Application and method of knocking out D53 gene of Populus tomentosa in improving wood yield

Abstract

The present invention discloses an application of knocking out the D53 gene of white poplar in improving wood yield and a method thereof. A D53 knockout plant vector is introduced into white poplar to obtain a D53 knockout transgenic plant. In the knockout plant, the proportion of stem xylem is significantly higher than that of WT, and the number of xylem layers is also significantly increased. The results indicate that the activity of stem cambium and xylem differentiation of poplar are promoted after the loss of D53, which is of great significance to the development and utilization of poplar.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of knocking out a D53 gene of white poplar in improving wood yield, and also to a method for improving wood yield. Background Art

[0002] During the growth and development of higher plants, secondary vascular tissues provide plants with necessary mechanical support and material transport functions, thereby maintaining their normal life activities (Fischer et al., 2019). Unlike herbaceous plants, perennial trees can undergo secondary growth based on primary growth, which eventually leads to thickening of the stems. The secondary growth of trees depends on the continuous division and differentiation of cambium cells, and their differentiation to both sides forms secondary phloem and secondary xylem. The secondary xylem of forest trees is wood, which is widely used in papermaking, construction, bioenergy and other aspects. It is an important renewable resource and has extremely important economic value for human production and life (Plomion et al., 2001). With the rapid development of my country's economy, people's demand for wood is increasing day by day. However, due to the long growth cycle and high heterozygosity of forest trees, traditional breeding is difficult to meet the growing demand for wood. Therefore, exploring the key genes for wood regulation (especially the regulatory factors of plant hormone signaling pathways), analyzing the molecular regulatory network of wood secondary development, and using molecular breeding methods to improve wood quality are considered to be one of the most effective ways to alleviate the contradiction between wood supply and demand.

[0003] Strigolactones (SLs) are terpene lactone compounds found in plant roots. As a new type of plant hormone, SLs have been reported to be involved in regulating developmental processes such as leaf senescence, taproot growth, root hair elongation, axillary bud initiation, stem secondary growth, and lateral branching (Brewer et al., 2013; Rubio-Moraga et al., 2014; Seto et al., 2012). In Arabidopsis, compared with the wild type, the stem cambium activity of SLs synthase mutants (max1, max2, max3, max4) was significantly reduced, and the marker genes related to the cambium and cell cycle were significantly downregulated, while MAX2 driven by a cambium-specific promoter could completely rescue the reduced secondary growth caused by the max2 loss-of-function mutant (Agusti et al., 2011). Agusti et al. (2011) also found that the cambium division activity was significantly inhibited in the pea rsm1 (i.e., MAX4 homologous gene) mutant, while the cambium cell division activity was enhanced after exogenous application of GR24 to the herbaceous plant Arabidopsis and the woody plant Eucalyptus globulus (Agusti et al., 2011). The above evidence shows that SL signaling is indeed involved in the development of stem vascular cambium, and its regulatory role is conserved in different plants. The latest report shows that SL signaling can regulate the proliferation of vascular cambium cells by regulating the stability of BES1, a key transcription factor in the BR signaling pathway, and then by changing the transcription level of WOX4 (Hu et al., 2021). These evidences strongly suggest that SL may play an important role in regulating the development of poplar vascular cambium.

[0004] At present, the research on SL in plant development is relatively clear, but its regulatory mechanism on wood secondary development still needs to be further explored. Carotenoids are the starting substrates for SL synthesis. Under the action of key enzymes such as D27, MAX3, and MAX4, their key precursors, caprolactone, are first synthesized (Zwanenburg et al., 2016; Booker et al., 2014). Caprolactone is further transported to the cytoplasm, and under the action of enzymes such as MAX1 and LBO, SL is finally synthesized and transported to the nucleus to play a role (Zwanenburg B et al., 2016). The expression of these SL synthases is precisely regulated in time and space to maintain their normal levels and dynamic distribution in plants. The signal transduction of SL in cells depends on the receptor-mediated "reinhibition release" model, that is, the binding of SL molecules to receptor D14 promotes the ubiquitination and degradation of D53 (SMXL6, 7, 8) proteins, relieves the inhibitory effect on downstream gene transcription, and then leads to the appearance of SL response phenotypes (Liang et al., 2013). D53, the core component of SL signal transduction, is considered to be the key site for cross-integration with other hormone signals in regulating different processes of plant growth and development (Dun et al., 2009). Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide an application of knocking out the D53 gene of Populus tomentosa in improving wood yield, and the second object of the present invention is to provide a method for improving the wood yield of Populus tomentosa.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Application of knockout Populus tomentosa D53 gene in improving timber yield, wherein the knockout Populus tomentosa D53 gene includes Populus tomentosa genes D53a and D53b, the nucleotide sequence of D53a is shown in SEQ ID NO.1; the nucleotide sequence of D53b is shown in SEQ ID NO.2.

[0008] Preferably, the method for knocking out the Populus tomentosa D53 gene is to use CRISPR / Cas9 technology.

[0009] Preferably, the knockout of Populus tomentosa D53 gene is to transform Populus tomentosa with a CRISPR / Cas9 gene editing vector of D53 to obtain a transgenic plant with an edited mutation of the D53 gene, and the wood yield of the obtained transgenic plant is increased.

[0010] Preferably, the method of increasing wood yield is to increase the number of stem xylem layers.

[0011] 2. A method for increasing the wood yield of Populus tomentosa, comprising transforming Populus tomentosa with the CRISPR / Cas9 gene editing vector of D53 to obtain transgenic plants with PtoD53 gene editing mutations.

[0012] Preferably, the method for transforming Populus tomentosa is mediated by Agrobacterium.

[0013] Preferably, the Agrobacterium of the present invention is Agrobacterium GV3101.

[0014] The beneficial effects of the present invention are as follows: the present invention provides an application of knocking out the D53 gene of Populus tomentosa in improving wood yield, and a D53 knockout transgenic plant is obtained by introducing a D53 knockout plant vector into Populus tomentosa; in the knockout plant, the proportion of stem xylem is significantly higher than that of WT, and the number of xylem layers is also significantly increased; this result indicates that the activity of the stem cambium and the differentiation of the xylem are promoted after the loss of D53. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0016] Figure 1 It is a small vector structure diagram of the gDNA expression cassette;

[0017] Figure 2 Identification of positive plants with D53 knockout in Populus tomentosa (A: PCR molecular detection of wild-type and Populus tomentosa d53 transgenic plants, PCR detection was performed using DNA of the wild-type strain and d53 transgenic strain as templates; M represents MarkerDL2000; WT is the wild-type plant, and L12-L16 are d53 transgenic plants to be identified; B: sequencing identification results of Populus tomentosa d53 transgenic plants).

[0018] Figure 3 The growth conditions of the knockout plant material of Populus tomentosa D53 (A: growth comparison between the knockout material and the wild type; B: statistical analysis of stem diameter between the knockout material and the wild type; C: statistical analysis of plant height between the knockout material and the wild type; D: statistics on the number of stem internodes between the knockout material and the wild type; E: statistics on internode length between the knockout material and the wild type).

[0019] Figure 4 Phenotypic analysis of secondary development of Populus tomentosa D53 knockout plants (A: Toluidine blue staining of knockout materials and wild type; B: analysis of xylem proportions of knockout materials and wild type; C: analysis of xylem layers of knockout materials and wild type; D: cambium phenotype analysis of knockout materials and wild type; E: analysis of cambium layers of knockout materials and wild type). DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0021] Example 1: Construction of plant expression vector

[0022] Gene knockout vector used in this study (pYL-CRISPR / Cas9-DH)

[0023] (1) Preparation of adapters: As shown in the D53a sequence (SEQ ID NO. 1) and D53b sequence (SEQ ID NO. 2) of the Populus tomentosa gene, primers that can specifically knock out the D53a and D53b targets were designed using the relevant knockout target design website, and the primers were synthesized by Shenzhen BGI. The specific primer sequences are as follows:

[0024] T1-F:5'-GCAGGTCACGCGCTCGACG-3' (SEQ ID NO.3);

[0025] T1-R:5'-CGTCGAGCGCGTGACCTGC-3' (SEQ ID NO.4);

[0026] T2-F:5'-TATTGTTAGGCCGTTACCGC-3' (SEQ ID NO.5);

[0027] T2-R:5'-GCGGTAACGGCCTAACAATA-3' (SEQ ID NO.6);

[0028] T3-F:5'-CTCGGTCTAGTGACACACCG-3' (SEQ ID NO.7);

[0029] T3-R:5'-CGGTGTGTCACTAGACCGAG-3' (SEQ ID NO.8);

[0030] T4-F:5'-CCATAAGCGAGTTCGAAAC-3' (SEQ ID NO.9);

[0031] T4-R:5'-GTTTCGAACTCGCTTATGG-3' (SEQ ID NO.10);

[0032] (2) The concentration of the target primer was diluted to 1 μM and annealed by PCR reaction. The reaction system is shown in Table 1:

[0033] Table 1. PCR system

[0034]

[0035]

[0036] (3) First Cutting and Ligating Method: Use BsaI restriction endonuclease and T4 DNA ligase to connect the target site to the target site using a variable temperature cycler (or PCR instrument). Figure 1 The BsaI restriction site of the small vector containing the gDNA expression cassette is shown. The construction process adopts the Golden Gate cloning (Engler et al., 2008; 2009) strategy. The T1 target annealing product is connected to obtain pYLsgRNA-PtoD53-1, the T2 target annealing product is connected to obtain pYLsgRNA-PtoD53-2, the T3 target annealing product is connected to obtain pYLsgRNA-PtoD53-3, and the T4 target annealing product is connected to obtain pYLsgRNA-PtoD53-4. The reaction system is shown in Table 2, and the reaction conditions are: 37℃5min, 20℃5min, and 5 cycles of reaction.

[0037] Table 2. PCR system

[0038]

[0039] (4) First round of PCR amplification: The ligation product in (2) was used as a template, and the gDNA expression cassette containing the PtoD53 specific target was PCR amplified using the universal primers UF / gRNA-R. The amplification system is shown in Table 3. The sequences of the primers UF / gRNA-R pair are as follows:

[0040] Primer UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO. 11);

[0041] Primer gRNA-R: 5′-CGGAGGAAAATTCCATCCAC-3′ (SEQ ID NO. 12);

[0042] Reaction procedure: 10 cycles at 98°C for 1 min, pre-denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 68°C for 10 s, 17-20 cycles: pre-denaturation at 94°C for 15 s, annealing at 60°C for 15 s, extension at 68°C for 10 s. After the reaction, take 3 μL for electrophoresis examination.

[0043] Table 3. PCR system

[0044]

[0045] (5) Second round of PCR amplification: The PCR product in step (3) was selected as a template, and the primers with adapters were used for the second round of PCR amplification. The amplification system is shown in Table 4. Reaction procedure: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 60°C extension for 15 s, 68°C post-extension for 30 s (35 cycles); 16°C storage. Furthermore, the successfully amplified PCR products were subjected to gel electrophoresis and gel excision recovery. The sequences of the primer pair B1' / B2' / B3' / B4'-F / B2 / B3 / B4 / BL-R are as follows:

[0046] Primer B1'-F: 5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3' (SEQ IDNO.13);

[0047] Primer B2-R: 5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 14);

[0048] Primer B2'-F: 5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3' (SEQ ID NO. 15);

[0049] Primer B3-R: 5′-AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC-3′ (SEQ ID NO. 16);

[0050] Primer B3′-F: 5′-TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG-3′ (SEQ ID NO. 17);

[0051] Primer B4-R: 5'-AGCGTGggtctcGagtcGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 18);

[0052] Primer B4'-F: 5'-AGCGTGggtctcGagtcGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 19);

[0053] Primer BL-R: 5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 20).

[0054] Table 4. PCR system

[0055]

[0056] (6) Second cutting and ligation: Select the mid-gel recovery product and pYL-CRISPR / Cas9-DN in (4), use BsaI restriction endonuclease and T4 DNA ligase, and connect the gDNA expression cassette containing the PtoD53 target to the pYL-CRISPR / Cas9-DN expression vector (see Ma, X. Let al. A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 24, pii: S1674-2052 (15) 00204-X (2015)) through a variable temperature cycler (or PCR instrument) to obtain a recombinant vector named PtoD53-Cas9. Reaction system: 37°C for 5 min, 20°C for 5 min, and 25 cycles of reaction.

[0057] (7) The ligation product in step (5) is transformed into E. coli DH5α competent cells to carry out the E. coli transformation process.

[0058] (8) Perform colony PCR experiment to screen positive clones.

[0059] The monoclonal colonies on the plate were numbered, and a small amount of bacteria were picked up in turn with a sterilized pipette tip and placed in a PCR tube as a template for amplification. The amplification system was shown in Table 5. In addition, the gene gel recovery fragments and ddH2O were used as templates, and positive and negative controls were set up respectively.

[0060] Table 5. PCR system

[0061]

[0062] The reaction conditions were set as follows: 98°C, 3 min pre-denaturation; 98°C, 15 s denaturation; 56°C, 20 s annealing; 72°C, 20 s extension; 35 cycles in total; 72°C, 5 min full extension; 16°C, 25 min cooling and storage. The product was detected by 1% agarose gel electrophoresis, and the colony that could amplify the same size band as the positive control was the positive clone.

[0063] (5) Positive clone plasmid extraction and sequence verification

[0064] The clones that were positive in PCR were picked and placed in LB liquid medium containing kanamycin or ampicillin, and cultured overnight at 37°C and 200 rpm / min. The plasmid was extracted using the alkaline lysis plasmid mini-extraction kit from BioFlux.

[0065] The plasmid is sent to the company for sequencing. After the sequence determination and comparison are correct, the vector construction is completed. The successfully constructed PtoD53-Cas9 recombinant plasmid is transferred into Agrobacterium.

[0066] The operation of transforming Agrobacterium is as follows:

[0067] 1) Take 3 μL of PtoD53-Cas9 recombinant plasmid and add it to 100 μL of Agrobacterium GV3101 competent cells and mix gently;

[0068] 2) Place on ice for 5-10 minutes, quickly freeze in liquid nitrogen for 1 minute, and immediately place in a 37°C water bath for 5 minutes;

[0069] 3) Add 800 μL YEP liquid medium to the competent cells in a clean bench, mix well, and then resuscitate and culture at 28°C and 200 rpm / min for 4 h;

[0070] 4) After the recovery, centrifuge at 3000 rpm / min for 10 min. In a clean bench, discard 800 μL of supernatant, mix the remaining 100 μL of bacterial solution, and evenly apply 100 μL of bacterial solution on YEP solid medium containing 40 mg / L rifampicin and 50 mg / L kanamycin using a high-temperature sterilized and cooled coating stick, and incubate inverted at 28°C for 48 h.

[0071] 5) The engineered bacteria was named (GV3101) PtoD53-Cas9, and sterilized glycerol was added to the bacterial solution to a final concentration of 20%. After quick freezing with liquid nitrogen, the solution was stored in a -80°C refrigerator for subsequent genetic transformation experiments.

[0072] Example 2: Genetic transformation of Populus tomentosa

[0073] (1) Double activation culture of Agrobacterium

[0074] 1) The engineered bacteria (GV3101) D53-Cas9 were inoculated on YEP solid medium containing 40 mg / L rifampicin and 50 mg / L kanamycin, and cultured in a constant temperature incubator at 28°C for 36 hours; a single colony was picked and inoculated into 10 mL of YEP+Rif+kan dual-antibody liquid medium;

[0075] 2) 28°C, 200 rpm / min shaking culture for 36-48 hours, until the bacterial solution concentration reaches OD600 = 0.8-1.0;

[0076] 3) According to the ratio of 1:1000, 50 μL of the first active liquid was pipetted into 50 mL of fresh YEP+Rif+kan dual-antibody liquid culture medium for second active liquid culture;

[0077] 4) Incubate at 28°C, 200 rpm / min for 12-16 hours until the bacterial solution concentration reaches OD600 = 0.3-0.4, and set aside.

[0078] (2) Preparation of Agrobacterium infection solution

[0079] 1) Use a 50 mL centrifuge tube to collect the second active liquid, 4000 rpm / min, 8 min, and collect the bacteria;

[0080] 2) Discard the culture medium supernatant, resuspend the Agrobacterium in 25 mL of WPM resuspension solution containing AS, and pour the resuspension into a sterile glass bottle;

[0081] 3) The resuspension was placed at 28°C and shaken at 200 rpm / min in the dark for 1-2 hours to enhance the infection activity of Agrobacterium.

[0082] (3) Leaf disk preparation

[0083] 1) In a clean bench, burn the sterilized scissors, tweezers, and surgical knife handles with an alcohol lamp for 15 seconds, and let them cool for later use;

[0084] 2) Use scissors to cut off 5-6 leaves of healthy wild-type tissue culture seedlings and place them in a culture dish. Add 1 / 3 volume of sterile water to the dish to keep the leaves moist;

[0085] 3) Place a sterile surgical blade into the handle, burn it with an alcohol burner for 15 seconds, and then let it cool. Use the blade to cut the leaves into 0.5 cm 2 Square leaf disk.

[0086] (4) Infection

[0087] 1) Use tweezers to clamp the leaf disc into the Agrobacterium resuspension, gently shake the glass bottle to make the resuspension evenly cover the leaf disc, and infect for 10 minutes;

[0088] 2) After the infection is complete, carefully remove the leaf disc with tweezers, place it on sterile paper, and absorb the excess infection fluid on the leaf disc;

[0089] 3) Place the leaf disc flatly on the co-culture plate, place it in a dark box, and culture it in the dark at 25°C for 36-48 hours.

[0090] (5) Leaf disc selection culture

[0091] 1) After dark culture, select appropriate plant resistance according to the carrier and prepare a selection medium containing antibiotics;

[0092] 2) In a clean bench, transfer the infected leaf disc to a selective culture medium to induce callus. Replace the leaf disc with a new culture medium every seven days for 3-4 weeks until white or light yellow callus grows on the edge of the leaf disc. The entire process is cultured in a dark environment at 25°C.

[0093] (6) Callus induction

[0094] The leaf disc with callus tissue is transferred to the budding medium containing the corresponding antibiotics, and cultured at 8000 Lux and 25°C for 5-6 weeks, with the medium replaced once a week. During this period, the callus tissue will fully grow and expand. Around the 5th week, buds will grow on the callus tissue, and clustered buds will grow.

[0095] (7) Root induction of clustered shoots

[0096] When the clustered buds grow to about 5 cm, cut them off with sharp scissors and carefully insert them into the rooting medium with tweezers. Cultivate them at 8000 Lux and 25°C for about 10 days to obtain rooted seedlings. These are candidate transgenic plants, which can be transplanted to soil after subsequent positive identification.

[0097] Example 3, knockout identification of d53 transgenic plants

[0098] (1) DNA extraction from wild-type and d53 transgenic Populus tomentosa

[0099] Select 10 to 15 transgenic resistant regenerated plants and extract the genomic DNA of Populus tomentosa. The method is as follows:

[0100] 1) Prepare CTAB buffer and preheat it in a 65°C water bath;

[0101] 2) Take about 0.5 g of leaves of wild-type and d53 transgenic Populus tomentosa, grind them into powder in liquid nitrogen, add them to 500 μL of the preheated CTAB extract, and mix well;

[0102] 3) Incubate in a 65°C water bath for 45 min, shaking gently every 15 min for 3 times.

[0103] 4) After the water bath, cool to room temperature, add equal volumes of chloroform:isoamyl alcohol (24:1), gently invert to mix, and then place flat to emulsify for 10 minutes. Centrifuge at 4°C, 12000 rpm / min for 10 minutes;

[0104] 5) Pipette the supernatant into a new sterile centrifuge tube, add an equal volume of -20°C pre-cooled isopropanol and mix by inversion until a white flocculent precipitate is visible;

[0105] 6) Centrifuge at 4°C, 12000 rpm / min for 10 min. Remove the supernatant, rinse the precipitate twice with 500 μL 75% (V / V) ethanol, rinse once with 500 μL anhydrous ethanol, and remove the liquid. Dry the precipitate in a rotary evaporator at 37°C until it becomes translucent;

[0106] 7) Add 25 μL of sterile water to dissolve the precipitate to obtain crude DNA extracts from wild-type and pYL-PtoD53 transgenic white poplar leaves;

[0107] 8) Add about 1 μl of RNase to the crude DNA extract to remove RNA. The reaction conditions are 37°C for 1 hour.

[0108] 9) Store the DNA sample in a -20°C refrigerator for future use.

[0109] (2) PCR amplification and knockout identification of positive plants

[0110] By introducing the D53-Cas9 knockout plant vector into Populus tomentosa, multiple d53 positive knockout transgenic plants were obtained. Using D53 specific amplification primers, WT and L12, L13 DNA were used as templates for amplification. The PCR identification results are as follows Figure 2 As shown in A. The amplified fragment was connected to the pMD19 vector and sequenced. The knockout results were identified by sequencing. Figure 2 As shown in B. The results showed that the D53a and D53b genes were knocked out in the L12 and L13 lines of the d53 transgenic plants. The d53 transgenic plants were named d53 L12 and d53 L13, respectively.

[0111] The screening was performed by using the vector PtoD53a / bF and the gene primer PtoD53a / bR for primer amplification. The sequences of the designed specific primers are as follows:

[0112] D53a detection-F: 5'-ATGCCTACGCCGGTAACTACAGC-3' (SEQ ID NO. 21);

[0113] D53a detection-R: 5'-CTTTGAAACGTGACGAGG-3' (SEQ ID NO. 22);

[0114] D53b detection-F: 5'-ATGCCGACGCCGGTAACTAC-3' (SEQ ID NO. 23);

[0115] D53b Detect-R: 5'-CTCTATCTGCATCCACGTG-3' (SEQ ID NO. 24).

[0116] The PCR reaction system was the same as that in Table 6. The reaction procedure was as follows: pre-denaturation at 94°C for 3 min, 1 cycle; denaturation at 94°C for 30 s, annealing for 30 s, extension at 72°C for 1 min, a total of 31 cycles; extension at 72°C for 10 min, and detection of amplification products by 1% agarose gel electrophoresis.

[0117] Table 6. PCR reaction system

[0118]

[0119] Example 4: Phenotypic analysis of Populus tomentosa D53 knockout plants

[0120] The four-month-old tissue culture seedlings were transplanted into soil pots and grown in a greenhouse at 25°C under long-day conditions (16 hours light / 8 hours dark, light intensity 10,000 lux) for three months ( Figure 3 Middle A). Parameters such as plant height, stem diameter, internode number and internode length of WT and d53 transgenic poplars were measured and statistically analyzed.

[0121] The results are as follows Figure 3 As shown in B, the stem diameter of d53 L12 and L13 plants was significantly increased compared with that of WT plants.

[0122] Example 5: Analysis of stem secondary development phenotypes of D53 knockout transgenic plants

[0123] This study also conducted stem cross-section and staining observation on Populus tomentosa d53 knockout plants and wild-type plants. Figure 4 As shown. By staining the stem tissue sections with toluidine blue, it was found that the number of stem xylem layers in the knockout plants was significantly higher than that in the WT, and the number of cambium layers was also significantly increased compared with the WT, which promoted the secondary development of the stem of Populus tomentosa and enhanced its wood yield.

[0124] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of knocking out the D53 gene of Populus tomentosa to improve wood yield, characterized by: The knocked-out Populus tomentosa D53 gene includes Populus tomentosa genes D53a and D53b, the nucleotide sequence of D53a is shown in SEQ ID NO.1; the nucleotide sequence of D53b is shown in SEQ ID NO.2; The method of increasing wood yield refers to increasing the number of stem xylem layers and cambium layers; the wood is Populus tomentosa.

2. The use of the knockout D53 gene of Populus tomentosa in improving wood yield according to claim 1, characterized in that: The method for knocking out the Populus tomentosa D53 gene is to use CRISPR / Cas9 technology.

3. The use of the knockout D53 gene of Populus tomentosa in improving wood yield according to claim 1, characterized in that: The knockout of the Populus tomentosa D53 gene is to transform the Populus tomentosa with a CRISPR / Cas9 gene editing vector of D53 to obtain a transgenic plant with an edited mutation of the D53 gene, and the wood yield of the obtained transgenic plant is increased.

4. A method for increasing the yield of white poplar wood, characterized in that: The specific method is to transform Populus tomentosa with the CRISPR / Cas9 gene editing vector of D53 to obtain a transgenic plant with an edited mutation of the PtoD53 gene; the knocked-out Populus tomentosa D53 gene includes Populus tomentosa genes D53a and D53b, the nucleotide sequence of D53a is shown in SEQ ID NO.1; the nucleotide sequence of D53b is shown in SEQ ID NO.2; The method of increasing wood yield refers to increasing the number of stem xylem layers and cambium layers; the wood is Populus tomentosa.

5. The method according to claim 4, characterized in that: The method for transforming Populus tomentosa is mediated by Agrobacterium.

6. The method according to claim 5, characterized in that: The Agrobacterium is Agrobacterium GV3101.

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