Modified RNA demethylase and encoding nucleic acid thereof, use thereof, plant cultivation method and product

Abstract

Provided are a modified RNA demethylase, a nucleic acid encoding the modified RNA demethylase, the use thereof, a plant cultivation method, and a product. Compared with an original RNA demethylase derived from algae or invertebrates, the modified RNA demethylase is obtained by means of adding a specific peptide fragment sequence at the N-terminus or C-terminus of the original RNA demethylase. The modified RNA demethylase or the nucleic acid encoding the modified RNA demethylase is introduced into an initial plant to obtain a target plant having optimized traits compared with the initial plant.

Description

Modified RNA demethylase and its encoding nucleic acid, application, plant cultivation method and product Technical Field

[0001] The present invention relates to the fields of genetic engineering and plant cultivation, and in particular to a modified RNA demethylase and nucleic acid encoding the modified RNA demethylase, as well as applications, plant cultivation methods, and products. Background Art

[0002] Artificial intervention methods for plant cultivation generally include a variety of approaches, such as genetic engineering, cell engineering, microbial engineering, etc. In order to obtain new plant varieties, the aforementioned methods are usually adopted; the new plant varieties here include the whole plant, plant organs (flowers, fruits, seeds, roots, stems, leaves, etc.), plant tissues, plant cells and other parts that can be obtained from plants.

[0003] The newly obtained plant varieties are usually expected to exhibit more optimized traits, such as optimized growth, increased yield, optimized biomass, optimized architecture, or optimized cell division. Among these optimized traits, increased yield can be achieved through various means, such as increasing the volume or weight of individual seeds / thousand seeds, increasing the number of root tillers, and other factors that can affect plant yield, especially increasing the yield of the desired part of the plant.

[0004] The above expectations are currently usually achieved through some transformation methods, such as by modifying the plant's DNA or histones to achieve the purpose of changing plant traits. Prior to this application, there have been studies on optimizing plant traits by demethylating the 6mA site of plant RNA.

[0005] Summary of the Invention

[0006] The aforementioned approach can indeed obtain plants with optimized traits, especially plants with increased yield. For details, please refer to the document CN108949806A previously applied for by the applicant and authorized in multiple countries. However, from the results of trait optimization, the trait optimization effect of the original RNA demethylase or its encoding DNA from algae or invertebrate sources on plants is significantly lower than that of the original RNA demethylase or its encoding DNA from humans or vertebrates. In addition, in terms of audience acceptance, the introduction of the original RNA demethylase from algae or invertebrate sources has a significant negative impact on the plant's trait optimization. The NA demethylase or its encoding DNA is significantly higher than the original RNA demethylase or its encoding DNA introduced from humans or other vertebrates; to this end, the applicant has found through research that by modifying the original RNA demethylase or its encoding DNA from algae or invertebrates using genetic engineering, the modified RNA demethylase or its encoding DNA after specific modification can achieve better trait optimization effects on plants, reaching or even exceeding the trait optimization effects of the original RNA demethylase or its encoding DNA from humans or other vertebrates on plants.

[0007] In view of this, the modified RNA demethylase and its encoding nucleic acid provided by the present invention can optimize the traits of the plant after being introduced into the plant and are more suitable for popularization.

[0008] Research has found that during plant growth and development, specific enzymes can promote the methylation of RNA bases. For example, the methyltransferase MTA can promote the 6-methylation of adenine bases in RNA. Here, 6- refers to the N that is connected to the RNA sequence as the starting point, and the N that is ranked 6th in order or in reverse order. The methylated RNA, through a series of processes such as subsequent expression, can promote the growth and development of plants by itself or its products. Experiments have confirmed that removing this specific methylating enzyme can affect the growth and development of plants (for example, the inability to produce viable seeds or the inability of seeds to germinate normally). However, research has found that after removing some of the methyl groups from the methylated bases, the RNA itself or its products are more conducive to the growth and development of plants, promoting the optimization of plant traits. The method of removing some of the methyl groups can be achieved by using certain inducing agents, such as demethylases.

[0009] The present application's research found that after the modified RNA demethylase of the present application is introduced into plants, the plant's own traits are optimized, especially the yield or biomass of the plant is increased.

[0010] This type of RNA demethylase typically targets RNA that has already been methylated at its bases. The chemical mechanism of its induction is shown in Figure 1, using the demethylation process of N6-methylated adenine as an example. Research has found that while the specific RNA sequences and locations of the demethylated sites induced by these inducing agents are currently unclear, it is clear that these inducing agents can indeed demethylate RNA that has already been methylated at its bases, thereby producing plants with optimized traits.

[0011] The present invention genetically modifies an original RNA demethylase derived from algae or invertebrates by adding at least one specific peptide sequence provided herein to at least one of its N-terminus or C-terminus, thereby obtaining a modified RNA demethylase. When the modified RNA demethylase or its encoding nucleic acid is introduced into a plant, the resulting plant can exhibit optimized traits, particularly increased yield or biomass.

[0012] The specific peptide sequence described in the present application contains at least three amino acids selected from P, K, or R. P is the abbreviation of the amino acid Pro, K is the abbreviation of the amino acid Lys, and R is the abbreviation of the amino acid Arg. Here, "at least three amino acids selected from P, K, or R" means that the amino acids selected from P, K, or R may be repeated or non-repeated, for example, a combination of P, K, K, a combination of R, R, R, a combination of P, K, R, and so on. In addition to the three specifically selected amino acids mentioned above, the other amino acids in the peptide sequence can be selected from currently available conventional amino acids, and the conventional abbreviations of amino acids are used herein.

[0013] More preferably, at least two of the three amino acids selected from P, K or R in the peptide sequence described in the present application are consecutive, which means that any two of the amino acids selected from P, K or R are consecutive, and the remaining amino acid may or may not be consecutive with the aforementioned two amino acids.

[0014] Optionally, there may be multiple peptide sequences as described in the present application, such as RPRK, RKVIK, RNKKKK, PKKKRKV, KRPAATKKAGQAKKKK, TLLLRETMNNLGVSDHAVLSRKTPQPY, KRTPTAEERERGAKKLRLLEELEDTWLPYL, etc.

[0015] As used herein, the term "plant" includes living whole plants, their ancestors and descendants, and plant parts, including seeds, branches, stems, leaves, roots, flowers, and tissues and organs. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores. The term "product obtained by processing a plant" is not limited to the aforementioned parts, but also extends to harvestable parts of the plants of the present invention, such as, but not limited to, the whole plant, flowers, fruits, seeds, roots, leaves, stems, and the like.

[0016] Plants that can be particularly used in the method of the present application include algae, ferns and all plants belonging to the superfamily Plantae, especially monocotyledons and dicotyledons, including plants selected from the following categories: food crops (such as rice, corn, soybean, etc.), feed crops (such as alfalfa, etc.), fiber crops (such as cotton, hemp, etc.), oil crops (such as sesame, peanuts, etc.), sugar crops (such as sugarcane, sugar beet, etc.), beverage crops, spice crops, seasoning crops, medicinal crops (such as ginseng, ganoderma lucidum, Fritillaria, etc.), dye crops, ornamental crops, fruit crops, vegetable crops, etc.; or selected from any one of the following plants: at least one of rapeseed (Brassica napus L.), tomato (Lycopersicon esculentum Mill.), lettuce (Lactuca sativa L. var. ramosa Hort.), sugar beet (Beta vulgaris L.); or preferably selected from rice (Oryza sativa L.), corn (Zea mays L.), soybean (Glycine max (Linn.) Merr.), potato (Solanum tuberosum), wheat (Triticum aestivum L.), millet (Setaria italica var. germanica (Mill.) Schred.), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor (L.) Moench), cassava (Manihot esculenta Crantz); or preferably at least one selected from tobacco (Nicotiana tabacum L.), alfalfa (Medicago Sativa Linn), rubber grass (Taraxacum kok-saghyz Rodin), cotton (Gossypium spp.), flax (Linum usitatissimum L.), sunflower (Helianthus annuus L.), flax mustard (Camelina sativa (L.) Crantz), sedge (Cyperus esculentus L.), hemp (Cannabis sativa L.), and poplar (Populus L.).

[0017] The term "initial plant" herein refers to a plant that has not been processed by the cultivation method of this application, and its structure and species can specifically include any of the aforementioned plants. The source of the "initial plant" can be a plant obtained from the wild or artificially cultivated.

[0018] The term "target plant" herein refers to the plant that is processed by the cultivation method of the present application and is expected to be obtained, and its structure and species can also include any of the aforementioned plants.

[0019] The term "products obtained by inactivation processing of plants" used in this article specifically refers to the products obtained after the aforementioned "plants" are processed, which are inactive; it is not limited to various industries, such as industrial, medicinal, edible, etc., nor is it limited to specific ingredient products, such as dry granules, powder, oil, fat, fatty acid, starch or protein, etc., and is also not limited to specific product forms, such as solid, liquid, gaseous or mixed states, etc.

[0020] The term "algae source" herein refers to algae, which refers to a type of eukaryotic organism in the kingdom Protistae, including multiple species, such as but not limited to blue algae, green algae, black algae, red algae, brown algae, and the algae used in the examples of this application.

[0021] The term "increased yield" as defined herein means an increased weight of a specific harvested part relative to the initial plant, e.g. for rice, an increase in the weight of rice grains is an increased yield, for potatoes, an increase in the weight of tubers is an increased yield, etc.

[0022] The term "increased biomass" as defined herein refers to the increased weight of the entire plant excluding the portion evaluated for yield increase; for example, for rice, the increased weight of the remaining portion excluding the rice grains is the increased biomass.

[0023] The yield and biomass described in this article are all compared with the dry weight of plants at the same period.

[0024] Therefore, the present invention provides a method for increasing plant yield and biomass, which comprises introducing the modified RNA demethylase obtained after modification in the present application or its encoding nucleic acid into an initial plant, and obtaining a target plant with optimized traits compared to the initial plant.

[0025] The traits of plants can be adjusted and optimized by introducing the modified RNA demethylase of the present application or its encoding nucleic acid. Alternatively, the traits of plants can also be adjusted and optimized by using homologues, variants / combinations of the modified RNA demethylase of the present application or its encoding nucleic acid with equivalent functions.

[0026] The term "modified RNA demethylase or its encoding nucleic acid" as defined herein refers to a medium that can induce demethylation of plant RNA. Specifically, it can refer to, for example, any one of catalysis and participation in a reaction, preferably catalysis. Furthermore, "RNA" includes one or more RNAs of a plant, such as ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), non-coding RNA, etc.; "demethylation" refers to the demethylation of already methylated RNA, which can be demethylation at one or more sites of RNA, such as the demethylation of already methylated bases of RNA, such as the demethylation of its N6-methylated bases. Here, 6- is the position that defines the methylation, and it can be, for example, the N starting position of the base-linked RNA is the 1-position, and the 6th position counting clockwise or counterclockwise is the 6-position. Here, "base" generally refers to adenine (A) and the like.

[0027] The source of the "original RNA demethylase" can be any one or more sources of algae or invertebrates, such as green algae; the "source" defined herein can be directly derived from algae or invertebrates, or indirectly derived from algae or invertebrates, such as an ortholog or paralog of algae or invertebrates, preferably an ortholog or paralog of green algae or cystoglossina.

[0028] The term "paralog" refers to the duplication of genes within the genome of a species, resulting in paralogous genes.

[0029] The term "orthologs" relates to homologous genes in different organisms resulting from speciation.

[0030] The term "homology" herein refers to homologous sequences that have equivalent functions to the "original RNA demethylase"; the term "variant / conjugate" herein refers to variants / conjugates with equivalent functions obtained by mutation, combination, etc. using the "original RNA demethylase".

[0031] The term "introduction" herein includes one or more aspects, such as introduction route, introduction area, introduction amount, and the like.

[0032] Introduction can occur in a variety of ways, such as by infecting a plant with a vector carrying the "original RNA demethylase" to insert the "original RNA demethylase" into the plant's DNA. Alternatively, the vector carrying the "original RNA demethylase" can be inserted into cells, with no particular limitation on the site of introduction, such as the nucleus or cytosolic region. Furthermore, introduction can be either a small amount or an excessive amount, depending on the characteristics of the plant and the gene being introduced.

[0033] The "nucleic acid encoding the modified RNA demethylase" described herein is a nucleic acid encoding the modified RNA demethylase that can be obtained directly by modifying the original nucleic acid, or by using the amino acid sequence of the modified RNA demethylase of the present application and various nucleic acids encoding its amino acid sequence obtained by conventional reverse engineering in the art.

[0034] The "target plant" mentioned herein may be any subsequent generation of plant grown from the initial plant.

[0035] In the present application method, any improvement or beneficial increase in the traits of a plant is considered trait optimization. In addition, the trait optimization degree Y defined herein can also be defined by a specific numerical value, which is based on the same trait of the initial plant at the same time as the comparison basis.

[0036] The degree of trait optimization, Y, can be calculated as a multiple of increase compared to the initial plant, where Y is a multiple of increase compared to the initial plant over the same period, Y≥3.50, preferably Y≥3.80, and more preferably Y≥4.00. Here, the multiple of increase can be, for example, a multiple of weight gain.

[0037] The term "DNA and / or protein, homologs or variants / combinations thereof" as defined herein refers to substances having a certain full sequence identity with DNA and / or protein, wherein the full sequence identity is, in increasing order of preference, homologous sequences, variants / combinations having equivalent functions compared to SEQ ID NOs: 1 to 48 in the sequence listing. The term also means that it can be a DNA or protein, or a substance that is a mixture of DNA and protein; it can even refer to paralogs or orthologs having the aforementioned homology with the DNA or protein, or to substances obtained by hybridization or fusion of the corresponding vectors. It should be understood that "DNA and / or protein, homologs or variants / combinations thereof having equivalent functions" are not limited to the sequences listed in the sequence listing.

[0038] "DNA and / or protein, functionally equivalent homologues or variants / combinations thereof" may be any natural or synthetic substance.

[0039] The term "hybridization" as defined herein is the process in which complementary nucleotide sequences of basic homology return to each other. The hybridization process can occur completely in solution, i.e., complementary nucleic acids are all in solution. The hybridization process can also be carried out in such a way that one of the complementary nucleic acids is fixed on a matrix such as magnetic beads, agarose beads or any other resin. In addition, the hybridization process can also be carried out in such a way that one of the complementary nucleic acids is fixed on a solid support such as nitrocellulose membrane or nylon membrane, or is fixed on a silica glass support such as by photolithography. In order to make hybridization occur, usually nucleic acid molecules are thermally denatured or chemically denatured so that the double-stranded nucleic acid is melted into two strands and / or hairpin or other secondary structures are removed from single-stranded nucleic acid. The stringency of hybridization is subject to the influence of conditions such as temperature, salt concentration and hybridization buffer composition.

[0040] The nucleic acid molecules or variants thereof may be derived from any natural or artificial source. Nucleic acids / genes or variants thereof may be isolated from non-mammalian sources. Nucleic acids may be modified from their native form in terms of composition and / or genomic environment by rigorous human manipulation. The nucleic acids are preferably of algal origin, particularly, for example, of green algae origin.

[0041] The term "homolog" encompasses two special forms of homology, orthologous sequences and paralogous sequences, which relate to evolutionary concepts used to describe the genetic relationships of genes.

[0042] Homologues can also be in the form of "insertion variants" of proteins, i.e., wherein one or more amino acid residues are introduced into a predetermined site in the protein. Insertion can include amino-terminal and / or carboxyl-terminal fusions, as well as insertions within the sequence of single or multiple amino acids. Typically, the insertion within the amino acid sequence will be smaller than the amino-terminal or carboxyl-terminal fusions, being approximately 1 to 10 residues. Examples of amino-terminal or carboxyl-terminal fusion proteins or peptides include binding domains or activation domains of transcriptional activators such as those used in yeast two-hybrid systems, phage coat proteins, (histidine) 6-tags, glutathione S-transferase-tags, protein A, maltose binding protein, dihydrofolate reductase, Tag.100 epitopes, c-myc epitopes, FLAG epitopes, lacZ, CMP (calmodulin binding peptide), HA epitopes, protein C epitopes, and VSV epitopes.

[0043] Functional variants that can be used in the present application's method also include optional nucleic acid molecules or gene splice variants.Term used herein " selectable splice variant " covers such nucleotide sequence variants, i.e. wherein selected introns and / or exons have been excised, replaced or added.Such variants are those in which the biological activity of the protein remains unaffected, and it can be obtained by selectively retaining the functional fragment of the protein.Such splice variants can be naturally found or can be artificial.Methods for producing such splice variants are well known in the art.

[0044] The "N-terminus" or "C-terminus" mentioned in this application specifically refers to the two ends of the original protein.

[0045] The "original protein" mentioned in this application specifically refers to a protein that has not been processed by the modification method of this application, and can be a protein of natural origin or an artificially modified protein.

[0046] Therefore, in summary, the solutions that may be provided by this application include but are not limited to the following solutions, and other solutions that do not deviate from the main idea of ​​this application may also be obtained by combining or transferring features based on the following solutions:

[0047] The first solution provided by this application is:

[0048] A modified RNA demethylase is obtained by adding at least one peptide sequence to at least one of the N-terminus and the C-terminus of the original RNA demethylase derived from algae or invertebrates to obtain the modified RNA demethylase.

[0049] Optionally, the peptide sequence has any of the following amino acid sequence characteristics:

[0050] a) contains at least three amino acids selected from the group consisting of H, K and R;

[0051] b) contains PY and at least one amino acid selected from the group consisting of H, K and R.

[0052] Optionally, when the peptide sequence has the amino acid sequence feature of a), at least two amino acids selected from the group consisting of H, K and R are consecutive, or contain V or I.

[0053] Optionally, the peptide sequence has any of the following amino acid sequence features: (K / R)(K / R)X 1-2 (K / R), (K / R)X 1-2 (K / R)(K / R), (K / R)(K / R)X 10-12 (K / R) 2-5 、(R / K / H)X 2-5 PY, or K(V / I)XKX 1-2 (K / H / R), where X is any amino acid.

[0054] Optionally, the peptide sequence includes at least one of RPRK, RKVIK, RNKKKK, PKKKRKV, KRPAATKKAGQAKKKK, TLLLRETMNNLGVSDHAVLSRKTPQPY, and KRTPTAEERERGAKKLRLLEELEDTWLPYL.

[0055] Optionally, the original RNA demethylase includes at least one of its homologues or variants / combinations with equivalent functions.

[0056] Alternatively, a proto-RNA demethylase promotes the demethylation of N6-methyladenine in RNA.

[0057] Optionally, the modified RNA demethylase is shown in Seq.ID.No:13 to Seq.ID.No:48.

[0058] Optionally, the invertebrate comprises at least one of a long-spined sea star or a cystoglossus.

[0059] Optionally, the algae include at least one of blue algae, red algae, cryptoalgae, dinoflagellates, golden algae, yellow algae, diatoms, brown algae, naked algae, green algae, and charophyte.

[0060] Optionally, the modified RNA demethylase promotes N6-methyladenine demethylation of RNA.

[0061] The second solution provided by this application is:

[0062] A nucleic acid encoding the aforementioned modified RNA demethylase.

[0063] Optionally, the nucleic acid is DNA.

[0064] The third solution provided by this application is:

[0065] Use of the aforementioned modified RNA demethylase or the aforementioned nucleic acid in introducing into plants.

[0066] Optionally, the application includes any one of increasing production and increasing biomass.

[0067] Optionally, the plant includes at least one of rapeseed, tomato, lettuce, beet, rice, corn, soybean, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, flax mustard, sedge, hemp, and poplar.

[0068] The fourth solution provided by this application is:

[0069] A plant breeding method comprises introducing the modified RNA demethylase or the nucleic acid into an initial plant, and obtaining a target plant having optimized traits compared to the initial plant.

[0070] Optionally, trait optimization includes at least one of increasing yield and increasing biomass.

[0071] Optionally, compared to the original plant, the modified RNA demethylase or the nucleic acid is introduced in excess.

[0072] Optionally, the initial plant is selected from at least one of rapeseed, tomato, lettuce, sugar beet, rice, corn, soybean, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, flax mustard, sedge, hemp, and poplar.

[0073] Optionally, the introduced plant region includes at least one of a plant organ, a plant tissue, and a plant cell.

[0074] Optionally, the introduced plant region is selected from plant meristems.

[0075] Optionally, the introduced plant region includes at least one of a cell nucleus and a cell sap of the plant.

[0076] Optionally, the introduction method includes introducing a vector encapsulating the aforementioned modified RNA demethylase or the aforementioned nucleic acid into the plant.

[0077] Optionally, compared to the initial plant, the degree of trait optimization is Y, where Y is the multiple increase compared to the initial plant over the same period, Y≥3.50, preferably Y≥3.80, and more preferably Y≥4.00.

[0078] The fifth solution provided by this application is:

[0079] The target plant obtained by the aforementioned plant cultivation method includes at least one of a living plant, a plant organ, a plant tissue, and a plant cell.

[0080] The sixth solution provided by this application is:

[0081] The product obtained by inactivating the target plant mentioned above. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG1 is a schematic diagram of the demethylation process of N6-methylated adenine;

[0083] FIG2 is a comparison of tubers obtained from the experimental group and the blank group, taking potatoes as an example;

[0084] FIG3 is a comparison diagram of the whole plant obtained from the experimental group and the blank group, taking rice as an example. DETAILED DESCRIPTION

[0085] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0086] Sequence modification of native RNA demethylases from algae or invertebrates

[0087] The original RNA demethylase obtained from various algae or invertebrates is sequenced, screened, and amplified to obtain the corresponding sequence samples. The sequences of the original RNA demethylases from specific algae sources are shown in the sequence table SEQ.ID.NO: 1 to 12. Each sequence is directly subjected to PCR and recombined into the p1307-FLAG vector to obtain p1307-RNA demethylase-FLAG. Thereafter, the RNA demethylase-FLAG fragment is amplified and recombined into the pGEX-His vector to obtain pGEX-RNA demethylase-FLAG-His, which can be used for subsequent in vitro experiments and peptide sequence introduction. Primers with peptide sequences are used to carry out P-loop recombination on the plasmid pGEX-RNA demethylase-FLAG-His, and finally the modified gene fragment is re-introduced into the p1307-FLAG vector. In this way, the modified RNA demethylase of the present application is obtained, and the specific sequence can be shown in the sequence table SEQ.ID.NO: 13 to 48. Specifically, the modified RNA demethylase obtained can introduce a peptide sequence at at least one of the N-terminus or C-terminus of the original protein. For example, to introduce a peptide sequence at the N-terminus, an NLS sequence can be inserted between the first amino acid M and the second amino acid at the N-terminus of the original protein. For example, to introduce a peptide sequence at the C-terminus, an NLS sequence can be added after the last amino acid at the C-terminus of the original protein.

[0088] Example 1: Method for obtaining target plants by infecting callus tissue with Agrobacterium

[0089] 1. Inducing callus tissue using plant seeds as test materials

[0090] 1) Disinfection:

[0091] Take mature seeds of the plant, shell them manually, select plump, smooth and sterile seeds, place the seeds in a 100ml sterile beaker, pour in 70% alcohol for disinfection for 2 minutes; pour out the alcohol, add 20% sodium hypochlorite (NaClO) solution, and soak for 30 minutes; pour out the sodium hypochlorite solution, wash the seeds with sterile distilled water 4 to 5 times, and finally soak the seeds in sterile distilled water for 30 minutes.

[0092] 2) Induction culture (sterile operation required):

[0093] After sterilizing, place the seeds on sterile filter paper to dry them. Place 12-14 seeds per dish in NB medium (pH 5.8) containing 2.0 mg / L 2,4-D. To ensure optimal induction efficiency, keep the seeds germinating parallel to the medium or slightly downward, rather than upward or vertically downward. Seal the dish with parafilm and incubate in a dark incubator at 30°C and approximately 50% humidity for 20-30 days until noticeably loose callus forms. Subculture or pre-culture transformation can then be performed.

[0094] 3) Subculture (sterile operation required):

[0095] Open a culture dish on a clean bench and use tweezers to select naturally split, vigorously growing, firm, tender yellow callus with a diameter of approximately 3 mm. Place 10 calli per dish in NB medium (pH 5.8) containing 2.0 mg / L 2,4-D and 0.5 mg / L 6-BA. Incubate at 30°C in the dark. If the callus becomes severely softened, subculture in the light is recommended. Subculture should last approximately 10-15 days (depending on the growth of the callus) for a total of two subcultures. Note: If excessive water is observed on the lid of the dish during culture, this is not recommended and may cause the culture to fail. This is because the bottom of the dish is heated, and the lid temperature is lower than the medium temperature, causing condensation on the lid.

[0096] 2. Agrobacterium culture

[0097] Experimental group: The vector carrying the modified DNA or modified protein of the present application and the hygromycin resistance gene Hygromycin is transferred into the parasitic bacteria, for example, the pCAMBIA 1307 vector carrying the modified DNA or modified protein of the present application and the hygromycin resistance gene Hygromycin is transferred into Agrobacterium LBA4404 and inoculated onto YEP solid culture medium containing 20 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kan).

[0098] Control group: The original DNA or original protein and the vector of the hygromycin resistance gene Hygromycin are transferred into the parasitic bacteria, for example, the pCAMBIA 1307 vector of the hygromycin resistance gene Hygromycin is transferred into Agrobacterium LBA4404, and inoculated onto YEP solid culture medium containing 20 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kan).

[0099] Blank group: The vector of the hygromycin resistance gene Hygromycin was transferred into the parasite, for example, the pCAMBIA 1307 vector of the hygromycin resistance gene Hygromycin was transferred into Agrobacterium LBA4404, and inoculated onto YEP solid medium containing 20 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kan).

[0100] After the control group, experimental group and blank group were cultured at 28°C for 2 days, single Agrobacterium clones were picked for colony PCR to verify whether the induction medium had been transferred into Agrobacterium, and positive single clones were selected and cultured in 4 ml YEP (containing 50 mg / L Kan and 20 mg / L Rif) culture medium at 28°C and 220 rpm for 20 to 36 hours until the OD600 of the bacterial solution was 0.8 to 1.0.

[0101] 3. Infection and Co-cultivation

[0102] 1) Centrifuge the cultured Agrobacterium at 4°C, 4000 rpm for 10 minutes, and discard the supernatant. Resuspend the culture in AAM medium containing 100 μmol / L acetosyringone to a final OD600 concentration of approximately 0.2.

[0103] 2) Pick out rice callus that has grown to a certain size and infect it with Agrobacterium suspension for 20 to 30 minutes.

[0104] 3) Remove the callus tissue and place it on sterile filter paper to drain for 20-30 minutes to prevent excessive growth of Agrobacterium during co-culture and excessive damage to the callus tissue.

[0105] 4) The callus tissue was placed in NB medium (pH 5.2) containing 2.0 mg / L 2,4-D and 100 μmol / L acetosyringone and cultured in the dark at 25°C for 48 to 72 hours.

[0106] Note: The co-culture should be followed up and observed. If Agrobacterium is obviously seen, it means that the infection is excessive and the subsequent screening will easily lead to bacterial growth.

[0107] 4. Screening of resistant callus

[0108] Remove the callus and wash it 5-6 times with sterile water, shaking it constantly. Then, place the callus on sterile filter paper to air dry, and evenly plate it on NB medium (pH 5.8) containing 50 mg / L hygromycin for the first round of selection. Incubate in the dark at 28°C. If mold or Agrobacterium growth is observed, transfer the callus to a new screening plate.

[0109] After new callus tissue grows after about 30 days of screening, transfer it to new NB medium (pH 5.8) containing 50 mg / L hygromycin and screen for another 7-10 days. If it grows obviously, it is a positive callus tissue. If it does not grow, even if it does not turn brown and die, it may be a false positive.

[0110] 5. Induction, differentiation and rooting of positive callus

[0111] Place the vigorously growing, yellow callus particles from the second screening (to ensure that the callus used for differentiation is free of defects) onto NB medium (pH 5.8) containing 2.0 mg / L 6-BA, 0.5 mg / L kinetin, and 50 mg / L hygromycin. Place two to three positive clones per bottle. A small amount of callus per clone is sufficient; the quality of the callus is of primary importance, not the quantity. Incubate in the dark at 27°C for 10 days. After 10-20 days of differentiation under light, rooting can begin after green leaves develop, using a light intensity of 4000 lux for 14 hours per day.

[0112] Induce rooting by placing robust seedlings from each clone in 1 / 2 N6 medium (pH 5.8) containing 0.5 mg / L naphthaleneacetic acid and 50 mg / L hygromycin, with 2-3 seedlings per bottle. Incubate at 27-30°C under 4000 lux for 14 hours per day. After 7-10 days, remove the sealing film and add an appropriate amount of sterile water. Transplant after 2-3 days.

[0113] Note: The callus cannot be subcultured during the differentiation process. The time from differentiation to transplantation of the target plant is about three months.

[0114] 6. Training and transplanting of target plants

[0115] Pick out the seedlings of the target plant with relatively complete differentiation of roots, stems and leaves (open the lid in time when the seedlings grow to the top of the test tube), open the sealing film, add an appropriate amount of distilled water or sterile water (to prevent bacteria from growing in the culture medium), harden the seedlings for about 3 days to a week, then wash off the agar, transplant them into soil pots in the greenhouse for growth, test, and obtain the target plants.

[0116] 7. Detection and verification of target plants

[0117] 1) PCR Assay: Design inducible primers, such as RNA demethylase-F: ATGAAGCGCACCCCGACTG; RNA demethylase-R: GGGTTTTGCTTCCAGAAGCTGA. PCR reaction program: 95°C for 5 min, 95°C for 15 s, 58°C for 15 s, 72°C for 30 s, 35 cycles, extension at 72°C for 10 min, and storage at 4°C. After completion of the reaction, analyze the PCR products by 1% agarose gel electrophoresis.

[0118] 2) Method for rapid detection of target plants with hygromycin: Cut and collect fresh green leaves about 1 cm long from the seedlings to be tested (with incisions left at both ends), place them flat on the test medium (0.7% agar, 1 ml / L 6-BA, 50 mg / L hygromycin), and incubate them at 28°C, 16 h light / 8 h dark for 48 h. Plants with leaves that remain bright green are positive, while negative seedlings have leaves that show blocky necrosis.

[0119] 8. Evaluation of the effect of induction media in target plants

[0120] Data was collected from mature target plants in the experimental and control groups. The data primarily reflects yield increases for each plant, expressed in percentages. The weights mentioned above are dry weights, obtained after oven-drying at 105°C for 20 minutes and then at 80°C for 20 hours.

[0121] Example 2: Method for obtaining target plants by infecting stem tip tissue with Agrobacterium

[0122] 1. Inducing callus tissue using mature plant embryos as test materials

[0123] Take mature seeds and soak them in clean water, change the water every 1 to 2 hours, and germinate and root them at 37℃ until the seeds burst and turn white. When the embryo grows to 1.5 to 2.0 cm, use a scalpel to cut off the embryo and coleoptile from the stem ring at the stem tip to expose the stem tip meristem.

[0124] 2. Agrobacterium culture (same as step 2 in Example 2)

[0125] 3. Infection and Co-cultivation

[0126] Place the treated plants in AAM resuspension (see Appendix of Example 1), add 200 l / L of surfactant Silwet L-77, vacuum treat at 1.5 kPa for 8 minutes, pour out the resuspension, place on clean and moist perlite, and culture in the dark at 28 ° C for 3 days.

[0127] 4. Screening, transplanting and management of target plants

[0128] After dark incubation, plants were transferred to a seedbed. Leaves from transformed plants at the three- to four-leaf stage were collected for PCR analysis of transformed seedlings. Designed primers for genes such as RNA demethylase-F: ATGAAGCGCACCCCGACTG; RNA demethylase-R: GGGTTTTGCTTCCAGAAGCTGA. The PCR reaction procedure was: 95°C for 5 minutes, 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 30 seconds, 35 cycles, extension at 72°C for 10 minutes, and storage at 4°C. PCR products were analyzed by 1% agarose gel electrophoresis. Seven days later, surviving plants were transplanted to a greenhouse at a transgenic plant demonstration base and maintained with standard fertilization and watering. Transformed seedlings were harvested for seed.

[0129] 5. Evaluation of target plant traits (same as step 8 in Example 2)

[0130] Example 3: Method for obtaining target plants by infecting with Agrobacterium and inducing meristem production

[0131] 1. Plant cultivation

[0132] The mature seeds were planted in nutrient soil and grown in a 24°C greenhouse environment with 16 h light / 8 h dark conditions until maturity (approximately 63 to 66 days).

[0133] 2. Agrobacterium culture

[0134] DRs (growth regulatory elements) are a class of growth regulatory elements that induce meristem formation in plants. Commonly used ones include BBM, ipt, ΔMP, STM, and Wus2. A vector carrying the induction medium, the hygromycin resistance gene, and DRs is transformed into Agrobacterium GV3101. The cells are cultured overnight at 28°C for 12 hours in growth medium (10 mM MES, pH 5.6, 20 μM acetosyringone, 50 mg / L kanamycin, and 50 mg / L gentamicin). After centrifugation at 5000 rpm for 10 minutes, the cells are harvested and resuspended in infiltration medium (10 mM MES, 150 μM acetosyringone, and 10 mM MgCl2) to an OD600 of 0.2-0.3. The Agrobacterium infection medium is incubated at room temperature (approximately 25°C) for 2-4 hours before inoculation.

[0135] 3. Infection and screening of target plants

[0136] Visible shoot meristems were removed from all plants, leaving 2-3 nodes and supporting leaves. The wound site was immediately inoculated with the Agrobacterium infection solution using a syringe and a 31G needle. Plants were observed 38-48 days after inoculation for bud formation at the cut site. Each injection site with newly formed tissue or meristems was counted as a single event. The presence of bud tissue was identified by genomic PCR (see Example 2, Step 7) as an indicator of transgene presence and expression.

[0137] 4. Transplantation and management of target plants

[0138] The newly formed bud tissue was cut and transferred to a rooting medium to root and form complete seedlings. The surviving plants were then transplanted to a greenhouse and fertilized and watered as usual. The transformed seedlings were harvested.

[0139] 5. Evaluation of target plant traits (same as step 8 in Example 2)

[0140] Example 4: Method for obtaining target plants by mechanical inoculation of PVX vectors

[0141] 1. Plant cultivation

[0142] The mature seeds were planted in nutrient soil and grown in a 24°C greenhouse environment with 16 h light / 8 h dark conditions until maturity (approximately 63 to 66 days).

[0143] 2. Agrobacterium culture

[0144] Transform Agrobacterium C58C1 with the induction vector pPZPVX. After culturing in Luria broth, harvest the cells and suspend them in infection buffer (10 mM MES [pH 5.8], 10 mM MgCl2, 100 μg / mL acetosyringone) to an OD600 of 0.2-0.3. Incubate the Agrobacterium infection solution at room temperature (approximately 25°C) for 2-4 hours before inoculation.

[0145] 3. Infection and screening of target plants

[0146] Tobacco leaves were infiltrated with Agrobacterium carrying the inducible mediator expression plasmid using a syringe. Seven days later, the inoculated leaves were homogenized in 10 mM NaPi buffer (pH 7.0) (Western blot analysis confirmed the expression of the inducible mediator). The homogenized leaves were centrifuged at 16,000 g for 3 minutes, and the supernatant was filtered through a 0.45 μm filter. The filtered supernatant was mixed with 600-mesh corundum and applied to the fourth or fifth true leaf. The leaves were then gently rubbed by hand to mechanically inoculate with PVX. After inoculation, the plants were grown at 16°C under a 16-hour light / 8-hour dark photoperiod. After 7-8 days, the inoculated sites were observed for new shoot formation.

[0147] Note: Western blot detection of induction mediator expression: Take the inoculated leaf tissue powder and mix it with 1×10- ...

[0148] 4. Transplantation and management of target plants

[0149] The newly formed bud tissue was cut and transferred to a rooting medium to root and form complete seedlings. The surviving plants were then transplanted to a greenhouse and fertilized and watered as usual. The transformed seedlings were harvested.

[0150] 5. Evaluation of target plant traits (same as step 8 in Example 2)

[0151] Example 5: Method for obtaining target plants through haploid induction-editing (HI-Edit) technology

[0152] Haploid induction (HI) technology can induce haploids by appropriately modifying CENTROMERIC HISTONE3 (CENH3) or knocking out MATRILINEAL (MATL) genes, followed by chromosome doubling. This allows homozygous candidate materials to be obtained quickly, fixing crop genotypes and holding significant significance in crop breeding practices. Haploid induction-editing (HI-Edit) technology combines haploid induction breeding with gene editing techniques, enabling direct improvement of a wide range of plants, including commercial varieties, in a short period of time, breaking free from the species and genotype limitations of existing gene editing systems. Since the chromosomes of the donor carrying the gene editing element are eliminated during hybridization after introduction into the haploid induction line, the resulting hybrids do not contain the genetic information of the paternal parent. Multiple backcrosses are eliminated; chromosome doubling of the target haploid plant alone can yield improved diploid lines, shortening the breeding cycle. HI-Edit can also be used to improve target crops through distant hybridization, making it widely applicable and enabling the generation of transgene-free lines.

[0153] 1. Construct an induction medium vector and introduce it into the non-haploid inbred line NP2222 to obtain stable transformed plants

[0154] Nine-day-old immature embryos isolated from the inbred line NP2222 were mixed with Agrobacterium tumefaciens strain LBA4404 carrying a vector containing an induction medium. The infected embryos were cultured on recovery medium and callus induction medium. Callus was selected on a medium containing mannose, and resistant callus was regenerated into the target plant to obtain stably transformed plants.

[0155] 2. Evaluation of target plant traits (same as step 8 in Example 2)

[0156] The above-mentioned Examples 1 to 5 can all be methods of introducing modified DNA or modified proteins into initial plants to obtain target plants, and the degree of trait optimization is measured. The yield and biomass data of the experimental group, control group and blank group are measured respectively. The blank group is used as the standard. The average data of the yield and biomass increase of the experimental group and the control group relative to the blank group are respectively based on dry weight. The data in the following table are displayed based on the data of Example 1. The data of the remaining examples are basically no more than 0.05 different from the data of Example 1.

[0157] Table 1—MRP_1-Mono-green flagellate Ostreococcus lucimarinus

[0158] The control group data in the above table can be compared with the original RNA demethylase sequence or its encoding nucleic acid shown in Seq. ID. NO: 1 and the blank group to calculate the average value. The experimental group data in the above table can be compared with the modified RNA demethylase sequences shown in Seq. ID. NO: 13-15 or their encoding nucleic acids and the blank group to calculate the average value. Figure 2 shows a comparison of the experimental and blank groups of potatoes in Table 1 above, wherein the left image in Figure 2 shows the blank group and the right image shows the experimental group. Figure 3 shows a comparison of the experimental and blank groups of rice in Table 1 above, wherein the left image in Figure 3 shows the blank group and the right image shows the experimental group.

[0159] Table 2—MRP_2-Mono-Proteobacteria Aphanomyces astaci

[0160] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 2 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 16 to 18 with the blank group to obtain the average value.

[0161] Table 3—MRP_3-Mono-Micromonas commoda

[0162] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 3 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 19 to 21 with the blank group to obtain the average value.

[0163] Table 4—MRP_4-Mono-Micromonas pusilla

[0164] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 4 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 22 to 24 with the blank group to obtain the average value.

[0165] Table 5—MRP_5-Mono-Polarella glacialis

[0166] The control group data in the above table can be compared with the original RNA demethylase sequence shown in Seq.ID.NO: 5 or its encoding nucleic acid and the blank group to calculate the average value. The experimental group data in the above table can be compared with the modified RNA demethylase sequences shown in Seq.ID.NO: 25 to 27 or their encoding nucleic acids and the blank group to calculate the average value.

[0167] Table 6—MRP_6-Mono-Guillardia theta

[0168] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 6 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 28 to 30 with the blank group to obtain the average value.

[0169] Table 7—MRP_7-Mono-Ostreococcus tauri

[0170] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO:7 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO:31 to 33 with the blank group to obtain the average value.

[0171] Table 8—MRP_11-mono-brown algae Ectocarpus sp

[0172] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 8 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 34 to 36 with the blank group to obtain the average value.

[0173] Table 9—MRP_12-mono-filamentous algae Conferva siliculosa

[0174] The control group data in the above table can be compared with the original RNA demethylase sequence shown in Seq.ID.NO: 9 or its encoding nucleic acid and the blank group to calculate the average value. The experimental group data in the above table can be compared with the modified RNA demethylase sequences shown in Seq.ID.NO: 37 to 39 or their encoding nucleic acids and the blank group to calculate the average value.

[0175] Table 10—Fragilariopsis cylindrus

[0176] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 10 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 40 to 42 with the blank group to obtain the average value.

[0177] Table 11 - Long-spined sea star (Acanthaster planci)

[0178] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 11 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 43 to 45 with the blank group to obtain the average value.

[0179] Table 12 - Saccoglossus kowalevskii

[0180] The control group data in the above table can be calculated by comparing the original RNA demethylase sequence or its encoding nucleic acid shown in Seq.ID.NO: 12 with the blank group to obtain the average value. The experimental group data in the above table can be calculated by comparing the modified RNA demethylase sequences or their encoding nucleic acids shown in Seq.ID.NO: 46 to 48 with the blank group to obtain the average value.

[0181] From the above data, it can be concluded that after the modified algae or invertebrate-derived RNA demethylase and its encoding nucleic acid are introduced into plants, new plants with increased yield and biomass can be obtained, and compared with the introduction effect of the original source RAN demethylase and its encoding nucleic acid, it has more advantages.

[0182] The above are merely preferred embodiments of the present invention. It should be noted that these preferred embodiments should not be construed as limiting the present invention. The scope of protection of the present invention shall be determined by the scope of the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

1. A modified RNA demethylase, characterized in that Compared with the original RNA demethylase derived from algae or invertebrates, at least one peptide sequence is added to at least one of the N-terminus and the C-terminus to obtain the modified RNA demethylase.

2. The modified RNA demethylase according to claim 1, wherein the peptide sequence has any of the following amino acid sequence characteristics: a) contains at least three amino acids selected from the group consisting of H, K and R; b) contains PY and at least one amino acid selected from the group consisting of H, K and R.

3. The modified RNA demethylase according to claim 1, characterized in that When the peptide sequence has the amino acid sequence feature of a), at least two amino acids selected from H, K or R are consecutive, or contain V or I.

4. The modified RNA demethylase according to claim 1, characterized in that The peptide sequence has any of the following amino acid sequence characteristics: (K / R)(K / R)X 1-2 (K / R), (K / R)X 1-2 (K / R)(K / R)、(K / R)(K / R)X 10-12 (K / R) 2-5 、(R / K / H)X 2-5 PY, or K(V / I)XKX 1-2 (K / H / R), where X is any amino acid.

5. The modified RNA demethylase according to claim 1, characterized in that The peptide sequence includes at least one of RPRK, RKVIK, RNKKKK, PKKKRKV, KRPAATKKAGQAKKKK, TLLLRETMNNLGVSDHAVLSRKTPQPY, and KRTPTAEERERGAKKLRLLEELEDTWLPYL.

6. The modified RNA demethylase according to claim 1, characterized in that The original RNA demethylase includes at least one of its homologues or variants / combinations with equivalent functions.

7. The modified RNA demethylase according to claim 1, characterized in that The original RNA demethylase promotes the demethylation of N6-methyladenine of RNA.

8. The modified RNA demethylase according to claim 1, characterized in that The modified RNA demethylase is shown in the sequence table Seq.ID.No:13 to Seq.ID.No:

48.

9. The modified RNA demethylase according to claim 1, characterized in that The invertebrate comprises at least one of a long-spined sea star or a cystoglossum.

10. The modified RNA demethylase according to claim 1, characterized in that The algae include at least one of blue algae, red algae, cryptophyte, dinoflagellates, golden algae, yellow algae, diatoms, brown algae, naked algae, green algae and charophyte.

11. A nucleic acid encoding the modified RNA demethylase according to claim 1.

12. The nucleic acid according to claim 11, characterized in that The nucleic acid is DNA.

13. Use of the modified RNA demethylase according to claim 1 or the nucleic acid according to claim 11 introduced into plants.

14. The use according to claim 13, characterized in that: The application includes any one of increasing production and increasing biomass.

15. The use according to claim 13, characterized in that: The plant comprises at least one of rapeseed, tomato, lettuce, beet, rice, corn, soybean, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, flax mustard, sedge, hemp, and poplar.

16. A plant cultivation method, characterized in that: The modified RNA demethylase according to claim 1 or the nucleic acid according to claim 11 is introduced into an initial plant, and a target plant with optimized traits compared to the initial plant is obtained.

17. The plant cultivation method according to claim 16, characterized in that Trait optimization includes at least one of increasing yield and increasing biomass.

18. The plant cultivation method according to claim 16, characterized in that Compared to the original plant, the modified RNA demethylase according to claim 1 or the nucleic acid according to claim 11 is introduced in excess.

19. The plant cultivation method according to claim 16, characterized in that The initial plant is selected from at least one of rapeseed, tomato, lettuce, sugar beet, rice, corn, soybean, potato, wheat, millet, sugarcane, sorghum, cassava, tobacco, alfalfa, rubber grass, cotton, flax, sunflower, flax mustard, sedge, hemp, and poplar.

20. The plant cultivation method according to claim 16, characterized in that The introduced plant region includes at least one of a plant organ, a plant tissue, and a plant cell.

21. The plant cultivation method according to claim 16, characterized in that The introduced plant region is selected from the plant meristem.

22. The plant cultivation method according to claim 16, characterized in that The introduced plant region includes at least one of a cell nucleus and a cell sap of the plant.

23. The plant cultivation method according to claim 16, characterized in that The introduction method includes introducing a vector encapsulating the modified RNA demethylase according to claim 1 or the nucleic acid according to claim 11 into a plant.

24. The plant cultivation method according to claim 16, characterized in that Compared with the initial plant, the degree of trait optimization is Y, where Y is the multiple increase compared with the initial plant in the same period, Y≥3.50, preferably Y≥3.80, and more preferably Y≥4.

00.

25. A product obtained by inactivating the target plant obtained by the plant breeding method according to claim 16.

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