Application of PagSUPa Gene and Its Encoded Protein in Optimizing Poplar Traits

Abstract

The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of the PagSUPa gene and its encoded protein in optimizing poplar traits. The optimized poplar traits include one or more of increasing the number of internodes of poplar, increasing the total biomass of poplar, and increasing the lignin content in poplar; the amino acid sequence of the encoded protein of the PagSUPa gene is shown in SEQ ID No.1. Verified by examples, knocking out the PagSUPa gene in Populus alba×Populus glandulosa 84K can effectively increase the ground diameter of the plant, increase the number of its internodes, and ultimately significantly increase the total biomass and lignin content of the plant, which is suitable for popularization and application in cultivating and improving forest tree varieties and ensuring the fast growth and high yield of forest trees.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the PagSUPa gene and the protein encoded thereby in optimizing the traits of poplar trees. Background Art

[0002] Poplar is a tree species widely planted in the world and is also one of the important fast-growing and high-yield timber tree species in China. Vigorously cultivating fast-growing and high-yield poplar plantations can not only relieve the pressure on natural forests, increase the biomass per unit area, but also slow down the greenhouse effect, promoting the mutual promotion and coordinated development of afforestation and environmental protection. Studying and revealing the changes in poplar in terms of biomass increase is of great significance for further tapping the production potential of poplar plantations and ensuring the fast growth and high yield of forest trees.

[0003] The SUP (SUPERMAN) gene is a key gene in Arabidopsis thaliana. It plays an important role in the flowering and reproductive development of Arabidopsis thaliana. The SUP gene encodes a transcription factor belonging to the C2H2 zinc finger protein family and plays a crucial role in regulating plant growth, development, and response to stress. Overexpression of SUP makes Arabidopsis thaliana plants dwarf, but there is no significant difference in cell size, indicating that SUP affects cell division; it may be involved in the regulation of cell division and differentiation, inhibiting plant growth and thus affecting the biomass of plants. However, there is currently little research on the role of the SUP gene in poplar development. Summary of the Invention

[0004] The purpose of the present invention is to provide a new application of the PagSUPa gene in poplar, develop a new use of the PagSUPa gene, and effectively increase the number of internodes, total biomass, and lignin content in poplar.

[0005] The present invention provides the application of the PagSUPa gene in optimizing the traits of poplar, wherein the optimization of poplar traits includes one or more of increasing the number of internodes, increasing the total biomass, and increasing the lignin content in poplar; the amino acid sequence of the protein encoded by the PagSUPa gene is as shown in SEQ ID No.1.

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

[0007] Preferably, the optimization of poplar traits also includes one or more of increasing the ground diameter of poplar, increasing the plant height of poplar, increasing the leaf area of poplar, increasing the leaf length of poplar, and increasing the leaf width of poplar;

[0008] The method for optimizing the traits of poplar includes: knocking out the PagSUPa gene in poplar.

[0009] The present invention also provides a recombinant vector for knocking out the PagSUPa gene in poplar, and the recombinant vector comprises a basic vector and a gRNA inserted into the basic vector; the nucleotide sequence of the gRNA is as shown in SEQ ID No.3.

[0010] The present invention also provides a method for constructing the recombinant vector described in the above technical solution, comprising the following steps:

[0011] Anneal the annealing primers for synthesizing the gRNA to obtain an annealing product;

[0012] Insert the annealing product into the basic vector to obtain the recombinant vector;

[0013] The annealing primers consist of PagSUPa-T1-F and PagSUPa-T1-R; the nucleotide sequence of PagSUPa-T1-F is as shown in SEQ ID No.4, and the nucleotide sequence of PagSUPa-T1-R is as shown in SEQ ID No.5.

[0014] Preferably, the basic vector is pYLCRISPR / Cas9-DH; the annealing temperature is 90 °C and the time is 30 s;

[0015] The annealing system comprises: 1 μL of pYLgRNA-AtU3d / U3b, 0.5 μL of BsaI, 1 μL of NEBrCutsmart buffer, 0.5 μL of the target site linker, 0.1 μL of T4 DNA ligase, 0.5 μL of T4 DNA ligase buffer, and 6.4 μL of ddH2O.

[0016] The present invention also provides a recombinant engineering bacterium for optimizing the traits of poplar, and the recombinant engineering bacterium comprises the recombinant vector and a basic strain described in the above technical solution.

[0017] Preferably, the basic strain is Agrobacterium.

[0018] The present invention also provides a method for optimizing the traits of poplar, which is characterized by comprising: culturing after introducing the recombinant engineering bacterium described in the above technical solution into a poplar receptor.

[0019] Preferably, the introducing method comprises: infecting the poplar receptor with the recombinant engineering bacterium for 10-20 min and then culturing under dark conditions, the culturing temperature is 22-26 °C, and the time is 2-3 d;

[0020] The poplar receptor comprises poplar callus.

[0021] Beneficial effects

[0022] The present invention provides the application of the PagSUPa gene and its encoded protein in optimizing the traits of poplar. The amino acid sequence of the encoded protein of the PagSUPa gene is shown as SEQ ID No.1. Verified by examples, knocking out the PagSUPa gene in poplar can effectively increase the number of internodes of the plant, and ultimately significantly improve the total biomass and lignin content of the plant, which is suitable for popularization and application in cultivating and improving forest tree varieties to ensure the rapid growth and high yield of forest trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0024] Figure 1 It is a schematic diagram of the recombinant vector; wherein, A is a schematic diagram of the design of the target site of the pagsupa mutant; B is a schematic diagram of the structure of the recombinant vector;

[0025] Figure 2 It is the editing result of the target site of the pagsupa mutant plant gene; wherein, the letter A represents the sequence of the PagSUPa protein in the plant of the Populus alba parent of Populus glandulosa 84K, and the letter G represents the sequence of the PagSUPa protein in the plant of the Populus glandulosa parent of Populus glandulosa 84K;

[0026] Figure 3 It is a phenotypic diagram of the poplar plant in Example 3;

[0027] Figure 4 It is a phenotypic diagram of the poplar leaf in Example 3;

[0028] Figure 5 It is a phenotypic diagram of the poplar stem in Example 3;

[0029] Figure 6 It is a comparison diagram of phloroglucinol staining of the 20th internode after 2 months of growth of the soil-cultured seedlings of non-transgenic poplar CK, transgenic poplar KO#48 and transgenic poplar KO#70; wherein, the scale bar is 600μm. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention provides the application of the PagSUPa gene in optimizing the traits of poplar, and the optimization of poplar traits includes increasing the number of internodes of poplar, increasing the total biomass of poplar and increasing the lignin content in poplar;

[0031] The amino acid sequence of the encoded protein of the PagSUPa gene is shown in SEQ ID No.1, specifically: MCIAMDRNYLQKSTTVKDKWEWNDVIFEGEHSTGISWPKRNYTCSFCKREFSSAQALGGHMNVHRRDRARLKQLPSWFFDCPKPTSMSNPKPLPYPSSTFSPYPDHTHDHSLLSPFLASFSSPSYPEKKSIVECPRSINSTRKIRHMRAVVGVGELKKNFVQDGDQLKVSRTSGIISLDLEMRCEDPKEVLDLELRLGCF.

[0032] In the present invention, the nucleotide sequence of the PagSUPa gene is preferably shown in SEQ ID No.2, specifically: 5'-ATGTGTATAGCCATGGACAGGAACTATCTGCAGAAAAGTACCAC TGTTAAAGACAAATGGGAGTGGAACGACGTGATCTTTGAAGGAGAACATTCAACTGGTATTTCATGGCCGAAAAGAAATTACACTTGCAGCTTTTGCAAGAGAGAATTCAGCTCTGCTCAAGCACTTGGGGGACATATGAATGTCCACAGGAGAGATAGAGCCAGGCTAAAACAGCTGCCTTCTTGGTTTTTTGATTGTCCAAAACCTACTTCCATGTCTAACCCTAAACCTTTACCTTATCCCTCATCCACGTTCTCTCCGTACCCTGATCACACTCATGATCACTCCTTGCTCTCTCCATTCCTCGCTTCTTTCTCTTCACCTTCTTACCCAGAAAAGAAATCCATAGTTGAATGTCCTCGGAGTATAAATTCAACAAGGAAGATAAGACATATGAGAGCTGTGGTTGGTGTTGGAGAATTGAAGAAGAATTTCGTACAAGATGGTGATCAGCTTAAAGTTTCGAGGACAAGTGGGATTATTAGCTTGGACTTGGAAATGAGATGTGAAGATCCGAAGGAGGTTTTGGATTTGGAGCTTCGACTTGGCTGTTTTTAG-3';

[0033] In the present invention, the optimization of poplar traits preferably further includes increasing the ground diameter of poplar, increasing the plant height of poplar, increasing the leaf area of poplar, increasing the leaf length of poplar, and increasing the leaf width of poplar, or one or more of them; the method for optimizing poplar traits preferably includes knocking out the PagSUPa gene in poplar.

[0034] The present invention also provides a recombinant vector for knocking out the PagSUPa gene in poplar, and the recombinant vector includes a basic vector and a gRNA inserted into the basic vector;

[0035] The nucleotide sequence of the gRNA is as shown in SEQ ID No.3, specifically 5'-AGCTCTGCTC AAGCACTTGG-3'. In the present invention, the basic vector is preferably the pYLCRISPR / Cas9-DH expression vector.

[0036] The present invention also provides a method for constructing the recombinant vector described in the above technical solution, including the following steps:

[0037] Anneal the annealing primers for synthesizing the gRNA to obtain an annealing product;

[0038] Insert the annealing product into the basic vector to obtain the recombinant vector;

[0039] The annealing primers are composed of PagSUPa-T1-F and PagSUPa-T1-R; the nucleotide sequence of PagSUPa-T1-F is as shown in SEQ ID No.4, specifically: 5'-gtcAGCTCTGCTCAAGCAC TTGG-3'; the nucleotide sequence of PagSUPa-T1-R is as shown in SEQ ID No.5, specifically: 5'-aaacCCAAGTGCTTGAGCAGAGC-3'.

[0040] In the present invention, the basic vector is preferably the pYLCRISPR / Cas9-DH expression vector; the annealing temperature is preferably 90°C; the annealing time is preferably 30 s; the annealing system preferably includes: 1 μL of pYLgRNA-AtU3d / U3b, 0.5 μL of BsaI, 1 μL of NEB rCutsmart buffer, 0.5 μL of the target linker, 0.1 μL of T4 DNA ligase, 0.5 μL of T4DNA ligase buffer, and 6.4 μL of ddH2O.

[0041] The present invention also provides a recombinant engineering bacterium for optimizing poplar traits, and the recombinant engineering bacterium includes the recombinant vector and a basic strain described in the above technical solution. In the present invention, the basic strain is preferably Agrobacterium, more preferably Agrobacterium GV3101.

[0042] The present invention also provides a method for optimizing the traits of poplar, including:

[0043] After introducing the recombinant engineering bacteria described in the above technical solution into the poplar receptor, culture it.

[0044] The recombinant vector constructed by the present invention can effectively edit the PagSUPa gene. Introducing the recombinant engineering bacteria containing the recombinant vector into poplar can effectively realize the optimization of poplar traits.

[0045] In the present invention, the preferred mode of introduction includes: infecting the poplar receptor with the recombinant engineering bacteria for 10 - 20 min, more preferably 15 min; then culturing under dark conditions; the preferred temperature for culturing is 22 - 26 °C, more preferably 25 °C; the preferred time for culturing is 2 - 3 d; the poplar receptor preferably includes poplar callus, and the poplar is preferably Populus alba×Populus glandulosa 84K.

[0046] In the present invention, the preferred mode of infecting the poplar receptor with the recombinant engineering bacteria is: mixing the recombinant engineering bacteria resuspension with the poplar callus; the preferred preparation method of the recombinant engineering bacteria resuspension is: performing colony PCR on the recombinant engineering bacteria, screening to obtain positive single colonies, expanding the culture of the positive single colonies to OD 600 = 0.4 - 0.6, then enriching the bacterial liquid, centrifuging and resuspending to OD 600 = 0.4 - 0.6. The preferred expanded culture is the first culture and the second culture carried out in sequence; the preferred medium for the first culture is LB liquid medium containing 50 mg / L Kanamycin, 10 mg / L Gentamicin and 17 mg / L Rifampicin antibiotics, the preferred conditions for the first culture are 28 °C, 200 rpm, and the preferred culture time is 12 - 14 h; the preferred medium for the second culture is LB liquid medium containing 50 mg / L Kanamycin, 10 mg / L Gentamicin and 17 mg / L Rifampicin antibiotics; the preferred second culture is to transfer the bacterial liquid obtained from the first culture and the medium for the second culture according to a volume ratio of 1:500, and the preferred conditions for the second culture are 28 °C, 200 rpm, and the preferred culture time is 13 h.

[0047] The preferred centrifugation is centrifuging at 3500 rpm for 15 min; the preferred resuspension is resuspending with a sterile resuspension, and the preferred resuspension solution is 100 μmol / L acetosyringone (AS) solution.

[0048] In the present invention, the cultivation is preferably carried out using a co-culture medium; the co-culture medium is preferably a 1 / 2 MS medium containing 100 μM AS. The present invention preferably continues to carry out differentiation induction culture and rooting culture after the cultivation, and after PCR verification, the pagsupa mutant plants are screened out.

[0049] To further illustrate the present invention, the application of the PagSUPa gene provided by the present invention and the protein encoded thereby in optimizing poplar traits will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] 1. Primer design

[0052] According to the analysis and design of the target site on the CRISPR-GE (http: / / skl.scau.edu.cn / ) online website, a 20-bp nucleotide sequence (PAM) was selected as the target sequence in the coding region sequence of the PagSUPa gene, specifically: 5'-AGCTCTGCTCAAGCACTTGG-3' (SEQ ID No. 3). The primer group PagSUPa-T1-F and PagSUPa-T1-R for silencing the expression of the PagSUPa gene was designed, and their sequences are as follows:

[0053] PagSUPa-T1-F: 5'-gtcAGCTCTGCTCAAGCACTTGG-3' (SEQ ID No. 4);

[0054] PagSUPa-T1-R: 5'-aaacCCAAGTGCTTGAGCAGAGC-3' (SEQ ID No. 5).

[0055] 2. Preparation of the target site adapter

[0056] The synthesized primers PagSUPa-T1-F and PagSUPa-T1-R were respectively dissolved in ddH2O to 10 μM, 1 μL of each was taken and added to 8 μL of ddH2O for mixing and diluted to 1 μM. The reaction was carried out at 90 °C for 30 s and then transferred to room temperature for cooling to complete annealing to obtain the target site adapter. The enzyme digestion and ligation reaction was carried out, and the reaction system is shown in Table 1, and its reaction conditions are 37 °C for 5 min; 20 °C for 5 min, for 5 cycles.

[0057] Table 1 Enzyme digestion and ligation reaction system

[0058]

[0059]

[0060] 3. Amplification of gRNA Expression Cassette

[0061] The first-round 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, for 30 cycles.

[0062] Table 2 First-round PCR Amplification System

[0063]

[0064] Note: U-F and gRNA-R in the table are fixed primers of the Cas9 system, and their sequences are specifically:

[0065] U-F: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No.6);

[0066] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No.7).

[0067] After the reaction, take 4 μL of the product for electrophoresis inspection and continue the second-round PCR reaction with the remaining product.

[0068] Second-round PCR amplification:

[0069] Take 1 μL of the first-round PCR reaction product, dilute it 10 times with ddH2O, and react at 95°C for 10 s. Then take 1 μL as the amplification template for the second-round PCR. The second-round PCR amplification system is shown in Table 3, 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, for 30 cycles.

[0070] Table 3 Second-round PCR Amplification System

[0071]

[0072]

[0073] Note: B1’ and BL in the table are fixed primers of the Cas9 system, and their sequences are specifically:

[0074] B1’: 5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3' (SEQ IDNo.8);

[0075] BL: 5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3' (SEQ IDNo.9).

[0076] 4. Purification of PCR products and ligation to expression vectors:

[0077] The products of the first-round PCR reaction and the second-round PCR reaction were mixed in equal amounts, purified using a PCR product purification kit, and then ligated to the pYLCRISPR / Cas9-DH final vector. The reaction system is shown in Table 4; the reaction conditions were: 37 °C for 2 min; 10 °C for 3 min, 20 °C for 5 min, 15 cycles; 37 °C for 2 min.

[0078] Table 4 Vector ligation system

[0079]

[0080] 5 μL of the mixed solution after the above reaction was taken to transform Escherichia coli and spread on a screening medium (LB solid medium containing 50 mg / L Kanamycin antibiotic). After culturing at 37 °C for 12 h, single colonies were picked from the screening plate for PCR detection and sequencing verification. It was confirmed that the gRNA expression cassette was successfully ligated to the pYLCRISPR / Cas9-DH expression vector, and the recombinant vector shown in Figure 1 B was obtained.

[0081] Example 2

[0082] The recombinant vector constructed in Example 1 was transferred into Agrobacterium tumefaciens GV3101, and through the Agrobacterium-mediated method, it was transferred into Populus alba×P. glandulosa 84K. The specific steps are as follows:

[0083] Preparation of bacterial cells

[0084] Single colonies were picked on a plate containing the recombinant vector for colony PCR, and positive single colonies were selected by electrophoresis inspection. They were inoculated into 1 mL of liquid LB medium (containing 50 mg / L Kanamycin, 10 mg / L Gentamicin, and 17 mg / L Rifampicin antibiotics), and cultured in a constant-temperature shaker at 28 °C and 200 rpm for 12 h to 14 h. 100 μL of the cultured bacterial liquid was transferred to 50 mL of LB liquid medium with resistance, and continued to be cultured at 28 °C and 200 rpm for 13 h until the OD 600 reached 0.4 - 0.6 to ensure good activity of the bacterial liquid. The bacterial liquid was concentrated, centrifuged at 3500 rpm for 15 min, and the bacterial cells were resuspended with a sterile resuspension solution (100 μmol / L acetosyringone was added before use). The OD of the resuspended bacterial liquid 600 was adjusted to 0.4 - 0.6 for genetic transformation.

[0085] Preparation of callus

[0086] Using two strains of Populus alba×Populus glandulosa 84K, KO#48 and KO#70, as experimental materials, leaves of 84K tissue culture seedlings in good growth condition were taken respectively. In a laminar flow hood, using sterile disinfection instruments, the leaves were scratched along the direction perpendicular to the veins, and the back of the leaves was laid flat on the callus induction medium. The callus induction medium contained 2 mg / L 2,4-D (2,4-Dichlorophenoxyacetic acid), 0.1 mg / L Kineti (KT, kinetin), and 0.2 mg / L NAA (naphthaleneacetic acid), and was cultured in the dark at 25 °C for 15 - 25 days.

[0087] Infection and identification

[0088] In a laminar flow hood, the cultured callus was divided into small pieces with forceps, transferred to the resuspended bacterial solution for infection for 15 min, and placed on sterile filter paper to dry. Then it was transferred to the co-culture medium (containing 100 μM AS) and co-cultured for 2 - 3 d under dark conditions at 25 °C.

[0089] After the co-culture, it was transferred to the differentiation induction medium (1 / 2 MS 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) to induce and screen for resistant adventitious buds; when adventitious buds differentiated from the leaves and reached about 2 cm in length, the adventitious buds were transferred to the rooting medium (1 / 2 MS medium containing 3 mg / L hygromycin B, 200 mg / L timentin, 0.05 mg / L IBA (indolebutyric acid), and 0.02 mg / L NAA) for rooting culture; DNA was extracted from the leaves of the rooted plants for PCR verification and gene sequencing; finally, KO#48pagsupa mutant plants and KO#70pagsupa mutant plants were obtained, and the editing results of the PagSUPa gene target sites are as Figure 2 shown. Populus alba×Populus glandulosa 84K is a hybrid poplar, Figure 2 in which A and G are used to represent its two parents, Populus alba and Populus glandulosa respectively. Genomically, the allelic loci of PagSUPa in the two parents, Populus alba (A) and Populus glandulosa (G), are different sequences.

[0090] By Figure 2It is known that the amino acid sequence of PagSUPa protein in the untransformed Populus alba (A) parent plant of Populus alba × Populus glandulosa 84K is: MCIAMDRNYLQKSTTVKDKWEWNDVIFEGEHSTGI SWPKRNYTCSFCKREFSSAQALGGHMNVHRRDRARLKQLPSWFFDCPKPTSMSNPKPLPYPSSKFSPYPDHTHDHSLLSPFLASFSSPSYPEKKSIVECSRSINSTRKISDTRAVVGVGELKKNFVQDGDQLKVSRTSGIISLDLEMRCEDPKEVLDLELRLGCF (SEQ ID No.10); and the amino acid sequence in the parent Populus glandulosa (G) plant is: MCIAMDRNYLQKSTTVKDKWEWNDVIFEGEHSTGISWPKRNYTCS FCKREFSSAQALGGHMNVHRRDRARLKQLPSWFFDCPKPTSMSNPKPLPYPS STFSPYPDHTHDHSLLSPFLASFSSPSYPEKKSIVECPRSINSTRKIRHMRAVVG VGELKKNFVQDGDQLKVSRTSGIISLDLEMRCEDPKEVLDLELRLGCF (SEQ ID No.1);

[0091] The amino acid sequence of PagSUPa protein in the parental Populus alba (A) plant of the KO#48 pagsupa mutant is: MCIAMDRNYLQKSTTVKDKWEWNDVIFEGEHSTGISWPKRN YTCSFCKREFSSAQALWGTYECPQER (SEQ ID No.11); the amino acid sequence in the parental Populus glandulosa (G) plant is: MCIAMDRNYLQKSTTVKDK WEWN DVIFEGEHSTGISWPKRNYTCSFCKREFSSAQAWGTYECPQER (SEQ ID No.12); the amino acid sequence of PagSUPa protein in the parental Populus alba (A) plant of the KO#70 pagsupa mutant is: MCIAMDRNYLQKSTTVKDKWEWNDVIFEGEHSTGISWPK RNYTCSFCKREFSSAQALFLGDI (SEQ ID No.13); the amino acid sequence in the parental Populus glandulosa (G) plant is: MCIAMDRNYLQKSTTVKDKWEWNDVIFEG EHSTGISWPKRNYTCSFCKREFSSAQALGDI (SEQ ID No.14).

[0092] Example 3

[0093] The KO#48 pagsupa mutant plants (denoted as supa48) and KO#70 pagsupa mutant plants (denoted as supa70) obtained in Example 2 and the non-transformed Populus alba × Populus glandulosa 84K plants (denoted as CK) were cultured for two months under the conditions of a photoperiod of 16 h light / 8 h dark and a temperature of 22 - 26 °C, and their traits were observed. The results are as Figures 3 to 5 shown, where "*" indicates P < 0.05 and "**" indicates P < 0.01; Figure 4 The leaves in are the leaves at different positions on the plants at the same stage. 1 - 15 in the figure represent different nodes of the plants. For example, the leaf on the first node is labeled 1, the leaf on the second node is labeled 2, and so on.

[0094] It can be seen from Figures 3 to 5 that compared with the control plants, the two lines (KO#48 and KO#70) of pagsupa mutant poplars are significantly taller. The ground diameter of the mutant plants is larger than that of the control, and the number of internodes also increases ( Figure 3 ). The leaves of pagsupa mutant poplars are significantly larger than those of the control, and the leaf area, leaf length, and leaf width are significantly greater than those of the control ( Figure 4 ). The stems of pagsupa mutant poplars become longer and thicker compared with the controlFigure 5 )。

[0095] Example 4

[0096] Using the mutant plants obtained by culturing in Example 3 and the untransformed Populus alba×P. glandulosa 84K plants as samples, their lignin contents were measured according to the method described in the literature "KNAT2 / 6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar (Zhao Y, Xueqin S, Zhou H, et al. New Phytologist, 2019, 225(4). DOI: 10.1111 / nph.16036.)".

[0097] The results are as Figure 6 shown. Among them, the scale bar in A is 600 μm, and B is the measurement and analysis result of lignin; the P value of the t-test is indicated by asterisks: "**" indicates P < 0.01; pf represents phloem fiber, Xy represents xylem, and Pi represents pith.

[0098] From the lignin content measurement results, it can be seen that compared with the control plants, the lignin contents of the two lines (KO#48 and KO#70) of pagsupa mutant poplars are significantly increased. It can be seen that knocking out the negative regulatory PagSUPa gene can effectively increase the lignin content in poplars.

[0099] From the above examples, it can be seen that by knocking out the PagSUPa gene in the present invention, compared with the untransgenic poplar (CK) plants, it is significantly taller and thicker, and the total biomass and lignin content are significantly improved, indicating that the PagSUPa gene can regulate the biomass of poplars, which has application significance for cultivating and improving forest tree varieties and ensuring the fast growth and high yield of forest trees.

[0100] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of PagSUPa gene in optimizing poplar traits, characterized in that the optimization of poplar traits includes one or more of increasing the internode number of poplar, increasing the total biomass of poplar, and increasing the lignin content in poplar; the amino acid sequence of the protein encoded by the PagSUPa gene is as shown in SEQ ID No.1; the method for optimizing poplar traits is: knocking out the PagSUPa gene in poplar.

2. The application according to claim 1, wherein the nucleotide sequence of the PagSUPa gene is as shown in SEQ ID No.

2.

3. The application according to claim 1, wherein the optimization of poplar traits also includes one or more of increasing the ground diameter of poplar, increasing the plant height of poplar, increasing the leaf area of poplar, increasing the leaf length of poplar, and increasing the leaf width of poplar.

4. The application according to claim 1, characterized in that Knocking out the PagSUPa gene in poplar using a recombinant vector, the recombinant vector includes a basic vector and a gRNA inserted into the basic vector; the nucleotide sequence of the gRNA is as shown in SEQ ID No.

3.

5. The application according to claim 4, characterized in that, The construction method of the recombinant vector includes the following steps: annealing the annealing primers for synthesizing gRNA to obtain an annealing product; inserting the annealing product into the basic vector to obtain the recombinant vector; the annealing primers consist of PagSUPa-T1-F and PagSUPa-T1-R; the nucleotide sequence of PagSUPa-T1-F is as shown in SEQ ID No.4, and the nucleotide sequence of PagSUPa-T1-R is as shown in SEQ ID No.

5.

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

7. The application according to claim 1, characterized in that, Optimizing poplar traits using a recombinant engineering bacterium, the recombinant engineering bacterium includes a recombinant vector and a basic strain; the recombinant vector includes a basic vector and a gRNA inserted into the basic vector; the nucleotide sequence of the gRNA is as shown in SEQ ID No.

3.

8. The application according to claim 7, wherein the basic strain is Agrobacterium.

9. A method for optimizing poplar traits, characterized in that, Including: culturing after introducing the recombinant engineering bacterium into a poplar receptor; the recombinant engineering bacterium includes a recombinant vector and a basic strain; the recombinant vector includes a basic vector and a gRNA inserted into the basic vector; the nucleotide sequence of the gRNA is as shown in SEQ ID No.3 the optimization of poplar traits includes one or more of increasing the internode number of poplar, increasing the total biomass of poplar, and increasing the lignin content in poplar.

10. The method according to claim 9, wherein the introduction method includes: infecting the poplar receptor with the recombinant engineering bacterium for 10 - 20 min and then culturing in the dark; the culture temperature is 22 - 26 °C and the time is 2 - 3 d; the poplar receptor includes poplar callus.

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