Gene editing technology system based on barley variety flower 22 immature embryo transformation

Abstract

The invention belongs to the technical field of genetic engineering. The invention provides a gene editing technology system based on barley variety flower 22 immature embryo transformation, which comprises the following steps: (1) taking barley immature embryos as explants, carrying out pretreatment, and optimizing infection and screening conditions in an agrobacterium tumefaciens transformation process; (2) designing aiming at an exon segment of a target gene to obtain a target sequence sgRNA, designing and synthesizing HvPDSCrp-F and HvPDSCrp-R by utilizing the target sequence sgRNA, and then constructing a TaU3-CAS9-pds gene editing vector; and (3) infecting barley immature embryos by using an agrobacterium mixed bacterium solution containing the gene editing carrier in the step (2), then carrying out induced culture, differentiating regenerated seedlings, and inducing rooting to obtain gene editing plants. The technical scheme provides a powerful way for functional gene mining, germplasm innovation and the like in barley.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene editing technology system based on the transformation of immature embryos of the barley variety Hua 22. Background Technology

[0002] Gene editing technology, capable of precisely editing genome sequences, has already been achieved in many plants, and its application has led to rapid advancements in plant molecular biology. The combination of gene editing technology and breeding can enable the targeted cultivation of disease-resistant, stress-resistant, high-quality, and high-yielding crop varieties, while avoiding the safety issues associated with conventional genetically modified organisms (GMOs). Establishing gene editing technology in more species and materials is essential.

[0003] Barley is the world's fourth largest cereal crop, highly adaptable to various environments and widely cultivated. Currently, the entire barley genome has been sequenced, which will greatly advance the development of barley molecular biology. Furthermore, extensive research into the function of target genes requires the support of barley gene editing technology.

[0004] Among the reported barley gene editing methods, most use a few barley varieties such as GoldenPromise as recipient materials, and gene editing research in conventional barley varieties in my country is scarce. Many excellent agronomic traits and functional genes have also been discovered in different barley materials. Therefore, establishing gene editing technology in more barley varieties is necessary for gene function research and molecular breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a gene editing technology system based on the transformation of the immature embryo of the barley variety Hua 22. Using this method, transgenic barley plants can be cultured, providing a powerful approach for gene function verification and germplasm innovation in barley.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a gene editing technology system based on the transformation of the immature embryos of the barley variety Hua 22. The method for constructing the gene editing technology system includes the following steps:

[0008] (1) Using barley embryos as explants, pretreatment was performed and screening conditions and Agrobacterium transformation and infection conditions were optimized;

[0009] (2) Target sequence sgRNA was designed for the exon region of the target gene, and HvPDScrp-F and HvPDScrp-R were designed and synthesized using the target sequence sgRNA. Then, the TaU3-CAS9-pds gene editing vector was constructed.

[0010] (3) Barley embryos were infected with Agrobacterium mixed bacterial solution containing the gene editing vector described in step (2), and then induced to grow, differentiate and regenerate seedlings and induce rooting to obtain transgenic plants.

[0011] Preferably, the pretreatment time of barley embryos in step (1) is 0.5-1.5 days; the infection time in step (1) is 35-45 minutes; and hygromycin is used for screening in step (1) at a concentration of 30-60 mg / L.

[0012] Preferably, the exon segment of the target gene in step (2) is the 62nd-81st segment of the first exon of the PDS gene.

[0013] Preferably, the sequence of the sgRNA in step (2) is shown in SEQ ID NO.2, the sequence of the HvPDScrp-F is shown in SEQ ID NO.3, and the sequence of the HvPDScrp-R is shown in SEQ ID NO.4.

[0014] Preferably, the construction process of the TaU3-CAS9-pds gene editing vector in step (2) includes the following steps:

[0015] (a) Linearized vectors were obtained by digesting TaU3-Cas9 as the original vector;

[0016] (b) The annealed products obtained by annealing HvPDScrp-F and HvPDScrp-R are subjected to Golden Gate enzyme digestion and ligation reaction with the linearized vector described in step (a) to obtain the final product.

[0017] Preferably, the annealing reaction system in step (b) includes 1 μL HvPDScrp-F, 1 μL HvPDScrp-R, and 8 μM Anneal Buffer; the annealing procedure includes: 95℃ for 10 min, then decreasing to 20℃ at 0.1℃ / s.

[0018] Preferably, step (b) after the Golden Gate enzyme digestion and ligation reaction further includes transforming the obtained ligation product into competent cells for culture, screening for positive clones, and identifying them by colony PCR.

[0019] Preferably, the primers used in the colony PCR identification process include M13F-47 and HvPDScrp-R, and the sequence of M13F-47 is shown in SEQ ID NO.5.

[0020] Preferably, the Agrobacterium used in steps (1) and (3) is EHA105.

[0021] This invention also provides the application of the gene editing technology system in site-directed mutagenesis of other genes in barley.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects:

[0023] This invention optimizes the genetic transformation process of barley by designing a target sequence targeting the coding region of the PDS gene, constructing a gene-editing vector, and using Agrobacterium-mediated transformation of barley embryos followed by tissue culture to obtain mutant plants. This provides a powerful approach for functional gene discovery and germplasm innovation in barley. Attached Figure Description

[0024] Figure 1 The effect of different hygromycin concentrations on callus formation in immature embryos;

[0025] Figure 2 To investigate the effect of different dark culture times on the infection efficiency of immature embryos in induction medium;

[0026] Figure 3 The effect of different shaking times on the infection efficiency of immature embryos;

[0027] Figure 4 The positive embryonic embryo after 3 days of co-culture as described in this invention (ruby gene as indicator gene);

[0028] Figure 5 This is a schematic diagram of the PDS gene editing vector TaU3-Cas9-pds described in this invention;

[0029] Figure 6 The regenerated seedlings obtained through infection as described in this invention;

[0030] Figure 7 This is a diagram showing the PCR positive verification results of the regenerated seedlings described in this invention.

[0031] Figure 8 This is a diagram showing the sequencing results of the regenerated seedling described in this invention. Detailed Implementation

[0032] This invention provides a gene editing technology system based on the transformation of the immature embryos of the barley variety Hua 22. The method for constructing the gene editing technology system includes the following steps:

[0033] (1) Using barley embryos as explants, pretreatment was performed and screening conditions and Agrobacterium transformation and infection conditions were optimized;

[0034] (2) Target sequence sgRNA was designed for the exon region of the target gene, and HvPDScrp-F and HvPDScrp-R were designed and synthesized using the target sequence sgRNA. Then, the TaU3-CAS9-pds gene editing vector was constructed.

[0035] (3) Use Agrobacterium mixed bacterial solution containing the gene editing vector described in step (2) to infect barley embryos, then induce culture, differentiate and regenerate seedlings, and induce rooting to obtain gene-edited plants.

[0036] In this invention, barley embryos are used as explants, which are pretreated and the concentration of hygromycin is optimized. The preferred barley variety in this invention is Hua 22 (Lang Shuping, Gong Laiting, Huang Jianhua. Breeding process and characteristics of new barley variety “Hua 22” [J]. Barley and Cereal Science, 2006, (04): 18-19. DOI: 10.14069 / j.cnki.32-1769 / s.2006.04.006.).

[0037] In this invention, the pretreatment process of the explant preferably includes: (1) selecting young spikelets 7-15 days after flowering and removing the glumes; (2) soaking the seeds in a disinfectant solution for shaking treatment, then removing the disinfectant solution and adding sterile water for continued shaking treatment, then removing the sterile water and rinsing, repeating step (2) once; (3) taking 1.5-2 mm embryos, cutting off the embryonic buds, placing the scutellum upwards on the induction medium, and culturing in the dark at 26°C for 3 days. In this invention, the disinfectant solution is preferably sodium hypochlorite, and the mass concentration of the disinfectant solution is preferably 8-12%, more preferably 9-11%, and even more preferably 10%.

[0038] In this invention, the pretreatment operation is preferably performed by placing the harvested barley embryos in an induction medium and culturing them in the dark at 26°C. The pretreatment time (dark culture time) of the barley embryos in this invention is 0.5-1.5 days, more preferably 0.8-1.2 days, and even more preferably 1 day.

[0039] In this invention, hygromycin is used for screening, and the screening concentration is preferably 30-60 mg / L, more preferably 35-50 mg / L, and even more preferably 40 mg / L.

[0040] In this invention, the preferred specific operation for optimizing Agrobacterium transformation and infection conditions is as follows: the pretreated explants are placed in a mixed Agrobacterium solution containing the ruby ​​gene for infection, and then the embryo infection time is optimized by using the marker gene ruby ​​(DONG WANG, YUZHONG, BIN FENG, et al. The RUBYreporter enables efficient haploid identification in maize and tomato[J]. Plant Biotechnology Journal,2023,21(8):1707-1715.DOI:10.1111 / pbi.14071.).

[0041] In this invention, the infection time is preferably 35-45 min, more preferably 38-42 min, and even more preferably 40 min.

[0042] In this invention, the *Agrobacterium* is preferably EHA105, and the preferred preparation process of the *Agrobacterium* mixed bacterial solution is as follows: the *Agrobacterium* bacterial solution is inoculated into YEB solid medium supplemented with kanamycin and rifampin, cultured for 2-3 days, the bacterial cells are collected and resuspended in liquid induction medium, and the OD of the bacterial solution is adjusted to the specified values. 600 ≈0.6; then add acetylsuccinone and Poloxamer 188 solution, and let stand for 30-60 minutes.

[0043] In this invention, the final concentration of kanamycin is preferably 45-55 mg / L, more preferably 48-52 mg / L, and even more preferably 50 mg / L; the final concentration of rifampin is preferably 35-45 mg / L, more preferably 38-42 mg / L, and even more preferably 40 mg / L.

[0044] In this invention, the final concentration of acetylsyleugenone is preferably 190-210 μmol / L, more preferably 195-205 μmol / L, and even more preferably 200 μmol / L. The Poloxamer 188 solution used in this invention is purchased from Sigma-Aldrich, catalog number Cat.No. P5556-100ML, with a concentration of 10% (100X). The final concentration (volume concentration) of the Poloxamer 188 solution is preferably 0.8-1.2%, more preferably 0.9-1.1%, and even more preferably 1%.

[0045] In this invention, a target sequence sgRNA is designed targeting an exon segment of the target gene, specifically the 62-81st exon of the first exon of the PDS gene. The PDS gene in this invention has the accession number HORVU.MOREX.r3.4HG0405810, and its sequence is shown in SEQ ID NO.1; the target sequence sgRNA is shown in SEQ ID NO.2, specifically the sequence CCGGACAACTTCATACGCAG.

[0046] In this invention, HvPDScrp-F and HvPDScrp-R are designed and synthesized using the target sequence sgRNA. The sequence of HvPDScrp-F is shown in SEQ ID NO.3, specifically aagcaCCGGACAACTTCATACGCAG; the sequence of HvPDScrp-R is shown in SEQ ID NO.4, specifically aaacCTGCGTATGAAGTTGTCCGGt.

[0047] In this invention, the construction process of the recombinant vector in step (1) includes the following steps:

[0048] (a) Linearized vectors were obtained by digesting TaU3-Cas9 as the original vector;

[0049] (b) The annealed products obtained by annealing HvPDScrp-F and HvPDScrp-R are coupled with the linearized vector described in step (a) to obtain the recombinant vector.

[0050] In this invention, TaU3-Cas9 was used as the original vector, and the original vector was digested with BsaI enzyme. The reaction was carried out at 37°C for 2-3 hours to obtain the vector digestion product. The vector digestion product was subjected to agarose gel electrophoresis, the target band was recovered and linearized, and then reacted at 37°C for 2-3 hours. After that, the target fragment was recovered by gel excision to obtain the linearized vector.

[0051] In this invention, the annealing reaction system comprises 1 μL HvPDScrp-F, 1 μL HvPDScrp-R, and 8 μM Anneal Buffer. The annealing procedure of this invention includes: 95°C for 10 min, followed by cooling to 20°C at a rate of 0.1°C / s.

[0052] In this invention, the annealed products obtained by annealing HvPDScrp-F and HvPDScrp-R are subjected to Golden Gate enzyme digestion and ligation reaction with the linearized vector described in step (a) to obtain the TaU3-CAS9-pds gene editing vector.

[0053] In this invention, after the Golden Gate enzyme digestion and ligation reaction, the product is further transformed into competent cells for culture, positive clones are screened, and colony PCR is used for identification.

[0054] In this invention, the primers used in the colony PCR identification process include M13F-47 and HvPDScrp-R. The sequence of M13F-47 is shown in SEQ ID NO.5, and the specific sequence is CGCCAGGGTTTTCCCAGTCACGAC.

[0055] In this invention, barley embryos are used as explants. The pretreated explants are placed in a mixed bacterial solution of Agrobacterium containing the recombinant vector for infection, followed by induced culture, differentiation and regeneration of seedlings, and then induced rooting to obtain transgenic plants. The pretreatment process of the barley embryos and the Agrobacterium mixed bacterial solution used in this invention are the same as described above.

[0056] This invention also provides the application of the gene editing technology system in site-directed mutagenesis of other genes in barley.

[0057] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1: Optimization of the genetic transformation system for barley embryos

[0059] (a) Treatment of barley 22 embryos

[0060] Select young spikelets 7-15 days after heading and remove the glumes; soak the seeds in a 10% sodium hypochlorite disinfectant solution at 200 rpm for 8 minutes on a shaker, remove the disinfectant solution with a pipette, add sterile water and shake at 200 rpm for 8 minutes, remove the liquid with a pipette, and rinse twice with sterile water; repeat the previous step; peel off the 1.5-2 mm translucent embryos, cut off the embryonic bud point, place them with the scutellum facing upward on the induction medium, and culture in the dark at 26℃ for 3 days.

[0061] The induction medium used water as a solvent and consisted of 4.3 g / L MS salt (M524), 30 g / L maltose, 1 g / L enzymatically hydrolyzed casein, 10 ml / L A solution, 2.5 mg / L Dicamba (D5417), and 3.5 g / L plant gel; the pH was 5.8.

[0062] (II) Preparation of Agrobacterium mixed bacterial solution

[0063] Agrobacterium tumefaciens EHA105 containing the ruby ​​gene vector (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number AC1010) was inoculated into YEB solid medium supplemented with kanamycin (50 mg / L) and rifampin (40 mg / L). After culturing for 2-3 days, the bacterial cells were collected and resuspended in liquid induction medium, and the culture was adjusted to the OD value of the bacterial culture. 600 ≈0.6; then add acetylsuccinone (final concentration 200 μmol / L) and 1% Poloxamer 188 solution 10%, and let stand for 30-60 min.

[0064] The liquid healing medium uses water as a solvent and consists of 4.3 g / L MS salt (M524), 30 g / L maltose, 1 g / L enzymatically hydrolyzed casein, 10 ml / L A solution, 2.5 mg / L Dicamba (D5417), and 3.5 g / L plant gel; the pH is 5.8.

[0065] Solution A uses water as a solvent and consists of 35 g / L inositol, 69 g / L proline, 0.12 g / L copper sulfate, and 0.1 g / L LVB1; the pH is 5.8.

[0066] (III) Optimization of barley embryo screening and transformation process

[0067] 1. Optimization of hygromycin screening concentration

[0068] After being cultured in the induction medium at 26°C in the dark for 3 days as described in step (I) above, barley 22 embryos were transferred to a selection medium supplemented with hygromycin at concentrations of 30 mg / L, 40 mg / L, 70 mg / L, 80 mg / L, and 100 mg / L. Callus formation efficiency was calculated after 2 weeks to determine the appropriate hygromycin concentration for subsequent system establishment.

[0069] The results are as follows Figure 1 As shown, when the hygromycin concentration is 40 mg / L, the callus formation rate is around 50%.

[0070] 2. Optimization of embryo pretreatment time

[0071] Following step (I) above, the extracted embryos were placed in induction medium and cultured in the dark at 26°C for 1, 2, 3, and 4 days. The emerging buds were then removed, and the embryos were incised with a scalpel to create wounds. The prepared Agrobacterium mixed bacterial solution was added, and the embryos were shaken at 200 rpm for 30 minutes. The bacterial solution was then removed, and the embryos were placed on sterile filter paper to dry off excess solution. The embryos were then transferred to a healing medium supplemented with 200 μmol / L acetylsyl syringone and co-cultured for 3 days. The ruby ​​gene was used as an indicator gene, and the infection efficiency was calculated after 3 days of co-culture.

[0072] The results are as follows Figure 2 As shown, the infection effect of the excised embryos cultured in the induction medium at 26°C in the dark for 1 day was relatively the best.

[0073] 3. Optimization of Agrobacterium infection time

[0074] Following step (I) above, the excised embryos were placed in induction medium and cultured in the dark at 26°C for 3 days. Then, the emerging buds were removed, and the embryos were incised with a scalpel to create wounds. The prepared Agrobacterium mixed bacterial solution was added, and the embryos were shaken at 200 rpm for 20 min, 40 min, and 60 min respectively. The bacterial solution was then aspirated, and the embryos were placed on sterile filter paper to dry off excess solution. The embryos were then transferred to induction medium supplemented with 200 μmol / L acetylsyl syringone and co-cultured for 3 days. Using the ruby ​​gene as an indicator gene, the infection efficiency was calculated after 3 days of co-culture.

[0075] See results Figure 3 As shown, the infection efficiency of the embryonic embryos was relatively highest when the shaking time was 40 minutes.

[0076] Example 2: PDS gene editing based on the CRISPR / Cas9 system

[0077] (I) Construction of barley PDS gene editing vector

[0078] 1. Primer design for PDS gene sgRNA

[0079] The PDS genome sequence was obtained by directly searching the barley genome (genome version Hordeum vulgare Morex V3) for the PDS gene accession number HORVU.MOREX.r3.4HG0405810 on the Phytozome website (https: / / phytozome-next.jgi.doe.gov / ).

[0080]

[0081] A target sequence sgRNA was designed targeting the region from exon 62 to 81 of the first exon of the PDS gene. The sequence of the target sgRNA is shown in SEQ ID NO.2, specifically CCGGACAACTTCATACGCAG. Primers for the sgRNA were designed: the sequence of HvPDScrp-F is shown in SEQ ID NO.3, specifically aagcaCCGGACAACTTCATACGCAG; and the sequence of HvPDScrp-R is shown in SEQ ID NO.4, specifically aaacCTGCGTATGAAGTTGTCCGca.

[0082] 2. Primer annealing

[0083] The annealing reaction system includes 1 μL HvPDScrp-F, 1 μL HvPDScrp-R, and 8 μM Anneal Buffer.

[0084] The annealing process includes: 95℃ for 10 min, then decreasing to 20℃ at a rate of 0.1℃ / s.

[0085] 3. Vector enzyme digestion

[0086] TaU3-Cas9 was used as the original vector, and the original vector was digested with BsaI enzyme. The reaction was carried out at 37℃ for 2-3 hours to obtain the vector digestion product. The vector digestion product was subjected to agarose gel electrophoresis at 160V and 290mA for 20 minutes to recover the target band, linearize it, and react it at 37℃ for 2-3 hours. Then, the target fragment was excised and recovered to obtain the linearized vector. The specific operation was carried out according to the FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novizan, catalog number DC301).

[0087] The enzyme digestion system includes 5 μL TaU3-Cas9 plasmid, 10 μL 10* buffer, 1 μL BsaI enzyme, and 84 μL water.

[0088] 4. Golden Gate reaction

[0089] The annealed product obtained above was diluted 25-fold and subjected to a golden gate reaction with the TaU3-Cas9 vector. The reaction mixture was digested at 37°C for 5 min, ligated at 16°C for 10 min, digested again at 37°C for 15 min, and treated at 85°C for 10 min. The reaction system consisted of 1 μL of diluted annealed product, 50 ng of linearized vector, 2 μL of 10x T4 buffer, 15 μL of T4 ligase, and 1 μL of BSA restriction endonuclease.

[0090] 5. Transformation

[0091] Add 5 μL of the Golden Gate reaction product to 100 μL of competent E. coli cells, incubate on ice for 30 min, heat shock at 42 °C for 45 s, incubate on ice for 2 min, add 400 μL of antibiotic-free LB, incubate at 37 °C for 1 h, centrifuge at 5000 rpm for 1 min, remove most of the supernatant, keep 100 μL of liquid, pipette it onto LB agar plates containing Kans, and incubate upside down at 37 °C overnight.

[0092] 6. Identification of positive monoclonal antibodies

[0093] Single colonies were picked up with a pipette tip and inoculated into LB liquid medium containing kanamycin. After incubation at 37°C and 200 rpm for 4-6 hours on a shaker, PCR verification was performed. Colonies were identified by PCR using primers M13F-47 and HvPDScrp-R. Positive clones were screened, and the recombinant vector was identified and named TaU3-Cas9-pds (e.g., ...). Figure 5 (As shown).

[0094] The sequence of M13F-47 is shown in SEQ ID NO.5, specifically CGCCAGGGTTTTCCCAGTCACGAC.

[0095] The PCR reaction system, in 10 μL, preferably includes the following components: 5 μL of 2×TaqMasterMix II reaction polymerase and buffer; the concentration of primers M13F-47 and HvPDScrp-R is preferably 10 μM, and the amount used is preferably 0.4 μL; 1 μL of DNA template; and ultrapure water to make up the remaining volume of the PCR reaction system.

[0096] The preferred PCR reaction procedure is: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 63℃ annealing for 15 seconds, 72℃ extension for 180 seconds, for a total of 35 cycles; the final extension is 72℃ for 5 minutes.

[0097] (II) Transformation of barley embryos using gene editing vectors

[0098] As described in Example 1, after 3 days of pretreatment, the buds that have grown out of the embryos were cut off, and the embryos were incised with a scalpel to create wounds. The prepared mixed bacterial solution containing Tau-Cas9-pds Agrobacterium was added, and the embryos were shaken at 200 rpm for 40 minutes. The bacterial solution was then aspirated, and the embryos were placed on sterile filter paper to dry off the excess bacterial solution. The embryos were then transferred to a healing medium containing 200 μmol / L acetylsyl syringone and co-cultured for 3 days.

[0099] The embryos were then transferred to a selection medium and cultured at 26°C in the dark for 6-8 weeks, with subculturing every 2 weeks. The callus was then transferred to a transition medium and induced to differentiate into regenerated plantlets under low light conditions at 22°C. Regenerated green shoots were observed after 7-15 days. Two weeks later, the callus was transferred to a regeneration medium and cultured at 22°C with a photoperiod of 12h light / 12h dark. After approximately 2 weeks, the robust regenerated plantlets were transferred to a rooting medium under the same conditions as the regeneration stage. Once the regenerated plantlets had well-developed root systems (roots longer than 1cm and with numerous roots), they were transplanted to a greenhouse for cultivation until maturity, yielding regenerated plants (e.g., ...). Figure 6 ).

[0100] The screening medium was prepared by adding 400 mg / L termethin and 40 mg / L hygromycin to the above-mentioned liquid healing medium.

[0101] The regeneration medium uses water as a solvent and consists of 2.7 g / L MS salt (M571), 20 g / L maltose, 400 mg / L termethin, 40 mg / L hygromycin, and 3.5 g / L plant gel; the pH is 5.8.

[0102] The transition medium is the above-mentioned regeneration medium supplemented with 2.5 mg / L 2,4-D, 0.1 mg / L 6-BA, and 40 mg / L hygromycin.

[0103] The root-strengthening culture medium uses water as a solvent and consists of 4.43 g / L 1 / 2 MS salt (M519), 30 g / L maltose, 0.05 mg / L NAA (naphthaleneacetic acid), 2 mg / L chlormequat chloride (CCC), and 3.5 g / L plant gel; the pH is 5.8.

[0104] (III) Target Site Editing Effect Analysis

[0105] 1. Positive identification of regenerated plants

[0106] Two regenerated seedlings, PDS-1 and PDS-2, obtained above, were used to extract DNA from the regenerated seedlings using the Tiangen Plant Genpmic DNA Kit (purchased from https: / / www.cassbuy.com / ). PCR DNA polymerase premix 2×Taq Master Mix II was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0107] Using the extracted regenerated seedling DNA as a template, PCR amplification was performed using primer M13F-47 and primer gRNAin designed based on the target sequence sgRNA, followed by gel electrophoresis. Wild-type flower 22 was used as a control group. PCR results are shown below. Figure 7 As shown.

[0108] The sequence of the gRNAin primer is shown in SEQ ID NO.6, specifically GCACCGACTCGGTGCCAC.

[0109] The PCR reaction system, in 10 μL, includes the following components: 5 μL of 2×Taq Master Mix II reaction polymerase and buffer; the preferred concentrations of primers M13F-47 and gRNAin are 10 μM, and the preferred amounts are 0.4 μL; 1 μL of DNA template; and ultrapure water to make up the remaining volume of the PCR reaction system.

[0110] The PCR reaction procedure was as follows: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 61℃ annealing for 15 seconds, 72℃ extension for 180 seconds, for a total of 35 cycles; the final extension was 72℃ for 5 minutes.

[0111] 2. Analysis of the effect of target site editing in regenerated plants

[0112] Sanger sequencing was performed on the PCR products of regenerated seedlings PDS-1, PDS-2 and wild-type flower 22 (PDS1-CK).

[0113] The sequencing primer was HvPDS-test-R1, and the sequence is shown in SEQ ID NO.7, specifically TGGTTTACTGGGGCGTTCAC.

[0114] Sequencing results as follows Figure 8 As shown.

[0115] After gene editing, compared to wild-type PDS-CK, the regenerated seedling PDS-1 produced a homozygous mutation with a T base insertion in the PDS gene, while PDS-2 produced a heterozygous mutation with a G to C insertion in the PDS gene.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene editing technology system based on the transformation of immature embryos of barley variety Hua 22, characterized in that, The method for constructing the gene editing technology system includes the following steps: (1) Using barley embryos as explants, pretreatment was performed and infection and screening conditions during Agrobacterium transformation were optimized; (2) Target sequence sgRNA was designed for the exon region of the target gene, and HvPDScrp-F and HvPDScrp-R were designed and synthesized using the target sequence sgRNA. Then, the TaU3-CAS9-pds gene editing vector was constructed. (3) Use Agrobacterium mixed bacterial solution containing the gene editing vector described in step (2) to infect barley embryos, then induce culture, differentiate and regenerate seedlings, and induce rooting to obtain gene-edited plants.

2. The gene editing technology system according to claim 1, characterized in that, The pretreatment time for barley embryos in step (1) is 0.5-1.5 days; In step (1), the time for Agrobacterium infection is 35-45 min; In step (1), hygromycin is used for screening, with a screening concentration of 30-60 mg / L.

3. The gene editing technology system according to claim 1, characterized in that, The exon segment mentioned in step (2) is the 62nd to 81st segment of the first exon of the PDS gene.

4. The gene editing technology system according to claim 1, characterized in that, The sequence of the sgRNA in step (2) is shown in SEQ ID NO.2, the sequence of the HvPDScrp-F is shown in SEQ ID NO.3, and the sequence of the HvPDScrp-R is shown in SEQ ID NO.

4.

5. The gene editing technology system according to claim 1, characterized in that, The construction process of the TaU3-CAS9-pds gene editing vector in step (2) includes the following steps: (a) Linearized vectors were obtained by digesting TaU3-Cas9 as the original vector; (b) The annealed products obtained by annealing HvPDScrp-F and HvPDScrp-R are subjected to Golden Gate enzyme digestion and ligation reaction with the linearized vector described in step (a) to obtain the final product.

6. The gene editing technology system according to claim 5, characterized in that, The annealing reaction system in step (b) includes 1 μL HvPDScrp-F, 1 μL HvPDScrp-R, and 8 μM Anneal Buffer; The annealing process includes: 95℃ for 10 min, then decreasing to 20℃ at a rate of 0.1℃ / s.

7. The gene editing technology system according to claim 5, characterized in that, Step (b) after the Golden Gate enzyme digestion and ligation reaction further includes transforming the obtained ligation product into competent cells for culture, screening for positive clones, and identifying them by colony PCR.

8. The gene editing technology system according to claim 7, characterized in that, The primers used in the colony PCR identification process include M13F-47 and HvPDScrp-R, and the sequence of M13F-47 is shown in SEQ ID NO.

5.

9. The gene editing technology system according to claim 1, characterized in that, The Agrobacterium mentioned in steps (1) and (3) is EHA105.

10. The application of the gene editing technology system according to any one of claims 1-9 in site-directed mutagenesis of other genes in barley.

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