Poa pratensis gene editing method

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to a poa pratensis gene editing method. The invention provides a bluegrass CRISPR / Cas9 gene editing system, which comprises a pCAMBIA1300-Cas9 vector, a bluegrass gene PpTCP4 target sequence and a primer pair for amplifying the bluegrass gene PpTCP4 target sequence, the gene PpTCP4 of the bluegrass is directionally edited by utilizing a CRISPR / Cas9 gene editing technology, gene editing of a base mutation type is realized, a gene editing plant is successfully obtained, and the gene editing effect is good. The gene editing efficiency is 0.67%, and new research basis and technical reference are provided for developing the directional improvement of the poa pratensis to create high-quality germplasm resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and in particular relates to a gene editing method for Kentucky bluegrass. Background Art

[0002] Kentucky bluegrass Poa pratensis L.) belongs to the genus Poa of the Poaceae family ( Poa L.) is a perennial herbaceous plant and a high-quality lawn grass and forage grass. At present, most of the high-quality meadow grass varieties in China rely on imports. Therefore, utilizing and developing state-owned meadow grass resources and cultivating high-quality new varieties that adapt to the local environment is one of the effective ways to solve the long-standing contradiction between the supply and demand of high-quality grass seeds.

[0003] Agrobacterium-mediated genetic transformation is currently the most commonly used genetic transformation method. Culture medium formulation, explant type and regeneration method selection are all important influencing factors in the establishment of the genetic transformation system. Currently, the establishment of the genetic transformation system of Kentucky bluegrass is still imperfect and needs further optimization.

[0004] CRISPR / Cas9 gene editing technology is the core of molecular breeding. Using this technology to create Kentucky bluegrass varieties is an effective way to improve the utilization of domestic Kentucky bluegrass germplasm resources. However, the currently imperfect Kentucky bluegrass genetic transformation system limits the development and utilization of this species. Summary of the invention

[0005] The present invention provides a gene editing method for Kentucky bluegrass, constructs a CRISPR / Cas9 gene editing system for Kentucky bluegrass and performs site-specific editing. PpTCP4 The gene was discovered and the gene-edited plants were identified. The gene-edited plants were successfully obtained and an efficient and complete genetic transformation system for Kentucky bluegrass was established.

[0006] In order to solve the above technical problems, the following technical solutions are proposed: The present invention provides PpTCP4 The application of genes in regulating tillering of Kentucky bluegrass, PpTCP4 The nucleotide sequence of the gene is shown in SEQ ID NO.8.

[0007] The present invention provides a targeted knockout as described in the above technical solution PpTCP4 Gene or reduce as described in the above technical solution PpTCP4 Application of gene expression reagents in promoting tillering of Kentucky bluegrass.

[0008] Preferably, the targeted knockout PpTCP4 Gene knockout agents include targeted PpTCP4CRISPR / Cas9 vector for targeting gene; the vector contains targeted knockout PpTCP4 sgRNA sequence of the gene.

[0009] The present invention provides a targeted knockout of Poa annua as described in the above technical solution PpTCP4 The sgRNA of the gene, the sgRNA sequence comprises PpTCP4- sgRNA-F and PpTCP4- sgRNA-R; PpTCP4- The nucleotide sequence of sgRNA-F is shown in SEQ ID NO. 4. PpTCP4- The nucleotide sequence of sgRNA-R is shown in SEQ ID NO. 5; the PAM sequence recognized by the sgRNA is AGG.

[0010] The present invention provides a bluegrass CRISPR / Cas9 gene editing system, comprising a pCAMBIA1300-Cas9 vector and the bluegrass gene targeted knockout method described in the above technical solution. PpTCP4 sgRNA sequence.

[0011] Preferably, the bluegrass gene PpTCP4 The nucleotide sequence of the target sequence is shown in SEQ ID NO.3.

[0012] The present invention provides a method for constructing the bluegrass CRISPR / Cas9 gene editing system described in the above technical solution, comprising the following steps: Using the above technical solution PpTCP4- sgRNA-F and PpTCP4- sgRNA-R was subjected to an annealing procedure to obtain sgRNA; Connecting the sgRNA and the pCAMBIA1300-Cas9 vector to obtain a connection product; The ligation product was transformed into Escherichia coli, and the positive colonies were expanded and cultured to extract the plasmid, and the gene editing pCAMBIA1300-Cas9- PpTCP4 system.

[0013] The present invention provides a gene editing method for bluegrass, wherein the CRISPR / Cas9 gene editing system described in the above technical solution or the gene editing pCAMBIA1300-Cas9-sgRNA- PpTCP4 The system was transformed into Agrobacterium competent cells for genetic transformation to obtain gene-edited plants of Poa annua.

[0014] Preferably, Agrobacterium-mediated genetic transformation is used, including: transforming an Agrobacterium solution containing the Poa annua CRISPR / Cas9 gene editing system into callus tissue of Poa annua; The explant for inducing the production of bluegrass callus tissue comprises mature seeds of bluegrass; the culture medium for culturing the bluegrass callus tissue comprises ZT culture medium; the composition of the ZT culture medium comprises: MS as the basic culture medium, and also comprises 200-250 mg / L TMT, 1.0-1.5 mg / L ZR, 25.0-35.0 g / L sucrose and 7.8-8.3 g / L agar.

[0015] Preferably, the method further comprises identifying the bluegrass gene-edited plants, wherein the identification comprises: using TCP4 -F and TCP4 -R primers were used to identify the target gene of the positive plants. PpTCP4 If there is a base mutation in the gene, the gene editing is considered successful; Said TCP4 - The nucleotide sequence of F is shown in SEQ ID NO.1; Said TCP4 The nucleotide sequence of -R is shown in SEQ ID NO.2.

[0016] Beneficial effects of the invention: The invention provides PpTCP4 Application of genes in regulating tillering of Kentucky bluegrass, a targeted knockout of Kentucky bluegrass genes PpTCP4 sgRNA and a bluegrass CRISPR / Cas9 gene editing system, the CRISPR / Cas9 gene editing system comprising a pCAMBIA1300-Cas9 vector and the targeted knockout bluegrass gene of the above technical solution PpTCP4 The present invention uses CRISPR / Cas9 gene editing technology to edit the gene of Kentucky bluegrass in a targeted manner. PpTCP4 ", achieved base mutation type gene editing, successfully obtained gene-edited plants, and the gene editing efficiency was 0.67%, providing new research basis and technical reference for the targeted improvement of meadow grass to create high-quality germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Kentucky bluegrass PpTCP4 Gene PCR amplification electrophoresis; Figure 2 This is an electrophoresis diagram for the identification of CRISPR / Cas9 system vectors; A is the electrophoresis diagram of the successfully constructed vector being transformed into Escherichia coli DH5α (i.e. PpTCP4- DH5α); B is to transfer the successfully constructed vector into Agrobacterium LBA4404 (i.e. PpTCP4-LBA4404); Figure 3 Figure 1 is a graph showing the effect of different culture media on stem segment growth; A is a graph showing stem culture on T culture medium; B is a graph showing stem culture on four culture media: ZT, GT, GZT and T; Figure 4 The figure shows the effects of different explants on callus induction; A shows the growth of mature seeds as explants in medium A; B shows the growth of stems as explants in medium A; C shows the callus produced by mature seeds as explants; D shows the callus produced by stems as explants; Figure 5 The effects of different regeneration methods on regeneration efficiency; A is direct regeneration of seedlings using stem segment explants; B is indirect regeneration of seedlings through callus tissue; Figure 6 Kentucky bluegrass PpTCP4 PCR identification and sequence analysis of gene-edited plants, where A is Kentucky bluegrass PpTCP4 Electrophoresis diagram of PCR identification of hygromycin marker gene in gene-edited plants; B is the sequencing result of PCR product and protein sequence analysis of gene-edited plants; Figure 7 This is a microscopic image of mature seeds of Kentucky bluegrass; Figure 8 The figure shows the transformation and cultivation process of the PpTCP4 gene-edited plants of Kentucky bluegrass. The left side shows the Kentucky bluegrass callus after infection; the middle shows the Kentucky bluegrass adventitious bud regeneration; and the right side shows the Kentucky bluegrass adventitious bud rooting culture. Figure 9 These are the rooting and transplanting pictures of Kentucky bluegrass during the transformation and cultivation of Kentucky bluegrass PpTCP4 gene-edited plants. The left side shows the rooting and transplanting of Kentucky bluegrass for one month; the middle shows the transplanting and culturing of Kentucky bluegrass for five months; and the right side shows the transplanting and culturing of Kentucky bluegrass for seven months. Figure 10 This is a diagram showing the growth process of adventitious buds in callus tissue after infection with Kentucky bluegrass during the transformation and cultivation of Kentucky bluegrass PpTCP4 gene-edited plants; the left side is a diagram of callus tissue, the middle is a diagram of adventitious bud points appearing after infection of Kentucky bluegrass callus tissue; the right side is a diagram of adventitious bud regeneration after infection of Kentucky bluegrass callus tissue. DETAILED DESCRIPTION

[0018] The present invention provides PpTCP4 The application of genes in regulating tillering of Kentucky bluegrass, PpTCP4 The nucleotide sequence of the gene is shown in SEQ ID NO.8, PpTCP4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.9.

[0019] SEQ ID NO.8:

[0020] SEQ ID NO.9: MGDAGQSHHHHGLQTQLLSFGGGHHMHQFTAQALPPAASQTRARGGRGGGELVSAATPTRVRGGGGGEIVAVQGGHIVRSTGRKDRHSKVCTARGPRDRRVRLSAHTAIQFYD VQDRLGYDRPSKAVDWLIKNAKDAIDSLDTLPAWQPTAVVPAASNAAAQPSSSTHPDSAENSDDQAQAITIAHTSFDFGGSGQGGGGGATGGISFLPPSLDSDSIADTIKSFFP MGGTAGGEASSSTVAAHSSAMSFQSYTPDLLSRTGSQSQELRLSLQPLPDPMFHQQQQQQEPHRSHGHESNGNAQQPIFPGGANYPFGGGAMWGEQAQSQRMLPWSVPDPGGG GGSTGGYLFNVSQQAAHMQAALSGQNQFFFQRGPLQSSNQPSDRGWPETVEADSNNSMQQYHQGGLNPSVSAVGFAPGVSFSGFRIPARIQGDEEHNGGNGDKPPSVSSASHH.

[0021] The gene of the present invention PpTCP4 To negatively regulate the tillering gene, gene editing PpTCP4 The base mutation or deletion can achieve gene knockout PpTCP4 It plays a role in promoting the tillering of Poa annua.

[0022] The present invention provides a targeted knockout as described in the above technical solution PpTCP4 Gene or reduce as described in the above technical solution PpTCP4 The use of a gene expression reagent in promoting tillering of Kentucky bluegrass. PpTCP4 Gene or reduce PpTCP4 Gene expression can include PpTCP4 base mutation or deletion.

[0023] In the present invention, the targeted knockout PpTCP4 Gene knockout agents include targeted PpTCP4 CRISPR / Cas9 vector for targeting gene; the vector contains targeted knockout PpTCP4 sgRNA sequence of the gene.

[0024] The present invention provides a targeted knockout of Poa annua as described in the above technical solution PpTCP4The sgRNA of a gene, wherein the sgRNA sequence comprises PpTCP4- sgRNA-F and PpTCP4- sgRNA-R; PpTCP4- The nucleotide sequence of sgRNA-F is shown in SEQ ID NO. 4. PpTCP4- The nucleotide sequence of sgRNA-R is shown in SEQ ID NO.5; the PAM sequence recognized by the sgRNA is AGG. The sgRNA target sequence of the present invention is PpTCP4- sgRNA-F and PpTCP4- sgRNA-R primer annealing is obtained; PpTCP4- The nucleotide sequence of sgRNA-F is shown in SEQ ID NO.4; PpTCP4- The nucleotide sequence of sgRNA-R is shown in SEQ ID NO.5.

[0025] The present invention provides a bluegrass CRISPR / Cas9 gene editing system, comprising a pCAMBIA1300-Cas9 vector and the bluegrass gene targeted knockout method described in the above technical solution. PpTCP4 sgRNA sequence.

[0026] As an practicable method, the bluegrass gene PpTCP4 The nucleotide sequence of the target sequence is shown in SEQ ID NO.3; The present invention provides a method for constructing the bluegrass CRISPR / Cas9 gene editing system described in the above technical solution, comprising the following steps: Using the PpTCP4- sgRNA-F and PpTCP4- sgRNA-R is subjected to an annealing procedure to obtain sgRNA; Connecting the sgRNA and the pCAMBIA1300-Cas9 vector to obtain a connection product; The ligation product was transformed into Escherichia coli, and the positive colonies were expanded and cultured to extract the plasmid, and the gene editing pCAMBIA1300-Cas9- PpTCP4 System. The present invention has no special limitation on the connection parameters, and conventional methods can be used. The present invention has no special limitation on the transformation parameters, and conventional methods can be used. The present invention has no special limitation on the plasmid extraction parameters, and conventional methods can be used.

[0027] The present invention provides a gene editing method for bluegrass, comprising the following steps: using the CRISPR / Cas9 gene editing system described in the above technical solution or the gene editing pCAMBIA1300-Cas9- PpTCP4The system was transformed into Agrobacterium competent cells for genetic transformation to obtain gene-edited plants of Poa annua.

[0028] As an practicable manner, the bluegrass described in the present invention may include bluegrass.

[0029] As an practicable method, the present invention uses Agrobacterium-mediated genetic transformation, which may include: transforming an Agrobacterium bacterial solution containing the bluegrass CRISPR / Cas9 gene editing system into the callus tissue of bluegrass. The present invention does not specifically limit the operation steps of the Agrobacterium-mediated genetic transformation, and conventional methods can be used. Using callus tissue as an infection receptor has high regeneration efficiency.

[0030] As an practicable manner, the explant for inducing the production of bluegrass callus in the present invention may include mature seeds of bluegrass. Mature seeds are more suitable as explants for inducing callus, and the induced callus is light yellow and dense, and the callus induction rate is about 59.1%, which is suitable as an explant for the genetic transformation system of meadow bluegrass.

[0031] As an practicable manner, the culture medium for culturing the bluegrass callus of the present invention includes a ZT culture medium; the composition of the ZT culture medium of the present invention may include: MS as a basic culture medium, and also includes 200-250 mg / L TMT, 1.2-1.5 mg / L ZR, 25.0-35.0 g / L sucrose and 7.8-8.3 g / L agar. The content of the MS culture medium in the ZT culture medium of the present invention may be 4.43 g / L; the TMT concentration in the ZT culture medium may be 200-250 mg / L, or 210-240 mg / L; in a specific embodiment of the present invention, the TMT concentration may be 200, 210, 220, 230, 240 or 250 mg / L. The ZR concentration in the ZT medium can be 1.0-1.5 mg / L, or 1.0-1.2 mg / L; in a specific embodiment of the present invention, the ZR concentration can be 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 mg / L. The sucrose concentration in the ZT medium can be 25.0-35.0 g / L, or 25-30 g / L; in a specific embodiment of the present invention, the sucrose concentration can be 25, 27, 30, 31 or 35 g / L. The agar concentration in the ZT medium can be 7.8-8.3 g / L, or 7.8-8.0 g / L; in a specific embodiment of the present invention, the agar concentration can be 7.8, 7.9, 8.1 or 8.3 g / L. ZT medium can improve the ability of plants to tolerate experimental operation damage. The germination rate of bluegrass seeds is high, and the stems are relatively sturdy, which is more suitable for subsequent transformation operations.

[0032] As an practicable method, the present invention further includes identifying the bluegrass gene-edited plants, wherein the identification includes: using TCP4 -F and TCP4 -R primers were used to identify the target gene of the positive plants. PpTCP4 If there is a base mutation in the gene, the gene editing is considered successful; Said TCP4 - The nucleotide sequence of F is shown in SEQ ID NO.1; Said TCP4 The nucleotide sequence of -R is shown in SEQ ID NO.2.

[0033] As an practicable method, before identifying the bluegrass gene-edited plants, the present invention also includes screening positive plants using Hyg-F and Hyg-R primer pairs, wherein the nucleotide sequence of Hyg-F is shown in SEQ ID NO.6; the nucleotide sequence of Hyg-R is shown in SEQ ID NO.7.

[0034] The present invention provides a gene editing method for Kentucky bluegrass, which utilizes the CRISPR / Cas9 gene editing system to edit the genes of Kentucky bluegrass. PpTCP4 The gene was site-specifically edited, and the base mutation type of gene editing was determined by sequencing analysis of the PCR products. Gene-edited plants were successfully obtained, and the gene editing efficiency was 0.67%, laying a good foundation for the gene function research and genetic breeding of meadow fescue.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] 1. Preparation of tissue culture medium TMT mother solution (100 mg / mL): Take 10 g of Timentin (meilunbio®, Dalian Bogreen Biotechnology Co., Ltd.) powder, add water to make up to 100 mL, divide into aliquots, and store at -20°C.

[0037] 6-BA mother solution (4 mg / mL): Dissolve 400 mg of 6-benzylpurine powder in a small amount (about 3 mL) of 1 mol / L NaOH solution, add water to make up to 100 mL, filter and sterilize using a 0.45-µm syringe filter (Minisart®Filter Unit, Sartorius Stedim Biotech GmbH, Goettingen, Germany), and store in aliquots at -20°C.

[0038] 2,4-D mother solution (1 mg / mL): Dissolve 200 mg of 2,4-dichlorophenoxyacetic acid powder in a small amount (about 4 mL) of anhydrous ethanol solution, add water to make up to 200 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0039] NAA mother solution (2 mg / mL): Dissolve 100 mg of naphthaleneacetic acid powder in a small amount (about 3 mL) of 1 mol / L NaOH solution, add water to make up to 50 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0040] KT stock solution (1 mg / mL): Dissolve 100 mg of kinetin powder in a small amount (about 3 mL) of 1 mol / L NaOH solution, add water to make up to 100 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0041] ZR mother solution (2 mg / mL): Dissolve 100 mg of zeatin riboside powder in a small amount (about 3 mL) of 1 mol / L NaOH solution, add water to make up to 50 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0042] GA 3 Mother solution (1 mg / mL): Dissolve 100 mg of gibberellin powder in a small amount (about 4 mL) of anhydrous ethanol solution, add water to make up to 100 mL, filter and sterilize using a 0.45 µm syringe filter, then divide into aliquots and store at -20°C.

[0043] CH mother solution (10 mg / mL): Dissolve 500 mg of hydrolyzed casein powder in ultrapure water, make up to 50 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0044] ABA stock solution (2 mg / mL): Dissolve 100 mg of abscisic acid powder in a small amount (about 3 mL) of 1 mol / L NaOH solution, add water to make up to 50 mL, filter and sterilize using a 0.45 µm syringe filter, then aliquot and store at -20°C.

[0045] In the present invention, MS medium was purchased from Phyto Tech LABS; 2,4-D, IAA, IBA, ZR, KT, NAA, 6-BA, CH, ABA and GA3 were purchased from BBI, and LB medium, sucrose and agar were purchased from Sangon (Sangon Biotechnology (Shanghai) Co., Ltd.).

[0046] 2. The preparation of infection solution and culture medium is illustrated using 1000 mL as an example.

[0047] Infection solution: 1000 mL ddH 2 O +4.43 g MS + 30 g sucrose, pH 5.8, autoclave and cool before use.

[0048] Complete MS medium: 1000 mL ddHO 2 O + 4.43g MS + 30g sucrose, adjust the pH to 5.8 and add 8g agar, sterilize by high pressure and cool to about 50℃, add the corresponding concentration of hormones in the clean bench and mix well, then pour into the sterilized culture dish.

[0049] The concentrations of hormones added to different culture media are as follows, see Table 1.

[0050] Table 1 Plant tissue culture medium formula

[0051] 3. Preparation of bacterial culture medium Cef mother solution (250 mg / mL): Weigh 25 g of ceftriaxone sodium powder, add water to make up to 100 mL, dispense into 1.5 mL EP tubes, place in sealed bags, and store at -20°C.

[0052] Kan + Mother solution (50 mg / mL): Weigh 5 g of kanamycin sulfate powder, add water to make up to 100 mL, filter and dispense into 1.5 mL EP tubes, place in sealed bags, and store at -20°C.

[0053] Hyg mother solution (50 mg / mL): Weigh 5 g of hygromycin B powder, add water to make up to 100 mL, filter and dispense into 1.5 mL EP tubes, place in a sealed bag, and store at -20°C.

[0054] AS mother solution (200 mM): Dispense the acetosyringone mother solution into 1.5 mL EP tubes and place them in sealed bags at -20°C.

[0055] Rif mother solution (50 mg / mL): Weigh 5 g of rifampicin powder, add water to make up to 100 mL, filter and dispense into 1.5 mL EP tubes, place in a sealed bag, and store at -20°C.

[0056] In the present invention, ceftriaxone sodium, ampicillin sodium, kanamycin sulfate, and hygromycin B were purchased from BBI Life Sciences Co., Ltd., LB medium, sucrose, and agar were purchased from Sangon Biotech (Shanghai) Co., Ltd., and acetosyringone and rifampicin were purchased from Beijing Coolbo Technology Co., Ltd.

[0057] 4. The preparation of culture medium is described using 100 mL as an example.

[0058] LB liquid medium: 100 mL ddHO 2 O + 2.5 g LB powder.

[0059] LB solid medium: 100 mL ddHO 2 O + 2.5 g LB powder + 1.5 g agar.

[0060] Kan + Liquid culture medium: Sterilize LB liquid culture medium and add 100 µL Kan + Mother liquor.

[0061] Kan + Solid medium: Sterilize LB solid medium and add 100 µL Kan + Mother liquor.

[0062] RK liquid medium: Sterilize LB liquid medium and add 100 µL Kan + Stock solution, 200 µL Rif stock solution.

[0063] RK solid medium: LB solid medium sterilized with 100 µL Kan + Stock solution, 200 µL Rif stock solution.

[0064] 5. The English abbreviations in this invention are as follows: 6-BA: 6-benzylaminopurine. GA 3 : Gibberellin. CH: Hydrolyzed casein. KT: Kinetin. ZR: Zeatin riboside. 2,4-D: 2,4-dichlorophenoxyacetic acid. ABA: Abscisic acid. TMT: Timentin. Rif: Rifampicin. MS: MS medium. LB: LB medium. PCR: polymerase chain reaction. ddH 2 O: ultrapure water. AS: acetosyringone.

[0065] Example 1 1. PpTCP4 sgRNA target site selection and primer design The full-length transcriptome sequence of Kentucky bluegrass was used to design the primers using the online software Primer3 (https: / / bioinfo.ut.ee / primer3-0.4.0 / ). PpTCP4 Gene-specific primers TCP4 -F: 5' -CACCACATGCACCAGTTCAC -3' (SEQ ID NO: 1) and TCP4-R: 5' -CATGGGGAAGAAGGACTTGA -3' (SEQ ID NO: 2). The parameters of primer design were set as follows: Product Size Ranges: 700-800; Primer Size: Max 25; Max Tm Difference: 2; Primer GC%: Min 40, Max 60. Then, PCR amplification was performed using DNA from four wild-type Kentucky bluegrass strains WT1, WT2, WT3, and WT4 as templates. PpTCP4 Gene fragments ( Figure 1 ), the sequencing results showed that the consistent wild-type Kentucky bluegrass 575 bp PpTCP4 The gene fragments are all located in the CDS region. The nucleotide sequence of the PpTCP4 gene is shown in SEQ ID NO:8, and the amino acid sequence of the protein encoded by the PpTCP4 gene is shown in SEQ ID NO:9. PCR amplification system: 2×mix 25 μL, TCP4 -F 2.5 μL, TCP4 -R 2.5 μL, DNA template 2.5 μL and dd H 2 O17.5 μL. PCR amplification program: 94°C, 5 min, 1 cycle; 95°C, 30 s, 32 cycles; 55°C, 30 s, 32 cycles; 72°C, 1 min, 32 cycles; 72°C, 10 min, 1 cycle; 12°C, storage.

[0066] PCR amplification yielded a 575 bp PpTCP4 Gene fragments, PpTCP4 The nucleotide sequence of the gene is shown in SEQ ID NO.8, PpTCP4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.9. The recovered product was subjected to Sanger sequencing, and the sequencing results were compared using MEGA7.0 software. Five CDS regions with consistent sequencing results were selected for subsequent sgRNA design.

[0067] Will get PpTCP4 The CDS gene sequence of the gene was input into the targetDesign online target design software (http: / / skl.scau.edu.cn / targetdesign / ), and the target site was designed according to the construction method of the gene editing vector pCAMBIA1300. A total of 15 suitable sgRNA target sequences were screened, and the target sequence and the full-length transcriptome of Kentucky bluegrass were locally blasted, and the sequence with high specificity was selected as the sgRNA target sequence to synthesize the corresponding primers. The final selected target sequence and guide RNA (sgRNA) are shown in Table 2 below.

[0068] Table 2 Kentucky bluegrass TCP4 Gene target sequence and sgRNA synthetic sequence

[0069] Note: The underlined area is the PAM box.

[0070] 2. Construction of CRISPR / Cas9 gene editing vector (1) Annealing and bonding The primers in Table 2 PpTCP4- sgRNA-F and PpTCP4- Take 1 μL of each sgRNA-R and add it to 8 μL of annealing buffer (Tris-EDTA buffer (TE for short) + 50 mM NaCl) and mix it by gently pipetting. Run the following annealing program (slowly cooling from 95°C to 16°C at a rate of 0.1°C / s) on a PCR instrument (Veriti-ABI Veriti 96-well gradient PCR amplifier, Applied Biosystems, Inc.), and connect the obtained annealed product sgRNA and the gene editing vector pCAMBIA1300-Cas9 after enzyme digestion to obtain a connection product. The nucleotide sequence of Cas9 is shown in SEQ ID NO.10:

[0071] Gene editing vector recombination system: 10×Cutsmart Buffer 1.5μL, T4 DNA Ligase Buffer 0.6 μL, vector 0.8μL, annealing product 4μL, Bsa1-HF 1μL, T4 DNA ligase 0.1μL and dd H2O 7 μL.

[0072] Gene editing vector recombination procedure: 37°C, 5 min, 15 cycles; 10°C, 5 min, 15 cycles; 20°C, 5 min, 15 cycles; 37°C, 5 min, 12 cycles; 12°C, store.

[0073] (2) E. coli transformation Take 10 μL of the ligation product and add it to 100 μL of Escherichia coli DH5α competent cells. Heat shock the cells in a 42℃ water bath. After 90 s of heat shock, immediately insert the cells on ice for 2 min and add 600 μL of liquid LB medium without antibiotics. Incubate the cells at 37℃ at 200 rpm for 60 min. Centrifuge at 5,000 rpm for 2 min to collect the bacteria. Use a pipette to aspirate 300 μL of the supernatant. Mix the remaining supernatant and the precipitate by pipetting. Apply 100 μL of the supernatant to Kan with sterile glass beads. + Solid culture medium. Incubate inverted in a 37°C incubator for 12-16 h. After bacterial plaques grow, pick a single colony and place it in 400 μL Kan + In liquid culture medium, shake at 37°C and 200 rpm for 5-6 h before using the identification primers TCP4 -F. TCP4 -R for bacterial liquid PCR identification (see Figure 2 A). Identification primers (SEQ ID NO: 1 and SEQ ID NO: 2), reaction system and procedure are the same as those of Example 1 PpTCP4 Gene amplification primers, PCR amplification system and PCR amplification procedures are the same. Figure 2 As shown in Figure A, the gene editing vector has been successfully transformed into Escherichia coli DH5α. Figure 2 The 1~4 in A represent WT1, WT2, WT3, and WT4 respectively.

[0074] (3) Plasmid extraction Preparation process: PCR identified positive bacterial liquid and sequenced it, and compared it with the predicted sequence of the final vector. The correct bacterial liquid was marked as PpTCP4 Take 100 μL of the identified bacterial solution and add it to 6 mL of Kan +The bacteria were shaken in liquid culture medium and the plasmid was extracted using the TIANprep Mini Plasmid Kit. The plasmid concentration and quality were detected using a NanoDrop1000 ultra-micro spectrophotometer and stored in a -20°C refrigerator. After sequencing analysis, the plasmid with successful vector construction was transferred into Agrobacterium LBA4404 for subsequent infection.

[0075] (4) Electrotransformation of Agrobacterium Preparation: 600 µL of LB liquid medium without resistance, Agrobacterium LBA4404 competent cells and a pre-cooled and dry clean electrode cup.

[0076] Electroporation process: Add 3 µL of the extracted plasmid to the LBA4404 competent cells melted on ice, gently pipette to mix, then add to the pre-cooled and dried electrode cup, set the electroporator (BIO-RAD MicroPulser, Bio-RadLaboratories, Inc.) gear to Agr gear for electroporation, then add 600 µL of LB liquid culture base to the electroporation cup, pipette to mix, transfer the bacterial liquid to a new 1.5 mL centrifuge tube, culture at 28°C, 200 rpm for 2 hours, centrifuge at 5000 rpm for 2 minutes, aspirate about 300 µL of supernatant, pipette the remaining supernatant and precipitate to mix, aspirate 10 µL to apply to RK solid culture medium, place in a 28°C incubator and invert for about 48 hours. Pick a single colony in 1 mL of RK liquid culture medium, culture at 200 rpm at 28°C for 36~48 hours, then take a sample for PCR identification, see Figure 2 According to B. Figure 2 From B, we can see that the gene editing vector has been successfully transformed into Agrobacterium LBA4404. Figure 2 The 1~4 in B represent WT1, WT2, WT3, and WT4 respectively.

[0077] The correctly identified bacterial solution was mixed with an equal volume of 50% glycerol and stored in a -80°C refrigerator.

[0078] 3. Cultivation of sterile seedlings of Kentucky bluegrass Select the seeds of Kentucky bluegrass with full seeds and pack them into 1.5 mL EP tubes (about 80 seeds per tube), add 1 mL of 4% sodium hypochlorite solution for disinfection for 5 min, and then use sterile ddH 2 After cleaning with sterile ddHO for 3 times, disinfect with 1 mL of 4% sodium hypochlorite for 5 min. 2O Wash the seeds until the water for washing the seeds is colorless and transparent. Use sterile filter paper to absorb the surface moisture of the seeds, and then evenly place the seeds on 4 germination media, namely, medium ZT, medium GT, medium GZT and medium T. The composition of the 4 germination media is shown in Table 1, and 3 replicates are set for each culture medium group. Culture conditions: temperature 25 ± 2℃, photoperiod of 16h / d light and 8h / d dark, and light intensity of about 2000lx. After 14 days of culture, the seed germination rate was counted, and the stem circumference of the meadow bluegrass seedlings 1 mm away from the root in the 4 culture media was measured, see Table 3.

[0079] The growth of the infected Kentucky bluegrass stem segments cultured in T medium was weak, and they were easily damaged by mechanical force and bacterial infection, and the mortality rate after infection was high ( Figure 3 The seeds of Kentucky bluegrass were cultured in ZT, GT, GZT and T medium with different plant hormone ratios. After 14 days of culture, it was found that the germination rate of Kentucky bluegrass seeds on ZT medium was higher, about 82.5%, and the plants grew well; the Kentucky bluegrass plants cultured in GT and GZT medium grew best, but the seed germination rate was relatively low, about 57.7%; the seed germination rate in T medium was the highest, about 87.3%, but the growth was weak (Table 3, Figure 3 Middle B).

[0080] Table 3 Effects of different culture media on germination and growth of Kentucky bluegrass

[0081] Note: Germination rate is the percentage of germinated seeds to the total number of seeds. Data were analyzed by one-way ANOVA (Duncan test), and lowercase letters indicate a significant level of a=0.05.

[0082] 4. Induction culture of Kentucky bluegrass callus (1) Callus culture using mature embryos of Kentucky bluegrass as explants Select the seeds of Kentucky bluegrass with full seeds, peel them and dispense them into 1.5 mL EP tubes (about 80 seeds per tube). Disinfection is the same as step 3. Use sterile filter paper to absorb the surface moisture of the seeds and evenly place them on callus induction medium A. Culture them in the dark at 25 ± 2℃. Callus tissue will grow after about one month. The selected volume is about 20mm 3 Infection of callus tissue.

[0083] (2) Callus induction culture using Kentucky bluegrass stem segments as explants According to the treatment method in step 3, the seeds of Kentucky bluegrass were cultured on medium ZT for about 14 days. When the seedlings grew to a height of about 6-8 cm, the seedling stem segments were cut about 1-2 mm away from the roots in the clean bench. Each segment was about 3-4 mm long, and about 3-4 segments were cut for each plant. The stem segments were evenly placed in medium A to induce callus tissue. They were cultured in the dark at 25 ± 2℃. Callus tissue grew after about 30 days. The selected volume was about 20mm 3 Infection of callus tissue.

[0084] Mature seeds and sterile stem segments of Kentucky bluegrass were cultured on medium A to induce callus tissue. The results are shown in the following figure ( Figure 4 As shown in A and B in the figure, the callus produced by mature seeds is light yellow in color and has a dense structure, while the callus induced by stem segments is water-soaked and loose ( Figure 4 C and D). The callus rate of mature seeds was about 59.1%, while that of stem segments was only about 8.2% (Table 4). The mature seeds of Kentucky bluegrass were more suitable as explants for callus induction.

[0085] Table 4 Callus induction rate of different explants

[0086] Note: The callus rate is the percentage of callus number to the number of explants. Data were analyzed by one-way analysis of variance (Duncan test), and lowercase letters indicate a significant level of a=0.05.

[0087] Regenerated plants can be obtained from callus induced from stem segment explants and seeds ( Figure 5 ), among which the regeneration efficiency of stem segment explants was about 2.52%, and the regeneration efficiency of callus tissue was about 4.67% (Table 5). Therefore, inducing the differentiation and budding of Kentucky bluegrass callus tissue is a more efficient regeneration method.

[0088] Table 5 Effect of different regeneration methods on regeneration efficiency

[0089] Note: The regeneration ratio is the percentage of regenerated plants to callus numbers; data were analyzed by one-way analysis of variance (Duncan test), and lowercase letters indicate a significant level of a=0.05.

[0090] 5. Agrobacterium tumefaciens-mediated transformation of Kentucky bluegrass (1) Method of infection of Kentucky bluegrass callus 1) Activation of Agrobacterium tumefaciens After thawing the successfully transformed Agrobacterium LBA4404 bacterial solution stored at -80°C in step 2 (4) of Example 1 on ice, pipette 1 mL into a 10 mL centrifuge tube, add 3 mL of RK liquid culture medium, and culture in a 28°C shaker at 200 rpm for 24 to 48 h. Then, pipette 100 μL of the bacterial solution onto RK solid culture medium and culture in a 28°C incubator for 24 to 48 h.

[0091] 2) Preparation of infection solution In the clean bench, add 4-5 mL of MS liquid medium to the RK solid medium full of Agrobacterium, gently scrape the Agrobacterium on the medium with a sterile pipette tip and blow to mix, then pipette about 600 μL of bacterial solution into a 50 mL centrifuge tube, add an appropriate amount of MS liquid medium to raise the bacterial solution OD 600 After adjusting the value to 0.7-0.8, add AS (50 mL bacterial solution + 50 μL AS mother solution) and shake evenly.

[0092] 3) Kentucky bluegrass callus infection and culture In a clean bench, take the Kentucky bluegrass callus (volume about 20 mm2) that has been dark-cultured for 30 days in step 4 (2) of Example 1. 3 ), put it into the prepared infection solution, and shake it gently for about 20 minutes to allow it to be fully infected. Gently wipe the infected callus dry with sterile filter paper and place it on medium A for dark culture for 2 days.

[0093] In a clean bench, add 500 mL of sterilized ddHO. 2 Add 1 mL of Cef stock solution to the O and shake well to make a washing solution. Place the callus that has been dark cultured for 2 days in a sterile 200 mL Erlenmeyer flask, add about 100 mL of washing solution, gently shake and wash for about 10 minutes, then move the callus to another clean Erlenmeyer flask and wash again, for a total of 5 times. Then take out the callus, wipe it dry with sterile filter paper, and place it on medium A for dark culture for 14 days.

[0094] 4) Bud differentiation culture After 14 days of dark culture, the callus was transferred to 6T medium for bud differentiation culture. Fresh 6T medium was replaced every 14 days. After about 3 months, part of the callus turned green and produced buds to grow adventitious buds.

[0095] The culture conditions for bud differentiation were as follows: temperature 25 ± 2°C, photoperiod of 16 h / d light and 8 h / d dark, and light intensity of 2000 lx.

[0096] 5) Screening and cultivation The callus tissue with adventitious buds was transferred to 6T+Hyg screening medium, and after 14 days, the surviving pratense seedlings were transferred to rooting medium T.

[0097] 6) Rooting culture and identification of gene-edited plants After about 30 days of rooting culture, leaves were cut to identify whether they were gene-edited plants.

[0098] (2) Direct infection of Kentucky bluegrass stems 1) Direct infection of Kentucky bluegrass stems The Kentucky bluegrass seeds were cultured for about 14 days. When the seedlings grew to a height of about 6-8 cm, 3-4 mm long stem segments were cut and immersed in the prepared infection solution (prepared in the same manner as in step 5 of Example 1, 1)-2)) in a clean bench, and gently shaken for about 10 minutes to allow for full infection. The infected stem segments were gently wiped dry with sterile filter paper and evenly placed on T culture medium for dark culture for 2 days.

[0099] 2) Bud induction culture Transfer the dark-cultured stem segments to T culture medium. After 13 to 20 days, some of the stem segments will swell, buds will appear, and small sprouts will grow.

[0100] 3) Screening and cultivation The seedlings grown on the stem segments were cut and transferred to the screening medium T + Hyg. After 14 days of culture, the surviving seedlings were transferred to T rooting medium for rooting culture.

[0101] 4) Rooting culture and identification of gene-edited plants After about 30 days of rooting culture, leaves were taken to identify whether they were gene-edited plants.

[0102] 6. DNA extraction and identification of gene-edited materials (1) Leaf DNA extraction The specific steps are as follows: The preparation method of CTAB extract is as follows: 2g CTAB, 2g PVP, 8.18g NaCl, 4mL EDTA solution (pH8.0), 10mL Tris-HCl (pH8.0) and 200 µL β-mercaptoethanol are diluted to 100mL with distilled water to obtain CTAB extract, wherein the molar concentration of EDTA solution is 20mM and the molar concentration of Tris-HCl is 100mM.

[0103] Weigh 0.5 g of Kentucky bluegrass leaves, place them in a 1.5 mL centrifuge tube containing 2-3 magnetic beads, freeze them in liquid nitrogen, and grind them for 90 s (60 Hz) using a grinder (Tissuelyser-24L multi-sample tissue grinder, Shanghai Jingxin Industrial Development Co., Ltd.). Then add 700 μL of CTAB extract to the centrifuge tube, mix thoroughly, and place in a 65°C water bath for 15-30 min, then add 700 μL of chloroform and isoamyl alcohol, the volume ratio of chloroform to isoamyl alcohol is 24:1, mix for 5 min, and centrifuge at 12,000 rpm for 10 min. Take the supernatant and put it in a new 1.5 mL centrifuge tube, add 500 µL isopropanol and mix well. After standing at -20℃ for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant and add 400 µL 70% alcohol to resuspend the precipitate, centrifuge at 12000 rpm for 2 min, discard the supernatant, and repeat the operation of adding 70% alcohol for more than 3 times. Discard the supernatant and dry it, add 50 µL ddH 2 O, and store in a -20℃ refrigerator after the precipitate is dissolved.

[0104] (2) Identification of gene-edited materials Using the extracted leaf DNA as a template, the universal primers (Hyg-F / R) of the hygromycin marker gene were used to preliminarily identify the gene-edited plants. The PCR products were detected by agarose gel electrophoresis. The gene-edited plants with positive preliminary identification were selected according to the results, and the relevant genes were cloned. Then, the positive PCR products with the same size as the target gene band were selected for sequencing. The sequencing results and the CDS sequence of the target gene were compared with the MAGE7.0 software. If the edited DNA sequence exists, the gene editing is judged to be successful. The primers used for the gene cloning involved in this step are: Hyg-F: 5'-ATGAAAAAGCCTGAACTCACCGCGACG-3' (SEQ ID NO: 6); Hyg-R: 5'-CTATTTCTTTGCCCTCGGACGAGTGCT -3' (SEQ ID NO: 7); TCP4 -F: 5' -CACCACATGCACCAGTTCAC -3' (SEQ ID NO: 1); TCP4 - R: 5' -CATGGGGAAGAAGGACTTGA-3' (SEQ ID NO: 2). 50µL reaction system: 2×Phanta Max Buffer 25µL, dNTP Mix (10 Mm each) 1µL, Phanta Max Super-Fidelity DNA Polymerase 1µL and ddH 2O 17µL, Hyg-F 2µL, Hyg-R 2µL, template 2µL. PCR cloning reaction program: 95℃, 3 min, cycle 1 time; 95℃, 30s, cycle 32 times; 55℃, 30s, cycle 32 times; 72℃, 50s, cycle 32 times; 72℃, 10min, cycle 1 time; 12℃, store.

[0105] The extracted DNA from Kentucky bluegrass leaves was used as a template, and the universal primers (Hyg-F / R) of the hygromycin marker gene were used to preliminarily identify the positive plants. According to the agarose gel electrophoresis detection results, the positive plants were preliminarily identified (the PCR product was consistent with the target gene band size for the positive plant, and the band could not be detected in the wild-type Kentucky bluegrass), and the gene was cloned and sequenced. The sequencing results and the CDS sequence of the target gene were compared with the MAGE7.0 software. If the edited DNA sequence was present, it was determined that the gene editing of the plant was successful and it was a gene-edited plant. In this embodiment, a total of 450 callus tissues were infected, and a total of 21 regenerated plants grew out, of which 11 were positive plants ( Figure 6 The gene editing efficiency was 0.67% (Table 6). PpTCP4 The sgRNA of the gene-edited plant has base mutations, but some mutation types do not lead to changes in amino acids ( Figure 6 The results showed that gene editing occurred near the PAM sequence, mainly base mutations. After that, the gene-edited seedlings that had been rooted for about 30 days were transplanted and planted in the soil. PpTCP4 The transformation and cultivation process of gene-edited plants can be found in Figures 7 - 10 . Kentucky bluegrass PpTCP4 The tillering number of gene-edited plants increased compared to those before editing. Figure 6 Kentucky bluegrass PpTCP4 PCR identification and sequence analysis of hygromycin marker genes in gene-edited plants, where A is Kentucky bluegrass PpTCP4 A is the electrophoresis diagram of PCR identification of hygromycin marker gene in gene-edited plants; B is the sequencing results of PCR products and protein sequence analysis of gene-edited plants. The calculation formulas for regeneration ratio, transformation efficiency and editing efficiency are as follows, and the results are shown in Table 6. Regeneration ratio = number of regenerated plants / number of calli × 100%; transformation efficiency = number of positive plants / total number of calli × 100%; editing efficiency = number of gene-edited plants / total number of calli × 100%.

[0106] Table 6 Gene editing efficiency

[0107] In summary, the present invention uses Kentucky bluegrass collected in Qinghai as the research object, and identifies the key factors regulating tillering from the full-length transcriptome. PpTCP4Based on the optimization of the genetic transformation system of Kentucky bluegrass, a CRISPR / Cas9 gene editing system for Kentucky bluegrass was established and site-directed editing was performed. PpTCP4 Gene, and finally the gene-edited plants were identified and successfully obtained.

[0108] 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. PpTCP4 The application of genes in regulating tillering of Kentucky bluegrass, PpTCP4 The nucleotide sequence of the gene is shown in SEQ ID NO.

8.

2. Targeted knockout as described in claim 1 PpTCP4 Gene or reduction as claimed in claim 1 PpTCP4 Application of gene expression reagents in promoting tillering of Kentucky bluegrass.

3. The use according to claim 2, characterized in that: The targeted knockout PpTCP4 Gene knockout agents include targeted PpTCP4 CRISPR / Cas9 vector for targeting gene; the vector contains targeted knockout PpTCP4 sgRNA sequence of the gene.

4. A targeted knockout of Poa annua as claimed in claim 1 PpTCP4 The sgRNA of a gene is characterized by: The sgRNA sequence includes PpTCP4- sgRNA-F and PpTCP4- sgRNA-R; PpTCP4- The nucleotide sequence of sgRNA-F is shown in SEQ ID NO.

4. PpTCP4- The nucleotide sequence of sgRNA-R is shown in SEQ ID NO.5; the PAM sequence recognized by the sgRNA is AGG.

5. A Poa annua CRISPR / Cas9 gene editing system, characterized in that: Comprising pCAMBIA1300-Cas9 vector and the targeted knockout bluegrass gene of claim 4 PpTCP4 sgRNA sequence.

6. The bluegrass CRISPR / Cas9 gene editing system according to claim 5, characterized in that Poa annua gene PpTCP4 The nucleotide sequence of the target sequence is shown in SEQ ID NO.

3.

7. The method for constructing the bluegrass CRISPR / Cas9 gene editing system according to claim 5 or 6, characterized in that: The following steps are involved: Using the method described in claim 4 PpTCP4- sgRNA-F and PpTCP4- sgRNA-R was subjected to an annealing procedure to obtain sgRNA; Connecting the sgRNA and the pCAMBIA1300-Cas9 vector to obtain a connection product; The ligation product was transformed into Escherichia coli, and the positive colonies were expanded and cultured to extract the plasmid, and the gene editing pCAMBIA1300-Cas9- PpTCP4 system.

8. A method for editing the gene of Poa annua, comprising the following steps: The CRISPR / Cas9 gene editing system of claim 5 or 6 or the gene editing pCAMBIA1300-Cas9-sgRNA- PpTCP4 The system was transformed into Agrobacterium competent cells for genetic transformation to obtain gene-edited plants of Poa annua.

9. The gene editing method of Poa annua according to claim 8, characterized in that: Agrobacterium-mediated genetic transformation is used, including: transforming Agrobacterium liquid containing the Poa annua CRISPR / Cas9 gene editing system into the callus tissue of Poa annua; The explant for inducing the production of bluegrass callus tissue comprises mature seeds of bluegrass; the culture medium for culturing the bluegrass callus tissue comprises ZT culture medium; the composition of the ZT culture medium comprises: MS as the basic culture medium, and also comprises 200-250 mg / L TMT, 1.0-1.5 mg / L ZR, 25.0-35.0 g / L sucrose and 7.8-8.3 g / L agar.

10. The gene editing method of Poa annua according to claim 8, characterized in that: The invention also includes identifying the bluegrass gene-edited plants, wherein the identification includes: using TCP4 -F and TCP4 -R primers were used to identify the target gene of the positive plants. PpTCP4 If there is a base mutation in the gene, the gene editing is considered successful; Said TCP4 - The nucleotide sequence of F is shown in SEQ ID NO.1; Said TCP4 The nucleotide sequence of -R is shown in SEQ ID NO.2.

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