U6 promoter derived from onobrychis and its application in onobrychis gene editing

Abstract

The application discloses a U6 promoter derived from Onobrychis viciafilia and application of the U6 promoter in gene editing of the Onobrychis viciafilia. The U6 promoter derived from the Onobrychis viciafilia comprises OvU6-1, OvU6-4 and OvU6-24 promoters. The application also discloses an Onobrychis viciafilia CRISPR / Cas9 gene editing vector constructed based on the U6 promoter. Experiments prove that, compared with AtU6, GmU6 and MsU6 promoters in the prior art, the gene editing efficiency of the Onobrychis viciafilia CRISPR / Cas9 gene editing vector constructed based on the U6 promoter derived from the Onobrychis viciafilia is significantly improved, and the Onobrychis viciafilia CRISPR / Cas9 gene editing vector can induce deletion and insertion mutations of various lengths, and has an advantage in producing diversified editing types. The application not only provides a more accurate and powerful tool for research on gene functions of the Onobrychis viciafilia, but also provides key technical support for construction of a molecular breeding system of the Onobrychis viciafilia.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the U6 promoter derived from red clover and its application in red clover gene editing. Background Technology

[0002] Onobrychis viciifolia Scop. is a perennial leguminous forage grass species with multiple advantages, including high protein content, good palatability, strong stress resistance, and nitrogen fixation, making it widely used in livestock production and ecological restoration. In recent years, with the increasing demands of grassland agriculture and ecological environment construction, the need for improving the stress resistance, yield, and nutritional quality of Onobrychis viciifolia has been growing. However, because Onobrychis viciifolia is an autotetraploid and self-incompatible, its genome is complex and highly heterozygous. Traditional genetic breeding methods are time-consuming and inefficient, severely hindering the progress of genetic improvement. Therefore, it is urgent to establish a molecular breeding technology system suitable for Onobrychis viciifolia to accelerate the analysis and utilization of its superior traits.

[0003] CRISPR / Cas systems are widely used in gene function research and trait improvement in plants such as Arabidopsis thaliana, rice, soybean, and alfalfa due to their simplicity, high specificity, and high efficiency. However, there are currently no published reports on CRISPR / Cas9 gene editing systems for *Alopecurus aequalis*. The main bottlenecks include: the lack of an endogenous U6 promoter adapted to *Alopecurus aequalis*, the lack of an efficient and stable genetic transformation system, and the lack of verification of the usability of exogenous U6 promoters in *Alopecurus aequalis*. In particular, the U6 promoter, as a core element driving sgRNA expression, exhibits significant sequence differences among different plants and strong species specificity; exogenous U6 promoters often show decreased driving efficiency or complete failure in non-model species. Therefore, the primary prerequisite for constructing a gene editing system for *Alopecurus aequalis* is to obtain its endogenous, highly active U6 promoter.

[0004] On the other hand, there is currently a lack of mature tissue culture and stable genetic transformation systems for *Alopecurus aequalis*. While existing leguminous crops such as soybeans and alfalfa often use cotyledonary nodes or callus tissue for genetic transformation, these methods are difficult to apply to *Alopecurus aequalis*, often resulting in problems such as difficulty in callus induction and low regeneration efficiency, making stable transgenic screening and mutant creation extremely challenging. Therefore, establishing a rapid, reproducible, and highly efficient gene editing verification system for *Alopecurus aequalis* has become a key technical problem urgently needing to be solved in this field. Hairy root transformation systems, due to their independence from plant regeneration cycles, ease of operation, and suitability for rapid verification of CRISPR editing efficiency, have been used in various leguminous crops, but currently there is no mature system suitable for *Alopecurus aequalis*. Therefore, constructing a hairy root CRISPR editing system for *Alopecurus aequalis* is of great significance. Summary of the Invention

[0005] Given the lack of suitable endogenous U6 promoter resources, CRISPR / Cas9 gene editing systems, and efficient and reliable genetic transformation systems for hairy roots of *Alopecurus aequalis* in the existing technology, this invention provides an endogenous, highly efficient U6 promoter suitable for *Alopecurus aequalis*, a CRISPR gene editing vector constructed based on the U6 promoter, and gene editing and genetic transformation methods for *Alopecurus aequalis* based on the CRISPR gene editing vector.

[0006] In a first aspect, the present invention provides a DNA molecule.

[0007] The provided DNA molecule is either A1) or A2) below. A1) The DNA molecule shown in sequence 4, 5, or 6; DNA molecules defined in A2) and A1) are DNA molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity and have the same function.

[0008] Those skilled in the art can readily mutate the nucleotide sequence of the DNA molecule of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the DNA molecule provided by the present invention, provided they possess promoter function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0009] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of the DNA molecule of the present invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0010] All of the above DNA molecules possess promoter activity.

[0011] Secondly, the present invention provides biomaterials related to the aforementioned DNA molecules.

[0012] The biomaterial is any one of the following B1)-B6): B1) Expression cassettes containing the aforementioned DNA molecules; B2) A recombinant vector containing the above-mentioned DNA molecules, or a recombinant vector containing the expression cassette described in B1); B3) Recombinant microorganisms containing the above-mentioned DNA molecules, or recombinant microorganisms containing the expression cassette described in B1), or recombinant microorganisms containing the recombinant vector described in B2); B4) Transgenic plant cell lines containing the above-mentioned DNA molecules, or transgenic plant cell lines containing the expression cassette described in B1); B5) Transgenic plant tissue containing the above-mentioned DNA molecules, or transgenic plant tissue containing the expression cassette described in B1); B6) Transgenic plant organs containing the above-mentioned DNA molecules, or transgenic plant organs containing the expression cassette described in B1).

[0013] In the aforementioned biological materials, the expression cassette (5' to 3') may include a promoter region (composed of the DNA molecule), a transcription initiation region, a target gene region, a transcription termination region, and optionally a translation termination region. The promoter region and the target gene region may be natural / similar to the host cell, or the promoter region and the target gene region may be natural / similar to each other, or the promoter region and / or the target gene region may be heterologous to the host or to each other. "Heterologous" means that the sequence is derived from a foreign species, or, if from the same species, the natural form has been substantially modified in terms of components and / or genomic sites through deliberate human intervention. The optionally included transcription termination region may be homologous to the transcription initiation region, homologous to the operatively linked target gene region, homologous to the host; or the target gene region and the host may be foreign or heterologous.

[0014] The expression box may also include a 5' guide sequence. The 5' guide sequence can enhance translation.

[0015] During expression cassette preparation, adapters may be used to ligate DNA fragments, or other operations may be involved to provide appropriate restriction enzyme sites, remove excess DNA, or remove restriction enzyme sites. To achieve this, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, and replacement, such as conversion and transversion, may be performed.

[0016] The expression cassette may also include a selective marker gene for screening transformed cells. The selective marker gene can be used to screen transformed cells or tissues. Marker genes include genes encoding antibiotic resistance. Other selective markers include phenotypic markers such as fluorescent proteins. The selective markers listed above are not limiting. Any selective marker gene can be used in this invention.

[0017] In the aforementioned biological materials, the vector refers to a vector that uses the aforementioned DNA molecule as a promoter to deliver the target gene into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned DNA molecule and the target gene with a plant expression vector. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb.

[0018] In the aforementioned biological materials, the microorganisms can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The recombinant microorganisms refer to those whose genes have been manipulated and modified to obtain recombinant microorganisms with altered functions. For example, recombinant microorganisms obtained by introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0019] In the aforementioned biological materials, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the DNA molecule into the recipient plant, but also its progeny. For transgenic plants, the DNA molecule can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties.

[0020] The transgenic plant tissues may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers. The transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant. The transgenic plant cell lines, the transgenic plant tissues, and the transgenic plant organs may or may not include propagation material.

[0021] Thirdly, this invention provides a CRISPR / Cas9 gene editing vector.

[0022] The CRISPR / Cas9 gene editing vector includes a Cas9 protein expression cassette and an sgRNA expression cassette; the sgRNA in the sgRNA expression cassette is transcribed by the aforementioned DNA molecules.

[0023] In the aforementioned CRISPR / Cas9 gene editing vector, the Cas9 protein is either C1 or C2. C1) The amino acid sequence of the protein is shown in sequence 9; C2) A protein that has the same function as the amino acid sequence shown in Sequence 9, but with one or more amino acid residues substituted and / or deleted and / or added.

[0024] The Cas9 protein-coding gene is either c1 or c2). c1) The DNA molecule shown in positions 11746-15846 of sequence 7; c2) has 75% or more identity with the nucleotide sequence defined by c1) and is a DNA molecule encoding the Cas9 protein.

[0025] In the aforementioned CRISPR / Cas9 gene editing vector, the sgRNA expression cassette includes a tRNA sequence and an sgRNA backbone.

[0026] The tRNA sequence is shown in positions 10173-10249 of sequence 7.

[0027] The nucleotide sequence of the sgRNA backbone is shown in positions 10270-10345 of sequence 7.

[0028] In the aforementioned CRISPR / Cas9 gene editing vector, the sgRNA expression cassette expresses one, two, or more sgRNAs.

[0029] In some embodiments, the sgRNA expression cassette expresses two sgRNAs. In this case, the sgRNA expression cassette sequentially includes a promoter for driving the transcription of the sgRNA, a tRNA sequence, a target 1 sequence, an sgRNA backbone, the tRNA sequence, a target 2 sequence, and the sgRNA backbone.

[0030] Fourthly, the present invention provides any one of the following applications (D1)-D6): D1) The application of the above DNA molecules as promoters; D2) The application of the above-mentioned DNA molecules or the above-mentioned biological materials in driving the expression of target genes; D3) Application of the above DNA molecules in the construction of CRISPR / Cas9 gene editing vectors; D4) Application of the above-mentioned DNA molecules, biological materials, or CRISPR / Cas9 gene editing vectors in gene editing of *Lysimachia foenum-graecum*. D5) Application of the above-mentioned DNA molecules, biological materials, or CRISPR / Cas9 gene editing vectors in the genetic transformation of red bean grass; D6) Application of the above-mentioned DNA molecules, biological materials, or CRISPR / Cas9 gene editing vectors in improving the gene editing efficiency of red bean grass.

[0031] Fifthly, the present invention provides any one of the following methods (F1)-F3): F1) A method for gene editing of red bean grass, comprising the following steps: introducing the above-mentioned CRISPR / Cas9 gene editing vector into red bean grass to achieve gene editing of red bean grass; F2) A method for improving the gene editing efficiency of red bean grass, comprising the following steps: introducing the above-mentioned CRISPR / Cas9 gene editing vector into red bean grass to improve the gene editing efficiency of red bean grass; F3) A method for genetic transformation of red bean grass, comprising the following steps: using Agrobacterium containing the above-mentioned CRISPR / Cas9 gene editing vector to infect red bean grass, thereby achieving genetic transformation of red bean grass.

[0032] The genetic transformation of any of the above-mentioned red bean grasses into the hairy root genetic transformation of red bean grasses.

[0033] The method described above for introducing the CRISPR / Cas9 gene editing vector into red bean grass involves infecting red bean grass with Agrobacterium containing the CRISPR / Cas9 gene editing vector.

[0034] The above-described method of infecting red clover with Agrobacterium containing a CRISPR / Cas9 gene editing vector includes the following steps: 1) After sterilizing the seeds by soaking them in sterile water, place them at 4℃ for 24 h, and then place the seeds on 1 / 2MS solid medium and culture them in a dark incubator at 25℃ for 48-60 h to obtain seedlings with a radicle length of about 2-3 cm. 2) After cutting off the root tip (about 0.5 cm) of the seedling, place it in the infection solution, sonicate for 30 seconds, and vacuum for 10 minutes to obtain the infected seedling; 3) The infected seedlings were placed on 1 / 2 MS solid medium and cultured for 3 days in a 20℃, 16h light / 8h dark incubator. Then they were transferred to 1 / 2 MS Re medium for recovery culture for 4 days, and then transferred to 1 / 2 MH screening medium for 10-14 days to obtain transgenic hairy roots.

[0035] In step 1) above, the disinfection method includes the following steps: first rinse twice with clean water, vacuum for 15 minutes to break the seed coat, then disinfect with 75% alcohol for 1 minute, then disinfect on a shaker with 6% sodium hypochlorite solution (with 0.1% surfactant Triton X-100) for 15 minutes, and finally rinse 5-6 times with sterile water until no foam remains.

[0036] The solvent for the 1 / 2MS solid culture medium is water, and the solvent and its concentration are as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L.

[0037] In step 2) above, the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0038] The preparation method of the infection solution is as follows: Agrobacterium containing the CRISPR / Cas9 gene editing vector is cultured overnight in YEP liquid medium (containing Rifampicin and Kanamycin) at 28°C and 220 rpm. The Agrobacterium, after being shaken to saturation, is centrifuged at 6000 rpm for 15 min, the supernatant is removed, and the solution is transferred to 1 / 2 MS liquid medium (with 100 mg / L of acetic acid added). Resuspend the sample in 1 mol / L acetylsalicylic acid and adjust the OD value to 0.3-0.5 to obtain the inoculum.

[0039] The YEP liquid culture medium (pH 7.0) is in water as the solvent, and the solutes and their concentrations are 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl.

[0040] The solvent for the 1 / 2MS liquid culture medium is water, and the solutes and their concentrations are as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L.

[0041] In step 3) above, if color screening markers are used, positive hairy roots can be obtained directly on 1 / 2 MSRe medium based on the color of the hairy roots without using 1 / 2MH medium.

[0042] In some implementations, a RUBY color screening marker is used to screen for positive hairy roots based on whether a red color is present.

[0043] The solvent for the 1 / 2 MSRe medium was water, and the solutes and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L, termethin 100 mg / L, cephalosporin 125 mg / L.

[0044] The screening medium 1 / 2MH was water as the solvent, and the solutes and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L, termethin 100 mg / L, cephalosporin 125 mg / L, hygromycin 3 mg / L.

[0045] The gene editing described above includes base substitution, base insertion, and / or base deletion of the genome sequence.

[0046] The bases include monobasic and / or polybasic bases.

[0047] The edited site may include one, two, or more sites. The site may be a neighboring site or a non-neighboring site.

[0048] The base deletions include deletions of less than 5 bp, deletions of 5-10 bp, deletions of 11-30 bp, and deletions of more than 30 bp.

[0049] The base insertions include insertions of less than 5 bp and insertions of 5-10 bp.

[0050] The red bean grass mentioned above can be common red bean grass.

[0051] This invention provides the U6 promoter derived from *Lysimachia foenum-graecum* and its application in gene editing within *Lysimachia foenum-graecum*. The U6 promoter derived from *Lysimachia foenum-graecum* includes the OvU6-1, OvU6-4, and OvU6-24 promoters. This invention also provides a *Lysimachia foenum-graecum* CRISPR / Cas9 gene editing vector constructed based on the aforementioned U6 promoter. Experiments have demonstrated that, compared to the AtU6, GmU6, and MsU6 promoters in existing technologies, the *Lysimachia foenum-graecum* CRISPR / Cas9 gene editing vector constructed based on the U6 promoter of this invention significantly improves gene editing efficiency and can induce deletion and insertion mutations of various lengths, showing advantages in generating diverse editing types. This invention not only provides a more precise and powerful tool for the study of *Lysimachia foenum-graecum* gene function but also provides crucial technical support for the construction of a *Lysimachia foenum-graecum* molecular breeding system. Attached Figure Description

[0052] Figure 1 The phylogenetic tree constructed by the OvU6 promoter together with the AtU6, GmU6, MtU6, and MsU6 promoters.

[0053] Figure 2 Conservative sequence analysis of the OvU6 promoter and the AtU6, GmU6, MtU6, and MsU6 promoters.

[0054] Figure 3 To identify the GUS activity of the OvU6 promoter in tobacco and red clover. In this study, A represents the identification of the GUS activity of the OvU6 promoter in tobacco; and B represents the identification of the GUS activity of the OvU6 promoter in red clover.

[0055] Figure 4 A shows schematic diagrams of the T1 and T2 target sites of OvWOX13 and schematic diagrams of the editing vector containing AtU6, GmU6, MsU6, and OvU6 promoters. B shows schematic diagrams of the editing vector containing AtU6, GmU6, MsU6, and OvU6 promoters.

[0056] Figure 5 The diagram shows the genetic transformation process and identification of hairy roots in *Sedum morganianum*. A is the genetic transformation process of hairy roots in *Sedum morganianum*. B is the DNA identification of hairy roots expressing RUBY in *Sedum morganianum* and a schematic diagram of red hairy roots.

[0057] Figure 6 This is a statistical result of gene editing efficiency.

[0058] Figure 7 This is a statistical result of gene editing types. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0061] The YEP liquid culture medium (pH 7.0) in the following examples uses water as the solvent, and the solutes and their concentrations are 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl.

[0062] The solvent for the 1 / 2MS solid culture medium in the following examples was water, and the solvents and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L.

[0063] The solvent for the 1 / 2MS liquid culture medium in the following examples was water, and the solutes and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L.

[0064] The solvent for the 1 / 2 MSRe medium in the following examples was water, and the solutes and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L, termethin 100 mg / L, cephalosporin 125 mg / L.

[0065] The solvent for the 1 / 2MH screening medium in the following examples was water, and the solutes and their concentrations were as follows: ammonium nitrate 825.0 mg / L, potassium nitrate 950.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, anhydrous calcium chloride 166.10 mg / L, anhydrous magnesium sulfate 90.35 mg / L, boric acid 3.10 mg / L, manganese sulfate monohydrate 8.45 mg / L, zinc sulfate heptahydrate 4.30 mg / L, potassium iodide 0.415 mg / L, sodium molybdate dihydrate 0.125 mg / L, cobalt chloride hexahydrate 0.0125 mg / L, copper sulfate pentahydrate 0.0125 mg / L, ferrous sulfate heptahydrate 13.90 mg / L, disodium EDTA (dihydrate) 18.630 mg / L, glycine 1.0 mg / L, myo-inositol 50.0 mg / L, and nicotinic acid 0.25 mg / L. mg / L, pyridoxine hydrochloride (vitamin B6) 0.25 mg / L, thiamine hydrochloride (vitamin B1) 0.05 mg / L, agar 7 g / L, termethin 100 mg / L, cephalosporin 125 mg / L, hygromycin 3 mg / L.

[0066] Example 1: Obtaining and Sequence Screening of the U6 Promoter from *Lysimachia christinae* I. Experimental Materials and Methods 1. Source of U6 sequence alignment The AtU6-26 (AT3G13855) sequence of Arabidopsis was retrieved and downloaded from the Arabidopsis genome Tair (https: / / www.arabidopsis.org). The sequence was then aligned with the sequence of the red clover genome (https: / / doi.org / 10.1038 / s42003-023-05754-6) using the BLAST function of TBtools, yielding 25 candidate snRNA sequences.

[0067] 2. Extraction of the starter sub-region Using the above snRNA as the transcription start site (+1 site), a 500-1000 bp region was extracted upstream to obtain the candidate red bean grass U6 promoter sequence.

[0068] 3. Sequence conservation analysis We used SnapGene to construct sequence alignment and phylogenetic trees (NJ method) to analyze promoter core elements such as USE, TATA-like box, and DSE in the 500bp region upstream of the snRNA transcription start site.

[0069] 4. Promoter activity screening (GUS transient expression) (1) Promoter cloning The AtU6-26 promoter was obtained by PCR amplification from the pRGB31 vector (https: / / doi:10.1093 / mp / sst119), the GmU6 promoter was obtained by PCR amplification from the p4708 vector (https: / / doi.org / 10.1016 / j.xinn.2024.100564), and the MsU6-7 promoter was obtained by PCR amplification from the pHSE401-MsU6 vector (https: / / doi.org / 10.1007 / s42994-025-00200-z). Multiple U6 promoters containing conserved elements were obtained by PCR amplification from *Hedysarum heterotropoides* genomic DNA and sequenced. The primer sequences are shown in Table 1.

[0070] Table 1

[0071] Different U6 promoter fragments derived from *Lysimachia foenum-graecum* were obtained through PCR amplification and cloning sequencing. The sequencing results were consistent with the predicted sequences of the *Lysimachia foenum-graecum* reference genome, with sequence lengths of approximately 400-500 bp. All fragments contained typical conserved elements of the U6 promoter (such as TATA boxes and USE elements), indicating that the cloned fragments can be used as promoter sequences for subsequent functional verification.

[0072] Evolutionary trees constructed based on different U6 promoter sequences, such as Figure 1 As shown, conserved sequence analysis is as follows Figure 2 As shown.

[0073] (2) Carrier construction The 35S promoter in the pMDC162-U6:GUS vector (Doi: 10.1104 / pp.103.027979) was replaced with different U6 promoters from red bean grass in step (1) to obtain vectors with different U6 promoters driving GUS expression.

[0074] (3) Transient expression of tobacco to verify GUS activity a. Activate Agrobacterium GV3101 and P19 of pMDC162-U6:GUS on YEP solid medium, and inoculate the plaques into YEP liquid medium and shake until saturated.

[0075] b. Centrifuge at 6000 g for 10 min, discard the supernatant, and resuspend the bacterial cells in tobacco conversion broth.

[0076] c. Mix the target protein and the P19 resuspension in a 1:1 ratio, and adjust the OD of the mixed bacterial culture. 600 The concentration is 1.0. After mixing, let it stand at room temperature in the dark for 2 hours.

[0077] d. Select tobacco leaves that have not flowered for 5-6 weeks and are growing well. Use a sterile syringe to inject the mixed bacterial solution into the tobacco leaves from the back. After placing them in the dark for 2-3 hours, wait for the tobacco to grow normally for 2-3 days, and then use a hole punch to make small holes of the same size.

[0078] e. Stain with X-Gluc (1 mg / mL), potassium ferricyanide / potassium ferrocyanide (1 mM each), and 0.1% Triton X-100 at 37℃ for 24 h, destain with 95% alcohol, and then take a picture.

[0079] (4) Transient expression of GUS activity by red bean grass a. Activate GV3101 Agrobacterium and P19 of pMDC162-U6:GUS on YEP solid medium, and inoculate the plaques into YEP liquid medium and shake until saturated.

[0080] b. Centrifuge at 6000 g for 10 min, discard the supernatant, and resuspend the bacterial cells in red bean grass transformation solution.

[0081] c. Mix the target protein and the resuspension of P19 in a 1:1 ratio, and adjust the OD of the mixed bacterial culture. 600 The concentration is 1.0. After mixing, let it stand at room temperature in the dark for 2 hours.

[0082] d. Select the second and third leaf positions from the top of the pinnate compound leaf of a healthy Hoagland plant that has been hydroponically grown for 3 weeks. Inject the mixed bacterial solution into the Hoagland plant from the underside of the leaf using a sterile syringe. After placing it in the dark for 2-3 hours, allow the Hoagland plant to grow normally for 3 days.

[0083] e. Use X-Gluc (1 mg / mL), potassium ferricyanide / potassium ferrocyanide 1 mM each, 0.1% Triton X-100 0.8 MPa vacuum for 30 min, stain at 37℃ for 48 h, decolorize with 95% alcohol and take a picture.

[0084] II. Experimental Results The results showed that promoters OvU6-1, OvU6-4, and OvU6-24 produced the deepest GUS staining in both tobacco and red clover. Figure 3 This indicates that it has the highest expression activity and can be used as a preferred promoter for the expression of red bean grass sgRNA.

[0085] Example 2: Construction of CRISPR / Cas9 editing vectors based on different U6 promoters and their application in gene editing of *Lysimachia foenum-graecum*. I. Construction of CRISPR / Cas9 editing vectors based on different U6 promoters This invention uses OvWOX13 as a target and artificially constructs the following CRISPR / Cas9 editing vectors (each vector is a circular plasmid): The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 is composed of the DNA molecules shown in Sequences 7 and 8. Specifically, positions 5007-5469 of Sequence 7 represent the nucleotide sequence of the CmYLCV promoter; positions 5480-9412 represent the RUBY protein-coding gene sequence; positions 9413-9661 represent the HSP terminator sequence; positions 9880-10172 represent the AtU6-26 promoter sequence; positions 10173-10249 represent the tRNA sequence; positions 10250-10269 represent the Target1 sequence; and positions 10270-10345 represent the sgRNA1 backbone sequence. Sequence 7, positions 10346-10422, is the tRNA sequence; positions 10153-10442, the Target2 sequence; positions 10443-10518, the sgRNA2 backbone sequence; positions 10519-10526, the AtU6t terminator sequence; positions 11132-11476, the 2×35S promoter sequence; positions 11746-15846, the Cas9 protein-coding gene sequence (encoding the Cas9 protein shown in Sequence 9); positions 58-689, the E9t terminator sequence; positions 1010-1686, the 35S promoter sequence; and positions 1753-2778, the Hyg protein-coding gene sequence.

[0086] The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-GmU6-OvWOX13 is obtained by replacing the AtU6-26 promoter sequence in the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 with the GmU6 promoter.

[0087] The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-MsU6-7-OvWOX13 is the sequence obtained by replacing the AtU6-26 promoter sequence in the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 with the MsU6-7 promoter.

[0088] The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-OvU6-1-OvWOX13 is the sequence obtained by replacing the AtU6-26 promoter sequence in the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 with the OvU6-1 promoter.

[0089] The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-OvU6-4-OvWOX13 is the sequence obtained by replacing the AtU6-26 promoter sequence in the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 with the OvU6-4 promoter.

[0090] The nucleotide sequence of the CRISPR / Cas9 editing vector pHSE401-RUBY-OvU6-24-OvWOX13 is the sequence obtained by replacing the AtU6-26 promoter sequence in the CRISPR / Cas9 editing vector pHSE401-RUBY-AtU6-26-OvWOX13 with the OvU6-24 promoter.

[0091] The OvWOX13 gene target sequence and structural diagrams of various CRISPR / Cas9 editing vectors are shown below. Figure 4 As shown.

[0092] II. Genetic transformation of hairy roots of *Lysimachia foenum-graecum* 1. Preparation of recombinant bacteria Each CRISPR / Cas9 editing vector constructed in step one was transformed into Agrobacterium tumefaciens GV3101, resulting in Agrobacterium tumefaciens GV3101 containing different CRISPR / Cas9 editing vectors. The specific steps are as follows: 100 ng of plasmid (CRISPR / Cas9 editing vector) was added to Agrobacterium tumefaciens GV3101 competent cells. After incubating on ice for 30 min, the cells were flash-frozen in liquid nitrogen for 1 min, heat-shocked at 37°C for 1 min, and incubated on ice for 5 min. Then, 500 μL of antibiotic-free LB liquid medium was added, and the cells were thawed at 28°C and 220 rpm for 2-3 h. The bacterial culture was then spread on YEP solid selection medium containing Rifampicin (50 mg / L) and Kanamycin (50 mg / L), and cultured in the dark at 28°C for 24-36 h. Single clones were picked for colony PCR identification. PCR amplification was performed using primers M13-F (5'-GTAAAACGACGGCCAGT-3') and Bsa-R (5'-CAGAAATTGAACGCCGAAGA-3'). Clones with a specific band of 1000 bp were considered positive clones. Positive clones were picked and placed into 2 mL of YEP liquid medium containing Rifampicin (50 mg / L) and Kanamycin (50 mg / L), and incubated overnight at 28°C and 220 rpm.

[0093] 2. Genetic transformation system of hairy roots of *Corydalis yanhusuo* The flowchart of genetic transformation of hairy roots of *Corydalis yanhusuo* is as follows: Figure 5 As shown in A. The specific steps are as follows: (1) Take several plump common red bean grass seeds into a 50 mL centrifuge tube and disinfect them according to the following steps: first rinse twice with clean water, vacuum for 15 min to break the seed coat, then disinfect with 75% alcohol for 1 min, rinse twice with clean water, then disinfect with 6% sodium hypochlorite solution (with 0.1% surfactant Triton X-100) on a shaker for 15 min, and finally rinse with sterile water 5-6 times until there is no foam (all operations from this step onwards must be performed in a clean bench). After disinfection, add an appropriate amount of sterile water to soak the seeds, place them at 4℃ for 24 h, then spread the seeds on 1 / 2 MS solid medium, absorb excess water, wrap with plastic wrap and place in a dark incubator at 25℃ for germination for 48-60 h. The radicles are about 2-3 cm long and can be used for infection.

[0094] (2) Centrifuge the Agrobacterium tumefaciens at 6000 rpm for 15 min after shaking until saturated, remove the supernatant, and use 1 / 2 MS liquid culture medium (add 100 mg / L) Resuspend the sample in 1 mol / L acetylsalicylic acid and adjust the OD value to 0.3-0.5 to obtain the inoculum.

[0095] (3) Add a small amount of 1 / 2MS liquid medium to a petri dish. Use tweezers to pick up seedlings with radicles about 2-3 cm long and gently fix them. Cut off about 0.5 cm of the root tip with a scalpel. Then place the seedlings in the inoculation solution, sonicate for 30 seconds, vacuum for 10 minutes, and dry the remaining Agrobacterium on filter paper to obtain infected seedlings. Place the infected seedlings on 1 / 2MS solid medium (with a filter paper to prevent excessive Agrobacterium infection), seal with plastic wrap, and incubate at 20℃ for 3 days in a 16h light / 8h dark incubator. Then transfer to 1 / 2MSRe medium for recovery culture for 4 days, and then transfer to 1 / 2MH selection medium to select for rooting for 10-14 days. Select positive hairy roots. If using RUBY or GFP as selection markers, 1 / 2MH medium can be omitted, and positive hairy roots can be selected directly on 1 / 2MSRe medium. RUBY can be seen with the naked eye as red hairy roots, and GFP can be seen as green fluorescence at a wavelength of 488 nm.

[0096] III. Analysis of Editing Efficiency and Editing Types of Different CRISPR / Cas9 Editing Vectors 1. Comparison of editing efficiency of CRISPR / Cas9 editing vectors containing different U6 promoters Genomic DNA was extracted from positive hairy roots and then identified by PCR. Primers Cas9-F (5'-GAGATGGCGAAGGTTGACGA-3') and Cas9-R (5'-CCACCTCCTCGAAGTTCCAC-3') were designed based on the Cas9 gene sequence at a length of 1000 bp. Then, primers were designed within 100 bp of the target site to amplify the target fragment, and the editing status of the target sequence in the positive hairy roots identified by PCR was detected. The specific primer sequences are as follows: WOX13-1.1&1.2-T1-F: 5'-ggagtgagtacggtgtgcATGGTGAACTTGATGGAATGG-3'; WOX13-1.1&1.2-T1-R: 5'-gagttggatgctggatggAGCAGAGAGTGTTTTGTGCATTT-3'; WOX13-1.1-T2-F: 5'-ggagtgagtacggtgtgcGTCAGCACGGTCCTATTTCTGA-3'; WOX13-1.1-T2-R: 5'-gagttggatgctggatggCGGGAAGCACAAATTTTCAGC-3'. Finally, the amplified target fragments were sent to the Hi-TOM platform (http: / / www.hi-tom.net) for NGS next-generation sequencing, with 5000 reads sequenced for each sample. In the Hi-TOM analysis, only edit types with a mutation frequency ((reads containing mutations / total sequencing reads at the target site) × 100%) higher than 5% were counted. The mutation frequencies of different edit types were summed to obtain the gene editing efficiency of each independent edit event. The gene editing efficiency of all edit events for two target sites (OvWOX13-Target1 and OvWOX13-Target2) driven by different promoters was statistically analyzed.

[0097] Gene editing efficiency statistics show that, for the Target1 target, the median gene editing efficiency of editing vectors containing the AtU6 promoter is approximately 0%, the median gene editing efficiency of editing vectors containing the GmU6 promoter is also approximately 0%, the median gene editing efficiency of editing vectors containing the MsU6 promoter is also approximately 0%, the median gene editing efficiency of editing vectors containing the OvU6-1 promoter is approximately 10%, the median gene editing efficiency of editing vectors containing the OvU6-4 promoter is approximately 8%, and the median gene editing efficiency of editing vectors containing the OvU6-24 promoter is... The median gene editing efficiency was approximately 6% for the Target2 target; for the Target2 target, the median gene editing efficiency of editing vectors containing the AtU6 promoter was approximately 52%, for those containing the GmU6 promoter it was approximately 14%, for those containing the MsU6 promoter it was approximately 30%, for those containing the OvU6-1 promoter it was approximately 60%, for those containing the OvU6-4 promoter it was approximately 55%, and for those containing the OvU6-24 promoter it was approximately 53%.

[0098] The results above show that, for both target sites, the gene editing efficiency of editing vectors containing the OvU6 promoter is higher than that of editing vectors containing the exogenous U6 promoter. Therefore, the OvU6-1, OvU6-4 and OvU6-24 promoters can be used as endogenous promoters to improve the gene editing efficiency of *Lysimachia nummularia*.

[0099] 2. Percentage analysis of gene editing types targeting different sites Taking two representative targets, Target1 and Target2, from *Lysimachia christinae* as examples, a statistical analysis was conducted on the relative proportion of the promoter editing events of different targets (based on the number of effective edited reads) according to the Left variation type. Based on the length of deletions and insertions, the editing types were classified as follows: <5 Deletion (deletion less than 5 bp), 5–10 Deletion (deletion 5–10 bp), 11–30 Deletion (deletion 11–30 bp), >30 Deletion (deletion greater than 30 bp), <5 Insertion (insertion less than 5 bp), 5–10 Insertion (insertion 5–11 bp), >10 Insertion (insertion greater than 10 bp), and SNP (single nucleotide base diversity).

[0100] Statistical results of gene editing types show that for Target1, the editing type distribution is as follows: <5 bp deletion accounts for 39.82%, 5–10 bp deletion accounts for 26.40%, 11–30 bp deletion accounts for 7.44%, >30 bp deletion accounts for 0.36%, <5 bp insertion accounts for 25.94%, 5–10 bp insertion accounts for 0.04%, and SNP accounts for 1.20%. For Target2, the editing type distribution is as follows: <5 bp deletion accounts for 32.44%, 5–10 bp deletion accounts for 32.04%, 11–30 bp deletion accounts for 20.18%, >30 bp deletion accounts for 2.76%, and <5 bp insertion accounts for 12.57%. No insertions >5 bp or single-base mutations were detected at this target.

[0101] The results show that the OvU6-driven CRISPR / Cas9 multi-target editing system can induce deletion and insertion mutations of various lengths in red clover, with short deletions (<10 bp) and small insertions (<5 bp) being the most common. This demonstrates the advantages of the system in generating diverse editing types and can meet the needs of gene knockout or frameshift mutations of different functions.

[0102] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A DNA molecule, as shown in A1) or A2). A1) The DNA molecule shown in sequence 4, 5, or 6; DNA molecules defined in A2) and A1) are DNA molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity and have the same function.

2. A biomaterial relating to the DNA molecule of claim 1, wherein the biomaterial is any one of B1)-B6): B1) An expression cassette containing the DNA molecule of claim 1; B2) A recombinant vector containing the DNA molecule of claim 1, or a recombinant vector containing the expression cassette of claim B1; B3) A recombinant microorganism containing the DNA molecule of claim 1, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2); B4) A transgenic plant cell line containing the DNA molecule of claim 1, or a transgenic plant cell line containing the expression cassette of claim B1; B5) Transgenic plant tissue containing the DNA molecule of claim 1, or transgenic plant tissue containing the expression cassette of claim B1; B6) A transgenic plant organ containing the DNA molecule of claim 1, or a transgenic plant organ containing the expression cassette of claim B1).

3. A CRISPR / Cas9 gene editing vector; the CRISPR / Cas9 gene editing vector comprises a Cas9 protein expression cassette and an sgRNA expression cassette; the sgRNA expression cassette is transcribed by the DNA molecule described in claim 1.

4. The CRISPR / Cas9 gene editing vector according to claim 3, characterized in that: The Cas9 protein is either C1 or C2. C1) The amino acid sequence of the protein is shown in sequence 9; C2) A protein that has the same function as the amino acid sequence shown in Sequence 9, but with one or more amino acid residues substituted and / or deleted and / or added.

5. The CRISPR / Cas9 gene editing vector according to claim 3 or 4, characterized in that: The sgRNA expression cassette includes a tRNA sequence and an sgRNA backbone; Alternatively, the tRNA sequence is shown as positions 10173-10249 of sequence 7; Alternatively, the nucleotide sequence of the sgRNA backbone is shown in positions 10270-10345 of sequence 7.

6. The CRISPR / Cas9 gene editing vector according to any one of claims 3-5, characterized in that: The sgRNA expression cassette expresses one, two, or more sgRNAs.

7. Any one of the following applications (D1)-D6): D1) The use of the DNA molecule of claim 1 as a promoter; D2) The application of the DNA molecule of claim 1 or the biomaterial of claim 2 in driving the expression of the target gene; D3) The application of the DNA molecule described in claim 1 in the construction of CRISPR / Cas9 gene editing vectors; D4) Application of the DNA molecule of claim 1, the biomaterial of claim 2, or the CRISPR / Cas9 gene editing vector of any one of claims 3-6 in gene editing of red bean grass; D5) Application of the DNA molecule of claim 1, the biomaterial of claim 2, or the CRISPR / Cas9 gene editing vector of any one of claims 3-6 in the genetic transformation of red bean grass; D6) The application of the DNA molecule of claim 1, the biological material of claim 2, or the CRISPR / Cas9 gene editing vector of any one of claims 3-6 in improving the gene editing efficiency of red bean grass.

8. The application according to claim 7, characterized in that: The genetic transformation of *Corydalis yanhusuo* into the genetic transformation of *Corydalis yanhusuo* hairy roots.

9. Any of the following methods (F1)-F3): F1) A method for gene editing of red bean grass, comprising the following steps: introducing the CRISPR / Cas9 gene editing vector as described in any one of claims 3-6 into red bean grass to achieve gene editing of red bean grass; F2) A method for improving the gene editing efficiency of red clover, comprising the following steps: introducing the CRISPR / Cas9 gene editing vector as described in any one of claims 3-6 into red clover to improve the gene editing efficiency of red clover; F3) A method for genetic transformation of red clover, comprising the following steps: infecting red clover with Agrobacterium containing the CRISPR / Cas9 gene editing vector as described in any one of claims 3-6 to achieve genetic transformation of red clover.

10. The method according to claim 9, characterized in that: The genetic transformation of *Corydalis yanhusuo* into the genetic transformation of *Corydalis yanhusuo* hairy roots.

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