Gene for regulating and controlling growth and development of poplar and application thereof

Abstract

The invention discloses a gene for regulating and controlling growth and development of poplars and application of the gene, and belongs to the technical field of agricultural biological genetic engineering. The invention discloses a gene for regulating and controlling growth and development of poplars. The gene comprises a PagZFP12A gene and a para-line homologous gene PagZFP12B of the PagZFP12A gene, the nucleotide sequence of the PagZFP12A gene is as shown in SEQ ID NO: 1, and the nucleotide sequence of the PagZFP12B gene is as shown in SEQ ID NO: 2. According to the invention, the PagZFP12A and / or PagZFP12B genes are / is knocked out to cause function deletion, so that the biomass accumulation and secondary growth of the forest trees are remarkably promoted, and a key molecular target and a technical means are provided for increasing the wood yield, optimizing the quality and improving the stress resistance of the forest trees.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural bioengineering technology, and particularly relates to a gene for regulating growth and development of poplar and application thereof. BACKGROUND

[0002] Wood, as an important renewable resource, plays an irreplaceable role in the fields of construction, papermaking, energy and the like, but the contradiction between supply and demand of wood is increasingly prominent with the development of social economy. The formation of wood is derived from the secondary growth of woody plants, which involves a series of dynamic biological events such as vascular cambium division, xylem and phloem differentiation, secondary cell wall formation and the like. As the main component of wood, the development of xylem directly determines the yield and quality of wood, and therefore, it is of great significance to analyze the molecular regulation mechanism of xylem development for promoting fast-growing and high-quality breeding of forest trees.

[0003] Zinc finger protein (ZFP) is an important transcription factor, which regulates gene expression through specific beta beta alpha tetrahedron structure (composed of C-terminal alpha helix, N-terminal two beta folds and central zinc atom) and participates in various biological processes such as plant growth and development, stress response and the like. Among them, C2H2 type zinc finger protein is widely distributed in plants and has diverse functions, for example, JcZFP8 in Jatropha curcas affects plant morphological establishment through gibberellin-related pathway, and its ectopic overexpression leads to tobacco dwarfing and leaf malformation. AtZFP2 in Arabidopsis thaliana, as a transcriptional repressor, inhibits xylem formation of funiculus cortical cells by targeting secondary cell wall (SCW) transcription factor NST1, and guarantees normal development of seeds; PagIDD15A in Populus alba x P. grijsii regulates xylem secondary wall thickening and vascular cambium activity by regulating genes related to lignin synthesis and cell proliferation.

[0004] Although some C2H2 type zinc finger proteins have been confirmed to be involved in plant secondary growth and secondary wall development, the molecular network of secondary growth of woody plants is still not completely clear, especially in poplar, the functions of many zinc finger proteins have not been characterized. Based on co-expression analysis of key genes of lignin synthesis, it is found that the transcription factor ZFP12 has a strong negative correlation with lignin monomer synthesis enzyme genes, and shows expression specificity in single-cell transcriptome sequencing of Populus alba x P. grijsii 84K, but the specific mechanism of ZFP12 in forest tree growth and xylem development has not been determined. SUMMARY

[0005] The application aims to provide a gene for regulating growth and development of poplar and application thereof, which can significantly promote biomass accumulation and secondary growth of forest trees by knocking out PagZFP12A and / or its paralog PagZFP12B , resulting in loss of function, and provides a key molecular target and technical means for improving wood yield, optimizing quality and improving stress resistance of forest trees.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A gene that regulates the growth and development of poplar trees, said gene comprising... PagZFP12A Genes and their paralogous genes PagZFP12B The PagZFP12A The nucleotide sequence of the gene is shown in SEQ ID NO:1. PagZFP12B The nucleotide sequence is shown in SEQ ID NO:2.

[0007] Preferably, the PagZFP12A The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:3. PagZFP12B The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:4.

[0008] The present invention also provides as described PagZFP12A Genes and / or PagZFP12B Application of genes in regulating poplar growth and xylem development.

[0009] Preferably, the regulation of poplar growth and xylem development is achieved by knocking down and / or knocking out... PagZFP12A Genes and / or PagZFP12B Genes promote the accumulation of forest biomass, secondary growth, and xylem development.

[0010] The present invention also provides a method for utilizing the above-mentioned PagZFP12A and / or PagZFP12B A method for genetically breeding transgenic poplar trees, characterized by comprising the following steps: S1, Cloned Poplar PagZFP12A Genes and / or PagZFP12B Gene; S2, Targeting poplar trees PagZFP12A Genes and / or PagZFP12A Gene-designed specific gRNAs were constructed and expressed in a CRISPR-Cas9 system expression vector to obtain... PagZFP12A Genes and / or Fig. 1 Gene knockout vector; S3. The knockout vector was transformed into poplar tissue culture seedlings using Agrobacterium-mediated transformation, and resistance screening was performed to obtain... Fig. 1 Genes and / or Fig. 2 Gene knockout transgenic positive plants.

[0011] Preferably, in S1, the cloned poplar tree Fig. 2 Genes and / or ProPagZFP12A::GUS The specific gene manipulation is as follows: Total RNA was extracted from poplar trees, reverse transcribed into cDNA, and used as a template to generate primers. Fig. 3 -F andFig. 3 -R is used for PCR amplification to obtain... Fig. 3 Genes; using primers Fig. 3 -F and Fig. 3 -R is used for PCR amplification to obtain... PagZFP12A Gene.

[0012] Preferably, the primer Fig. 4 The nucleotide sequence of -F is shown in SEQ ID NO:5, and the primer... Fig. 4 The nucleotide sequence of -R is shown in SEQ ID NO:6; the primer PtrZFP12 in The nucleotide sequence of -F is shown in SEQ ID NO:7, and the primer... Fig. 4 The nucleotide sequence of -R is shown in SEQ ID NO:8.

[0013] The present invention also provides as described PagZFP12A Genes and / or Fig. 4 Application of genes in regulating timber yield and improving timber quality.

[0014] Preferably, by knockdown and / or knockout proPagZFP12A::GUS Genes and / or Fig. 5 The gene was reduced, its expression level was decreased, the biomass of poplar wood was increased, and the wood quality was improved.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a gene that regulates the growth and development of poplar trees and its application. This invention clarifies... Fig. 5 As a transcriptional activator, the gene negatively regulates cambium activity and xylem development in poplar by modulating downstream repressive target genes, revealing its crucial role in the molecular network of secondary growth in woody plants and providing new theoretical basis for elucidating the molecular mechanisms of xylem development. Knockout Fig. 6 and / or PagZFP12 The gene can cause its function to be lost, which significantly promotes the accumulation of poplar biomass and secondary growth. Three-month-old knockout plants have increased plant height, increased ground diameter, and enlarged pith cavity. At the same time, the thickness of the xylem wood fiber cell wall increases and the amount of lignin deposition increases, which effectively improves timber yield and improves the material.

[0016] Should Fig. 6 The application of gene editing provides novel gene targets and molecular design ideas for improving poplar timber yield and quality, through targeted editing. Fig. 7 Genes can directionally regulate tree growth and xylem development, laying an important foundation for promoting the breeding of fast-growing and high-quality trees.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Fig. 7 A schematic diagram of the pDe-Cas9-npt-eYGFP knockout vector constructed in Example 1; PagZFP12A, PagZFP12B The results of gene editing analysis of the double-deficient mutant plant in Example 1 are shown. ​ This refers to the genetic transformation process of 84K poplar, in which... ​ In this context, A represents the infection process of callus tissue induced by Agrobacterium infection of leaves. ​ In this context, B represents the induction of callus bud differentiation. ​ In this context, C represents the seedlings that have developed from buds after screening with the resistant rooting medium. ​ for ​ The analysis of expression patterns, among which, ​ A in the text is ​ In the spatiotemporal dynamics of the AspWood database, Phloem represents phloem, Cambium represents cambium, Expanding xylem represents developing xylem, and Lignified xylem represents lignified xylem. ​ B in the text is ​ Relative transcription levels in different tissues of *Populus alba* ​ C in the text is ​ Reporter gene localization in *Populus alba* tissues; ​ for ​ The results of growth index measurements of double-mutant transgenic lines and wild-type plants, among which... ​ In the figure, A represents the morphological comparison result. ​ In the figure, B represents the plant height measurement result. ​ In this context, C represents the diameter measurement result; ​ for ​ Phenotypic analysis results of regulation of xylem development and lignin deposition, among which, ​ In the figure, A represents a cross-section of the stem of IN13 from 3-month-old wild-type, KO#6, KO#9, and KO#14 plants stained with toluidine blue. ​ In the figure, B represents the statistical result of the xylem width. ​ In this context, C represents the statistical result of the medullary cavity size. ​ In the diagram, D represents the number of xylem cell layers. Red dashed lines mark xylem regions, yellow lines mark xylem ray cells, and red dots mark vessel cells. Scale bar = 50 μm. ​ In this context, E represents the total number of cell layers in the xylem region. ​ In the figure, F represents the statistical result of the number of vessel cell layers in the xylem region. ​G in FIG. 1 is the result of the number of layers of wood fiber cells in the xylem region, ​ H in FIG. 2 is the cell division phenotype of the cambium of IN13 stem, scale bar = 20 μm, ​ I in FIG. 3 is a frequency distribution diagram of the number of cambium cells of IN13, ​ J in FIG. 4 is a cross section of the stem after staining with phloroglucinol and Maule staining, scale bar = 50 μm; ​ is ​ The area of the single vessel of the double mutant transgenic line is larger than that of the wild type, and the results of the cell wall of the wood fiber, wherein, ​ A in FIG. 5 is a scanning electron microscope image at 500 times and a local enlarged image of a single vessel cell thereof, ​ B in FIG. 6 is a statistical result of the area of a single vessel cell, ​ C in FIG. 7 is a scanning electron microscope image of the cell wall thickness of the wood fiber, scale bar = 2 μm, n = 300, ​ D in FIG. 8 is a statistical result of the cell wall thickness of the wood fiber, n = 300. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0020] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0021] Source of test material: Acetyl-syringone (AS) formulation: 392.4 mg of acetyl-syringone powder was dissolved in 20 mL of dimethyl sulfoxide (DMSO), filtered with a 0.22 μm filter into a sterile centrifuge tube to obtain a 100 mM stock solution.

[0022] Agrobacterium resuspension: 2.4 g / L WPM449 (Phyto Technology #L4490) and 20 g / L sucrose were dissolved in deionized water, pH adjusted to 5.4, and autoclaved. Before use, 100 μM acetyl-syringone was added on the clean bench.

[0023] Co-culture medium: 2.4 g / L WPM449 (Phyto Technology #L449), 0.5 g / L morpholinoethanesulfonic acid (MES), and 20 g / L sucrose were dissolved in deionized water, pH adjusted to 5.9, 3.2 g / L plant gel was added and sterilized. After cooling to below 60°C, 100 μM acetyl-syringone was added on the clean bench.

[0024] Differentiation selection medium: 2.4 g / L WPM449 (Phyto Technology #L449), 0.5 g / L MES, 0.1 mg / L naphthaleneacetic acid (NAA), 0.5 mg / L 6-benzyladenine (6-BA), 20 g / L sucrose, pH adjusted to 5.9, 3.2 g / L plant gel added and then sterilized. After cooling to below 60°C, 200 mg / L termethin and 1.5 mg / L hygromycin were added.

[0025] Callus induction medium: Dissolve 2.4 g / L WPM449 (Phyto Technology #L449), 0.5 g / L LMES, 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg / L NAA, 0.1 mg / L furanoaminopurine (KT) and 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 3.2 g / L plant gel and sterilize.

[0026] Rooting medium: Dissolve 2.2 g / L MS519 (Phyto Technology #M519), 0.5 g / L MES, 0.05 mg / L NAA, 0.02 mg / L indolebutyric acid (IBA), and 20 g / L sucrose in deionized water, adjust the pH to 5.9, add 7.8 g / L agar, and sterilize. After cooling to below 60°C, add 200 mg / L termethin.

[0027] Unless otherwise specified, the materials, reagents, enzymes, competent cells, plasmids, and instruments used in this invention are all conventional experimental materials in the field and can be purchased through commercial channels.

[0028] Example 1 Total RNA was extracted from 84K poplar trees, and cDNA was obtained using a reverse transcription kit. PCR amplification was performed using designed specific primers. ​ The full-length gene sequence was obtained, and the PCR product was cloned into a vector to construct the vector. The correctness of the vector was verified through bacterial testing and sequencing, using primers... ​ -F and ​ -R is used for PCR amplification to obtain... ​ Genes; using primers ​ -F and ​ -R is used for PCR amplification to obtain... ​ Genes. Among them, primers ​ The nucleotide sequence of -F is shown in SEQ ID NO:5, primer ​ The nucleotide sequence of -R is shown in SEQ ID NO:6; primers ​ The nucleotide sequence of -F is shown in SEQ ID NO:7, primer ​The nucleotide sequence of -R is set forth in SEQ ID NO: 8.

[0029] ​ The gene is the nucleotide sequence set forth in SEQ ID NO: 1.

[0030] SEQ ID NO: 1:

[0031] ​ The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0032] SEQ ID NO: 2:

[0033] ​ The protein expressed by the gene is the amino acid sequence set forth in SEQ ID NO: 3.

[0034] SEQ ID NO: 3: MMSGDAFSLPSLIAGFAQDQNNANPNPNPNPAVAKRKRNLPGTPDPDAEVIALSPKTLMATNRFICEICNKGFQRDQNLQLHRRGHNLPWKLKQRTNKEVRKKVYICPEKTCVHHDSSRALGDLTGIKKHFSRKHGEKKWKCEKCSKKYAVQSDWKAHSKTCGTREYKCDCGTLFSRKDSFITHRAFCDALADESARITSVQDTDLNFRNDTVNLPHGFSNRPGVPDIAGISQFSAGFRPDFSGMSTPGNSLGADQQKTGLSLWINQANSHITPNSNLYVPPISTGLPEMVQMVANLYSSSSSANLGNLTSSGLPHELKEEGINKANMVDTSASLYSDRIQNKQLKLAAVPMSATALLQKAAQMGSTRSNQPFFGNSYGLMSSSSSSSPTTNHICLNQNPNELYHVFQNVKQPASESLTATYRVAMSDAVMGTSSNLDQLVMQTSGNLQNDPTQLKLHRGSNSTESGLTRDFLGMSSESGRLFLPHDLAKFASISSAMSLNHFTANH.

[0035] ​ The protein expressed is the amino acid sequence set forth in SEQ ID NO: 4.

[0036] SEQ ID NO: 4: MMSGDAFSLPSSIAGFAQDQNNANPNPNPKTNPNQAAKRKRNLPGTPDPDAEVIALSPKSLMATNRFICEICNKGFQRDQNLQLHRRGHNLPWKLKQRTNKEVRKKVYICPENTCVHHDPSRALGDLTGIKKHFSRKHGEKKWKCEKCSKKYAVQSDWKAHSKTCGTREYKCDCGTLFSRKDSFITHRAFCDALAEESARITSVPAAANLNFRNDTVDLPHGFSDRPGVPDVAGIPQFNSSFGPDFSGMTPGNSLGADQQKPGLSLWLNQANSHISPNLNLYVPSSSSGLPEMVQIGSASLYGSSSTANFGNLTLSGLPHGLKEEGSSSKANMVNTSASLYSDSIQNKQSKPAAVPNMSATALLQKAAQMGSTRSNQSFFGNSYGLMSCSSSSPTTNPVSLNQNPNELHQVFQNVKQTAAESLTTTNSTVAMSDAMMGSNLDQLMMQTSGKLQNDQTQLKHQRGSNSIESGLTRDFLGMSSESSRPFLPQDLAKFASISSAMSLNHFTVNP.

[0037] SEQ ID NO: 5: ATGATGTCTGGTGATGCCTTT.

[0038] SEQ ID NO: 6: TCAATGATTGGCAGTGAAATGGTTC.

[0039] SEQ ID NO: 7: ATGATGTCTGGTGATGCTTTCTCAC.

[0040] SEQ ID NO: 8: TCAAGGATTGACAGTGAAATGG.

[0041] PCR reaction program: 95℃, 3 min; (95℃: 30 s; 58℃: 30 s; 72℃: 1.6 min for one cycle, 34 cycles in total); 72℃, 5 min; 12℃, ∞. After amplification, the PCR product was used for subsequent experimental analysis.

[0042] ​ Gene reaction system: 2×Phanta Flash Master Mix 25 μL; ​ -F (10 μM) 1 μL;​ - R (10 mM) 1 pL; cDNA 1 pL; sterile water 22 pL.

[0043] After amplification of the PCR product, it needs to be separated by agarose gel electrophoresis and the gel is recovered: 8 pL of 6x Loading Buffer was added to the amplified PCR product and mixed, and the mixed sample and 2000 bp Maker were added to the agarose gel hole, respectively, with a voltage of 135V and a time of 15 min. After electrophoresis, it was placed in a gel imager to observe, and the target band was cut and placed in a 2 mL PCR tube. The target gel was recovered using a gel extraction kit (Omega Gel Extraction Kit D2500), and finally the product concentration was measured using a spectrophotometer.

[0044] Vector construction: The vector construction method uses CRISPR-Cas9 technology to construct a pDe-Cas9-npt-eYGFP knockout vector ​ )。

[0045] The nucleotide sequences of the two knockout target sites are shown in SEQ ID NO: 9 and SEQ ID NO: 10: SEQ ID NO: 9: GAAGAGGTCACAATCTTCCA.

[0046] SEQ ID NO: 10: AGACTTGCGGGACTAGAGAG.

[0047] The primers of the target gene and the reference gene are designed, and the nucleotide sequences are shown in SEQ ID NO: 11-SEQ ID NO: 14.

[0048] gRNA1-F: SEQ ID NO: 11: ATTGGAAGAGGTCACAATCTTCCA.

[0049] gRNA1-R: SEQ ID NO: 12: AAACTGGAAGATTGTGACCTCTTC.

[0050] gRNA2-F: SEQ ID NO: 13: ATTGCTCTCTAGTCCCGCAAGTCT.

[0051] gRNA2-R: SEQ ID NO: 14: AAACAGACTTGCGGGACTAGAGAG.

[0052] The specific experimental scheme is as follows: Enzymatic digestion of the intermediate vector pEn-C1.1 The reaction system is as follows: pEn-C1.1 1 μg; 10×NEB CutSmart buffer 5 μL; NEB BbsI-HF 1 μL; sterile water added to 50 μL.

[0053] After incubation at 37°C for 5-6 h, the enzyme digestion product was purified.

[0054] The specific experimental method of the ligation reaction is as follows: 5 μL of the enzyme-digested pEn-C1.1, 15 μL of the annealed oligonucleotide, 2.5 μL of 10×T4 buffer, and 25 μL of T4 ligase. Incubate overnight at room temperature, transform the ligation product into DH5α competent cells, and take 100 μL of bacterial solution and spread on an LB plate containing ampicillin. After single colonies grow, perform colony PCR identification.

[0055] gRNA1 related operation gRNA1 fragment PCR amplification (1) Amplify the gRNA1 expression cassette using pEn-C1.1-gRNA1 as the template.

[0056] (2) Reaction system: 9.5 μL ddH2O, 1 μL 200 ng / μL DNA template, 1 μL 10 μM gRNA1-F, 1 μL 10 μM gRNA1-R, 12.5 μL 2×Phanta Max Master Mix.

[0057] (3) PCR program: 98°C pre-denaturation for 5 min; 98°C denaturation for 30 s, 58°C annealing for 40 s, 72°C extension for 40 s, 30 cycles; 72°C final extension for 10 min. The target fragment size is 580 bp, and the target fragment is purified after agarose gel electrophoresis.

[0058] (4) Enzymatic digestion: The final vector pDe-Cas9-npt-eYGFP was subjected to enzyme digestion, and the reaction system was as follows: 1 μg of plasmid, 5 μL of 10×CutSmart buffer, 1 μL of Bsu36I, 1 μL of Mlul-HF, and ddH2O added to 50 μL. Incubate at 37°C for 3 h, and purify the enzyme digestion product.

[0059] (5) Recombination reaction: reaction system: 200 ng of pDe-Cas9-npt-eYGFP after enzyme digestion, 30 ng of inserted DNA (580 bp), 4 μL of 5×CE II buffer, 2 μL of Exnase II, and ddH2O to 20 μL. After incubation at 37°C for 30 min, ice bath for 5 min. Transform the recombination product into DB3.1 competent cells, take 100 μL of bacterial solution and spread on LB plate containing spectinomycin. After single colony growth, perform colony PCR identification.

[0060] gRNA2 related operation (1) Gateway reaction: reaction system: 100 ng of pEn-C1.1-gRNA2, 100 ng of pDe-Cas9-gRNA1, add TE buffer to 5 μL, and add 0.5 μL of LR clonase II enzyme mix. After vortexing, centrifuge. Incubate at room temperature for more than two hours.

[0061] (2) Transformation and colony PCR identification: transform all reaction products into DH5α competent cells, take 100 μL of bacterial solution and spread on LB plate containing spectinomycin. Pick 3-6 colonies for detection. Primer uses Crispr_F and gRNA2-R (SEQ ID NO: 14). PCR conditions: annealing at 58°C, extension for 1 min, 30 cycles.

[0062] Crispr_F primer is used for sequencing gRNA2, and the sequence of Crispr_F is shown in SEQ ID NO: 15: SEQ ID NO: 15: CTCCCTAGGCCTGTTATCCCT.

[0063] from ​ It can be known that the pDe-Cas9-npt-eYGFP vector core is used for CRISPR / Cas9 gene editing. It contains regulatory elements such as lac promoter to drive Cas9 expression, and Cas9 coding sequence is the key of gene cutting; gRNA related elements and multiple cloning sites (such as AflII, ApaI, etc.) are set for constructing targeted gRNA expression cassette; NeoR / KanR resistance gene is used for screening, and eYGFP can be used as a reporter gene. Integrating expression regulation, gene editing, and screening markers provides vector support for gene editing experiments.

[0064] 84K poplar genetic transformation: After the pDe-Cas9-npt-eYGFP knockout vector was transferred into Agrobacterium GV3101 strain, 84K poplar genetic transformation experiment was carried out. The leaf of Populus alba var. pyramidalis 84K was placed in callus induction medium, and callus was induced in dark culture; the callus or the leaf was infected with Agrobacterium liquid, and after two days of dark culture on co-culture medium, it was transferred to differentiation culture medium for light culture until adventitious buds were differentiated; the adventitious buds were screened by kanamycin (Kan) containing screening medium, and then rooted.

[0065] The un-identified resistant seedlings were used to extract leaf DNA with CTAB for identification, and the extraction method was as follows: (1) Sample pretreatment: In the clean bench, select the plant to be identified on the resistant rooting medium, number it, take its leaf, put it in a 2 mL centrifuge tube containing sterilized steel balls, freeze it in liquid nitrogen for a moment, and then crush it to powder by high-throughput tissue grinder (set the frequency of the grinder to 50 Hz and the time to 120 s).

[0066] (2) Add 500 μL of 2x CTAB lysis buffer to the powdered sample, mix well by vortexing, and incubate at 65°C for 30 min, during which time mix it up and down every 10 min to promote cell lysis.

[0067] (3) After lysis, add an equal volume of chloroform, vortex to emulsify, and centrifuge at 12000 rpm for 10 min.

[0068] (4) Transfer the supernatant to a new centrifuge tube, and add 2 times the volume of absolute ethanol to the supernatant in the centrifuge tube, mix well by vortexing, and centrifuge at 12000 rpm for 10 min. After centrifugation, a white precipitate can be seen.

[0069] (5) Discard the supernatant, collect the precipitate, wash the precipitate with 75% ethanol 1-2 times (12000 rpm, 5 min / time), dry at room temperature, and then dissolve the DNA in water.

[0070] (6) The purity of the DNA was detected by NanoDrop microspectrophotometer (A260 / A280 ratio 1.8-2.0 is ideal), and the qualified samples were stored at -20°C and could be used for subsequent experiments.

[0071] The wild type plant DNA was used as negative control. The Cas9 gene band was detected by 1% agarose gel electrophoresis to preliminarily screen the candidate strains with the expected size. Then the strains with the expected size were subjected to PCR amplification with the primers designed according to the target site near PagZFP12 (about 150-300 bp fragment including the target site). The forward primer was added with a bridge sequence, as shown in SEQ ID NO: 16. The reverse primer was added with a bridge sequence, as shown in SEQ ID NO: 17. The DNA of the mutant plants determined to be positive was subjected to PCR amplification, and the PCR amplification product was subjected to high-throughput sequencing alignment analysis. The double mutant plants with base deletion or addition and causing frame shift of amino acid sequence to cause premature termination were selected for further mass propagation as the materials for subsequent experiments. The results of gene editing analysis of the double mutant plants are shown in ​ .

[0072] SEQ ID NO: 16: 5'-ggagtgagtacggtgtgc-3'.

[0073] SEQ ID NO: 17: 5'-gagttggatgctggatgg-3'.

[0074] As shown in Table 1, among the three different gene editing strains, KO#6, #9 and #14 caused deletion or premature termination of protein sequence due to base deletion or insertion. ​ The poplar stem tissue sections were prepared according to the following test scheme: tissue structure analysis: the stem sections of the gene editing type and the wild type at the same development stage were fixed and sectioned, and the changes in the structure of the stem sections were observed under a microscope.

[0075] GUS activity detection: the poplar 90% acetone was fixed on ice, and X-Gluc staining and elution were performed, and the expression pattern of the tissue was observed under a microscope.

[0076] ​ The test results are as follows: screening and identification of double mutant plants and expression analysis, and the genetic transformation process of 84K poplar is shown in .

[0077] As shown in Table 1, among the three different gene editing strains, KO#6, #9 and #14 caused deletion or premature termination of protein sequence due to base deletion or insertion. ​ As shown in Table 1, among the three different gene editing strains, KO#6, #9 and #14 caused deletion or premature termination of protein sequence due to base deletion or insertion.

[0078] ​ As shown in Table 1, among the three different gene editing strains, KO#6, #9 and #14 caused deletion or premature termination of protein sequence due to base deletion or insertion. ​ A in the above table is the callus formed by Agrobacterium infection and induction of leaf differentiation; ​ B in the above table is the bud differentiated from the callus; ​ C in the above table is the seedling formed by rooting of the bud.

[0079] ​​ Expression pattern analysis, such as ​ As shown.

[0080] ​ In this context, A represents Populus tomentosa in the AspWood database of micron-sized slices representing wood formation. ​ Gene expression trends at different developmental stages (T1-T4: phloem-cambium-developing xylem-mature xylem) reveal their temporal regulatory characteristics during vascular tissue differentiation.

[0081] Depend on ​ From B, we can know that the silver gland is in Yang Zhong ​ It has a relatively high expression level in the phloem, cambium, and leaf blade.

[0082] Depend on ​ As can be seen from C in the code, in its bootstrap driver... ​ After observing transgenic poplar sections stained with GUS, GUS active signals (blue staining) were observed in the petioles, veins, roots, and internodes (IN3, IN5, IN7) of the stem. However, the signal intensity and distribution varied in different parts: the staining was more obvious in the petioles and veins, and GUS signals were also detected in specific areas of the roots. The GUS signal was strong in the xylem of the young internodes of the stem.

[0083] ​ The results of growth index measurements of double mutant transgenic lines and wild-type plants were obtained from... ​ It can be seen that, compared with the wild type, the 3-month-old double mutant knockout plants have increased plant height, increased ground diameter, and enlarged medullary cavity.

[0084] ​ for ​ Phenotypic analysis results of regulation of xylem development and lignin deposition, by ​ It can be seen that the width of the xylem is significantly narrowed, the number of xylem cell layers is significantly reduced, the thickness of the wood fiber cell wall is increased, the cambium region is expanded, and the amount of lignin deposition is increased.

[0085] ​ The double mutant transgenic lines showed increased single vessel area and wood fiber cell wall size compared to wild-type lines, resulting from... ​ It can be seen that knockout ​ This leads to an increase in the area of ​​individual vessels and a thickening of the cell walls of wood fibers.

[0086] It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A gene that regulates the growth and development of poplar trees, characterized in that, The genes include PagZFP12A Genes and their paralogous genes PagZFP12B The PagZFP12A The nucleotide sequence of the gene is shown in SEQ ID NO:

1. PagZFP12B The nucleotide sequence is shown in SEQ ID NO:

2.

2. The gene for regulating the growth and development of poplar trees according to claim 1, characterized in that, The PagZFP12A The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

3. PagZFP12B The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

4.

3. PagZFP12A Genes and / or PagZFP12B The application of genes in regulating poplar growth and xylem development is characterized by, The PagZFP12A The nucleotide sequence of the gene is shown in SEQ ID NO:

1. PagZFP12B The nucleotide sequence is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The regulation of poplar growth and xylem development is achieved by knocking down and / or knocking out PagZFP12A Genes and / or PagZFP12B Genes promote the accumulation of forest biomass, secondary growth, and xylem development.

5. A method for cultivating transgenic poplar trees, characterized in that, Includes the following steps: S1, Cloned Poplar PagZFP12A Genes and / or PagZFP12B Genes, the ones mentioned PagZFP12A The nucleotide sequence of the gene is shown in SEQ ID NO:

1. PagZFP12B The nucleotide sequence is shown in SEQ ID NO:2; S2, Targeting poplar trees PagZFP12A Genes and / or PagZFP12B Gene-designed specific gRNAs were constructed and expressed in a CRISPR-Cas9 system expression vector to obtain... PagZFP12A Genes and / or PagZFP12B Gene knockout vector; S3. The knockout vector was transformed into poplar tissue culture seedlings using Agrobacterium-mediated transformation, and resistance screening was performed to obtain... PagZFP12A Genes and / or PagZFP12B Gene knockout transgenic positive plants.

6. The method according to claim 5, characterized in that, In S1, the cloned poplar PagZFP12A Genes and / or PagZFP12B The specific gene manipulation is as follows: Total RNA was extracted from poplar trees, reverse transcribed into cDNA, and used as a template to generate primers. PagZFP12A -F and PagZFP12A -R is used for PCR amplification to obtain... PagZFP12A Genes; using primers PagZFP12B -F and PagZFP12B -R is used for PCR amplification to obtain... PagZFP12B Gene.

7. The method according to claim 6, characterized in that, The primers PagZFP12A The nucleotide sequence of -F is shown in SEQ ID NO:5, and the primer... PagZFP12A The nucleotide sequence of -R is shown in SEQ ID NO:6; the primer PagZFP12B The nucleotide sequence of -F is shown in SEQ ID NO:7, and the primer... PagZFP12B The nucleotide sequence of -R is shown in SEQ ID NO:

8.

8. PagZFP12A Genes and / or PagZFP12B The application of genes in regulating timber yield and improving timber quality is characterized by, The PagZFP12A The nucleotide sequence of the gene is shown in SEQ ID NO:

1. PagZFP12B The nucleotide sequence is shown in SEQ ID NO:

2.

9. The application according to claim 8, characterized in that, By knocking down and / or knocking out PagZFP12A Genes and / or PagZFP12B The gene was reduced, its expression level was decreased, the biomass of poplar wood was increased, and the wood quality was improved.

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