Application of knockout Nanlin 895 poplar DOF20 gene in increasing wood yield and method of knockout Nanlin 895 poplar DOF20 gene

Abstract

The invention discloses an application and a method for knocking out a DOF20 gene of poplar in NL895 to increase the wood yield, and the method comprises the following steps: introducing a DOF20 knockout plant vector into NL895 to obtain a dof20 knockout transgenic plant; the ratio of stem xylem in a knockout plant is obviously higher than that of WT, and the number of xylem layers and the thickness of secondary fiber cell walls are also obviously increased; the result shows that the xylem differentiation of the poplar stem is promoted after the DDOF20 is deleted, and the method has important significance on development and utilization of the poplar.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of knocking out the DOF20 gene of Populus deltoides cv. 'Nanlin895' in improving wood yield, and also relates to a method for improving wood yield. Background Art

[0002] Poplar is a fast-growing and high-yielding forest tree species worldwide and is also a model species for genetic breeding research of woody plants. Populus deltoides cv. 'Nanlin895' is an excellent poplar clone variety independently selected and bred in China, which has the characteristics of fast growth, high yield, excellent wood quality and strong adaptability. It is an important tree species resource for the construction of artificial forests and the wood industry in China, and is suitable for manufacturing fiberboards, pulp and structural timber. However, it still has problems such as low wood density, low yield, small volume, limited stress resistance, and wet heartwood. Therefore, taking Populus deltoides cv. 'Nanlin895' as the object of scientific research, by targeting the improvement of its wood properties and increasing its biomass, combined with precise site management, high yield and high efficiency can be achieved on the premise of ensuring ecological safety, providing a sustainable solution for forestry resource development.

[0003] The stem vascular tissue is an important material transport channel for woody plants, and at the same time provides necessary mechanical support for their growth, playing a decisive role in the normal growth and development of vascular plants. Different from herbaceous plants, perennial trees can carry out secondary growth on the basis of primary growth, ultimately resulting in stem thickening. Generally, the vascular tissue is composed of three types of cells, phloem, cambium and xylem, which are highly organized and arranged in an orderly manner, and each type of cell has specific biological functions (Nieminen et al., 2015; Ruonala et al., 2017). 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 many aspects such as papermaking, construction and bioenergy, and is an important renewable resource with extremely important economic value for human production and life (Plomion et al., 2001).

[0004] The DOF (DNA binding with One Finger) protein family is an important transcription factor family in plants and belongs to the classical proteins in the ZF superfamily (Yang et al., 2022; Zhang et al., 2022). DOF proteins are composed of 200 - 400 amino acids. Different from other ZF proteins, DOF TFs contain only one Cys2 / Cys2 ZF, and the ZF structure specifically recognizes the upstream core sequence 5’-(T / A) / AAAG-3’ of target genes (Umemura et al., 2004; Kim et al., 2010). At present, a large number of DOF family transcription factors have been identified in different plants, and it has been found that they play important regulatory roles in developmental processes such as cell division, tissue differentiation, metabolic regulation, and seed germination, and respond to various signals such as plant hormones and light (Yanagisawa, 2000; Park et al., 2003; Ward et al., 2005; Li et al., 2009; Chen et al., 2012; Ahmad et al., 2013; Noguero et al., 2013). In Arabidopsis thaliana, it has been reported that AtDOF2.4 and AtDOF5.8 are expressed in the procambium and early leaf veins respectively, and both are involved in the establishment of primary vascular tissues in their respective tissues (Konishi & Yanagisawa, 2007). However, there is no report on the function of Populus DOF transcription factors. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide the application of knocking out the DOF20 gene of Populus deltoides cv. 'Nanlin895' in increasing wood yield; another objective of the present invention is to provide a method for increasing wood yield.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] 1. The application of knocking out the DOF20 gene of Populus deltoides cv. 'Nanlin895' in increasing wood yield, wherein the DOF20 gene is DOF20a and DOF20b, and the nucleotide sequence of DOF20a is as shown in SEQ ID NO.1; the nucleotide sequence of DOF20b is as shown in SEQ ID NO.2.

[0008] Preferably, the CRISPR / Cas9 editing technology is used to knock out the DOF20 gene of Populus deltoides cv. 'Nanlin895' in the present invention.

[0009] Preferably, the target sequences of the CRISPR / Cas9 editing technology are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] Preferably, in the present invention, the improvement of wood yield is achieved by increasing the number of xylem layers and the thickness of the fiber secondary wall.

[0011] 2. A method for improving wood yield, comprising the following steps: transforming the CRISPR / Cas9 gene editing vector of the Populus deltoides cv. Nanlin895 DOF20 gene into Populus deltoides cv. Nanlin895 to obtain transgenic plants with gene editing mutations of the DOF20 gene.

[0012] Preferably, in the present invention, the method for transforming Populus deltoides cv. Nanlin895 is mediated by Agrobacterium.

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

[0014] The beneficial effect of the present invention is that the present invention provides the application of knocking out the NL895 DOF20 gene in improving wood yield. By introducing the DOF20 knockout plant vector into NL895, transgenic plants with DOF20 knockout were obtained; in the knockout plants, the proportion of stem xylem was significantly higher than that of the WT, the number of xylem layers was also significantly increased, and the fiber cell wall thickness was also significantly increased compared with the WT; this result indicates that the differentiation of the poplar stem xylem is promoted after the deletion of DOF20. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0016] Figure 1 Identification of positive NL895 knockout plants (A, C: Alignment and editing efficiency of reads of dof20 knockout lines IF-389 and IF-390 detected by high-throughput Hi-TOM; the editing efficiency is determined by the percentage and number of sequence reads showing unmodified and modified alleles; A shows the results of the IF-389 line; C shows the results of the IF-390 line; B, D: Visual distribution of identified alleles around the sgRNA cleavage site. Different colors represent different nucleotides; A = green; C = red; G = yellow; T = purple; substitution edits are shown in bold; insertion edits are highlighted by red rectangles; deletion edits are represented by horizontal dotted lines; the predicted cleavage site is represented by a vertical dotted line; B shows the results of the IF-389 line; D shows the results of the IF-390 line).

[0017] Figure 2 Growth of NL895 knockout plant materials (A: Comparison of growth between knockout materials and wild type; B: Statistical analysis of the number of stem internodes between knockout materials and wild type; C: Statistical analysis of plant height between knockout materials and wild type; D: Statistical analysis of stem diameter between knockout materials and wild type; E: Statistical analysis of internode length between knockout materials and wild type; WT is the wild type).

[0018] Figure 3 Phenotypic analysis of secondary development of NL895 knockout plants (A: Toluidine blue staining of the 12th, 14th, and 16th internodes of the stems of the knockout material and the wild type; the red double-headed arrow indicates the xylem width; B: Analysis of the number of xylem layers in the knockout material and the wild type).

[0019] Figure 4 Observation results of the 16th internode of the stem of NL895 knockout plants by scanning electron microscopy (A: Results shown under scanning electron microscopy at 3000X and 16000X for the knockout material and the wild type; Xf represents secondary fiber cells; Xv represents vessel cells; B: Analysis of the secondary fiber cell wall thickness in the mature xylem of the knockout material and the wild type). Specific implementation manners

[0020] The present invention will be 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 be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0021] Example 1: Construction of a plant expression vector

[0022] The gene knockout vector (BGK012-CRISPR / Cas9-DH) used in this study was purchased from Bagger CRISPR / Cas vector (product number BGK012).

[0023] (1) Adapter preparation: As shown in the DOF20a sequence (SEQ ID NO.1) and DOF20b sequence (SEQ ID NO.2) of the NL895 gene, using relevant knockout target design websites, targets that can specifically knockout DOF20a and DOF20b were designed and synthesized by BGI Shenzhen Co., Ltd. The specific primer sequences are as follows:

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

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

[0026] (2) Dilute the concentration of the target primers to 1 μM and complete annealing through PCR reaction. The reaction system is shown in Table 1:

[0027] Table 1. PCR system

[0028]

[0029] (3) The first method of cutting and ligating simultaneously: The construction process adopts the Golden Gate cloning (Engler et al., 2008; 2009) strategy. Among them, the annealing product of the T1 target site is ligated to obtain BGK012sgRNA-PdeDOF20-1, and the annealing product of the T2 target site is ligated to obtain BGK012sgRNA-PdeDOF20-2. The reaction system is shown in Table 2, and the reaction conditions are: 37°C for 5 min, 20°C for 5 min, and 5 cycles of reaction.

[0030] Table 2. PCR system

[0031]

[0032] (4) The first round of PCR amplification: Take the ligation product in (2) as the template, and use the universal primers U-F / gRNA-R to perform PCR amplification on the gDNA expression cassette containing the specific target site of PdeDOF20. The amplification system is shown in Table 3. The sequences of the primers U-F / gRNA-R are as follows:

[0033] Primer U-F: 5’-CTCCGTTTTACCTGTGGAATCG-3’ (SEQ ID NO.5);

[0034] Primer gRNA-R: 5’-CGGAGGAAAATTCCATCCAC-3’ (SEQ ID NO.6);

[0035] Reaction procedure: 98°C for 1 min for 10 cycles, 98°C for pre-denaturation for 15 s, 55°C for annealing for 15 s, 68°C for extension for 10 s, 17 - 20 cycles: 94°C for pre-denaturation for 15 s, 60°C for annealing for 15 s, 68°C for extension for 10 s. After the reaction, take 3 μL for electrophoresis inspection.

[0036] Table 3. PCR system

[0037]

[0038] (5) The second round of PCR amplification: Select the PCR product in step (3) as the template, and use the primers with adapters to perform the second round of PCR amplification. The amplification system is shown in Table 4. Reaction procedure: 98°C for pre-denaturation for 3 min; 98°C for denaturation for 10 s, 60°C for extension for 15 s, 68°C for post-extension for 30 s (35 cycles); store at 16°C. Further, perform gel electrophoresis and gel extraction and recovery on the successfully amplified PCR product. The sequences of the primers B1’ / BL-R are as follows:

[0039] Primer B1’-F: 5’-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3’ (SEQ IDNO.7);

[0040] Primer BL-R: 5’-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3’ (SEQ ID NO.8).

[0041] Table 4, PCR system

[0042]

[0043] (6) Second edge digestion and ligation: Select the gel extraction product in (4) and BGK012-CRISPR / Cas9-DN. Using BsaI restriction endonuclease and T4 DNA ligase, connect the gDNA expression cassette containing the PdeDOF20 target site to the BGK012-CRISPR / Cas9-DN expression vector through a temperature cycling instrument (or PCR instrument) (see Ma, X.L. et 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).) to obtain the recombinant vector, named Pdedof20-Cas9. Reaction system: 37°C for 5 min, 20°C for 5 min, react for 25 cycles.

[0044] (7) Transform the ligation product in step (5) into competent Escherichia coli DH5α cells and perform the Escherichia coli transformation process.

[0045] (8) Perform colony PCR experiments to screen for positive clones.

[0046] Number the monoclonal colonies on the plate, and sequentially pick a small amount of bacterial cells with a sterilized pipette tip into a PCR tube as the amplification template. The amplification system is shown in Table 5. In addition, use the gene gel extraction fragment and ddH2O as templates, and set positive and negative controls respectively.

[0047] Table 5, PCR system

[0048]

[0049] Set the reaction conditions as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s; annealing at 56°C for 20 s; extension at 72°C for 20 s; a total of 35 cycles; complete extension at 72°C for 5 min; cooling and preservation at 16°C for 25 min. The products are detected by 1% agarose gel electrophoresis. The colonies that can amplify a band of the same size as the positive control are positive clones.

[0050] (9) Extraction of Positive Cloning Plasmid and Verification by Sequencing

[0051] Pick the clones that are positive by PCR detection and inoculate them into LB liquid medium containing kanamycin or ampicillin. Incubate overnight at 37°C with shaking at 200 rpm / min. Use the alkaline lysis plasmid mini-prep kit from BioFlux to extract the plasmid.

[0052] Send the plasmid to the company for sequencing. After the sequence alignment is correct, the vector construction is completed. The successfully constructed Pdedof20-Cas9 recombinant plasmid is then transferred into Agrobacterium tumefaciens.

[0053] The operation of transforming Agrobacterium tumefaciens is as follows:

[0054] 1) Add 3 μL of Pdedof20-Cas9 recombinant plasmid to 100 μL of Agrobacterium tumefaciens GV3101 competent cells and mix gently.

[0055] 2) Incubate on ice for 5 - 10 min, then quickly freeze in liquid nitrogen for 1 min, and immediately place in a 37°C water bath for 5 min.

[0056] 3) In a laminar flow hood, add 800 μL of YEP liquid medium to the competent cells, mix well, and then incubate at 28°C on a shaker at 200 rpm / min for 4 h for recovery.

[0057] 4) After the recovery, centrifuge at 3000 rpm / min for 10 min. In a laminar flow hood, discard 800 μL of the supernatant, mix the remaining 100 μL of the bacterial solution, and use a sterilized and cooled spreading rod to evenly spread 100 μL of the bacterial solution on YEP solid medium containing 40 mg / L rifampicin and 50 mg / L kanamycin. Incubate upside down at 28°C for 48 h.

[0058] 5) Name the engineered bacteria as (GV3101)Pdedof20-Cas9. Add sterilized glycerol to the bacterial solution to a final concentration of 20%, quickly freeze in liquid nitrogen, and store at -80°C in the refrigerator for subsequent genetic transformation experiments.

[0059] Example 2: Genetic Transformation of NL895

[0060] (1) Two - step Activation Culture of Agrobacterium tumefaciens

[0061] 1) Streak the engineered bacteria (GV3101)Pdedof20-Cas9 on YEP solid medium containing 40 mg / L rifampicin and 50 mg / L kanamycin, and incubate in a 28°C constant temperature incubator for 36 h. Pick a single colony and inoculate it into 10 mL of YEP + Rif + kan double - antibiotic liquid medium.

[0062] 2) Incubate with shaking at 28 °C and 200 rpm / min for 36 - 48 hours until the cell density reaches OD 600 = 0.8 - 1.0;

[0063] 3) Pipette 50 μL of the first-stage activated solution into 50 mL of fresh YEP + Rif + kan double-antibiotic liquid medium at a ratio of 1:1000 for the second-stage activated solution culture;

[0064] 4) Incubate with shaking at 28 °C and 200 rpm / min for 12 - 16 hours until the cell density reaches OD 600 = 0.3 - 0.4 for later use.

[0065] (2) Preparation of Agrobacterium infection solution

[0066] 1) Use a 50 mL centrifuge tube to collect the second-stage activated solution, centrifuge at 4000 rpm / min for 10 min to collect the cells;

[0067] 2) Discard the supernatant of the medium, resuspend the Agrobacterium with 40 mL of WPM resuspension containing AS, and pour the resuspension into a sterile glass bottle;

[0068] 3) Place the resuspension at 28 °C and shake it in the dark at 200 rpm / min for 45 min to enhance the infection activity of Agrobacterium.

[0069] (3) Preparation of leaf discs

[0070] 1) In the laminar flow hood, sterilize scissors, forceps, and the surgical knife handle by burning them in the outer flame of an alcohol lamp for 15 seconds, and let them cool for later use;

[0071] 2) Use scissors to cut 5 - 6 leaves from healthy wild-type tissue culture seedlings and place them in a petri dish. Add 1 / 3 volume of sterile water to the dish to keep the leaves moist;

[0072] 3) Install the sterile surgical blade on the handle, sterilize it by burning it in the alcohol lamp flame for 15 s and then let it cool. Use the blade to evenly cut the leaves into 0.5 cm 2 square leaf discs.

[0073] (4) Infection

[0074] 1) Use forceps to pick up the leaf discs and place them into the Agrobacterium resuspension. Gently shake the glass bottle to evenly coat the leaf discs with the resuspension, and infect for 6 - 8 min, shaking once every 3 min during this period;

[0075] 2) After the infection, carefully pick out the leaf discs with forceps and place them on sterile paper to blot excess infection solution on the leaf discs;

[0076] 3) Place the leaf discs flat with the back side down on the co-culture plate, put them in a dark box, and incubate them in the dark at 25 °C for 36 - 48 h.

[0077] (5) Selection and culture of leaf discs

[0078] 1) After co - culture, prepare WPM selection medium containing antibiotics according to the vector (add 90 μl / L Hyg, 0.5 mg / L KT, 1.0 mg / L 2,4 - D, 300 mg / L TMT, 300 mg / L Cef);

[0079] 2) Transfer the infected leaf discs to the selection medium in a laminar flow hood to induce callus; every seven days during this period, transfer the leaf discs to a new medium and continue to change for 3 - 4 weeks until white or light yellow callus grows at the edge of the leaf discs; the whole process is cultured at 25 °C in the dark environment.

[0080] (6) Inducing shoots from callus

[0081] Transfer the leaf discs with callus to WPM shoot - induction medium containing corresponding antibiotics (add 90 μl / L Hyg, 0.02 mg / L TDZ, 300 mg / L TMT, 300 mg / L Cef), culture under 8000 Lux and 25 °C light for 5 - 6 weeks, and change the medium once a week. During this period, the callus will grow and expand fully. Around the 5th week, bud points will grow on the callus and produce cluster shoots.

[0082] (7) Elongation of cluster shoots

[0083] After the cluster shoots grow to a length of 2 cm, transfer the cluster shoots to the WPM elongation medium (300 mg / L TMT, 300 mg / L Cef) by cluster, culture under 8000 Lux and 25 °C light for 4 - 5 weeks, and the cluster shoots will grow taller quickly.

[0084] (8) Inducing roots from cluster shoots

[0085] When the cluster shoots grow to about 5 cm, cut the shoots with a sharp scissors, carefully insert the cluster shoots into the WPM rooting medium (add 90 μl / L Hyg, 0.02 mg / L TDZ, 300 mg / L TMT, 300 mg / L Cef), culture under 8000 Lux and 25 °C light for about 10 days to obtain rooted seedlings. These are the candidate transgenic plants, and they can be transplanted to soil for cultivation only after subsequent positive identification.

[0086] Example 3. Knock - out identification of dof20 transgenic plants

[0087] (1) DNA extraction of wild - type and dof20 transgenic NL895

[0088] Select 10 - 15 transgenic resistant regenerated plants respectively and extract the genomic DNA of NL895. The method is as follows:

[0089] 1) Prepare CTAB buffer and preheat it in a 65 °C water bath in advance for later use.

[0090] 2) Take about 0.5 g of leaves from wild-type and dof20 transgenic NL895, grind them into powder in liquid nitrogen, and add them to 500 μL of the preheated CTAB extraction solution mentioned above, then mix well.

[0091] 3) Incubate in a 65 °C water bath for 45 min, and gently shake and mix every 15 min during the process, for a total of 3 times.

[0092] 4) After the water bath, cool to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert and mix well, then let it stand for emulsification for 10 min. Centrifuge at 4 °C, 12000 rpm / min for 10 min.

[0093] 5) Pipette the supernatant into a new sterile centrifuge tube, add an equal volume of isopropanol pre-cooled at -20 °C and invert to mix well. White flocculent precipitates can be seen after mixing.

[0094] 6) Centrifuge at 4 °C, 12000 rpm / min for 10 min. Discard the supernatant, rinse the precipitate twice with 500 μL of 75% (V / V) ethanol and once with 500 μL of absolute ethanol, then discard the liquid. Dry the precipitate in a rotary evaporator at 37 °C until it becomes semi-transparent.

[0095] 7) Add 25 μL of sterile water to dissolve the precipitate to obtain crude DNA extracts from wild-type and BGK012-Pdedof20 transgenic NL895 leaves.

[0096] 8) Add about 1 μl of RNase to the crude DNA extract to remove RNA, and the reaction conditions are at 37 °C for 1 h.

[0097] 9) Store the DNA samples in a -20 °C refrigerator for later use.

[0098] (2) PCR amplification and knockout identification of positive plants

[0099] By introducing the dof20-Cas9 knockout plant vector into NL895, multiple dof20 positive knockout transgenic plants were obtained. Using DOF20-specific amplification primers, PCR amplification was carried out with the DNA of WT, IF-389, and IF-390 as templates. After PCR amplification, the amplified stock solution was sent to Shanghai Personal Biotechnology Co., Ltd. for NGS high-throughput sequencing. The results proved that in the IF-389 and IF-390 lines of dof20 transgenic plants, both the DOF20a and DOF20b genes were knocked out. The dof20 transgenic plants were named IF-389 and IF-390 respectively.

[0100] Screening was carried out by amplifying using the vector PdeDOF20a / b-F and the gene primer PdeDOF20a / b-R primer. The designed specific primers had the following sequences:

[0101] DOF20a / b detection-F: 5’-TGCGAGAGGGCTTTGTTTTG-3’ (SEQ ID NO.9);

[0102] DOF20a / b detection-R: 5’-GCAAAAGTACCTTGGCTGAG-3’ (SEQ ID NO.10);

[0103] The PCR reaction system was the same as in Table 6, and 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 the amplified products were detected by 1% agarose gel electrophoresis.

[0104] And the amplified products were sequenced, and the results were as Figure 1 shown. The results showed that efficient editing occurred in the knockout strains.

[0105] Table 6. System of PCR reaction

[0106]

[0107] Example 4. Phenotypic analysis of NL895 dof20 knockout plants

[0108] The appropriate-age tissue culture seedlings were transplanted into soil culture pots and grown in a greenhouse at 25°C under long-day conditions (16 hours of light / 8 hours of darkness, light intensity 10000 lux) for four months ( Figure 2 in A). Parameters such as plant height, stem diameter, internode number, and internode length of WT and dof20 transgenic poplars were measured and statistically analyzed.

[0109] The results were as Figure 2 shown in B - E. Compared with WT plants, the plant height and stem diameter of dof20 plants were significantly increased. Among them, the plant height increased by 76.64%; the internode number increased by 9; the stem diameter of the 16th internode increased by 18.97% and the internode length increased by 29.03%.

[0110] Example 5. Phenotypic analysis of secondary stem development of dof20 knockout transgenic plants

[0111] In this study, stem transverse sections and staining observations were also carried out on NL895 dof20 knockout plants and wild types, and the results were as Figure 3 and 4As shown in the figure. Toluidine blue staining was performed on stem tissue sections, and it was found that the number of layers of stem xylem in the knockout plants was significantly higher than that in the WT. Among them, the 12th internode was thickened by 11.81%, the 14th internode was thickened by 14.81%, and the 16th internode was thickened by 10.96%. Ultra-thin sections were made of the stem tissue material, and the secondary wall thickness of fibers in the transgenic lines and the WT was observed by scanning electron microscopy. It was found that the secondary wall thickness of xylem fibers in the dof20 transgenic lines was significantly increased compared with the WT, with an increase of 14.78%.

[0112] The review shows that knocking out the DOF20 gene line promotes the secondary development of the stem of NL895 and enhances its wood yield.

[0113] The above-described embodiments are only preferred embodiments cited to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all 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 DOF20 gene of Populus deltoides cv. 'Nanlin895' in increasing wood yield, characterized in that: The DOF20 genes are DOF20a and DOF20b, and the nucleotide sequence of DOF20a is shown in SEQ ID NO.1; the nucleotide sequence of DOF20b is shown in SEQ ID NO.

2.

2. Use of knocking out the DOF20 gene of Populus deltoides cv. 'Nanlin895' according to claim 1 in increasing wood production, characterized in that: The DOF20 gene of Populus deltoides cv. 'Nanlin895' was knocked out using the CRISPR / Cas9 editing technology.

3. Use of knocking out the DOF20 gene of Populus deltoides cv. 'Nanlin895' according to claim 1 in increasing wood yield, characterized in that: The target sequences of the CRISPR / Cas9 editing technology are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. Use of the knockout of the DOF20 gene in Populus deltoides cv. 'Nanlin895' according to claim 1 in increasing wood production, characterized in that: The increase in wood production is achieved by increasing the number of xylem layers and the thickness of the fiber secondary wall.

5. A method for increasing wood production, characterized in that: It includes the following steps: transforming the CRISPR / Cas9 gene editing vector of the DOF20 gene of Populus deltoides cv. 'Nanlin895' into Populus deltoides cv. 'Nanlin895' to obtain transgenic plants with gene editing mutations of the DOF20 gene.

6. The method according to claim 5, characterized in that: The method for transforming Populus deltoides cv. 'Nanlin895' is mediated by Agrobacterium.

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

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