A PagSUPb gene for optimizing poplar traits and its application

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a PagSUPb gene for optimizing poplar traits and its application. The amino acid sequence encoded by the PagSUPb gene of the present invention is shown in SEQ ID No. 1. It has been verified by the examples that knocking out the PagSUPb gene in poplars can significantly increase the total biomass and lignin content of poplar plants, which is suitable for cultivating improved forest varieties and ensuring fast growth and high yield of forests, and is of great significance for meeting the needs of different industrial materials.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a PagSUPb gene for optimizing poplar traits and an application thereof. Background Art

[0002] Poplar is one of the important fast-growing and high-yield timber species in my country. Different timbers have different requirements for lignin content. For example, pulpwood requires wood with low lignin content, high cellulose content and long fiber length, while building materials, decorative materials and large-diameter materials require increased lignin content to improve the mechanical properties of wood. How to improve the quality of wood, improve the quality of artificial timber forest resources and increase wood yield according to the needs of different timbers is an urgent problem to be solved in forest breeding.

[0003] SUP (SUPERMAN) genes play a vital role in regulating plant growth, development and response to stress. Studying the role of SUP genes in poplar development may help to achieve engineering regulation of wood properties in order to cultivate improved tree varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a PagSUPb gene for optimizing poplar traits and its application, so as to effectively increase the total biomass of poplars and the lignin content in poplars.

[0005] The present invention provides a PagSUPb gene for optimizing poplar traits. The amino acid sequence encoded by the PagSUPb gene is shown as SEQ ID No.1.

[0006] Preferably, the nucleotide sequence of the PagSUPb gene is shown as SEQ ID No.2.

[0007] The present invention also provides a gRNA for silencing the PagSUPb gene described in the above technical solution, and the nucleotide sequence of the gRNA is shown in SEQ ID No.3.

[0008] The present invention also provides a recombinant vector for optimizing poplar traits, wherein the recombinant vector comprises a basic vector and a gRNA inserted into the basic vector; the nucleotide sequence of the gRNA is shown in SEQ ID No.3.

[0009] The present invention also provides a method for constructing the recombinant vector described in the above technical solution, comprising the following steps:

[0010] Annealing the annealing primer used to synthesize gRNA to obtain an annealing product;

[0011] Inserting the annealing product into a basic vector to obtain the recombinant vector;

[0012] The annealing primer consists of PagSUPb-T2-F and PagSUPb-T2-R; the nucleotide sequence of PagSUPb-T2-F is shown in SEQ ID No.4, and the nucleotide sequence of PagSUPb-T2-R is shown in SEQ ID No.5.

[0013] Preferably, the basic vector is pYLCRISPR / Cas9-DH; the annealing temperature is 90°C and the time is 30s;

[0014] The annealing system includes: pYLgRNA-AtU3d / U3b 1 μL, BsaI 0.5 μL, NEBrCutsmartbuffer 1 μL, target linker 0.5 μL, T4 DNAligase 0.1 μL, T4 DNAligase buffer 0.5 μL, and ddH2O 6.4 μL.

[0015] The present invention also provides a recombinant engineering bacterium for optimizing poplar traits, wherein the recombinant engineering bacterium comprises the recombinant vector and the basic strain described in the above technical solution.

[0016] The present invention also provides a method for optimizing poplar traits, comprising: introducing the recombinant engineering bacteria described in the above technical solution into a poplar receptor and then culturing it.

[0017] Preferably, the recombinant engineered bacteria are used to infect a poplar receptor for 10 to 20 minutes and then cultured in the dark; the culture temperature is 22 to 26° C. and the culture time is 2 to 3 days; and the poplar receptor includes poplar callus.

[0018] The present invention also provides the use of the PagSUPb gene or the gRNA described in the above technical solution or the recombinant vector described in the above technical solution or the recombinant engineered bacteria described in the above technical solution in optimizing poplar traits.

[0019] Beneficial Effects

[0020] The present invention provides a PagSUPb gene for optimizing poplar traits, and the amino acid sequence encoded by the PagSUPb gene is shown in SEQ ID No. 1. It is verified by the examples that knocking out the PagSUPb gene in poplars can significantly increase the total biomass and lignin content of poplar plants, which is suitable for cultivating improved forest varieties and ensuring fast growth and high yield of forest trees, and is of great significance for meeting the needs of different industrial materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 Schematic diagram of pagsupb mutant; A is a schematic diagram of target site design of pagsupb mutant; B is a schematic diagram of expression vector structure;

[0023] Figure 2 The results of identifying the target sites of the pagsupb mutant plants, wherein the letter A represents the sequence of the PagSUPb protein in the plants of Populus alba, the parent of Populus alba 84K, and the letter G represents the sequence of the PagSUPb protein in the plants of Populus glandulosa, the parent of Populus glandulosa 84K;

[0024] Figure 3 This is the poplar phenotype of the pagsupb mutant;

[0025] Figure 4 This is the stem phenotype of the pagsupb mutant plant;

[0026] Figure 5 This is a comparison of phloroglucinol staining of the 11th internode of the soil-cultured poplar seedlings grown for 2 months in Example 3; wherein, the scale bar in A is 600 μm, pf represents bast fiber, Xy represents xylem, and Pi represents pith; B is the measurement and analysis result of lignin, and ** represents P<0.01. DETAILED DESCRIPTION

[0027] The present invention provides a gene PagSUPb for optimizing poplar traits. The amino acid sequence of a protein encoded by the PagSUPb gene is shown in SEQ ID No. 1, specifically: MVKKYSSTVKDKWEKNN AIFKGEHSSVCSWPQRNYSCSFCKRQFISAQALGGHMNVHRRDRAKLRQLPSWFFKCPKYPTSNPNPDHLLSSSSKFLPYPDDHTHDHSPYLSSFSSPCCREKKSIVECQEGKDLTKKKSNAGAVFGVGELKKNFAQECDQLEVLRRSEIINLDMEMGCEDPKEVLDLELRLGL.

[0028] In the present invention, the nucleotide sequence of the PagSUPb gene is preferably as shown in SEQ ID No. 2, specifically: 5'-ATGGTCAAGAAATATAGCAGCACGGTTAAAGACAAATGGGAGA -3'.

[0029] The present invention also provides a gRNA for silencing the PagSUPb gene described in the above technical solution, and the nucleotide sequence of the gRNA is shown in SEQ ID No. 3, specifically: 5'-GGGGCCATATGAACG TTCAC-3'.

[0030] The present invention also provides a recombinant vector for optimizing poplar traits, the recombinant vector comprising a basic vector and a gRNA inserted into the basic vector;

[0031] The nucleotide sequence of the gRNA is shown in SEQ ID No.3.

[0032] The present invention also provides a method for constructing the recombinant vector described in the above technical solution, comprising the following steps:

[0033] Annealing the annealing primer used to synthesize gRNA to obtain an annealing product;

[0034] Inserting the annealing product into a basic vector to obtain the recombinant vector;

[0035] The annealing primer consists of PagSUPb-T2-F and PagSUPb-T2-R; the nucleotide sequence of PagSUPb-T2-F is shown in SEQ ID No.4, specifically: 5'-gtcAGGGGCCATATGAACG TTCAC-3'; the nucleotide sequence of PagSUPb-T2-R is shown in SEQ ID No.5, specifically: 5'-aaacGTGAACGTTCATATGGCCCC-3'.

[0036] In the present invention, the basic vector is preferably pYLCRISPR / Cas9-DH; the annealing temperature is preferably 90° C., and the annealing time is preferably 30 s;

[0037] The annealing system preferably includes: pYLgRNA-AtU3d / U3b 1 μL, BsaI 0.5 μL, NEBrCutsmartbuffer 1 μL, target linker 0.5 μL, T4 DNAligase 0.1 μL, T4 DNAligase buffer 0.5 μL, and ddH2O 6.4 μL.

[0038] The present invention also provides a recombinant engineering bacterium for optimizing poplar traits, wherein the recombinant engineering bacterium comprises the recombinant vector and the basic strain described in the above technical solution.

[0039] In the present invention, the basic strain is preferably Agrobacterium, more preferably Agrobacterium GV3101.

[0040] The present invention also provides a method for optimizing poplar traits, comprising: introducing the recombinant engineering bacteria described in the above technical solution into a poplar receptor and then culturing it.

[0041] In the present invention, it is preferred to use the recombinant engineered bacteria to infect a poplar receptor and then culture it in the dark. The infection time of the present invention is preferably 10 to 20 minutes, more preferably 15 minutes; the culture temperature is preferably 22 to 26°C, more preferably 25°C, and the culture time is preferably 2 to 3 days; the poplar receptor includes poplar callus; the poplar is preferably silver gland poplar 84K.

[0042] In the present invention, the method of infecting the poplar receptor with the recombinant engineered bacteria is preferably: mixing the recombinant engineered bacteria resuspension with the poplar callus; the method of preparing the recombinant engineered bacteria resuspension is preferably: performing colony PCR on the recombinant engineered bacteria to screen for positive single colonies, and expanding and culturing the positive single colonies to OD 600=0.4-0.6, then enrich the bacterial solution, centrifuge and resuspend to OD 600 =0.4~0.6. The enlarged culture is preferably a first culture and a second culture carried out in sequence; the culture medium of the first culture is preferably an LB liquid culture medium containing 50mg / L Kanamycin, 10mg / L Gentamicin and 17mg / L Rifampicin antibiotics, the conditions of the first culture are preferably 28°C, 200rpm, and the culture time is preferably 12~14h; the culture medium of the second culture is preferably an LB liquid culture medium containing 50mg / L Kanamycin, 10mg / L Gentamicin and 17mg / L Rifampicin antibiotics; the second culture is preferably a transfer of the bacterial liquid obtained from the first culture and the culture medium of the second culture at a volume ratio of 1:500, the conditions of the second culture are preferably 28°C, 200rpm, and the culture time is preferably 13h.

[0043] The centrifugation is preferably performed at 3500 rpm for 15 min; the resuspension is preferably performed using a sterile resuspension solution, and the resuspension solution is preferably a 100 μmol / L acetosyringone (AS) solution.

[0044] In the present invention, the culture is preferably carried out using a co-culture medium; the co-culture medium is preferably a 1 / 2MS medium containing 100 μM AS.

[0045] The present invention also provides the use of the PagSUPb gene or the gRNA or the recombinant vector or the recombinant engineering bacteria described in the above technical solution in optimizing poplar traits.

[0046] In the present invention, the optimization of poplar traits preferably includes one or more of increasing the ground diameter of poplars, increasing the number of internodes of poplars, increasing the stem length of poplars, increasing the stem diameter of poplars, increasing the total biomass of poplars and increasing the lignin content in poplars; more preferably, increasing the total biomass of poplars and increasing the lignin content in poplars.

[0047] To further illustrate the present invention, the PagSUPb gene for optimizing poplar traits and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] 1. Primer Design

[0050] The target site was designed based on the analysis of the CRISPR-GE (http: / / skl.scau.edu.cn / ) online website. A 20 bp nucleotide sequence (PAM) was selected from the coding region of the PagSUP gene as the target sequence, specifically: 5'-GGGGCCATATGAACGTTCAC-3' (SEQ ID No. 3). The primer sets PagSUPb-T1-F and PagSUPb-T1-R for silencing the expression of the PagSUPb gene were designed, and their sequences are as follows:

[0051] PagSUPb-T2-F: 5'-gtcAGGGGCCATATGAACGTTCAC-3' (SEQ ID No. 4);

[0052] PagSUPb-T2-R: 5'-aaacGTGAACGTTCATATGGCCCC-3' (SEQ ID No. 5).

[0053] 2. Preparation of target linker

[0054] The synthesized primers PagSUPb-T2-F and PagSUPb-T2-R were dissolved in ddH2O to 10 μM, 1 μL of each was added to 8 μL ddH2O, and diluted to 1 μM. The mixture was reacted at 90°C for 30 s, and then cooled to room temperature to complete annealing to obtain the target linker. The enzyme digestion and ligation reaction was performed, and the system was shown in Table 1. The reaction conditions were 37°C for 5 min; 20°C for 5 min, and 5 cycles.

[0055] Table 1 Enzyme ligation reaction system

[0056]

[0057] 3. Amplification of gRNA expression cassette

[0058] The first round of PCR amplification system is shown in Table 2, and the reaction conditions are 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 68°C for 10 s, and 30 cycles.

[0059] Table 2 The first round of PCR amplification system

[0060]

[0061] Note: UF and gRNA-R in the table are fixed primers of the Cas9 system, and their sequences are as follows:

[0062] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No. 6);

[0063] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No. 7).

[0064] After the reaction was completed, 4 μL of the product was taken for electrophoresis and the remaining product was subjected to a second round of PCR reaction.

[0065] Second round of PCR amplification:

[0066] Take 1 μL of the first round PCR reaction product, dilute it 10 times with ddH2O, and react at 95℃ for 10 seconds. Then take 1 μL as the amplification template for the second round of PCR. The second round PCR amplification system is shown in Table 3. The reaction conditions are: 95℃ for 3 minutes; 95℃ for 15 seconds, 55℃ for 15 seconds, 68℃ for 10 seconds, and 30 cycles.

[0067] Table 3 Second round PCR amplification system

[0068]

[0069] Note: B1' and BL in the table are fixed primers of the Cas9 system, and their sequences are as follows:

[0070] B1': 5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3' (SEQ IDNo. ​​8);

[0071] BL: 5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3' (SEQ ID No. 9).

[0072] 4. PCR product purification and expression vector ligation:

[0073] The first round PCR reaction products and the second round PCR reaction products were mixed in equal amounts, purified using a PCR product purification kit, and then connected to the pYLCRISPR / Cas9-DH final vector. The reaction system is shown in Table 4; the reaction conditions are: 37°C for 2 min; 10°C for 3 min, 20°C for 5 min, 15 cycles; 37°C for 2 min.

[0074] Table 4 Vector connection system

[0075]

[0076] Take 5 μL of the mixture after the above reaction to transform E. coli and spread it on the screening medium (LB solid medium containing 50 mg / L Kanamycin antibiotics). After culturing at 37°C for 12 hours, pick a single clone from the screening culture plate for PCR detection and sequencing verification. Confirm that the gRNA expression cassette is successfully connected to the pYLCRISPR / Cas9-DH expression vector, and obtain the following Figure 1 The recombinant vector shown.

[0077] Example 2

[0078] The recombinant vector constructed in Example 1 was transferred into Agrobacterium GV3101, and then transferred into Populus argentea 84K by Agrobacterium-mediated method. The specific steps are as follows:

[0079] Bacteria preparation

[0080] Pick a single clone on the plate containing the recombinant vector for colony PCR, and select the positive single colony by electrophoresis. Inoculate it into 1mL of liquid LB medium (LB solid medium containing 50mg / L Kanamycin antibiotics), and culture it at 28℃ constant temperature shaker, 200rpm for 12-14h. Take 100μL of the cultured bacterial solution and transfer it to 50mL of LB liquid medium with resistance, and continue to culture it at 28℃, 200rpm for 13h until OD 600 =0.4~0.6, to ensure that the bacterial solution has good activity. The bacterial solution was enriched, centrifuged at 3500rpm for 15min, and the bacteria were resuspended in a sterile resuspension solution (100μmol / L acetosyringone). The OD of the resuspended bacterial solution was 600 To 0.4-0.6, used for genetic transformation.

[0081] Callus preparation

[0082] Silver gland poplar 84K was used as the experimental material. Leaves of 84K tissue culture seedlings with good growth status were taken respectively. In the clean bench, sterile disinfection instruments were used to scratch the leaves along the direction perpendicular to the leaf veins. The back of the leaves were spread flat on callus induction medium (1 / 2MS medium containing 2 mg / L2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid), 0.1 mg / L Kineti (KT, kinetin) and 0.2 mg / L NAA (naphthaleneacetic acid)), and cultured in the dark at 25°C for 15 to 25 days.

[0083] Infection and identification

[0084] In the clean bench, use tweezers to cut the cultured callus into small pieces, transfer them to the resuspended bacterial solution and infect for 15 minutes, place them on sterile filter paper and blow dry, transfer them to the co-culture medium (1 / 2MS medium containing 100μM AS), and co-culture them at 25℃ in the dark for 2-3 days.

[0085] After the co-cultivation, the leaves were transferred to a differentiation induction medium (containing 3 mg / L hygromycin B, 200 mg / L timentin, 0.5 mg / L 6-BA (6-benzyl aminopurine) and 0.05 mg / L NAA in 1 / 2MS medium) to induce and screen resistant adventitious buds. When the leaves differentiated adventitious buds and the length reached about 2 cm, the adventitious buds were transferred to a rooting medium (containing 3 mg / L hygromycin B, 200 mg / L timentin, 0.05 mg / LIBA (indolebutyric acid) and 0.02 mg / L NAA in 1 / 2MS medium) for rooting culture. The leaves of the rooted plants were selected to extract DNA for PCR verification and gene sequencing. Finally, KO#63pagsupb mutant plants and KO#76pagsupb mutant plants were obtained, and the editing results of the PagSUPb gene target site were as follows: Figure 2 Silver gland poplar 84K is a hybrid poplar. Figure 2 The A and G in the sequence represent its two parents, Populus alba and Populus glandulosa, respectively. In the genome, PagSUPb has different sequences at the allele loci of the two parents, Populus alba (A) and Populus glandulosa (G).

[0086] Depend on Figure 2It can be seen that the amino acid sequence of PagSUPb protein in the untransformed parent Populus alba plant of Populus alba 84K is: MVKKYPQGSSTVKDKWEKNNAIFKGEHSSVFSWPQR NYSCSFCKRQFISAQALGGHMNVHRRDRAKLRQLPSWFFKCPKYPTSNPNPDHLLSSSSKLLPYPDYHTHDHSPYLSSFSSPCCREKKSIVECHEVKDLTKKKSNAGAVFGVGELKKNFAQECDQLEVLRRSEIINLDMEMGCEDPKEVLDLELRLGL (SEQ ID No.10); the amino acid sequence in the parent Populus glandulosa plant is: MVKK YSSTVKDKWEKNNAIFKGEHSSVCSWPQRNYSCSFCKRQFISAQALGGHMNVHRRDRAKLRQLPSWFFKCPKYPTSNPNPDHLLSSSSKFLPYPDDHTHDHSPYLSFSSPCCREKKSIVECQEGKDLTKKKSNAGAVFGVGELKKNFAQECDQLEVLRRSEIINLDMEMGCEDPKEVLDLELRLGL(SEQ ID No. 1);

[0087] The amino acid sequence of the PagSUPb protein in the KO#63pagsupb mutant parent Populus alba plant is: MVKKYPQGSSTVKDKWEKNNAIFKGEHSSVFSWPQRNYSCSFCKR QFISAQALGGHMNVSQER (SEQ ID No. 11); the amino acid sequence in the parent Populus glandulosa plant is: MVKKYSSTVKDKWEKNNAIFKGEHSSVCSWPQRNYSCSFCKRQFISAQER (SEQ ID No. 12);

[0088] The amino acid sequence of PagSUPb protein in the KO#76pagsupb mutant parent Populus alba plant is: MVKKYPQGSSTVKDKWEKNNAIFKGEHSSVFSWPQRNYSCSFCKR QFISAQALGGHMNVAQER (SEQ ID No. 13); the amino acid sequence in the parent Populus glandulosa plant is: MVKKYSSTVKDKKWEKNNCKRQFISAQALGGHMNVTG IEKGEHSSVCSWPQRNYSCSFTGEIEPS (SEQ ID No. 14).

[0089] Example 3

[0090] The KO#63pagsupb mutant plants (denoted as supb63) and KO#76pagsupb mutant plants (denoted as supb76) obtained in Example 2 and the untransformed Populus argentea 84K plants (denoted as CK) were cultured for two months under a 16 h light / 8 h dark photoperiod, a temperature of 22-26°C, and a humidity of 40-60%, and their properties were observed and measured. Then, their lignin content was detected according to the method described in the literature KNAT2 / 6b, a class I KNOX gene, impedes xylem differentiation by regulating NACdomain transcription factors inpoplar (Zhao Y, Xueqin S, Zhou H, et al. New Phytologist, 2019, 225(4). DOI: 10.1111 / nph.16036.).

[0091] The results are as follows Figure 3 to Figure 5 As shown, Figure 5 It is a comparison of phloroglucinol staining of the 20th internode of the soil-cultured seedlings of non-transgenic poplar CK and transgenic poplars KO#63 and KO#76 of the present invention grown for 2 months; the scale bar in A is 600 μm, B is the measurement and analysis results of lignin, the t-test P value is represented by an asterisk: t-test P value is represented by an asterisk:, "**" indicates P < 0.01, pf indicates bast fiber, Xy indicates xylem, and Pi indicates pith.

[0092] Depend on Figure 3 to Figure 5 It can be seen that compared with the control plants, the two strains of pagsupb mutant poplar (KO#63 and KO#76) were significantly taller. The ground diameter of the mutant plants was larger than that of the control plants, and the number of internodes was also significantly increased ( Figure 3 The stems of pagsupb mutant poplars became longer and thicker than those of the control ( Figure 4). The lignin content was significantly higher than that of the control ( Figure 5 ).

[0093] It can be seen from the above examples that the expression of the PagSUPb gene silenced in the present invention significantly increased the height and thickness of the poplar (CK) plants compared to the non-transgenic plants, and the lignin content was significantly increased, indicating that the PagSUPb gene can regulate the development of poplar wood and change the lignin content of poplar, which has application significance for cultivating tree varieties suitable for different industrial timber needs.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A PagSUPb gene for optimizing poplar traits, wherein the amino acid sequence encoded by the PagSUPb gene is shown in SEQ ID No.

1.

2. The PagSUPb gene according to claim 1, characterized in that The nucleotide sequence of the PagSUPb gene is shown in SEQ ID No.

2.

3. A gRNA for silencing the PagSUPb gene according to claim 1 or 2, wherein the nucleotide sequence of the gRNA is shown in SEQ ID No.

3.

4. A recombinant vector for optimizing poplar traits, characterized in that: The recombinant vector is obtained by inserting gRNA into the basic vector; The nucleotide sequence of the gRNA is shown in SEQ ID No.

3.

5. The method for constructing the recombinant vector according to claim 4, characterized in that: The following steps are involved: Annealing the annealing primer used to synthesize gRNA to obtain an annealing product; Inserting the annealing product into a basic vector to obtain the recombinant vector; The annealing primer consists of PagSUPb-T2-F and PagSUPb-T2-R; the nucleotide sequence of PagSUPb-T2-F is shown in SEQ ID No.4, and the nucleotide sequence of PagSUPb-T2-R is shown in SEQ ID No.

5.

6. The construction method according to claim 5, characterized in that: The basic vector is pYLCRISPR / Cas9-DH; the annealing temperature is 90°C and the time is 30s; The annealing system includes: pYLgRNA-AtU3d / U3b 1 μL, BsaI 0.5 μL, NEB rCutsmart buffer 1 μL, target linker 0.5 μL, T4 DNA ligase 0.1 μL, T4 DNA ligase buffer 0.5 μL, and ddH2O 6.4 μL.

7. A recombinant engineered bacterium for optimizing poplar traits, characterized in that: The recombinant engineered bacteria are obtained by transferring the recombinant vector described in claim 4 into a basic strain.

8. A method for optimizing poplar traits, characterized in that: include: The recombinant engineered bacteria according to claim 7 are introduced into a poplar recipient and then cultured.

9. The method according to claim 8, characterized in that Infecting a poplar receptor with the recombinant engineering bacteria for 10 to 20 minutes and then culturing in the dark; The culture temperature is 22-26°C and the culture time is 2-3 days; The poplar recipient includes poplar callus.

10. Use of the PagSUPb gene according to claim 1 or 2, the gRNA according to claim 3, the recombinant vector according to claim 4, or the recombinant engineered bacteria according to claim 7 in increasing the total biomass of poplar plants and / or increasing the lignin content in poplars.

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