Method for creating fragrant mian with crisper / cas12i.3 system and application thereof

Abstract

The application discloses a method for creating a fragrant glutinous millet by using a CRISPR / Cas12i.3 system and application thereof, and belongs to the technical field of genetic engineering. The application first applies a miniaturized CRISPR / Cas12i.3 gene editing system with independent intellectual property rights of China to the genetic improvement of millet, and establishes an efficient and precise gene editing technology system suitable for the crop. Compared with commonly used Cas9 and other systems, the Cas12i.3 protein has a smaller volume, is easier to construct a plant expression vector, significantly improves the transformation efficiency, and avoids foreign patent restrictions, thereby providing a key tool for functional gene research and molecular design breeding of millet.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a method for creating fragrant millet using the CRISPR / Cas12i.3 system and its application. Background Technology

[0002] Millet (Panicum miliaceum L.) is an important specialty grain crop in my country, rich in nutrients and with good environmental adaptability. With increasing consumer demand for high-quality agricultural products, millet, with its distinctive aroma, has attracted much attention due to its unique flavor and higher commercial value. Research shows that the aroma trait in plants is usually closely related to the function of the betaine aldehyde dehydrogenase 2 (BADH2) gene. The loss of function or reduced expression of this gene can lead to the accumulation of aromatic substances such as 2-acetyl-1-pyrroline (2-AP), thus producing aroma. However, as a non-model crop, the genetic transformation and gene editing technologies for millet are still underdeveloped, severely restricting the precise and efficient molecular breeding process for superior agronomic traits, including aroma.

[0003] In existing technologies, studies have employed the CRISPR / Cas9 system for gene editing in cereal crops to improve aroma traits. For example, by targeting and knocking out the key gene BADH2, which controls aroma synthesis in rice, with Cas9, rice materials with an aromatic phenotype have been successfully obtained. This method typically involves constructing plant expression vectors containing the Cas9 protein-coding sequence and specific sgRNA, which are then introduced into plant cells via Agrobacterium-mediated transformation or gene gun methods to achieve site-directed mutagenesis of the target gene. Similar strategies have also been attempted to improve the quality of other gramineous crops.

[0004] However, the existing CRISPR / Cas9 system for plant gene editing has the following shortcomings: On the one hand, the Cas9 protein is relatively large (approximately 1368 amino acids), requiring high vector loading capacity, which limits its application in capacity-limited delivery systems (such as certain viral vectors or simplified expression vectors), hindering efficient transformation and multi-gene editing; on the other hand, the core patents related to Cas9 are mainly held by foreign institutions, posing intellectual property risks in commercial applications in my country, thus restricting the construction and promotion of an independent and controllable agricultural bio-breeding technology system.

[0005] In recent years, CRISPR / Cas gene editing technology has provided a revolutionary tool for crop genetic improvement. Although systems such as Cas9 and Cas12a have been successfully applied to some major crops, their editing efficiency is often low in specialty minor crops such as millet. Cas12i.3, a novel CRISPR / Cas system independently discovered and developed by Chinese researchers, has advantages such as short recognition sites, high editing efficiency, and strong specificity, providing new possibilities for overcoming the technical bottlenecks in millet gene editing.

[0006] Currently, there are no reports on using the CRISPR / Cas12i3 system for gene function research and aroma germplasm creation in millet. Therefore, establishing a high-efficiency gene editing platform for millet based on Cas12i3 and applying it to the targeted knockout of the key aroma gene BADH2 is of significant theoretical and practical value for rapidly creating new aroma-enhancing millet germplasm, promoting quality improvement, and advancing industrialization of millet. Summary of the Invention

[0007] The technical problem this application aims to solve is how to use gene editing tools to regulate the aroma of millet and prepare millet with enhanced aroma. To solve this technical problem, this application provides the following technical solution: In a first aspect, this application provides a method for preparing gene-edited millet, the method comprising at least one of A1) and A2) below: A1) Knock out the gene encoding betaine aldehyde dehydrogenase 2 in millet seeds to be edited using a genome editing system, wherein the genome editing system comprises: (i) Protein components, namely, a protein or its conjugate with the amino acid sequence shown in SEQ ID NO:8, and (ii) A nucleic acid component comprising the RNA sequence shown in SEQ ID NO:9 from the 5' to 3' direction. The protein component and the nucleic acid component combine to form a complex; A2) Perform at least one of the following mutations on the genome of the millet seed to be edited: A2-1) Delete the DNA fragment with nucleotide sequence of SEQ ID NO:1 from position 1355 to 1364 and the DNA fragment with nucleotide sequence of SEQ ID NO:4 from position 1243 to 1255 in the millet seed to be edited; A2-2) Delete the DNA fragment with nucleotide sequence of SEQ ID NO:1 from position 1344 to 1370 and the DNA fragment with nucleotide sequence of SEQ ID NO:4 from position 1228 to 1256 in the millet seed to be edited; A2-3) Delete the DNA fragment from positions 1354 to 1365 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:1; A2-4) Delete the DNA fragment from positions 1345 to 1370 of the nucleotide sequence of the millet seed to be edited, such as SEQ ID NO:1; A2-5) Delete the DNA fragment from positions 1245 to 1254 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:4; A2-6) Delete the DNA fragment from positions 1247 to 1257 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:4; The millet seed to be edited contains the encoding gene for betaine aldehyde dehydrogenase 2, which includes PmBADH2a and PmBADH2b. The amino acid sequence of PmBADH2a is shown in SEQ ID NO:3, and the amino acid sequence of PmBADH2b is shown in SEQ ID NO:6.

[0008] In this application, the gene encoding PmBADH2a is shown below: (1) The encoded sequence is shown in SEQ ID NO:2; (2) The nucleotide sequence is shown in SEQ ID NO:1.

[0009] In this application, the gene encoding PmBADH2b is shown below: (1) The encoded sequence is shown in SEQ ID NO:5; (2) The nucleotide sequence is shown in SEQ ID NO:4.

[0010] Secondly, this application also provides the application of the genome editing system and related biological materials in at least one of C1) to C4), wherein the biological materials are at least one of B1) to B4): B1) Isolated nucleic acid molecules, said nucleic acid molecules expressing the protein components and / or nucleic acid components described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); Application of C1 in the preparation of gene-edited millet; C2) Application in the preparation of gene-edited millet products; C3) Application in millet breeding or assisted breeding; C4) Application in the preparation of millet breeding or assisted breeding products.

[0011] Wherein, the nucleic acid molecule expressing the above-mentioned protein components is a DNA molecule or an RNA molecule transcribed from said DNA molecule, at least one of g1) and g2) below: g1) DNA molecules with nucleotide sequences such as positions 3143 to 6286 of SEQ ID NO:7; g2) A DNA molecule that has more than 70% identity with the DNA molecule at positions 3143 to 6286 of SEQ ID NO:7 and encodes the protein component; The nucleic acid molecule expressing the nucleic acid component of claim 1 is a DNA molecule as described in at least one of the following g3) and g4): g3), DNA molecules with nucleotide sequences such as positions 427 to 474 of SEQ ID NO:7; g4) A DNA molecule that has more than 70% identity with the DNA molecule at positions 427 to 474 of SEQ ID NO:7 and that transcribes the nucleic acid components thereon.

[0012] Thirdly, this application also provides the following substances as described in D1) or D2): D1) The genome editing system described above; D2), the aforementioned biological materials.

[0013] Fourthly, this application provides gene-edited millet, said gene-edited millet is named as follows: pmbadh2a / b- 1 , pmbadh2a / b-2, pmbadh2a-1 , pmbadh2a-2 , pmbadh2b-1 and pmbadh2b-2 At least one of the gene-edited millet varieties, The pmbadh2a / b-1 DNA fragments that do not contain nucleotide sequences such as those in positions 1355 to 1364 of SEQ ID NO:1 and nucleotide sequences such as those in positions 1243 to 1255 of SEQ ID NO:4; The pmbadh2a / b-2 DNA fragments that do not contain nucleotide sequences such as those in positions 1344 to 1370 of SEQ ID NO:1 and nucleotide sequences such as those in positions 1228 to 1256 of SEQ ID NO:4; The pmbadh2a-1 It does not contain DNA fragments with nucleotide sequences such as those at positions 1354 to 1365 of SEQ ID NO:1; The pmbadh2a-2It does not contain DNA fragments with nucleotide sequences such as those from positions 1345 to 1370 of SEQ ID NO:1; The pmbadh2b-1 It does not contain DNA fragments with nucleotide sequences such as those at positions 1245 to 1254 of SEQ ID NO:1; The pmbadh2b-2 It does not contain DNA fragments with nucleotide sequences such as positions 1247 to 1257 of SEQ ID NO:1.

[0014] The gene-edited millet seed is obtained by gene editing of the millet seed to be edited, and the millet seed to be edited contains the encoding gene of betaine aldehyde dehydrogenase 2 mentioned above.

[0015] The gene-edited millet seeds were prepared using the method described above.

[0016] Fifthly, this application also provides the use of the above-mentioned millet in at least one of the following: E1) Application in enhancing the aroma of millet; Application of E2 in millet breeding or assisted breeding.

[0017] The application is achieved by hybridizing the gene-edited millet seed as a hybrid parent with the millet seed to be improved.

[0018] This application also provides millet with enhanced aroma, which is obtained by hybridizing the above-mentioned gene-edited millet as a hybrid parent with the millet to be improved.

[0019] In this application, the genome editing system is a CRISPR / Cas12i.3 system, in which the protein component is Cas12i.3. Cas12i.3 protein is smaller in size, making it easier to construct plant expression vectors and significantly improving transformation efficiency.

[0020] In this application, the expression cassette of the protein component contains a DNA fragment with a nucleotide sequence as shown in positions 3143 to 6286 of SEQ ID NO:7; the transcribed DNA molecule (expression cassette) of the nucleic acid component contains a DNA fragment with a nucleotide sequence as shown in positions 427 to 474 of SEQ ID NO:7.

[0021] In this application, the expression box further includes a promoter and a terminator.

[0022] The expression cassette of the protein component is promoted by the Ubi promoter.

[0023] The promoter of the transcribed DNA molecule of the nucleic acid component is the U3 promoter, and the terminator is the U3t terminator.

[0024] In this application, the genome editing system is introduced into the immature embryo of the millet grain to be edited in the form of a plasmid.

[0025] In this application, the plasmid is transformed into the immature embryo of millet grains to be edited via Agrobacterium-mediated transformation.

[0026] The recombinant microorganisms described in this application can be yeast, bacteria, algae, or fungi. The bacteria can be Gram-positive or Gram-negative. The Gram-negative bacteria can be *Agrobacterium tumefaciens*. Specifically, *Agrobacterium tumefaciens* can be *Agrobacterium tumefaciens* EHA105.

[0027] In this application, the evaluation indicators for millet breeding include the aroma of millet grains.

[0028] In this application, the purpose of millet breeding is to cultivate millet with improved grain aroma.

[0029] In this application, compared to the unedited millet, the seed aroma of the gene-edited millet is significantly improved.

[0030] In this application, compared to the millet to be improved, the aroma-enhanced millet has a significantly improved grain aroma.

[0031] In some embodiments of this application, the aroma of millet grains is manifested by the content of the aroma substance 2-acetyl-1-pyrrolidone (2-AP).

[0032] In some embodiments of this application, the aroma of millet grains is determined through human sensory evaluation.

[0033] The aroma enhancement described in this application is a comparison conducted under comparable conditions.

[0034] "Comparable conditions" refer to the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly promote or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus).

[0035] As used herein, “gene-edited millet” and / or “flavor-enhanced millet” includes plant cells, explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0036] As used herein, "plant part" can refer to any organ or intact tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof. The plant part in this application can be viable, non-viable, renewable, and / or non-renewable. "Propagule" can include any plant part that can grow into a whole plant.

[0037] Plant cells are biological cells of plants, which are taken from plants or derived from cultures obtained by culturing cells taken from plants. Cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0038] The beneficial technical effects achieved by this application are as follows: 1. First successful application of the CRISPR / Cas12i.3 system in millet: This application marks the first successful application of a miniaturized CRISPR / Cas12i.3 gene editing system with independent intellectual property rights in my country to millet. Panicum miliaceum Through genetic improvement, a highly efficient and precise gene editing technology system suitable for this crop was established. Compared with commonly used systems such as Cas9, the Cas12i.3 protein is smaller in size, making it easier to construct plant expression vectors, significantly improving transformation efficiency, and circumventing foreign patent restrictions, providing a key tool for the study of functional genes and molecular design breeding in millet.

[0039] 2. Through targeted knockout PmBADH2a and / or PmBADH2b Genetically Created Fragrant Millet Grains: This application clearly identifies a key negative regulatory gene in millet that regulates the synthesis of the aroma compound 2-acetyl-1-pyrrolline (2-AP). PmBADH2a and PmBADH2b Furthermore, by using the Cas12i.3 system to knock out the aforementioned genes individually or in pairs, millet grains with significantly increased 2-AP content (more than 10 times that of the wild type) and a stable, rich aroma were successfully obtained. This method achieves targeted improvement of millet quality traits, and the obtained fragrant millet grains and their preparation method are the core achievements and protected objects of this application. Attached Figure Description

[0040] Figure 1 This is a structural diagram of a gene editing vector.

[0041] Figure 2 The results of BADH2 gene editing.

[0042] Figure 3 This is an agarose gel electrophoresis image of T1 generation plants.

[0043] Figure 4 Standard operating curve.

[0044] Figure 5 To determine the content of aroma compound 2-AP in gene-edited millet grains.

[0045] Figure 6 Gas chromatography-mass spectrometry detection of 2-AP in different millet varieties.

[0046] Figure 7 The aroma intensity of the gene-edited millet grains was scored. Detailed Implementation

[0047] I. Terminology in this application: Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0048] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0049] For ease of understanding this application, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0050] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency.

[0051] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0052] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "including" and "comprise" are used interchangeably.

[0053] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least 3 amino acids in length. Proteins, peptides, or polypeptides can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.

[0054] The terms "nuclear localization signal," "nuclear localization sequence," or "NLS," used interchangeably in this article, refer to the amino acid sequence that "tags" a protein for transport into the nucleus via nuclear transport. Typically, this signal consists of a short sequence of one or more positively charged lysine or arginine residues exposed on the protein surface. Different nuclear localization proteins may share the same NLS. The NLS functions in the opposite way to the nuclear export signal, aiming to expel the protein from the nucleus.

[0055] As used in this article, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thus forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively.

[0056] The term "biomaterial" refers to any material that carries genetic information and is capable of self-replication or replication within a biological system, such as genes, plasmids, microorganisms, animals, and plants.

[0057] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0058] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0059] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0060] In this application, the genetically modified plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the genetically modified plant.

[0061] In this application, the transgenic plant cells, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0062] The expression cassette described in this application refers to a DNA molecule capable of expressing the IbNAC90 protein in a host cell. This DNA molecule may include not only a promoter to initiate transcription of the target gene, but also a terminator to terminate transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this application include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin and soybean beta-conglycin (Beachy et al. (1985) EMBO J). 4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627.

[0063] The recombinant vectors described in this application include the expression cassettes mentioned above. Further, recombinant vectors containing the gene-encoding expression cassettes can be constructed using plant expression vectors. The plant expression vectors can be Gateway system vectors or binary Agrobacterium vectors, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-GFP, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant vectors using IbNAC90, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter or the ubiquitin gene Ubiqutin promoter (pUbi). These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes described in this application, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are broad, and they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0064] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0065] The recombinant microorganisms described in this application can be yeast, bacteria, algae, or fungi. The bacteria can be Gram-positive or Gram-negative bacteria.

[0066] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0067] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0068] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0069] In this application, "editing" or "genome editing" means using targeted genome editing technology to produce a targeted mutation, deletion, inversion, or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, or at least 10,000 nucleotides of endogenous plant genome nucleic acid sequence.

[0070] In this application, “editing” or “genome editing” also covers the use of targeted genome editing technology to target and insert or site-specifically integrate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides into the endogenous genome of a plant.

[0071] In this application, a “target site” for genome editing refers to a location within a plant genome of a polynucleotide sequence that is targeted and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone and / or its complementary DNA strand. The site-specific nuclease may bind to the target site, for example, via a non-coding guide RNA (e.g., but not limited to CRISPR RNA (crRNA) or single-strand guide RNA (sgRNA)). The non-coding guide RNA provided herein may be complementary to the target site (e.g., complementary to the strand of a double-stranded nucleic acid molecule or the chromosome of the target site). A “target site” also refers to a location within the plant genome of a polynucleotide sequence that is bound and cleaved by another site-specific nuclease, which may not be guided by a non-coding RNA molecule, such as a broad-spectrum nuclease, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN), to introduce a double-strand break (or single-strand nick) into the polynucleotide sequence and / or its complementary DNA strand.

[0072] II. Implementation Examples This application provides a method for creating fragrant millet using the CRISPR / Cas12i.3 gene editing system. A specific sgRNA targeting the key millet aroma gene BADH2 is designed and constructed into a plant vector along with a proprietary Cas12i3 protein expression element. The vector is then transformed into millet callus tissue using Agrobacterium tumefaciens to obtain T0 generation edited plants. Molecular identification and screening revealed a homozygous mutant of the BADH2 gene, exhibiting a significantly increased content of 2-acetyl-1-pyrroline (2-AP) and a stable aroma phenotype. This method is the first to apply the Cas12i.3 system to millet, achieving efficient and precise targeted improvement of aroma traits, providing core technology and germplasm support for the breeding of new non-transgenic fragrant millet varieties.

[0073] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.

[0074] Unless otherwise specified, the experimental methods used in the following examples 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 examples are commercially available.

[0075] The Longmi No. 4 in the following embodiments is owned by the applicant and is described in the following published literature: "Shi J, Ma X, Zhang J, Zhou Y, Liu M, Huang L, Sun S, Zhang X, Gao X, Zhan W, Li P, Wang L, Lu P, Zhao H, Song W, Lai J. Chromosome conformation capture resolved nearcomplete genome assembly of broomcorn millet. Nat Commun. 2019 Jan 25;10(1):464. doi: 10.1038 / s41467-018-07876-6. PMID: 30683940; PMCID: PMC6347627." The public can apply to obtain this biological material from the applicant. The obtained biological material can only be used to verify the technical solution of this application and cannot be used for other purposes.

[0076] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0077] The following examples used GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used. Different letters indicate that there is a significant difference between the two groups of data. P <0.05), the same letter indicates that there is no significant difference between the two groups of data ( P >0.05).

[0078] Example 1. Preparation and phenotypic detection of gene-edited millet. 1.1 Target design and vector construction: Based on the millet genome database, the coding sequences of the key aroma genes PmBADH2a (longmi055384) and PmBADH2b (longmi045889) were cloned.

[0079] The genomic sequence of the PmBADH2a gene is SEQ ID NO:1 (including introns), its coding sequence is shown in SEQ ID NO:2, and the amino acid sequence encoding the protein is shown in SEQ ID NO:3.

[0080] The genomic sequence of the PmBADH2b gene is SEQ ID NO:4 (including introns), its coding sequence is shown in SEQ ID NO:5, and the amino acid sequence encoding the protein is shown in SEQ ID NO:6.

[0081] Based on the PAM recognition characteristics (such as TTN) of the Cas12i.3 system, target sequences were designed for screening the exon regions of the PmBADH2a and PmBADH2b genes, respectively. The target sequences for the PmBADH2a and PmBADH2b genes are 5'-AGTACTTTGCGGATCAGGCAGA-3', located at positions 1342 to 1363 of SEQ ID NO:1 or positions 1232 to 1253 of SEQ ID NO:4.

[0082] DNA fragments targeting the PmBADH2a and PmBADH2b genes were synthesized based on the target sequences and constructed into a binary plant expression vector containing the proprietary Cas12i.3 protein expression cassette. Figure 1The vector, named Cas12i.3-badh2, is a circular DNA. One strand of the circular DNA is composed of segment 1 (shown in SEQ ID NO:7) and segment 2 (shown in SEQ ID NO:10) linked together from the 5' end to the 3' end. Specifically, positions 27 to 426 of SEQ ID NO:7 represent the nucleotide sequence of the promoter U6p, positions 427 to 474 represent the nucleotide sequence of crRNA, and positions 475 to 811 represent the nucleotide sequence of the terminator TaU3t. Positions 831 to 3014 of SEQ ID NO:7 represent the nucleotide sequence of the promoter UBi, positions 3143 to 6286 represent the coding sequence of Cas12i.3, and positions 6373 to 6275 represent the nucleotide sequence of the terminator NOS.

[0083] The Cas12i.3-badh2 vector can be used to express gene editing systems targeting the PmBADH2a and PmBADH2b genes, including: (i) Cas12i.3, whose amino acid sequence is shown in SEQ ID NO:8, is an effector protein of the gene editing system; (ii) crRNA with nucleotide sequences as shown in SEQ ID NO:9, wherein positions 1 to 26 of the crRNA are repeat sequences and positions 27 to 48 hybridize with the target sequences of the PmBADH2a and PmBADH2b genes; Cas12i.3 binds to crRNA to form a complex.

[0084] After being introduced into the receptor, the transcribed crRNA can target the PAM sequence of the PmBADH2a and PmBADH2b genes through base complementarity pairing. The Cas12i.3 protein forms a genome editing effect complex with the crRNA, causing double-strand breaks in the DNA near the target sites of the PmBADH2a and PmBADH2b genes. Through the organism's own DNA damage repair response mechanism, gene mutations occur in the cleaved regions during the repair process, thereby knocking out the PmBADH2a and PmBADH2b genes.

[0085] Agrobacterium EHA105 was transformed with the Cas12i.3-badh2 vector, positive clones were screened and cultured to obtain Agrobacterium culture for later use.

[0086] 1.2 Seed sterilization and explant preparation: Mature millet seeds (Longmi 4) with lemma and husk removed were used to induce embryogenic callus. The dehulled seeds were first surface sterilized with 75% (v / v) ethanol aqueous solution for 2 minutes, then transferred to sterile filter paper to absorb excess moisture. Ten seeds were placed in each dish on callus induction medium (CIM, formulation: 4.33 g / L MS salt, 30 g / L sucrose, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L kinetin, 2 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, and 2 mg / L AgNO3, solvent: water, pH 5.8) and cultured in the dark at 28 ℃. Eighteen days later, the callus tissue was transferred to fresh CIM medium and subcultured for two weeks under the same conditions. Embryogenic callus tissue was then selected for genetic transformation.

[0087] 1.3 Agrobacterium tumefaciens-mediated genetic transformation: Callus tissue was cut into small pieces of about 5 mm and placed in liquid co-culture medium (CoIM, formulation: 4.33 g / L MS salt, 30 g / L sucrose, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L kinetin, 2 mg / L 2,4-dichlorophenoxyacetic acid and 200 µM acetylsylgenone, solvent: water, pH 5.4). The medium was pre-cooled on ice for 20 minutes, and then fresh Agrobacterium tumefaciens culture (OD600 = 0.8-1.0) was added. The medium was then co-cultured at 26 ℃ and 100 rpm on a shaker for 30 minutes. The callus tissue was then placed on sterile filter paper to remove excess Agrobacterium tumefaciens culture, and transferred to solid CoIM medium supplemented with 3 g / L Phytagel. The culture was then carried out in the dark at 22°C for 3 days.

[0088] After co-culture, the callus tissue was transferred to CIM medium containing 150 mg / L Timentin and cultured in the dark at 28°C for 2 weeks. Then it was transferred to screening medium (SM, formula: CIM + 100 mg / L Timentin + 50 mg / L Hygromycin B, solvent: water, pH 5.8, 3 g / L Phytagel) and screened for 10 days under the same conditions to select resistant callus tissue.

[0089] 1.4 Plant regeneration: Resistant callus was transferred to pre-regeneration medium (PRM, formulation: 4.43 g / L MS salt, 15 g / L sucrose, 15 g / L glucose, 3 mg / L 6-benzylaminopurine, 0.2 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 3 g / L Gelzan and 100 mg / L Timentin, solvent: water, pH 5.8) and cultured for 10 days at 28℃ under a 16-hour light / 8-hour dark light cycle. The regenerated shoots were then transferred to regeneration medium (RM, formulated as follows: 4.43 g / L MS salt, 15 g / L sucrose, 15 g / L glucose, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 3 g / L Gelzan and 50 mg / L Timentin, in water, pH 5.8) and cultured for 20-30 days under the same light and temperature conditions.

[0090] When the regenerated shoots reach 3-5 cm in length, they are transferred to a seedling culture medium (PM, formulated as follows: 2.21 g / L MS salt, 15 g / L sucrose, 100 mg / L inositol, 2.6 g / L Gelzan, and 50 mg / L Timentin, with water as the solvent, pH 5.8), and cultured at 28 ℃ under 16 hours of light / 8 hours of darkness for 10-14 days. Complete T0 generation candidate regenerated plants are obtained after approximately 2-3 weeks. The obtained T0 generation candidate regenerated plants can be directly transplanted into pots or in the field.

[0091] 1.5 Mutant Identification and Screening: For molecular identification of T0 generation candidate regenerated plants, genomic DNA was first extracted from fresh leaves using the CTAB method or commercially available plant genomic DNA extraction kits (such as TIANGEN, Omega, etc.). The integrity, concentration, and purity of the DNA were then detected by agarose gel electrophoresis and spectrophotometry (such as NanoDrop) (A260 / A280 ratio controlled between 1.8 and 2.0). Subsequently, based on the millet... BADH2Gene sequence was obtained, and specific primers were designed approximately 150-200 bp upstream and downstream of the Cas12i.3 target site. Using extracted DNA as a template, PCR amplification was performed using a high-fidelity DNA polymerase (such as Phanta Max or Q5). The PCR products were verified by 1.5% agarose gel electrophoresis, then excised and recovered for Sanger bidirectional sequencing. Sequencing results were compared with wild-type sequences using software such as SnapGene, Chromas, or DSDecodeM to analyze the presence of insertions, deletions, or base substitutions at the target site and to determine the mutation type (homozygous, heterozygous, or chimeric). All sequences containing... BADH2 Plants that produce frameshift mutations or premature stop codons in the coding region are considered successfully edited T0 generation regenerated plants, which are used for subsequent aroma phenotype verification and genetic analysis.

[0092] The results (Table 1) show that Cas12i.3 exhibits excellent editing efficiency; among T0 plants, PmBADH2a The editing efficiency is 76%. PmBADH2b The editing efficiency was 64%, and the efficiency of simultaneous editing of two genes reached 52%.

[0093] Table 1. Cas12i3 Editing Efficiency

[0094] Successfully edited T0 generation regenerated plants were self-pollinated to obtain edited line T1 plants, from which Casfree ( Figure 3 And pure editing () Figure 2 T1 generation gene-edited millet was used to measure phenotype.

[0095] (1) The detection method for edit type is as follows: Genomic DNA was extracted from leaves at the three-leaf stage and used... PmBADH2a -F:5'-AGTGTGCATTTTTCCAGCAC-3' / PmBADH2a -R: 5'-AAGGAAGGCCGACTTCTTTA-3' detection PmBADH2a ;use PmBADH2b -F:5'-GCATTTTTTCCAGCTTGAAG-3' / PmBADH2b -R: 5'-TTCACGCAAGCGATAAATAA-3' detection PmBADH2b Primer detection of gene editing type, results as follows: Figure 2 .

[0096] The results show that: (1) Compared with the wild-type Longmi 4, the nucleotide sequences at positions 1355 to 1364 of SEQ ID NO:1 and positions 1243 to 1255 of SEQ ID NO:4 in the pmbadh2a / b-1 genome are deleted, resulting in the knockout of the coding genes for betaine aldehyde dehydrogenase 2 (PmBADH2a and PmBADH2b) in pmbadh2a / b-1.

[0097] (2) Compared with the wild-type Longmi 4, the nucleotide sequences at positions 1344 to 1370 of SEQ ID NO:1 and positions 1228 to 1256 of SEQ ID NO:4 in the pmbadh2a / b-2 genome are deleted, resulting in the knockout of the coding genes for betaine aldehyde dehydrogenase 2 (PmBADH2a and PmBADH2b) in pmbadh2a / b-1.

[0098] (3) Compared with the wild-type Longmi 4, the nucleotide sequence from position 1354 to 1365 of SEQ ID NO:1 in the pmbadh2a-1 genome is deleted, resulting in the knockout of the gene encoding betaine aldehyde dehydrogenase 2 (PmBADH2a) in pmbadh2a / b-1.

[0099] (4) Compared with the wild-type Longmi 4, the nucleotide sequence from position 1345 to 1370 of SEQ ID NO:1 in the pmbadh2a-2 genome is deleted, resulting in the knockout of the gene encoding betaine aldehyde dehydrogenase 2 (PmBADH2a) in pmbadh2a / b-1.

[0100] (5) Compared with the wild-type Longmi 4, the nucleotide sequence from position 1245 to 1254 of SEQ ID NO:4 in the pmbadh2b-1 genome is deleted, resulting in the knockout of the gene encoding betaine aldehyde dehydrogenase 2 (PmBADH2b) in pmbadh2b-1.

[0101] (6) Compared with the wild-type Longmi 4, the nucleotide sequence from position 1247 to 1257 of SEQ ID NO:4 in the pmbadh2b-2 genome is deleted, resulting in the knockout of the gene encoding betaine aldehyde dehydrogenase 2 (PmBADH2b) in pmbadh2b-1.

[0102] (2) The method for screening Casfree T1 generation gene-edited millet that was removed from the Cas12i.3 system is as follows: Fresh leaves from T1 generation seedlings (three-leaf stage) were selected as samples. Primers used were primer pairs specifically designed for the coding region of the hygromycin phosphotransferase gene (hpt) (upstream primer: 5'-GCCTGAACTCACCGCGACGT-3', downstream primer: 5'-TCGGACGAGTGCTGGGGCGT-3'). The detection method involved PCR amplification after genomic DNA extraction using the CTAB method, and the results were observed by agarose gel electrophoresis.

[0103] Electrophoretic patterns as follows Figure 3 The result interpretation criteria were as follows: the presence of a specific band (approximately 950 bp) in the electrophoresis pattern corresponding to the positive control position was considered positive (i.e., still carrying the T-DNA backbone), while the absence of a band indicated a Casfree-negative plant. The positive control consisted of plasmid DNA containing the hpt gene or confirmed positive transgenic millet genomic DNA; the negative control was wild-type millet genomic DNA; and the blank control was a PCR reaction system using sterile water instead of a template. (The marker was a DL2000 DNA marker consisting of 6 bands, from bottom to top: 100, 250, 500, 750, 1,000, and 2,000 bp.) according to Figure 2 and Figure 3 The results were filtered to select Casfree ( Figure 3 And pure editing () Figure 2 T1 generation gene-edited millet pmbadh2a / b-1, pmbadh2a / b-2, pmbadh2a-1, pmbadh2a-2, pmbadh2b-1 and pmbadh2b-2.

[0104] Figure 3 The edited strain names corresponding to each lane are: pmbadh2a / b-1 (lane 3), pmbadh2a / b-2 (lane 13), pmbadh2a-1 (lane 19), pmbadh2a-2 (lane 23), pmbadh2b-1 (lane 26) and pmbadh2b-2 (lane 37).

[0105] Example 2: Measurement of millet phenotype The T1 generation gene-edited millet (pmbadh2a / b-1, pmbadh2a / b-2, pmbadh2a-1, pmbadh2a-2, pmbadh2b-1 and pmbadh2b-2) prepared in Example 1 and Longmi No. 4 were planted, and the harvested grains were used for phenotypic testing.

[0106] Pot cultivation: Millet seeds used for phenotypic testing were planted in pots with a diameter of 15-20 cm, with 3-5 plants per pot. Ten replicates were set up for each millet variety. The plants were then cultured in a greenhouse, and the millet grains were harvested at maturity. Field cultivation: Millet seeds used for phenotypic testing were planted in experimental fields located in Ordos, Inner Mongolia. Each millet variety was planted in 4 rows with a row spacing of 50 cm; 10 plants were planted per row with a plant spacing of 15-20 cm. The millet grains were harvested at maturity.

[0107] 2.1 Determination of 2-AP content: 2-AP standard (Weiye Measurement, CAS: 85213-22-5, concentration 1000 μg / mL) was diluted with ethanol to a series of standard concentrations of 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 ng / mL, respectively. The solutions were then analyzed using an instrument, and a standard working curve was plotted. Figure 4 ).

[0108] Weigh a certain amount of mature seeds and place them in a mortar. Add liquid nitrogen and grind rapidly into powder. Weigh approximately 1.0 g of the powder sample into a 10 mL centrifuge tube. During the weighing process, the sample should be placed on dry ice to prevent aroma evaporation. Add 3 mL of anhydrous ethanol to the sample, immediately tighten the centrifuge tube cap, and seal with sealing film. Sonicate in a 68 ℃ water bath for 2 h, centrifuge at 10000 r / min and 4 ℃ for 10 min, collect the supernatant, and filter through a 0.22 μm filter membrane. Analyze using a gas chromatography-mass spectrometry (GC-MS) instrument (8890+7000D, Agilent). Chromatographic parameters: column HP-5MS; injection port temperature: 250 ℃; flow rate: 1 mL / min; injection volume: 1 μL; temperature program: 1: 45 ℃, hold for 1 min; 2: rate 8 ℃ / min, 100 ℃, hold for 0 min; 3: rate 50 ℃ / min, 250 ℃, hold for 1 min. Mass spectrometry parameters: electron impact source: 70 eV; ionization mode: electron impact ion source: EI source; interface temperature: 250℃; ion source temperature: 250℃.

[0109] The gas chromatography-mass spectrometry chromatograms of 2-AP for each sample are shown below. Figure 6 The 2-AP content is shown in Table 2 and Figure 5 .

[0110] Table 2. 2-AP content in BADH2 gene-edited millet

[0111] 2.2 Sensory Evaluation of Aroma: Mature seeds from wild-type and mutant plants were ground into a fine powder. Approximately 1.0 g of seed powder was placed in a 15 mL centrifuge tube, and 1 mL of a 1.7 g / 100 mL KOH aqueous solution was added. The mixture was incubated at 28 °C for 15 minutes. Subsequently, a panel of at least five evaluators with normal olfactory senses assessed the released aroma (blind evaluation). Specific modifications in this study were as follows: The T1 generation gene-edited millet seed powder was independently scored by five members of the laboratory using a 10-point scoring system (0 points = no aroma; 1-5 points = faint aroma; 6-10 points = strong aroma). The scores from all evaluators were summarized and presented in tabular form (Table 3).

[0112] Experimental results showed that the wild-type control variety "Longmi 4" had an average aroma score of 0.4, indicating virtually no aroma perception; among the single-gene knockout lines, pmbadh2b-1 and pmbadh2b-2 The average scores were 3.0 and 2.8 respectively, indicating a faint to medium fragrance; while pmbadh2a-1 and pmbadh2a-2 The average scores significantly improved to 7.4 and 7.2, demonstrating a pronounced aroma. This was particularly evident in the double-gene knockout strains. pmbadh2a / b-1 and pmbadh2a / b-2 Their average aroma scores reached 9.4 and 9.0 respectively, and were unanimously rated by volunteers as having a rich, typical popcorn-like aroma, significantly better than single-gene knockout lines and wild types. p <0.01)( Figure 7 ).

[0113] The sensory evaluation results above are highly consistent with the 2-AP content determination data, further confirming that targeted knockout via the CRISPR / Cas12i.3 system... PmBADH2a and PmBADH2b The gene can effectively remove its inhibitory effect on the synthesis of aroma substances, significantly improving the aroma intensity of millet grains. Among them, the double gene knockout strain has the most prominent aroma and has excellent eating quality and market application potential.

[0114] Table 3 BADH2 Aroma rating of gene-edited millet

[0115] This application utilizes the CRISPR / Cas12i.3 system to create fragrant millet, which has significant advantages over the traditional Cas9 system. The Cas12i.3 protein is significantly smaller than Cas9, making it easier to load into plant expression vectors, thus improving genetic transformation efficiency. It is particularly suitable for multi-gene editing or delivery systems sensitive to vector capacity. Furthermore, this system possesses independent intellectual property rights in China, effectively circumventing the restrictions of foreign Cas9-related patents, and providing a safe and controllable technical path for gene editing breeding in Chinese agriculture.

[0116] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented 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 are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing gene-edited millet, characterized in that, The method includes at least one of the following A1) and A2): A1) Knock out the gene encoding betaine aldehyde dehydrogenase 2 in millet seeds to be edited using a genome editing system, wherein the genome editing system comprises: (i) Protein components, namely, a protein or its conjugate with the amino acid sequence shown in SEQ ID NO:8, and (ii) A nucleic acid component comprising the RNA sequence shown in SEQ ID NO:9 from the 5' to 3' direction. The protein component and the nucleic acid component combine to form a complex; A2) Perform at least one of the following mutations on the genome of the millet seed to be edited: A2-1) Delete the DNA fragment with nucleotide sequence of SEQ ID NO:1 from position 1355 to 1364 and the DNA fragment with nucleotide sequence of SEQ ID NO:4 from position 1243 to 1255 in the millet seed to be edited; A2-2) Delete the DNA fragment with nucleotide sequence of SEQ ID NO:1 from position 1344 to 1370 and the DNA fragment with nucleotide sequence of SEQ ID NO:4 from position 1228 to 1256 in the millet seed to be edited; A2-3) Delete the DNA fragment from positions 1354 to 1365 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:1; A2-4) Delete the DNA fragment from positions 1345 to 1370 of the nucleotide sequence of the millet seed to be edited, such as SEQ ID NO:1; A2-5) Delete the DNA fragment from positions 1245 to 1254 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:4; A2-6) Delete the DNA fragment from positions 1247 to 1257 of the nucleotide sequence in the millet seed to be edited, such as SEQ ID NO:4; The millet seed to be edited contains the encoding gene for betaine aldehyde dehydrogenase 2, which includes PmBADH2a and PmBADH2b. The amino acid sequence of PmBADH2a is shown in SEQ ID NO:3, and the amino acid sequence of PmBADH2b is shown in SEQ ID NO:

6.

2. The use of the genome editing system and related biological materials as described in claim 1 in at least one of C1) to C4), wherein the biological materials are at least one of B1) to B4): B1) Isolated nucleic acid molecules, said nucleic acid molecules expressing the protein components and / or nucleic acid components described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); Application of C1 in the preparation of gene-edited millet; C2) Application in the preparation of gene-edited millet products; C3) Application in millet breeding or assisted breeding; C4) Application in the preparation of millet breeding or assisted breeding products.

3. The application according to claim 2, characterized in that, The nucleic acid molecule expressing the protein component of claim 1 is a DNA molecule or an RNA molecule transcribed from the DNA molecule, as described in at least one of g1) and g2) below: g1) DNA molecules with nucleotide sequences such as positions 3143 to 6286 of SEQ ID NO:7; g2) A DNA molecule that has more than 70% identity with the DNA molecule at positions 3143 to 6286 of SEQ ID NO:7 and encodes the protein component; The nucleic acid molecule expressing the nucleic acid component of claim 1 is a DNA molecule as described in at least one of the following g3) and g4): g3), DNA molecules with nucleotide sequences such as positions 427 to 474 of SEQ ID NO:7; g4) A DNA molecule that has more than 70% identity with the DNA molecule at positions 427 to 474 of SEQ ID NO:7 and that transcribes the nucleic acid components thereon.

4. Any of the following substances: D1) The genome editing system as described in claim 1; D2), the biomaterials described in claim 2 or 3.

5. Gene-edited millet, characterized in that, The gene-edited millet is named as follows: pmbadh2a / b-1 , pmbadh2a / b-2, pmbadh2a-1 , pmbadh2a-2 , pmbadh2b-1 and pmbadh2b-2 At least one of the gene-edited millet varieties, The pmbadh2a / b-1 DNA fragments that do not contain nucleotide sequences such as those in positions 1355 to 1364 of SEQ ID NO:1 and nucleotide sequences such as those in positions 1243 to 1255 of SEQ ID NO:4; The pmbadh2a / b-2 DNA fragments that do not contain nucleotide sequences such as those in positions 1344 to 1370 of SEQ ID NO:1 and nucleotide sequences such as those in positions 1228 to 1256 of SEQ ID NO:4; The pmbadh2a-1 It does not contain DNA fragments with nucleotide sequences such as those at positions 1354 to 1365 of SEQ ID NO:1; The pmbadh2a-2 It does not contain DNA fragments with nucleotide sequences such as those from positions 1345 to 1370 of SEQ ID NO:1; The pmbadh2b-1 It does not contain DNA fragments with nucleotide sequences such as those at positions 1245 to 1254 of SEQ ID NO:1; The pmbadh2b-2 It does not contain DNA fragments with nucleotide sequences such as positions 1247 to 1257 of SEQ ID NO:

1.

6. The gene-edited millet seed according to claim 5, characterized in that, The gene-edited millet seed is obtained by gene editing of millet seed to be edited, and the millet seed to be edited contains the encoding gene of betaine aldehyde dehydrogenase 2 as described in claim 1.

7. The gene-edited millet according to claim 5 or 6, characterized in that, The gene-edited millet is prepared by the method described in claim 1.

8. The use of millet according to any one of claims 5 to 7 in at least one of the following: E1) Application in enhancing the aroma of millet; Application of E2 in millet breeding or assisted breeding.

9. The application according to claim 8, characterized in that, The application is achieved by hybridizing the gene-edited millet seed of any one of claims 4 to 6 with the millet seed to be improved.

10. Millet with enhanced aroma, characterized in that, The millet seed is obtained by hybridizing the gene-edited millet seed of any one of claims 4 to 6 with the millet seed to be improved.

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