure primarily composed of cellulose microfibrils as the framework, and hemicellulose and lignin as binding and filling components. As a secondary metabolite produced during the evolution of vascular plants, lignin plays a crucial role in upright growth and water transport. However, the presence of lignin limits the conversion rate of forest biomass in large-scale conversion and utilization, such as biofuel production, and increases production and environmental costs in pulping and papermaking. To reduce lignin's resistance to depolymerization, there has been great interest in engineering projects to reduce its content. In recent years, through continuous research and exploration, our understanding of the lignin monomer synthesis network has become clearer, and we have gradually realized that interfering with G-type lignin synthesis within the xylem vessels affects plant water transport safety and biomass accumulation. Current modification strategies mainly focus on controlling S-type lignin synthesis within wood fiber cells, including inhibiting the expression of key enzyme genes or upstream transcription factors in its synthesis pathway, thereby reducing lignin content or altering its composition. Therefore, identifying the key genetic factors for lignin synthesis in tree fiber cells can not only enrich the molecular basis of wood formation, but also has important value for the efficient separation and degradation of lignocellulose and the genetic improvement of the quality of broad-leaved wood.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a PtoPASPA3A gene that regulates lignin synthesis in tree cell walls and its application, in order to solve the problem of the lack of genes regulating lignin synthesis in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a PtoPASPA3A gene that regulates lignin synthesis in tree cell walls, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] This invention provides the application of the PtoPASPA3A gene in regulating lignin synthesis in tree cell walls, and achieves regulation of lignin synthesis in tree cell walls by overexpressing the PtoPASPA3A gene.

[0007] Preferably, the tree includes white poplar.

[0008] The present invention provides a protein encoded by the PtoPASPA3A gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] This invention provides the application of the protein in regulating lignin synthesis in tree cell walls, including Populus tomentosa.

[0010] This invention provides a recombinant vector for expressing the PtoPASPA3A gene, the recombinant vector comprising the PtoPASPA3A gene, the PtoPASPA3A gene promoter, and an empty vector; The nucleotide sequence of the PtoPASPA3A gene is shown in SEQ ID NO.1; the nucleotide sequence of the PtoPASPA3A gene promoter is shown in SEQ ID NO.3; the empty vector includes pGWB10.

[0011] This invention provides the application of the recombinant vector in regulating lignin synthesis in tree cell walls, wherein the tree includes Populus tomentosa.

[0012] This invention provides a recombinant bacterium expressing the PtoPASPA3A gene, the recombinant bacterium comprising the recombinant vector and the host bacterium; The host bacteria include Agrobacterium GV3101.

[0013] This invention provides the application of the recombinant bacteria in regulating lignin synthesis in tree cell walls, wherein the trees include Populus tomentosa.

[0014] This invention provides a method for regulating lignin synthesis in tree cell walls by transferring the PtoPASPA3A gene or a product containing the PtoPASPA3A gene into trees. The product containing the PtoPASPA3A gene is the recombinant vector or the recombinant bacteria described above. The trees mentioned include white poplars.

[0015] The present invention has the following technical effects and advantages: This invention obtained Populus tomentosa plants overexpressing the PtoPASPA3A gene through an Agrobacterium-mediated genetic transformation system. Analysis using a series of techniques revealed that overexpression of the PtoPASPA3A gene affects the thickening process of the secondary cell wall in xylem fibers, thereby influencing the proportions of wood components. Specifically, overexpression of PtoPASPA3A does not affect the plant's external growth phenotype, but it does thin the secondary cell walls of xylem fibers, ultimately leading to decreased lignin content and increased cell wall saccharification capacity. This indicates that the PtoPASPA3A gene plays a regulatory role in the thickening of the secondary cell wall and the accumulation of lignin in trees, providing important theoretical guidance for the breeding of new pulpwood varieties. Attached Figure Description

[0016] Figure 1 The growth status of OE-2 and OE-9 resistant Populus tomentosa and wild-type plants after 3 months of growth; Figure 2 The results of transcriptional level identification between OE-1~9 resistant Populus tomentosa lines and wild-type plants; Figure 3 The results of growth phenotypic analysis of OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants; Figure 4 The results of tissue section observation of OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants; Figure 5 Results of wood composition analysis of OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants Figure 6 Analysis of cell wall saccharification capacity in OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants. Detailed Implementation

[0017] This invention provides a PtoPASPA3A gene that regulates lignin synthesis in tree cell walls, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0018] This invention provides the application of the PtoPASPA3A gene in regulating lignin synthesis in tree cell walls, and achieves regulation of lignin synthesis in tree cell walls by overexpressing the PtoPASPA3A gene.

[0019] In this invention, the trees include white poplar.

[0020] The present invention provides a protein encoded by the PtoPASPA3A gene, the amino acid sequence of which is shown in SEQ ID NO.2; The amino acid sequence of SEQ ID NO.2 is:

[0021] This invention provides the application of the protein in regulating lignin synthesis in tree cell walls, including Populus tomentosa.

[0022] This invention provides a recombinant vector for expressing the PtoPASPA3A gene, the recombinant vector comprising the PtoPASPA3A gene, the PtoPASPA3A gene promoter, and an empty vector; The nucleotide sequence of the PtoPASPA3A gene is shown in SEQ ID NO.1; the nucleotide sequence of the PtoPASPA3A gene promoter is shown in SEQ ID NO.3; the empty vector includes pGWB10;

[0023] This invention provides the application of the recombinant vector in regulating lignin synthesis in tree cell walls, wherein the tree includes Populus tomentosa.

[0024] This invention provides a recombinant bacterium expressing the PtoPASPA3A gene, the recombinant bacterium comprising the recombinant vector and the host bacterium; The host bacteria include Agrobacterium GV3101.

[0025] This invention provides the application of the recombinant bacteria in regulating lignin synthesis in tree cell walls, wherein the trees include Populus tomentosa.

[0026] This invention provides a method for regulating lignin synthesis in tree cell walls by transferring the PtoPASPA3A gene or a product containing the PtoPASPA3A gene into trees. The product containing the PtoPASPA3A gene is the recombinant vector or the recombinant bacteria described above. The trees mentioned include white poplars.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1: Obtaining the PtoPASPA3A gene from Populus tomentosa and constructing its expression vector

[0029] (1) Cloning of the PtoPASPA3A gene sequence

[0030] Total RNA was extracted from wild-type Populus tomentosa using pBIOZOL plant total RNA extraction reagent (purchased from Hangzhou Borui Technology Co., Ltd., Cat#: BSC55M1). Reverse transcription was performed using a reverse transcription kit (purchased from Borui Biotechnology (Beijing) Co., Ltd., catalog number: RR047A). The obtained cDNA was aliquoted and stored at -20℃ for later use.

[0031] Total DNA was extracted from the leaves of wild-type Populus tomentosa using a one-step rapid plant genomic DNA extraction reagent developed by Biotech (purchased from Beijing Biotech Biotechnology Co., Ltd., catalog number DP3211). The obtained DNA was aliquoted and stored at -20℃ for later use.

[0032] Referring to the information on Populus tomentosa PtoPASPA3A (Potom04G0005800) provided on the NCBI website, gene-specific primers were designed at both ends of the PtoPASPA3A gene promoter (SEQ ID NO.2) and the PtoPASPA3A gene coding region sequence (SEQ ID NO.1). The primers were synthesized by Shanghai Baiqu Biomedical Technology Co., Ltd., and the upstream and downstream primers used for PtoPASPA3A gene cloning were obtained. The specific sequences are shown in Table 1.

[0033] Table 1 Primers and sequences used for PtoPASPA3A cloning

[0034] The promoter fragment of PtoPASPA3A (SEQ ID NO.3) was cloned by polymerase chain reaction (PCR) using leaf DNA as a template, with proPASPA3A-F and PASPA3A-PC-mid-R paired together. Then, the promoter fragment of PtoPASPA3A (SEQ ID NO.1) was cloned by polymerase chain reaction (PCR) using PASPA3A-PC-mid-F and PASPA3A-R paired together. Finally, the promoter and coding region sequences of PtoPASPA3A (i.e., the fragment sequentially linked from SEQ ID NO.3 and SEQ ID NO.1, PASPA3A-PC) were cloned by polymerase chain reaction (PCR) using proPASPA3A-F and PASPA3A-R paired together and using both the promoter fragment and the PtoPASPA3A fragment as templates.

[0035] The obtained PCR products were separated into target bands by agarose gel electrophoresis. The target fragment was recovered from the gel using the Thermo SilicaBead DNA Gel Extraction Kit, and the recovered product was ligated with the PQB vector at its ends. The ligation system of the PQB vector is shown in Table 2.

[0036] Table 2 PQB Carrier Linkage System

[0037] Thaw DH5α competent cells on ice, add all the ligation products to the competent cells, and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add antibiotic-free LB medium, and activate at 37°C and 200 rpm for 1 hour. Collect the cells at low speed and spread them on solid LB medium (containing 50 mg / L chloramphenicol), and incubate overnight at 37°C. The next day, perform PCR identification of the obtained single-point clones using primers for the target gene to obtain the PASPA3A-PC-PQB plasmid.

[0038] (2) Construction of expression carrier

[0039] The obtained PASPA3A-PC-PQB plasmid and pGWB10 expression vector were subjected to LR reaction. The specific reaction system and reaction conditions are shown in Table 3.

[0040] Table 3 LR Switching System

[0041] Thaw DH5α competent cells on ice. Add all the reaction products to the competent cells and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add antibiotic-free LB medium, and activate at 37°C and 200 rpm for 1 hour. Collect bacterial cells at low speed and spread them on solid LB medium (50 mg / L kanamycin + 50 mg / L hygromycin), and incubate overnight at 37°C. The next day, perform PCR identification on the obtained single-point clones using identification primers, as shown in Table 4. Table 4 shows the primer sequences used for expression vector construction and monoclonal detection.

[0042] PCR products were analyzed by electrophoresis to identify positive clones. Positive single clones were picked and cultured overnight in 5 mL LB medium (50 mg / L kanamycin + 50 mg / L hygromycin) at 37°C and 200 rpm. The next day, the bacterial strain was preserved and collected by centrifugation. Plasmids were extracted and sequenced to obtain the recombinant vector expressing the PtoPASPA3A gene.

[0043] Sequencing analysis revealed that the obtained PtoPASPA3A gene is 1689 bp (SEQ ID NO.1), encoding 563 amino acids (SEQ ID NO.2), and the obtained PtoPASPA3A promoter fragment is 1872 bp (SEQ ID NO.3).

[0044] Example 2: Construction of recombinant bacteria expressing the PtoPASPA3A gene

[0045] The method for constructing recombinant bacteria expressing the PtoPASPA3A gene is as follows: ① Melt Agrobacterium GV3101 competent cells in an ice bath; ② Add 3 μL of the recombinant vector expressing the PtoPASPA3A gene obtained in Example 1, incubate on ice for 30 min, freeze in liquid nitrogen for 1 min, and then incubate in water at 37°C for 5 min; ③ Add 950 μL of antibiotic-free YEP medium, incubate at 28℃ and 200 rpm for 4 h with shaking to obtain bacterial culture; ④ Centrifuge at 3000 rpm for 5 min to concentrate the bacterial culture, and then rehydrate the bacterial cells with 100 μL of YEP; ⑤ Spread the bacterial cells on solid YEP medium supplemented with 50 mg / L kanamycin, 50 mg / L hygromycin, 50 mg / L gentamicin and 50 mg / L rifampin, and incubate at 28°C for 48 h.

[0046] Positive clones were detected by bacterial culture PCR, and recombinant bacteria expressing the PtoPASPA3A gene (recombinant Agrobacterium) were obtained.

[0047] Example 3: Obtaining PtoPASPA3A transgenic plants of Populus tomentosa

[0048] (1) Genetic transformation of Populus tomentosa mediated by Agrobacterium was carried out using the following method: ① Select recombinant Agrobacterium expressing the PtoPASPA3A gene and add it to 20 mL of LB medium containing resistant (kanamycin 50 mg / L + hygromycin 50 mg / L + hygromycin 50 mg / L + gentamicin 50 mg / L + rifampin 50 mg / L) and incubate overnight at 28°C and 220 rpm.

[0049] ②The next day, take 1000μL of bacterial culture and add it to 50mL of resistant LB liquid medium (50mg / L kanamycin + 50mg / L hygromycin + 50mg / L gentamicin + 50mg / L rifampin), and incubate at 28℃ and 220rpm until the OD600 is 0.6.

[0050] ③ Take leaves from a 1-month-old aseptic tissue culture seedling of Populus tomentosa and place them in the cultured bacterial solution for 20 minutes.

[0051] ④ Remove the leaves and place them on a co-culture medium. Incubate in the dark for 2 days.

[0052] ⑤ Two days later, thoroughly wash the leaves with sterile water and place them on the proliferation medium for normal culture.

[0053] ⑥ After the resistant buds emerged, they were inserted into the rooting medium to induce rooting, resulting in 9 independent resistant Populus tomentosa lines overexpressing the PtoPASPA3A gene, which were named OE-1 to OE-9.

[0054] (2) Identification of transgenic overexpression plants

[0055] ①Plant seedlings of the OE-1~9 resistant Populus tomentosa line along with wild-type plants of the same condition into the soil and cultivate them until they are 3 months old. Collect the developing xylem tissue of the resistant plants and the wild-type control.

[0056] ② RNA was extracted from developing xylem tissue using a plant RNA extraction kit (pBIOZOL).

[0057] ③ The RNA was reverse transcribed using the plant Takara reverse transcription kit (RR047A) to obtain cDNA.

[0058] ④ Dilute the cDNA 10-fold and perform RT-qPCR, using PtoActin as the internal control.

[0059] The RT-qPCR reaction system (20 μL) contained 10 μL of 2×TB Green Premix Ex Taq II, 0.8 μL of forward and reverse primers, 0.4 μL of ROX Reference Dye II, 1 μL of template, and 7 μL of sterile water. PCR parameters were: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 60°C annealing for 15 s, 72°C extension for 30 s, for 40 cycles. PtActin was used as an internal control, and each analysis was performed in triplicate.

[0060] The primer sequences in the RT-qPCR reaction system are shown in Table 5.

[0061] Table 5 Primer sequences for RT-qPCR reaction system

[0062] The growth status of OE-2 and OE-9 resistant Populus tomentosa lines overexpressing the PtoPASPA3A gene and wild-type plants after 3 months of growth is as follows: Figure 1 As shown in the figure. The transcriptional level identification results of the PtoPASPA3A gene overexpression OE-1~9 resistant Populus tomentosa lines and wild-type plants are as follows. Figure 2 In the figure shown, the horizontal axis represents the plant and the vertical axis represents the transcription level of the PtoPASPA3A gene.

[0063] according to Figures 1-2 It can be seen that, compared with wild-type Populus tomentosa, the transcription level of the PtoPASPA3A gene in the OE-1~9 resistant Populus tomentosa lines was significantly increased, indicating that the OE-1~9 resistant Populus tomentosa lines overexpressing the PtoPASPA3A gene were successfully obtained.

[0064] Example 4: Application of the PtoPASPA3A gene in regulating the secondary growth process of Populus tomentosa

[0065] (1) Growth phenotype analysis was performed on the OE-2 and OE-7 resistant Populus tomentosa lines obtained in Example 3 and the wild-type plants.

[0066] Growth phenotypic analysis includes the following indicators: Populus tomentosa resistant lines OE-2 and OE-7 overexpressing the PtoPASPA3A gene, along with wild-type plants, were simultaneously transplanted into sterilized black soil and placed in a greenhouse. After 30 days, a mark was made 10 cm above the soil surface; this point served as the reference for basal stem measurement. At least six trees from each line were transplanted.

[0067] Wild-type and OE-2 and OE-7 resistant Populus tomentosa plants grown in greenhouses for 3 months were collected to measure tree height (cm), stem diameter (mm), number of stem nodes, average stem node length (cm), fiber length (μm), fiber width (μm), vessel length (μm), and vessel width (μm). The method for measuring the number of stem nodes and average stem node length was as follows: the stem node containing the first fully unfolded leaf at the top was taken as the first stem node (IN1), and the number of nodes from the first node to the reference point below was counted, which was the number of internodes of the plant. The average length of the 10th, 11th, and 12th nodes was counted as the average stem node length of the plant. At least 4 trees were collected from each line. The method for measuring fiber length (μm), fiber width (μm), vessel length (μm), and vessel width (μm) was as follows: IN22 of 4-month-old plants was taken, peeled, and then immersed in xylem cell dissociation solution and bathed in water at 60°C for 6 hours. Rinse the stem segments six times with clean water to remove the xylem cells attached to the surface. Then gently resuspend the stem segments to allow the mature xylem cells to detach. Take an equal volume of the xylem resuspension and 0.1% acid fuchsin, mix well, prepare a slide, and observe and measure the cell fiber length (μm), fiber width (μm), vessel length (μm), and vessel width (μm) under an upright microscope. Measure at least 200 cells of each cell type from the same plant, and at least 4 trees from each line.

[0068] The results of growth phenotypic analysis of OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants are as follows: Figure 3 As shown.

[0069] according to Figure 3 It was found that, compared with wild-type plants, the OE-2 and OE-7 resistant Populus tomentosa lines did not show significant changes in tree height, stem diameter, number of stem nodes, or average stem node length; similarly, cell fiber length, fiber width, vessel length, and vessel width also did not show significant changes. This indicates that overexpression of the PtoPASPA3A gene does not affect tree height and diameter growth.

[0070] (2) Histological observation of the OE-2 and OE-7 resistant Populus tomentosa lines obtained in Example 3 and the wild-type plants.

[0071] Take the 12th node of the stem of wild-type and OE-2 and OE-7 resistant Populus tomentosa plants grown in greenhouse for 3 months and quickly cut it into rectangular strips 1 mm wide (based on the position near the phloem, the width of the cross section radiating to the xylem) with a bottom of 0.5 mm square. Then fix them in PBS buffer containing 2.5% glutaraldehyde and 4% paraformaldehyde; vacuum for 5 min, negative pressure for 30 min, repeat this step 6 times, and then incubate the fixed strips at 4°C overnight. On the second day, the wooden strips were washed three times with PBS buffer (5 min each time), soaked in 0.5% osmium tetroxide for 2 h, washed three times with PBS buffer (5 min each time), and then dehydrated with a gradient concentration of ethanol (30%→50%→70%→90%→100%, twice; 5 min each time). The samples were then transferred to a mixture of acetone and a gradient concentration of resin (0→50%→70%→100%; 3 h each time) for embedding, and finally soaked in pure resin overnight. On the third day, the embedded samples were polymerized in a 72°C oven. After 48 h, the resin-embedded samples were cut into 70 nm thick sections using an EMUC7 microtome. The sections were stained with uranyl acetate and lead citrate, and finally photographed using an HT-7700 electron transmission microscope. At least four trees were used for each line, and three slides (at least 15 cells) were randomly selected from each tree for cell wall thickness analysis. Cell wall thickness was measured using ImageJ. The results of tissue section observations from OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants are as follows: Figure 4 As shown.

[0072] according to Figure 4 It was found that, compared with wild-type plants, the cell wall thickness of OE-2 and OE-7 resistant Populus tomentosa lines was thinner. This indicates that overexpression of the PtoPASPA3A gene affects the thickening of xylem fiber cell walls, ultimately leading to thinner cell walls.

[0073] (3) Wood composition analysis was performed on the OE-2 and OE-7 resistant Populus tomentosa lines obtained in Example 3 and the wild-type plants.

[0074] A 10 cm long basal stem segment was taken from a 4-month-old Populus tomentosa tree. Stem segments from three trees were combined to form one sample replicate, with three biological replicates per lineage. After peeling, the remaining xylem tissue was dried at 55°C, and then the dried wood was ground into fine particles using a ball mill. 70 mg of wood flour was weighed from each sample and washed sequentially with 70% ethanol, chloroform / methanol (1:1 v / v) solution, and acetone (each step involving shaking for 2 min followed by centrifugation at 10000 g for 10 min). The resulting insoluble residue was air-dried at room temperature and labeled as cell wall material for component determination. 1.5 mg of cell wall material was required for each crystalline cellulose content determination, and 2 mg of cell wall material was required for each lignin content determination. Each content in each sample was measured three times, and the average of the three measurements was taken as the crystalline cellulose or total lignin content of that sample.

[0075] The detailed sequencing method for lignin is as follows: ① Take 2.0 mg of the prepared cell wall material into a centrifuge tube.

[0076] ② Rinse the tube wall with 500 μL of acetone and collect the cell wall material at the bottom of the tube. The acetone evaporates very gently under the airflow.

[0077] ③ Gently add 1.0 mL of freshly prepared acetyl bromide solution (25% v / v acetyl bromide in glacial acetic acid) along the tube wall.

[0078] ④ Cover with the cap and heat at 50°C for 2 hours.

[0079] ⑤ Shake for 15 minutes, then heat for 1 hour.

[0080] ⑥ Cool to room temperature on ice and centrifuge (10000g, 15 minutes).

[0081] ⑦ Add 0.5 mL of the solution to a tube containing 2.5 mL of HAc and 1.5 mL of 0.3 M NaOH.

[0082] ⑧ After shaking the sample well, add 0.5 mL of 0.5 M hydroxylamine hydrochloride solution and add HAc to bring the volume to 10 mL.

[0083] ⑨ Measure the optical density three times at 280 nm, and determine the percentage (%ABSL) of acetyl bromide-soluble lignin using an appropriate coefficient (poplar = 18.21g). -1 ·cm -1 The formula for %ABSL (μg / mg) is as follows: %ABSL (μg / mg) = 100 × (ASample - Ablank)V / (coeff × 1.0 × W); V is 10 mL; W is half of 2.0 mg.

[0084] The results of wood composition analysis of OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants are as follows: Figure 5As shown in the figure, A represents the secondary wall thickness of OE-2 and OE-7 resistant Populus tomentosa plants and wild-type plants, B represents the crystalline cellulose content of OE-2 and OE-7 resistant Populus tomentosa plants and wild-type plants, and C represents the total lignin content of OE-2 and OE-7 resistant Populus tomentosa plants and wild-type plants.

[0085] according to Figure 5 It was found that, compared with wild-type plants, the secondary cell wall thickness of OE-2 and OE-7 resistant Populus tomentosa lines was significantly thinner, while the crystalline cellulose content showed no significant difference, and the total lignin content was significantly reduced. This indicates that overexpression of the PtoPASPA3A gene leads to a decrease in the lignin content of wood cell walls.

[0086] (4) Cell wall saccharification capacity was analyzed in the OE-2 and OE-7 resistant Populus tomentosa lines obtained in Example 3 and in wild-type plants.

[0087] The xylem from the basal stems of wild-type and OE-2 and OE-7 resistant Populus tomentosa plants were collected in April, dried at 55°C, and then ground into powder using a ball mill. 10 mg of the wood powder was placed in 2 mL test tubes, and 400 μL of hot water or 350 μL of dilute alkali (1% NaOH, w / v) was added for pretreatment. All samples were incubated in a 30°C water bath for 30 min, followed by autoclaving at 120°C for 60 min. After cooling to room temperature, the pretreated samples were neutralized with 50 μL of HCl (2.5 M), and 600 μL of cell wall saccharification solution was added. Saccharification was initiated at 50°C / 200 rpm. After shaking incubation for 12, 24, 36, 48, 60, and 72 h, the samples were centrifuged at 14000 g for 10 min, and 20 μL of the supernatant was used for reducing sugar content determination (DNS method). Reaction procedure: Mix 20 μL of sample and 200 μL of DNS reagent in a centrifuge tube, incubate at 95°C for 6 minutes, and after cooling, measure the absorbance (OD540) using a microplate reader. Construct a glucose standard curve and determine the amount of sugar released per milligram of cell wall (μg). Perform at least three biological replicates per plant. The results of cell wall saccharification capacity analysis for OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants are shown below. Figure 6 As shown, Figure A represents the amount of sugar released per milligram of cell wall in OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants pretreated with dilute alkali, while Figure B represents the amount of sugar released per milligram of cell wall in OE-2 and OE-7 resistant Populus tomentosa lines and wild-type plants pretreated with hot water.

[0088] according to Figure 6It was found that, compared with wild-type plants, the OE-2 and OE-7 resistant Populus tomentosa lines showed a significant increase in the amount of sugar released per milligram of cell wall. This indicates that overexpression of the PASPA3A gene leads to increased saccharification efficiency of wood cell walls, especially after hot water pretreatment.

[0089] As shown in the above embodiments, this invention provides a PtoPASPA3A gene that regulates lignin synthesis in tree cell walls and its application. This invention obtained Populus tomentosa plants overexpressing the PtoPASPA3A gene through an Agrobacterium-mediated genetic transformation system. Analysis using a series of techniques revealed that overexpression of the PtoPASPA3A gene affects the thickening process of the secondary cell wall of fiber cells, thereby affecting the proportion of wood components. Specifically, overexpression of PtoPASPA3A does not affect the plant's external growth phenotype, but it does thin the secondary cell wall of the xylem fiber cells, ultimately leading to a decrease in lignin content and an increase in cell wall saccharification capacity. This indicates that the PtoPASPA3A gene has a certain regulatory role in the thickening of the secondary cell wall and the accumulation of lignin in trees, providing important theoretical guidance for the breeding of new pulpwood varieties.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PtoPASPA3A gene that regulates lignin synthesis in tree cell walls, characterized in that, The nucleotide sequence of the PtoPASPA3A gene is shown in SEQ ID NO.

1.

2. The application of the PtoPASPA3A gene as described in claim 1 in regulating lignin synthesis in tree cell walls, wherein the regulation of lignin synthesis in tree cell walls is achieved by overexpressing the PtoPASPA3A gene.

3. The application according to claim 2, characterized in that, The trees mentioned include white poplars.

4. The protein encoded by the PtoPASPA3A gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

5. The application of the protein of claim 4 in regulating lignin synthesis in tree cell walls, wherein the tree includes Populus tomentosa.

6. A recombinant vector expressing the PtoPASPA3A gene of claim 1, characterized in that, The recombinant vector includes the PtoPASPA3A gene, the PtoPASPA3A gene promoter, and an empty vector. The nucleotide sequence of the PtoPASPA3A gene is shown in SEQ ID NO.1; the nucleotide sequence of the PtoPASPA3A gene promoter is shown in SEQ ID NO.3; the empty vector includes pGWB10.

7. The application of the recombinant vector according to claim 6 in regulating lignin synthesis in tree cell walls, wherein the tree includes Populus tomentosa.

8. A recombinant bacterium expressing the PtoPASPA3A gene of claim 1, characterized in that, The recombinant bacteria include the recombinant vector and host bacteria as described in claim 6; The host bacteria include Agrobacterium GV3101.

9. The application of the recombinant bacteria of claim 8 in regulating lignin synthesis in tree cell walls, wherein the tree includes Populus tomentosa.

10. A method for regulating lignin synthesis in tree cell walls, characterized in that, Transferring the PtoPASPA3A gene or products containing the PtoPASPA3A gene into trees. The product containing the PtoPASPA3A gene is the recombinant vector of claim 6 or the recombinant bacteria of claim 8; The trees mentioned include white poplars.

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