A gene regulating tree cell wall lignin synthesis and its constructed recombinant vector and application

Abstract

The present invention relates to the field of genetic engineering technology. The present invention provides a gene for regulating the synthesis of lignin in tree cell walls, a recombinant vector constructed therefrom, and its application. The present invention obtains transgenic plants overexpressing the PtrXCP2A gene of Populus trichocarpa through an Agrobacterium-mediated Populus trichocarpa genetic transformation system. Analyses using a series of technical means revealed that overexpression of the PtrXCP2A gene affects the thickening process of the secondary wall of fiber cells, thereby affecting the proportion of wood components. This is mainly reflected in the fact that overexpression of PtrXCP2A does not affect the external growth phenotype of the plant, while the secondary wall of the xylem fiber cells of the transgenic plant becomes thinner, ultimately leading to a decrease in lignin content and an increase in the saccharification capacity of the cell wall. The above results indicate that the PtrXCP2A gene has a certain regulatory effect on the thickening of the secondary wall of trees and the accumulation of lignin. The research content of the present invention has important theoretical guiding significance for the cultivation of high-quality timber and new forest varieties.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a gene for regulating the synthesis of lignin in tree cell walls, a recombinant vector constructed therefrom, and applications thereof. Background Art

[0002] Wood is an essential raw material for pulp and papermaking, building construction, and other applications, and is also a major renewable resource worldwide. Wood formation is a highly ordered developmental process, with its structure primarily composed of cellulose microfibrils as the skeleton and hemicellulose and lignin as the binding and filling components. Lignin, a secondary metabolite produced during the evolution of vascular plants, plays a crucial role in plant growth and water transport. However, the presence of lignin limits the conversion rate of forest biomass for large-scale conversion and utilization, such as biofuel production, and increases production and environmental costs in pulping and papermaking. To mitigate this recalcitrance towards depolymerization, engineering methods to reduce lignin content have shown significant interest. Recent research and exploration has led to a clearer understanding of the lignin monomer synthesis network, with the increasing recognition that interfering with G-type lignin synthesis within the vessels can compromise plant water transport safety and biomass accumulation. Current engineering strategies primarily target controlling S-type lignin synthesis within wood fiber cells, including inhibiting the expression of key enzyme genes or upstream transcription factors involved in its synthesis pathway, thereby reducing lignin content or altering its composition. Therefore, identifying the key genetic factors for lignin synthesis in forest fiber cells can not only enrich the molecular basis of wood formation, but also has important value in breaking the bottleneck of efficient separation and degradation of lignocellulose and genetic improvement of the quality of broadleaf wood.

[0003] PLCP proteases are one of the four major protease families in plants and play important regulatory roles in a variety of biological processes. Among them, the PtrXCP2A gene in Populus trichocarpa is highly abundantly expressed in the developing xylem of the stem, but the biological function of this gene has never been studied. Summary of the Invention

[0004] The purpose of the present invention is to provide a gene for regulating the synthesis of lignin in tree cell walls and a recombinant vector constructed therefrom and its application, which has important theoretical guiding significance for the cultivation of high-quality timber and new forest tree varieties.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a gene for regulating the synthesis of lignin in tree cell walls. The gene for regulating the synthesis of lignin in tree cell walls is PtrXCP2A. The nucleotide sequence of the PtrXCP2A gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of the PtrXCP2A gene in regulating the synthesis of lignin in tree cell walls.

[0008] The present invention also provides the use of the PtrXCP2A gene in preparing a recombinant vector containing the PtrXCP2A gene.

[0009] The present invention also provides a recombinant vector containing the PtrXCP2A gene, wherein the recombinant vector comprises the PtrXCP2A gene and the PtrXCP2A gene promoter;

[0010] The nucleotide sequence of the PtrXCP2A gene promoter is shown in SEQ ID NO.2.

[0011] The present invention also provides a method for constructing a recombinant vector containing the PtrXCP2A gene, comprising the following steps: constructing the PtrXCP2A gene and the PtrXCP2A gene promoter into a plant expression vector to obtain a recombinant vector;

[0012] The plant expression vector is pGWB10.

[0013] The present invention also provides application of the recombinant vector in regulating the synthesis of lignin in tree cell walls.

[0014] The present invention also provides a method for regulating the synthesis of lignin in tree cell walls using the recombinant vector, comprising the following steps:

[0015] (1) transforming the recombinant vector into Agrobacterium;

[0016] (2) Inoculate Agrobacterium into trees to regulate the synthesis of lignin in the tree cell walls.

[0017] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0018] The present invention also provides a protein encoded by the PtrXCP2A gene, the amino acid sequence of which is shown in SEQ ID NO.3.

[0019] The present invention also provides application of the protein in regulating the synthesis of cell wall lignin.

[0020] The present invention provides a gene for regulating the synthesis of lignin in tree cell walls, a recombinant vector constructed therefrom, and its application. The present invention obtains transgenic plants overexpressing the PtrXCP2A gene of Populus trichocarpa through an Agrobacterium-mediated genetic transformation system of Populus trichocarpa. Utilizing a series of technical means for analysis, it was found that overexpression of the PtrXCP2A gene affects the thickening process of the secondary wall of fiber cells, thereby affecting the proportion of wood components. This is mainly reflected in that overexpression of PtrXCP2A does not affect the external growth phenotype of the plant, while the secondary wall of the xylem fiber cells of the transgenic plant becomes thinner, ultimately leading to a decrease in lignin content and an increase in the saccharification capacity of the cell wall. The above results indicate that the PtrXCP2A gene has a certain regulatory effect on the thickening of the secondary wall of trees and the accumulation of lignin. The research content of the present invention has important theoretical guiding significance for the cultivation of high-quality timber and new forest varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the growth status of PtrXCP2A mutants, overexpressing P. trichocarpa plants, and wild-type P. trichocarpa plants after 3 months of greenhouse growth. In the figure, proXCP2A::XCP2A-line-2 / 8 / 11 represent three different lines of overexpressing plants, WT represents wild-type P. trichocarpa, and the scale bar is 10 cm.

[0022] Figure 2 Figure 2 shows the results of transcriptional level identification in PtrXCP2A-overexpressing (proXCP2A::XCP2A) Populus trichocarpa plants. Lines 2 / 8 / 11 represent two different lines with high XCP2A overexpression abundance, WT represents wild-type Populus trichocarpa, error bars represent standard errors calculated from at least three biological replicates, and asterisks represent ANOVE test results, *P<0.05, **P<0.01.

[0023] Figure 3 Figure 2 shows the height changes of PtrXCP2A-overexpressing Populus trichocarpa plants and wild-type Populus trichocarpa plants after 4 months of greenhouse growth. In the figure, proXCP2A::XCP2A-line-2 / 8 / 11 represent three different lines of overexpressing plants, and WT represents wild-type Populus trichocarpa.

[0024] Figure 4 Figure 2 shows the results of growth phenotype analysis of PtrXCP2A-overexpressing and wild-type Populus trichocarpa plants after 4 months of greenhouse growth. In the figure, proXCP2A::XCP2A-line-2 / 8 / 11 represent three different lines of overexpressing plants, WT represents wild-type Populus trichocarpa, error bars represent standard errors calculated from at least 6 biological replicates, and asterisks represent the results of ANOVE test, *P<0.05, **P<0.01.

[0025] Figure 5 Figure 3 shows the morphological observation results of the cell wall thickening process in the 12th stem node of PtrXCP2A-overexpressing and wild-type Populus trichocarpa plants. In the figure, proXCP2A::XCP2A-2 represents the overexpression plant line, and WT represents the wild type. Scale bar = 10 μm.

[0026] Figure 6 This is a statistical graph showing the cell wall thickening process at the 12th stem node of PtrXCP2A-overexpressing Populus trichocarpa plants and wild-type Populus trichocarpa plants of the present invention; wherein, proXCP2A::XCP2A-line-2 / 8 / 11 in the figure represent three different strains of overexpressing plants, and WT represents wild-type Populus trichocarpa. At least three trees were included in each strain, and three cross sections were randomly selected from each tree. The cell wall thickness of at least six cells in each layer was counted using ImageJ;

[0027] Figure 7 Figure 1 is a statistical graph of wood component content in PtrXCP2A-overexpressing Populus trichocarpa plants and wild-type Populus trichocarpa plants of the present invention; wherein, proXCP2A::XCP2A-line-2 / 8 / 11 in the figure represents three different strains of overexpressing plants, and WT represents wild-type Populus trichocarpa; stem segments of three trees were mixed as a sample replicate, each strain had three biological replicates, each content in each sample was measured three times, and the mean of the three measurements was used as the crystalline cellulose or total lignin content of the sample. Asterisks represent ANOVE test results, *P<0.05, **P<0.01;

[0028] Figure 8 This is a statistical graph of the saccharification efficiency of wood cell walls of PtrXCP2A-overexpressing Populus trichocarpa plants and wild-type Populus trichocarpa plants of the present invention; wherein, proXCP2A::XCP2A-line-2 / 8 / 11 in the figure represents three different strains of overexpressing plants, and WT represents wild-type Populus trichocarpa; stem segments of three trees were mixed as one sample replicate, and each transgenic material was biologically replicated at least three times. Asterisks represent ANOVE test results, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0029] The present invention provides a gene for regulating the synthesis of lignin in tree cell walls. The gene for regulating the synthesis of lignin in tree cell walls is PtrXCP2A. The nucleotide sequence of the PtrXCP2A gene is shown in SEQ ID NO.1.

[0030]

[0031] The present invention also provides the use of the PtrXCP2A gene in regulating the synthesis of lignin in tree cell walls.

[0032] The present invention also provides the use of the PtrXCP2A gene in preparing a recombinant vector containing the PtrXCP2A gene.

[0033] The present invention also provides a recombinant vector containing the PtrXCP2A gene, wherein the recombinant vector comprises the PtrXCP2A gene and the PtrXCP2A gene promoter;

[0034] The nucleotide sequence of the PtrXCP2A gene promoter is shown in SEQ ID NO.2.

[0035]

[0036] The present invention also provides a method for constructing a recombinant vector containing the PtrXCP2A gene, comprising the following steps: constructing the PtrXCP2A gene and the PtrXCP2A gene promoter into a plant expression vector to obtain a recombinant vector;

[0037] The plant expression vector is pGWB10.

[0038] The present invention also provides application of the recombinant vector in regulating the synthesis of lignin in tree cell walls.

[0039] The present invention also provides a method for regulating the synthesis of lignin in tree cell walls using the recombinant vector, comprising the following steps:

[0040] (1) transforming the recombinant vector into Agrobacterium;

[0041] (2) Inoculate Agrobacterium into trees to regulate the synthesis of lignin in the tree cell walls.

[0042] In the present invention, the Agrobacterium is preferably Agrobacterium GV3101.

[0043] The present invention also provides a protein encoded by the PtrXCP2A gene, the amino acid sequence of which is shown in SEQ ID NO.3.

[0044] The amino acid sequence of the protein encoded by the PtrXCP2A gene is: MALSSLSLMFLLAFSFMSFFANSGLARDFSIVG YTPEDLTSGDKIIDLFESWISKHGKIYESMEEKWLRFEIFKDNLFHIDETNKKVVNYWLGLNEFSDLSHEEFKNKYLGLKVDMSERRECSQEFNYKDVMSIPKSVDWRKKGAVTDVKNQGSCGSCWAFSTVAAVEGINQIVTGNLTSLSEQELVDCDTTNNYGCNGGLMDYAFSYIISNGGLHKEVDYPYIMEEGTCEMRKEESEVVTISGYHDVPQNSEESLLKALANQPLSVAIEASGRDFQFYSGGVFDGHCGTQLDHGVAAVGYGSTNGLDYIIVKNSWGSKWGEKGYIRMKRNTGKPAGLCGINKMASYPTKKK.

[0045] The present invention also provides application of the protein in regulating the synthesis of cell wall lignin.

[0046] The technical solutions provided by the present invention are 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.

[0047] Example 1 Obtaining the Populus trichocarpa PtrXCP2A gene and constructing a plant expression vector

[0048] (1) Cloning of the PtrXCP2A gene sequence

[0049] RNA was extracted from Populus trichocarpa using the pBIOZOL plant RNA extraction kit.

[0050] Total RNA of wild-type plants of Nisqually-1 genotype was reverse transcribed using Takara reverse transcription kit (RR047A), and the obtained cDNA was aliquoted and stored in a -20°C refrigerator for future use.

[0051] The total DNA from leaves of wild-type plants of the Nisqually-1 genotype of Populus trichocarpa was extracted using Biotek one-step plant genomic DNA rapid extraction reagent. The eluted DNA was aliquoted and stored in a -20°C refrigerator for later use.

[0052] Referring to the genome sequence information of Populus trichocarpa provided by the phytozome website, gene-specific primers were designed at both ends of the promoter and coding region of the target gene and synthesized by a biological company. The upstream and downstream primer sequences for PtrXCP2A gene cloning were obtained and are shown in Table 1.

[0053] Table 1 Primers and sequences used for PtrXCP2A cloning

[0054]

[0055] The proXCP2A promoter fragment was obtained by pairing proXCP2A-F and XCP2A-PC-mid-R, and leaf DNA was used as a template. The XCP2A fragment was then cloned by polymerase chain reaction (PCR) using XCP2A-PC-mid-F and XCP2A-R and xylem cDNA as a template. Finally, the recombinant promoter and coding region sequence of XCP2A (XCP2A-PC) was obtained by pairing proXCP2A-F and XCP2A-R and the diluted target fragment as a template.

[0056] The obtained PCR products were subjected to agarose gel electrophoresis to separate the target bands. The target fragments were recovered using Thermo's Silica Bead DNA Gel Extraction Kit and ligated to the blunt-ended PQB vector.

[0057] Table 2 PQB vector connection system

[0058]

[0059] Thaw DH5α competent cells on ice. Add all ligation products to the competent cells and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add resistance-free LB medium, and activate at 37°C, 200 rpm for 1 hour. Harvest the cells at low speed and spread them on solid LB medium (containing 50 mg / L chloramphenicol). Incubate at 37°C overnight. The next day, perform PCR analysis on the resulting single clones using target gene primers.

[0060] (2) Construction of plant overexpression vector

[0061] The obtained XCP2A-PC-PQB plasmid and the prepared pGWB10 plant expression vector were subjected to LR reaction. The specific reaction system and reaction conditions are as follows:

[0062] Table 3 LR exchange system

[0063]

[0064] Thaw the DH5α competent cells on ice. Add all of the above reaction products to the competent cells and mix thoroughly. Heat shock at 42°C for 1 minute, place on ice for 3 minutes, add resistance-free LB medium, and activate at 37°C, 200 rpm for 1 hour. Collect the cells at low speed and spread them on solid LB medium (kanamycin 50 mg / L + hygromycin 50 mg / L) and culture at 37°C overnight. The next day, perform PCR identification on the obtained single point clones using the identification primers shown in Table 4:

[0065] Table 4 Primer sequences for monoclonal detection of plant expression vectors

[0066] Primername Sequence(5'-3') serial number IdentifyXCP2A-PC-F GTCATACATGATCATGTCATATGC SEQ ID NO.8 XCP2A-R CTTCTTTTTAGTGGGATAAGAAGCC SEQ ID NO.9

[0067] PCR products were analyzed by electrophoresis to identify positive clones. Positive single clones were selected and cultured in 5 mL of LB medium (50 mg / L kanamycin and 50 mg / L hygromycin) at 37°C, 200 rpm, overnight. The next day, the strains were preserved and centrifuged to collect the cells for plasmid extraction and sequencing.

[0068] Sequencing analysis revealed that the gene we obtained was 1059 bp (SEQ ID NO. 1), encoding 352 amino acids (SEQ ID NO. 3), and the promoter fragment we obtained was 2617 bp (SEQ ID NO. 2), which was consistent with the sequence information of Populus trichocarpa PtrXCP2A provided by the phytozome website, indicating that we successfully obtained the Populus trichocarpa PtrXCP2A gene and its promoter sequence.

[0069] Example 2 Obtaining transgenic plants of Populus trichocarpa PtrXCP2A

[0070] (1) Freeze-thaw transformation of Agrobacterium

[0071] ① Thaw the Agrobacterium GV3101 competent cells in an ice bath;

[0072] ② Add 3 μL of expression vector plasmid, place on ice for 30 minutes, freeze in liquid nitrogen for 1 minute, and then place in a 37°C water bath for 5 minutes;

[0073] ③ Add 950 μL of antibiotic-free YEP medium and culture at 28°C, 200 rpm, with shaking for 4 h;

[0074] ④ Centrifuge at 3000 rpm for 5 min to concentrate the bacterial solution, and then use 100 μL YEP to lyse the cells.

[0075] ⑤ Plate the cells on solid YEP medium supplemented with 50 mg / L kanamycin, 50 mg / L hygromycin, 50 mg / L gentamicin, and 50 mg / L rifampicin and incubate at 28°C for 48 hours. Detect positive clones by PCR using primers used for identification.

[0076] (2) Agrobacterium-mediated genetic transformation of Populus trichocarpa

[0077] ① Pick the Agrobacterium strain containing the plant expression vector and add it to 20 mL of LB medium containing resistance (kanamycin 50 mg / L + hygromycin 50 mg / L + hygromycin 50 mg / L + gentamicin 50 mg / L + rifampicin 50 mg / L), and culture it at 28°C, 220 rpm, and culture overnight.

[0078] ② The next day, 1000 μL of the bacterial solution was added to 50 mL of resistant LB liquid medium (kanamycin 50 mg / L + hygromycin 50 mg / L + gentamicin 50 mg / L + rifampicin 50 mg / L) and cultured at 28°C and 220 rpm until OD600 reached 0.6.

[0079] ③ Take the stem segment of a one-month-old sterile tissue culture seedling of Populus trichocarpa and place it in the cultured bacterial solution for 20 minutes.

[0080] ④ Remove the stem segments, place them on co-cultivation medium, and culture in the dark for 2 days.

[0081] ⑤ Two days later, wash the explant stem segments thoroughly with sterile water and place them on proliferation culture medium for normal culture.

[0082] ⑥ After the resistant buds grow, insert them into the rooting medium to induce rooting.

[0083] (3) Identification of transgenic overexpressing plants

[0084] ① Plant the resistant plants together with wild-type plants of the same condition in the soil and culture them until they are 3 months old. Collect the developed xylem tissues of the resistant plants and wild-type controls.

[0085] ② Extract RNA. For specific steps, refer to the instructions of the Plant RNA Extraction Kit (pBIOZOL).

[0086] ③ Obtain cDNA by reverse transcription. The specific steps are as follows: Plant Takara Reverse Transcription Kit (RR047A).

[0087] ④ Dilute the cDNA 10-fold and perform RT-qPCR. The internal reference is PtrActin 2. The primer sequences are shown in Table 6.

[0088] Each 20 μL reaction 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 denaturation for 30 s; 95°C denaturation for 5 s, 60°C annealing for 15 s, and 72°C extension for 30 s, for 40 cycles. PtActin2 was used as an internal control, and each analysis was performed in triplicate.

[0089] Table 5 PCR detection system

[0090] Reagents volume program 2×TBGreenPremixExTaqII 10 μL Pre-denaturation at 95°C for 30 seconds ROXReferenceDyeII 0.4μL 35cycle: 95℃ / 5s Primer 1 (10 mM) 0.8μL 60℃ / 15s Primer 2 (10 mM) 0.8μL 72℃ / 30s template 1 μL Extension at 72°C for 7 min <![CDATA[ddH2Oupto]]> 20 μL Store at 16℃

[0091] Table 6 Quantitative PCR detection system

[0092] Primername Sequence(5'-3') serial number RT-XCP2A-F CATGGGAAGATTTATGAGAGCATGG SEQ ID NO.10 RT-XCP2A-R CATTGCACCCATAGTTATTAGTAGTG SEQ ID NO.11 PtActin2-F AACATGGGATTGTTAGCAACTGG SEQ ID NO.12 PtActin2-R TCCATCACCAGAATCCAGCACA SEQ ID NO.13

[0093] A total of 13 independent resistant strains were obtained through Agrobacterium-mediated genetic transformation, named ProXCP2A::XCP2A-1~13. The growth status of PtrXCP2A overexpressing and wild-type plants after 3 months of greenhouse growth is shown in the figure. Figure 1 As shown. After transcriptional level identification, compared with wild-type plants of Populus trichocarpa, the transcription level of PtrXCP2A gene in most resistant plants was significantly increased ( Figure 2), indicating that transgenic Populus trichocarpa plants overexpressing the PtrXCP2A gene were successfully obtained.

[0094] Example 3 Application of Populus trichocarpa PtrXCP2A gene in regulating the secondary growth of trees

[0095] (1) Growth phenotype analysis of the independent resistant strains obtained in Example 2 and wild-type Populus trichocarpa plants

[0096] Growth phenotypic analysis includes the following indicators:

[0097] Plant height and base stem measurement: Transgenic and wild-type plants were transplanted simultaneously into sterilized black soil in a greenhouse. After 30 days, a mark was made at 20 cm above the ground. This point served as the reference point for plant height and base stem measurement. A minimum of six trees were collected for each line.

[0098] The wild-type and transgenic plants expressing high abundance of PtrXCP2A were taken from greenhouses for 3 months. By comparing the wild-type and transgenic plants of PtrXCP2A grown in greenhouses, it was found that the height and basal stem thickness of the plants overexpressing PtrXCP2A were not significantly different from those of the wild-type (see Figure 1 、 Figure 3 and Figure 4 ), these data indicate that overexpression of the PtrXCP2A gene does not affect the height and diameter growth of trees.

[0099] (2) Observation of tissue sections of the independent resistant strains obtained in Example 2 and wild-type Populus trichocarpa plants

[0100] The 12th stem node of wild-type and PtrXCP2A transgenic plants grown in the greenhouse for 3 months were sliced, and the IN12 of wild-type and transgenic plants were quickly fixed in FAA fixative; vacuum was applied for 5 minutes, negative pressure was applied for 30 minutes, and this step was repeated 3 times; fresh FAA fixative was replaced and incubated at 4°C overnight. The next day, the fixed stem segments were gently shaken and dehydrated in alcohol with gradient concentrations (50% → 60% → 70%, 2 hours per step); then the samples were fixed on the electron microscope stage and placed in a vacuum desiccator for 48 hours. On the fourth day, the cross-section of the stem segment was sprayed with gold using an ion sputtering instrument (10mA / 60s), and then transferred to a desktop scanning electron microscope to analyze the cell wall structure and thickness of mature xylem cells. There were 3 trees in each line, and 3 cross-sections were randomly selected from each tree. The thickness of 30 cell walls was counted using ImageJ. The results showed that overexpression of PtrXCP2A affected the thickening process of the xylem fiber cell wall, ultimately leading to thinning of the cell wall (see Figure 5 and Figure 6 ).

[0101] (3) Wood component analysis of the independent resistant strains and wild-type plants obtained in Example 2

[0102] Four-month-old basal stem segments (10 cm long) of Populus trichocarpa were collected. Segments from three trees were pooled to form a single replicate sample, with three biological replicates per strain. After debarking, the remaining xylem tissue was oven-dried at 55°C and then finely ground using a ball mill. 70 mg of wood powder was weighed from each sample and washed sequentially with 70% ethanol, chloroform / methanol (1:1 v / v), and acetone (each step followed by shaking for 2 minutes and centrifugation at 10,000 g for 10 minutes). The resulting insoluble residue was air-dried at room temperature and labeled as cell wall material for component analysis. Each crystalline cellulose content determination required 1.5 mg of cell wall material, and each lignin content determination required 2 mg of cell wall material. Each content was measured in triplicate for each sample, and the mean of the three measurements was used as the crystalline cellulose or total lignin content for that sample. Results showed that overexpression of XCP2A reduced the lignin content of wood cell walls.

[0103] The detailed lignin sequencing method is as follows:

[0104] ① Take 2.0 mg of prepared cell wall material and place it into a centrifuge tube.

[0105] ② Rinse the tube wall with 500 μL of acetone and collect the cell wall material at the bottom of the tube. The acetone is evaporated very gently under air flow.

[0106] ③ Gently add 1.0 mL of freshly prepared acetyl bromide solution (25% v / v acetyl bromide in glacial acetic acid) along the wall of the tube.

[0107] ④ Cover the tube cap and heat at 50℃ for 2 hours.

[0108] ⑤ Shake for 15 minutes and then heat for 1 hour.

[0109] ⑥ Cool to room temperature on ice and centrifuge (10000g, 15 minutes).

[0110] 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.

[0111] ⑧ After shaking the sample, add 0.5 mL of 0.5 M hydroxylamine hydrochloride solution and add HAc to make the volume up to 10 mL.

[0112] ⑨ The optical density was measured three times at 280 nm and the percentage of acetyl bromide soluble lignin (% ABSL) was determined using the appropriate coefficient (poplar = 18.21 g -1 cm -1 L), the formula 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.

[0113] (4) Analysis of cell wall saccharification ability of the independent resistant strains and wild-type plants obtained in Example 2

[0114] Xylem samples from the basal stems of four-month-old wild-type and XCP2A-overexpressing plants were dried at 55°C and ground into powder using a ball mill. 10 mg of wood powder was placed in a 2 mL test tube and pretreated with either 400 μL of water or 350 μL of dilute alkali (1% NaOH, w / v). All samples were incubated at 30°C for 30 min and then autoclaved at 120°C for 60 min. After cooling to room temperature, the dilute alkali-pretreated samples were neutralized with 50 μL of 2.5 M HCl. Saccharification was then initiated at 50°C / 200 rpm. After incubation with shaking for 12, 24, 36, 48, 60, and 72 h, the samples were centrifuged at 14,000 g for 10 min, and 20 μL of the supernatant was collected for reducing sugar content determination (DNS method). Reaction procedure: Mix 20 μL of sample with 200 μL of DNS reagent in a centrifuge tube, incubate at 95°C for 6 minutes, and measure absorbance (OD540) in a microplate reader after cooling. A glucose standard curve was generated, with at least three biological replicates performed for each transgenic material. Saccharification results revealed that overexpression of XCP2A increased wood cell wall saccharification efficiency, particularly after hot water pretreatment.

[0115] As can be seen from the above examples, the present invention provides a gene for regulating the synthesis of lignin in tree cell walls, a recombinant vector constructed therefrom, and its application. The present invention obtained transgenic plants overexpressing the PtrXCP2A gene of Populus trichocarpa through an Agrobacterium-mediated Populus trichocarpa genetic transformation system. Utilizing a series of technical means for analysis, it was found that overexpression of the PtrXCP2A gene affects the thickening process of the secondary wall of fiber cells, thereby affecting the proportion of wood components. This is mainly reflected in that overexpression of PtrXCP2A does not affect the external growth phenotype of the plant, while the secondary wall of the xylem fiber cells of the transgenic plant becomes thinner, ultimately leading to a decrease in lignin content and an increase in the saccharification capacity of the cell wall. The above results indicate that the PtrXCP2A gene has a certain regulatory effect on the thickening of the secondary wall of trees and the accumulation of lignin. The research content of the present invention has important theoretical guiding significance for the cultivation of high-quality timber and new varieties of forest trees.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of the PtrXCP2A gene in regulating the synthesis of lignin in tree cell walls, characterized in that: The nucleotide sequence of the PtrXCP2A gene is shown in SEQ ID NO.1; the tree is Populus trichocarpa.

2. Application of a recombinant vector containing the PtrXCP2A gene in regulating the synthesis of lignin in the cell wall of Populus trichocarpa, characterized in that: The recombinant vector comprises the PtrXCP2A gene and the PtrXCP2A gene promoter; the nucleotide sequence of the PtrXCP2A gene is shown in SEQ ID NO.1, and the nucleotide sequence of the PtrXCP2A gene promoter is shown in SEQ ID NO.

2.

3. A method for regulating the synthesis of lignin in the cell wall of Populus trichocarpa using the recombinant vector according to claim 2, characterized in that: The following steps are involved: (1) transforming the recombinant vector into Agrobacterium; (2) Agrobacterium was inoculated into Populus trichocarpa to regulate the synthesis of lignin in the tree cell walls.

4. The method according to claim 3, characterized in that The Agrobacterium is GV3101 Agrobacterium.

5. Application of the protein encoded by the PtrXCP2A gene in regulating the synthesis of lignin in the cell wall of Populus trichocarpa, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.3.

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