GRF4-GIF1 chimeric gene for improving genetic transformation and gene editing efficiency of oat immature embryos as well as vector and application of GRF4-GIF1 chimeric gene

Abstract

The invention discloses a GRF4-GIF1 chimeric gene for improving genetic transformation and gene editing efficiency of an oat immature embryo as well as a vector and application of the GRF4-GIF1 chimeric gene. The GRF4-GIF1 chimeric gene is constructed into a transformation vector or a gene editing vector, so that the synergistic expression of the chimeric gene in recipient cells is realized. The method comprises the following steps: firstly, stripping an oat immature embryo, performing agrobacterium infection and co-culture, and then transferring into a recovery culture medium for culture; then, transferring well-grown calluses to a screening culture medium for screening, sequentially transferring the screened positive calluses into a differentiation and proliferation culture medium and a rooting culture medium, and culturing to obtain differentiated seedlings with certain root length; and finally, hardening seedlings, and culturing in vermiculite to obtain complete regenerated plants. According to the method, the GRF4-GIF1 chimeric gene is integrated in the carrier construction stage, and the immature embryo of the oat is selected as the explant, so that the genetic transformation efficiency of the oat is effectively improved, and the gene editing of the quality character of the feeding oat is successfully completed.

Description

Technical Field

[0001] This invention belongs to the field of gene editing breeding technology, specifically relating to a GRF4-GIF1 chimeric gene and its vector and application for improving the efficiency of genetic transformation and gene editing of immature oat embryos. Background Technology

[0002] Oats, as a dual-purpose crop for both grain and forage, possess excellent characteristics such as high forage yield, drought resistance, cold resistance, and tolerance to poor soil. They also boast high nutritional content, good palatability, high digestibility, and suitability for green fodder or haymaking. Furthermore, oats play a vital role in ensuring food security, protecting the ecological environment, and promoting agricultural efficiency. Therefore, oats are a forage species with significant development potential.

[0003] Plant genetic transformation involves introducing exogenous genes or specific DNA sequences into plant recipient tissues to precisely improve target traits. The selection of suitable explants is the first step in establishing tissue culture technology and directly affects the success of subsequent cultures. However, in oat genetic transformation systems, mature embryos and other explants still suffer from low efficiency and poor regeneration capacity. Immature embryos, on the other hand, are in an active division phase, with low cell differentiation, full totipotency expression, and a looser cell wall structure, making it easier to introduce exogenous genes. Therefore, using immature embryos as superior explants will contribute to the development of oat genetic transformation and gene editing.

[0004] This invention, based on immature oat embryos, leverages the promoting effect of GRF-GIF chimeric genes on callus regeneration and combines hygromycin screening to successfully overcome the challenge of low oat conversion efficiency, providing an efficient approach for the precise editing of oat target genes. Summary of the Invention

[0005] In view of the problems existing in the above-mentioned background technology, the purpose of this invention is to provide a GRF4-GIF1 chimeric gene, its vector and application for improving the efficiency of genetic transformation and gene editing of oat immature embryos. By utilizing the characteristics of oat immature embryos and the promoting effect of GRF-GIF chimeric protein on callus regeneration, the efficiency of oat genetic transformation and gene editing can be significantly improved.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A GRF4-GIF1 chimeric gene that improves the efficiency of genetic transformation and gene editing in immature oat embryos is constructed by linking the oat growth regulator GRF4 (nucleotide sequence as shown in SEQ ID NO.1) and the oat transcription coactivator GIF1 (nucleotide sequence as shown in SEQ ID NO.2) through the gene sequence GCAGCAGCAGCA. The sequence of the GRF4-GIF1 chimeric gene is shown in SEQ ID NO.3.

[0007] GRF4-GIF1 chimeric gene expression vector: The 3' and 5' ends of the GRF4-GIF1 chimeric gene were inserted into the BamHI and KpnI sites of the pUN1301-EGFP vector, respectively, to construct the GRF4-GIF1 chimeric gene expression vector.

[0008] The aforementioned GRF4-GIF1 chimeric gene expression vector can be used to improve the genetic transformation efficiency of immature oat embryos.

[0009] GRF4-GIF1 chimeric gene editing vector: A GRF4-GIF1 chimeric gene editing vector was constructed by combining the GRF4-GIF1 chimeric gene with an oat gene editing vector, including the following GRF4-GIF1 chimeric gene editing vector: (1) GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9: By cloning the gene sequences of ARE1, APP1, IPA1, SPDT1 and AKT1 in oats, gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the multi-gene editing vector pMMGE-Cas9. (2) GRF4-GIF1 chimeric gene-single-base gene editing vector rABE8e: By cloning the EPSPS gene sequence in oats, gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the single-base editing vector rABE8e. (3) GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j: The TCP19 gene sequence from oats was cloned, and gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the AsU6-Cas12i / Cas12j vector.

[0010] Choosing one of the aforementioned GRF4-GIF1 chimeric gene editing vectors can improve the efficiency of gene editing in immature oat embryos.

[0011] The methods for using GRF4-GIF1 chimeric gene expression vectors to improve the genetic transformation efficiency of immature oat embryos or GRF4-GIF1 chimeric gene editing vectors to improve the gene editing efficiency of immature oat embryos are as follows: S1. The peeled immature oat seeds are disinfected, washed, and the immature oat embryos are removed and placed in a tissue infection solution; the tissue infection solution is: MS base salts 0.43 g / L + sucrose 30 g / L, pH=5.8; S2. The plasmid of the GRF4-GIF1 chimeric gene expression vector or the GRF4-GIF1 chimeric gene editing vector is transformed into competent cells of Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens bacterial suspension for transformation; the Agrobacterium tumefaciens bacterial suspension for transformation is centrifuged at room temperature, washed and resuspended with the tissue infection solution to obtain the resuspended bacterial suspension; S3. Aspirate the tissue infection solution containing immature oat embryos from step S1, and then add the resuspended bacterial solution from step S2 for infection. S4. After infection, remove the immature oat embryos and allow the surface bacterial solution to dry. Then, transfer the immature oat embryos to a co-culture medium for further culture. The co-culture medium consists of: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + acetylsylgenone 200 M / L, pH=5.8. S5. The co-cultured immature oat embryos were transferred to a recovery medium for further culture. The recovery medium consisted of L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L, pH=5.8. S6. After recovery culture, immature oat embryos were sequentially transferred to screening medium 1 and screening medium 2 for screening culture. Both screening media 1 and screening media 2 consist of L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L + hygromycin, pH=5.8; wherein the concentration of hygromycin in screening media 1 is 5 mg / L and the concentration of hygromycin in screening media 2 is 10 mg / L. S7. The positive callus tissue obtained after screening is transferred to differentiation and proliferation medium and cultured until the seedling length is 2-3 cm; then transferred to rooting medium and cultured until the root length of the seedling to be differentiated is 5-7 cm. The differentiation and proliferation culture medium was: basal medium + 6-BA 2 mg / L + NAA 0.5 mg / L, pH=5.7; The rooting medium used is a basal medium, which is: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L, pH=5.7; S8. Remove the seedlings to be differentiated, open the bottle to harden the seedlings, then wash the roots, and culture them in sterilized vermiculite to obtain complete regenerated plants.

[0012] Preferably, in step S4, the culture conditions of the co-culture medium are: placed at a constant temperature of 26±1℃ and cultured in the dark for 2 days.

[0013] Preferably, in step S5, the culture conditions for the recovery culture medium are: dark culture at a constant temperature of 26±1℃ for 5 days.

[0014] Preferably, in step S6, the culture conditions for both screening medium 1 and screening medium 2 are: dark culture at a constant temperature of 26±1℃ for 30 days.

[0015] Preferably, in step S7, the culture conditions for the differentiation and proliferation culture medium and the rooting culture medium are: light culture, with a light intensity of 4300~4800 Lux and a culture time of 14 h·d. -1 The incubation temperature was 26±1℃.

[0016] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: (1) Based on the GRF4-GIF1 chimeric gene, this invention improves the genetic transformation and gene editing system of oat immature embryos. It uses immature embryos with low cell differentiation and full totipotency expression as excellent explants, shortens the callus induction time, and accelerates differentiation into seedlings, which can reduce the risk of contamination and the probability of variation during the culture process, thereby constructing an efficient and stable oat genetic transformation and gene editing system. (2) In this invention, the method for improving the genetic transformation and gene editing efficiency of immature oat embryos based on the GRF4-GIF1 chimeric gene utilizes the GRF4-GIF1 chimeric gene to regulate the transformation of stem cells into over-expansion cells, thereby reserving sufficient cells for tissue differentiation and accelerating shoot regeneration. Combined with hygromycin screening, this method successfully overcomes the difficulty of low oat transformation efficiency. Simultaneously, by incorporating the GFP gene in the vector during the genetic transformation of immature oat embryos, successfully transformed callus tissue exhibits fluorescence, enabling rapid screening of positive materials and reducing screening costs and errors. This method has advantages such as rapid propagation and high production efficiency, providing important research basis for the smooth progress of gene editing and oat germplasm innovation. Attached Figure Description

[0017] Figure 1This is a schematic diagram of the construction of the GRF4-GIF1 chimeric gene expression vector pUN1301-EGFP provided in the embodiments of the present invention. In the figure, a is a schematic diagram of the GRF4-GIF1 chimeric gene sequence, b is a schematic diagram of the pUN1301-EGFP vector structure, and c is a map of the pUN1301-EGFP vector.

[0018] Figure 2 This is a schematic diagram of the construction of the GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9 provided in this embodiment of the invention. In the figure, A is a schematic diagram of the AsARE1 gRNA target site, B is a schematic diagram of the AsAPP1 gRNA target site, C is a schematic diagram of the AsIPA1 gRNA target site, D is a schematic diagram of the AsSPDT gRNA target site, E is a schematic diagram of the AsAKT1 gRNA target site; F is the combination design of PCR amplification of small fragments of the five-gene knockout vector, and G is a schematic diagram of the structure of the multi-gene editing vector pMMGE-Cas9.

[0019] Figure 3 This is a schematic diagram of the construction of the GRF4-GIF1 chimeric gene-single-base gene editing vector rABE8e provided in the embodiments of the present invention. In the figure, A is the single-base editing site of the AsEPSPS gene, and B is the map of the single-base editing vector rABE8e for feed oats.

[0020] Figure 4 This is a schematic diagram illustrating the construction of the GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j provided in this embodiment of the invention.

[0021] Figure 5 This is the Agrobacterium tumefaciens bacterial solution that has been shaken overnight, as provided in the embodiments of the present invention.

[0022] Figure 6 This is a schematic diagram of the oat immature embryo removal process provided in an embodiment of the present invention.

[0023] Figure 7 This is a state diagram of immature embryos after 2 days of co-culture provided in an embodiment of the present invention.

[0024] Figure 8 This is a diagram showing the state of an immature embryo after 5 days of recovery culture, as provided in an embodiment of the present invention.

[0025] Figure 9 This is a schematic diagram of positive callus tissue after screening and culture provided in an embodiment of the present invention.

[0026] Figure 10 This is a schematic diagram of positive callus tissue observed under a stereofluorescence microscope, provided in an embodiment of the present invention.

[0027] Figure 11This is a gel image of oat seedlings identified based on the GRF4-GIF1 chimeric gene expression vector pUN1301-EGFP, provided in an embodiment of the present invention.

[0028] Figure 12 The results are PCR identification results of gene-edited plants obtained based on the GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9 provided in this embodiment of the invention.

[0029] Figure 13 This invention provides an embodiment for the phenotypic identification of AsU6-Cas12i / Cas12j positive plants. Figure 13 A) and crude protein content ( Figure 13 B) Result.

[0030] Figure 14 The results are Sanger assays of the AsDQ-1 gene-edited plant provided in this embodiment of the invention.

[0031] Figure 15 The results are Sanger assays of the AsDQ-2 gene-edited plants provided in this embodiment of the invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Example 1 Construction of the GRF4-GIF1 chimeric gene: The gene sequences of the oat growth regulator GRF4 and the transcriptional coactivator GIF1 were cloned. The nucleotide sequence of GRF4 is shown in SEQ ID NO.1, and the nucleotide sequence of GIF1 is shown in SEQ ID NO.2. GRF4 and GIF1 were ligated using the gene sequence GCAGCAGCAGCA to construct a GRF4-GIF1 chimeric gene, the sequence of which is shown in SEQ ID NO.3.

[0034] Example 2 Carrier construction: (1) GRF4-GIF1 chimeric gene expression vector: The 3' and 5' ends of the GRF4-GIF1 chimeric gene were inserted into the BamHI and KpnI sites of the pUN1301-EGFP vector, respectively, to construct the GRF4-GIF1 chimeric gene expression vector. The vector construction process was described in detail below. Figure 1 ; (2) GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9: The gene sequences of ARE1, APP1, IPA1, SPDT1, and AKT1 from oats were cloned, gRNA targets were designed, and the gRNA was co-constructed with the GRF4-GIF1 chimeric gene into the multi-gene editing vector pMMGE-Cas9. Vector construction details are as follows. Figure 2 ; (3) GRF4-GIF1 chimeric gene-single-base gene editing vector rABE8e: The EPSPS gene sequence from oats was cloned, a gRNA target was designed, and the gene was co-constructed with the GRF4-GIF1 chimeric gene into the single-base editing vector rABE8e. Vector construction details are as follows. Figure 3 ; (4) GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j: The TCP19 gene sequence from oats was cloned, a gRNA target was designed, and the gRNA was co-constructed with the GRF4-GIF1 chimeric gene into the AsU6-Cas12i / Cas12j vector. Vector construction details are as follows. Figure 4 .

[0035] Example 3 1. Pretreatment of immature oat seeds (1) Peeling treatment: Select immature oat seeds that are free from mold and disease, plump and uniform in size, peel off the oat seed coat, select immature seeds that are soft and yellow in color, and wait for the next step. (2) Disinfection treatment: Take the peeled immature oat seeds into a 50 mL centrifuge tube, soak them in 75% C2H5OH for 5 min, then wash the oat seeds with ultrapure water 4-6 times; then soak them in 5% NaClO for 6 min, and then place the centrifuge tube containing the oat seeds into a laminar flow hood and wash the oat seeds with sterile ultrapure water 5-7 times in the laminar flow hood. During the two soaking processes, the 50 mL centrifuge tube should be placed on a rotary culture mixer to keep it in a rotating state.

[0036] 2. Prepare the culture medium (1) Tissue infection solution: MS base salts 0.43 g / L + sucrose 30 g / L, pH=5.8; autoclave at 115℃ for 30 min; (2) Co-culture medium: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + acetylsylgenone 200 M / L, pH=5.8; autoclave at 115℃ for 30 min; (3) Recovery medium: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L, pH=5.8; autoclave at 115℃ for 30 min; (4) Screening medium 1: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L + hygromycin 5 mg / L, pH=5.8; autoclave at 115℃ for 30 min; (5) Screening medium 2: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L + hygromycin 10 mg / L, pH=5.8; autoclave at 115℃ for 30 min; (6) Basic culture medium: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L; autoclave at 115℃ for 30 min; (7) Differentiation and proliferation medium: basal medium + 6-BA 2 mg / L + NAA 0.5 mg / L, pH=5.7; (8) Rooting medium: The above-mentioned basic medium was used, pH=5.7.

[0037] 3. Agrobacterium tumefaciens transformation The plasmids of the four vectors constructed in Example 2 were transformed into competent Agrobacterium tumefaciens EHA105 cells for four parallel experiments. After the cells grew into single colonies, the colonies were picked and transferred to 4 ml of LB liquid medium (with 50 mg / L kanamycin and rifampin added), and incubated overnight at 28°C with shaking to obtain four Agrobacterium bacterial suspensions for transformation. Shake the Agrobacterium-containing bacterial solution used for transformation until it turns orange-yellow (refer to...). Figure 5 Centrifuge at 6000 rpm for 10 min at room temperature, discard the supernatant, wash and resuspend twice with tissue infection solution (pH 5.8) to obtain 4 kinds of resuspended bacterial solutions, which are left for infection.

[0038] 4. Transformation of immature embryos (1) Embryo removal treatment of immature oat seeds: Under a stereomicroscope, the embryos of pretreated immature oat seeds were removed (refer to...). Figure 6 Place it in the tissue infection solution for the next step; (2) Infection: Use a pipette to remove the infection solution from the tissue containing the pretreated immature oat embryos. Perform four parallel experiments, adding the four resuspended bacterial solutions obtained after transformation by Agrobacterium tumefaciens and infecting for 10 min respectively. (3) Co-culture: After inoculation, the immature embryos were removed and placed on sterile filter paper. After the surface bacterial solution was dried, the immature embryos were transferred to co-culture medium and cultured in the dark for 2 days at a temperature of 26±1℃. The state of the immature embryos after 2 days of co-culture was as follows: Figure 7 (Transformation of immature embryos based on GRF4-GIF1 chimeric gene expression vector); (4) Recovery culture: After co-culture, the immature embryos were transferred to recovery culture medium and cultured in the dark in a tissue culture room at a constant temperature of 26±1℃ for 5 days. The state of the immature embryos after 5 days of recovery culture was as follows: Figure 8 (Transformation of immature embryos based on GRF4-GIF1 chimeric gene expression vector); (5) Screening culture: The immature embryos after recovery culture were transferred to screening medium 1 and cultured in the dark in a tissue culture room at a constant temperature of 26±1℃ for 30 days; after the end of the culture, they were transferred to screening medium 2, and the positive calli were selected as references. Figure 9 (Transformation of immature embryos based on the GRF4-GIF1 chimeric gene expression vector), cultured in the dark for 30 days in a tissue culture room at a constant temperature of 26±1℃; positive callus reference observed under a stereofluorescence microscope. Figure 10 (Transformation of immature embryos based on GRF4-GIF1 chimeric gene expression vector); (6) Transfer to differentiation and proliferation medium and continue culturing until the seedlings are 2-3 cm long. The culturing process is carried out under light with a light intensity of 4300-4800 Lux and a culturing time of 14 h·d. -1 The incubation temperature was 26±1℃; (7) Transfer the seedlings to the rooting medium and culture them until the roots of the seedlings to be differentiated are about 5 cm long; the culture process is carried out under light, with a light intensity of 4300~4800 Lux and a culture time of 14 h·d. -1 The incubation temperature was 26±1℃; (8) Remove the seedlings from the tissue culture room, add tap water, and harden them off under natural light. Then, count the rooting rate. After opening the culture bottle and cleaning the roots, plant them in vermiculite sterilized in a high-pressure steam sterilizer to obtain complete regenerated plants.

[0039] 5. Identification of positive seedlings Using the DNA from the screened positive plants as templates, approximately 620 bp upstream and downstream of each gene target site was amplified by PCR using gene-specific primers. The specific reaction system and reaction procedure are shown in Table 1 and Table 2, respectively.

[0040] The gel image for identifying positive seedlings obtained by transforming immature embryos based on the GRF4-GIF1 chimeric gene expression vector is shown below. Figure 11 As shown. PCR identification results of gene-edited plants obtained by transformation of immature embryos based on the GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9 are as follows. Figure 12 As shown. Using the wild type as a control, the phenotype and crude protein content of positive plants transformed from immature embryos based on the GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j were identified as follows. Figure 13 As shown.

[0041] 6. Identification results of gene-edited plants (Sanger) For gene-edited plants obtained by transformation of immature embryos based on the GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9, primer sequences of approximately 500 bp in length were designed upstream and downstream of each gene target site. Using DNA from positive plants as templates, PCR amplification of the gene targets was performed. After successful band extraction and gel recovery, plasmids were extracted for Sanger sequencing identification. The Sanger sequencing results for AsDQ-1 and AsDQ-2 gene-edited plants are as follows: Figure 14 and Figure 15 As shown.

[0042] 7. Statistical analysis of implementation results The conversion rate of immature oat embryos based on the GRF4-GIF1 chimeric gene expression vector was 33.4%–34.2%; the multi-gene editing efficiency of oats based on the GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9 reached 23%. Oat single-base gene editing based on the GRF4-GIF1 chimeric gene-single-base gene editing vector rABE8e was performed in 27 batches using the feed oat single-base gene editing technology system, with an oat single-base editing efficiency of 21.4%. The editing types of the feed oat single-base gene editing technology system are shown in Table 3. The feed oat gene editing technology system based on the GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j achieved an editing efficiency of 20%.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A GRF4-GIF1 chimeric gene that improves the efficiency of genetic transformation and gene editing in immature oat embryos, characterized in that, The GRF4-GIF1 chimeric gene is constructed by linking the oat growth regulator GRF4 (nucleotide sequence as shown in SEQ ID NO.1) and the oat transcription coactivator GIF1 (nucleotide sequence as shown in SEQ ID NO.2) through the gene sequence GCAGCAGCAGCA. The GRF4-GIF1 chimeric gene sequence is shown in SEQ ID NO.

3.

2. A GRF4-GIF1 chimeric gene expression vector, characterized in that, The GRF4-GIF1 chimeric gene expression vector was constructed by inserting the 3' and 5' ends of the GRF4-GIF1 chimeric gene as described in claim 1 into the BamHI and KpnI sites on the pUN1301-EGFP vector, respectively.

3. A GRF4-GIF1 chimeric gene editing vector, characterized in that, The GRF4-GIF1 chimeric gene of claim 1 is combined with an oat gene editing vector to construct a GRF4-GIF1 chimeric gene editing vector, comprising the following GRF4-GIF1 chimeric gene editing vector: (1) GRF4-GIF1 chimeric gene-multi-gene editing vector pMMGE-Cas9: By cloning the gene sequences of ARE1, APP1, IPA1, SPDT1 and AKT1 in oats, gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the multi-gene editing vector pMMGE-Cas9. (2) GRF4-GIF1 chimeric gene-single-base gene editing vector rABE8e: By cloning the EPSPS gene sequence in oats, gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the single-base editing vector rABE8e. (3) GRF4-GIF1 chimeric gene-gene editing vector AsU6-Cas12i / Cas12j: The TCP19 gene sequence from oats was cloned, and gRNA targets were designed and co-constructed with the GRF4-GIF1 chimeric gene into the AsU6-Cas12i / Cas12j vector.

4. The application of the GRF4-GIF1 chimeric gene expression vector as described in claim 2 in improving the genetic transformation efficiency of immature oat embryos.

5. The application of the GRF4-GIF1 chimeric gene editing vector as described in claim 3 in improving the gene editing efficiency of immature oat embryos.

6. A method for implementing the application as described in claim 4 or 5, characterized in that, Includes the following steps: S1. The peeled immature oat seeds are disinfected, washed, and the immature oat embryos are removed and placed in a tissue infection solution; the tissue infection solution is: MS base salts 0.43 g / L + sucrose 30 g / L, pH=5.8; S2. The plasmid of the GRF4-GIF1 chimeric gene expression vector or the GRF4-GIF1 chimeric gene editing vector is transformed into competent cells of Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens bacterial suspension for transformation; the Agrobacterium tumefaciens bacterial suspension for transformation is centrifuged at room temperature, washed and resuspended with the tissue infection solution to obtain the resuspended bacterial suspension; S3. Aspirate the tissue infection solution containing immature oat embryos from step S1, and then add the resuspended bacterial solution from step S2 for infection. S4. After infection, remove the immature oat embryos and allow the surface bacterial solution to dry. Then, transfer the immature oat embryos to a co-culture medium for further culture. The co-culture medium consists of: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + acetylsylgenone 200 M / L, pH=5.

8. S5. The co-cultured immature oat embryos were transferred to a recovery medium for further culture. The recovery medium consisted of L3 basesalts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L, pH=5.

8. S6. After recovery culture, immature oat embryos were sequentially transferred to screening medium 1 and screening medium 2 for screening culture. Both screening media 1 and screening media 2 consist of L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + termethin 200 mg / L + hygromycin, pH=5.8; wherein the concentration of hygromycin in screening media 1 is 5 mg / L and the concentration of hygromycin in screening media 2 is 10 mg / L. S7. The positive callus tissue obtained after screening is transferred to differentiation and proliferation medium and cultured until the seedling length is 2-3 cm; then transferred to rooting medium and cultured until the root length of the seedling to be differentiated is 5-7 cm. The differentiation and proliferation culture medium was: basal medium + 6-BA 2 mg / L + NAA 0.5 mg / L, pH=5.7; The rooting medium used is a basal medium, which is: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L, pH=5.7; S8. Remove the seedlings to be differentiated, open the bottle to harden the seedlings, then wash the roots, and culture them in sterilized vermiculite to obtain complete regenerated plants.

7. The implementation method as described in claim 6, characterized in that, In step S4, the culture conditions for the co-culture medium are: to be placed at a constant temperature of 26±1℃ and cultured in the dark for 2 days.

8. The implementation method as described in claim 6, characterized in that, In step S5, the culture conditions for the recovery culture medium are: to be placed at a constant temperature of 26±1℃ and cultured in the dark for 5 days.

9. The implementation method as described in claim 6, characterized in that, In step S6, the culture conditions for both screening medium 1 and screening medium 2 are: to be placed at a constant temperature of 26±1℃ and cultured in the dark for 30 days.

10. The implementation method as described in claim 6, characterized in that, In step S7, the culture conditions for the differentiation and proliferation media and the rooting media are as follows: light culture, light intensity of 4300~4800 Lux, and culture time of 14 h·d. -1 The incubation temperature was 26±1℃.

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