Use of ZmHY5.1 protein and its encoding gene in regulating corn stem and leaf biomass

Abstract

The application discloses ZmHY5.1 protein and application of a coding gene thereof in regulating corn stem and leaf biomass, and belongs to the technical field of biotechnology.The technical problem solved by the application is how to regulate plant biomass.The ZmHY5.1 protein disclosed by the application is a protein with an amino acid sequence of SEQ ID No.1.It is found by the application that overexpression of the coding gene of the ZmHY5.1 protein in a corn plant can significantly increase the fresh weight of the corn aboveground part, especially the biomass of the stem and leaves below the ear (an increase of 40%-56%), which shows that the expression abundance of the gene in the corn can regulate the biomass of the corn. ZmHY5.1 The application provides a good theoretical basis and germplasm resource for breeding high-quality silage corn.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of ZmHY5.1 protein and its encoding gene in regulating the biomass of maize stalks and leaves. Background Technology

[0002] Silage corn is a type of corn silage that utilizes corn ( Zea mays The above-ground parts of the plant are used to make high-yield, high-quality feed for herbivorous livestock. With the rapid development of China's animal husbandry industry, the silage corn industry in China has also developed rapidly. The demand for silage corn is increasing daily; therefore, improving the biomass of silage corn is a major issue for China's silage corn industry.

[0003] Silage corn is categorized into foliage silage, straw silage, and whole-plant silage. Foliage silage, also known as corn straw silage, consists only of foliage and contains little to no corn kernels; it has a high yield but low nutritional value. Straw silage, also called yellow silage, uses dry or semi-dry straw as raw material and is sealed and fermented with the addition of a fermenting agent, making it highly valuable for development and utilization. Whole-plant silage is one of the most important sources of roughage in China. It utilizes the fresh, green whole plant, including the ears, harvested at the early milk stage of corn kernels. After chopping and fermentation, it is used as feed for herbivorous livestock, representing the future direction of high-quality silage corn. Currently, researchers have conducted extensive studies on silage corn variety selection, cultivation methods, and nitrogen fertilizer application methods. However, the use of genetic engineering to breed high-yield and high-quality silage corn varieties offers more possibilities for the development of the silage corn industry.

[0004] Photomorphogenesis transcription factors HY5 These genes belong to the bZIP family of transcription factors and play important roles in physiological processes such as biotic and abiotic stress responses and development in plants. Maize has a large number of bZIP family protein factors, each with different biological functions, participating in multiple biological processes and playing a major and extensive role in biological regulation. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate plant biomass.

[0006] To address the aforementioned technical problems, the present invention first provides at least one of the following applications of proteins or substances that regulate the content or activity of said proteins: D1) Regulates plant biomass; D2) Prepare products that regulate plant biomass; D3) Cultivating plants with altered biomass; D4) Preparation of products from plants with altered biomass; The protein is derived from corn ( Zea maysIts name is ZmHY5.1 protein, which is listed as A1), A2), or A3). A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has more than 98% identity with A1) and has the same function as the amino acid sequence shown in SEQ ID No. 1 in the sequence listing, after substitution and / or deletion and / or addition of amino acid residues; A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] In the proteins described in A2) above, having more than 98% identity means having 98% or 99% identity. Identity refers to the identity of the amino acid sequence. The identity of amino acid 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, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gapexistence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity calculation for a pair of amino acid sequences, the identity value (%) can then be obtained.

[0008] The proteins mentioned in A2 above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0009] The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in positions 110-622 of SEQ ID No. 3, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ and / or 3′ ends. The DNA molecule shown in positions 110-622 of SEQ ID No. 3 encodes the protein shown in SEQ ID No. 1.

[0010] The tag described in A3) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0011] In the above applications, the substance may be any one of B1) to B7) below: B1) Nucleic acid molecules encoding the ZmHY5.1 protein; 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), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0012] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0013] Those skilled in the art can readily mutate the nucleotide sequence encoding the ZmHY5.1 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity with the nucleotide sequence of the ZmHY5.1 protein isolated according to this invention, provided they encode and function the ZmHY5.1 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0014] In the above applications, B1) the nucleic acid molecule may be a DNA molecule whose coding sequence is the 110-622nd position of SEQ ID No. 3 in the sequence listing.

[0015] Specifically, the nucleic acid molecule described in B1) may be the DNA molecule shown in positions 110-622 of SEQ ID No. 3, SEQ ID No. 3, or SEQ ID No. 2 in the sequence listing.

[0016] In the above application, the expression cassette (ZmHY5.1 gene expression cassette) containing a nucleic acid molecule encoding the ZmHY5.1 protein described in B2) refers to DNA capable of expressing the ZmHY5.1 protein in host cells. This DNA may include not only a promoter to initiate ZmHY5.1 gene transcription but also a terminator to terminate ZmHY5.1 gene transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention 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: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically induced 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 methyl jasmonic acid); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)); and promoters specific to seed storage proteins (e.g., beta-carotene, napin, etc.). The promoters of 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.

[0017] Recombinant vectors containing the ZmHY5.1 gene expression cassette can be constructed using existing expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). NosThe untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing plant expression vectors using the genes of this invention, 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 wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. 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 encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer resistance to glyphosate) or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0018] In the above applications, the vector can be a plasmid, granule, bacteriophage, or viral vector.

[0019] In the above applications, the microorganisms may be yeast, bacteria, algae, or fungi.

[0020] In the above applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation material.

[0021] In the above applications, the substance that regulates the content or activity of ZmHY5.1 protein can be a substance that increases the content or activity of ZmHY5.1 protein, the substance that regulates plant biomass can be a substance that increases the biomass of plant stems and leaves, and the substance that cultivates plants with altered biomass can be a plant with increased biomass.

[0022] The present invention also provides any of the following methods: X1) Methods to increase plant biomass include: expressing ZmHY5.1 protein in plants, or increasing the content or activity of ZmHY5.1 protein in plants to increase plant biomass; X2) Methods for cultivating plants with increased biomass include: expressing ZmHY5.1 protein in plants, or increasing the content or activity of ZmHY5.1 protein in plants to obtain plants with increased biomass.

[0023] In the above methods, X1) and X2) can be achieved by introducing the gene encoding the ZmHY5.1 protein into the plant.

[0024] The gene encoding the ZmHY5.1 protein can be introduced into the plant via a recombinant expression vector containing the gene encoding the ZmHY5.1 protein.

[0025] In the above method, the encoding gene can be the nucleic acid molecule described in B1).

[0026] In the above method, the gene encoding the ZmHY5.1 protein can be modified as follows before being introduced into the recipient plant to achieve better expression: 1) Modify and optimize according to actual needs to enable efficient gene expression; for example, the codons of the gene encoding the ZmHY5.1 protein described in this invention can be changed to conform to plant preference while maintaining the amino acid sequence of the gene. During the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to achieve the best high-level expression of the introduced gene in the plant. The GC content can be 35%, more than 45%, more than 50%, or more than about 60%. 2) Modify the gene sequence adjacent to the initiation methionine to enable efficient translation initiation; for example, by using a sequence known to be effective in plants. 3) Linked to promoters of various plant expression to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-selective, and tissue-specific promoters; the selection of promoters will vary with the time and space requirements of expression, and also depends on the target species; for example, tissue or organ-specific expression promoters, depending on the stage of development of the target receptor; although it has been shown that many promoters derived from dicotyledons are functional in monocotyledons and vice versa, ideally, dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons; 4) Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked with the gene of the present invention. 5) Introduce enhancer sequences, such as intron sequences (e.g., derived from Adhl and Bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0027] The recombinant expression vector can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

[0028] The target plant is understood to include not only first-generation plants containing the gene encoding the ZmHY5.1 protein, but also their progeny. For the target plant, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus tissue, intact plants, and cells.

[0029] In this invention, the plant may be M1, M2, or M3. M1) Monocotyledons; M2) Gramineae plants; M3) corn.

[0030] In this invention, the biomass can be the biomass of the aboveground parts. The biomass can also be the biomass of the aboveground stems and / or leaves.

[0031] This invention has found that overexpressing the gene encoding the ZmHY5.1 protein in maize plants can significantly increase the aboveground fresh weight of maize, especially the biomass of the stems and leaves below the ear (an increase of 40%-56%). This indicates that by altering... ZmHY5.1 The expression abundance of genes in maize can regulate maize biomass. This invention provides a sound theoretical basis and germplasm resources for cultivating high-quality silage maize.

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0033] Picture 1Transcriptome sequencing of leaves from homozygous transgenic plants and wild-type seedlings cultured for 20 days was conducted. ZmHY5 Gene FPKM values. FPKM stands for Fragments Per Kilobase of exon model per Million mapped fragments, meaning the number of fragments read per million mapped fragments per thousand bases of transcription. An asterisk indicates significance compared to wild type (***, p < 0.001), and red indicates a significant increase. HY5.1 and HY5.2 They represent ZmHY5.1 Genes and ZmHY5.2 Gene.

[0034] Picture 2 for ZmHY5.1 A schematic diagram of the silking phenotype of gene overexpression materials.

[0035] Picture 3 This is a graph showing the fresh weight distribution of different parts of the aboveground plant during the grain-filling stage. Red asterisks in the graph indicate significant differences compared to the same part in the wild type (p<0.05). Each bar represents the total fresh weight of the corresponding part of the plant as indicated in the graph.

[0036] Picture 4 This is a statistical chart showing the fresh weight of different parts of the aboveground plant during the grain-filling stage. Red asterisks in the chart indicate significant differences compared to the same part in the wild type (p<0.05). Each bar represents the total fresh weight of the corresponding part of the plant as indicated in the chart. Detailed Implementation

[0037] 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, reagents, instruments, etc., used in the following examples are commercially available.

[0038] In the quantitative experiments described below, at least three replicates were performed. Data were processed using SPSS 11.5 statistical software. Experimental results are expressed as mean ± standard error. One-way ANOVA was used, with P < 0.05 (*) indicating a significant difference, P < 0.01 (**) indicating a highly significant difference, and P < 0.001 (***) indicating a highly significant difference.

[0039] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0040] Example 1: ZmHY5.1 protein can regulate maize biomass. This embodiment discovered an inbred line derived from maize KN5585. ZmHY5 The gene (Zm00001eb235510) regulates maize biomass. It was found in the maize genome database (https: / / maizegdb.org). ZmHY5 The gene has two transcripts in the KN5585 inbred line. ZmHY5 The genome sequence of the gene is shown in SEQ ID No. 2, and its transcript... ZmHY5.1 The cDNA sequence of (Zm00001eb235510_T001) is shown in SEQ ID No. 3. Positions 110-622 of SEQ ID No. 3 represent the CDS sequence, encoding the ZmHY5.1 protein (Zm00001eb235510_P001) shown in SEQ ID No. 1. Transcript ZmHY5.2 The cDNA sequence of (Zm00001eb235510_T002) is shown in SEQ ID No.4. Positions 381-803 of SEQ ID No.4 are the CDS sequence, which encodes the ZmHY5.2 protein (Zm00001eb235510_P002) shown in SEQ ID No.5.

[0041] 1. Corn ZmHY5.1 Preparation of gene overexpression materials A gene expression vector suitable for maize genetic transformation was constructed containing the gene sequence shown in positions 110-622 of SEQ ID No. 3. ZmHY5.1 An overexpression vector for a gene expression cassette, containing a Basta-based resistance selection tag, capable of expressing the ZmHY5.1 protein. ZmHY5.1 Gene expression is driven by the UBI promoter. Using the obtained overexpression vector, genetic transformation was performed on the maize KN5585 inbred line as the recipient plant to obtain transgenic genes. ZmHY5.1 Genetically modified corn seeds.

[0042] DNA level identification: DNA was extracted from maize plant leaves using the CTAB method. Basta was used as the selection resistant agent, and the amplification primers were bar-F and bar-F, HY5OE-F and Vector-R. A double-primer identification method was used to select the resistant DNA. ZmHY5.1 Two homozygous overexpression lines, OE3 and OE21, were obtained from the gene-overexpression-positive plants. The primer sequences are as follows: bar-F: 5'-ACCATCGTCAACCACTACATCG-3'; bar-R: 5'-GCTGCCAGAAACCCACGTCATG-3'; HY5OE-F: 5'-CACAAGCGCCTCAAAAGGTT-3'; Vector-R: 5'-TAATCATCGCAAGACCGGCA-3'.

[0043] RNA level identification: Leaves from maize plants cultured for 20 days (including wild type and OE3, OE21) were collected, flash-frozen in liquid nitrogen, and sent to Beijing Novogene Technology Co., Ltd. for transcriptome sequencing. Gene FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values ​​were obtained through quality control, alignment, and quantification. Picture 1 As shown, ZmHY5 Although the gene has other alternative splicing versions, another transcript... ZmHY5.2 The FPKM of (SEQ ID No. 4) was significantly lower than that of (SEQ ID No. 4). ZmHY5.1 The FPKM value of (SEQ ID No. 3) indicates that... ZmHY5.1 The gene was overexpressed.

[0044] 2. Phenotypic observation of maize materials Wild-type KN5585 (WT) and homozygous overexpression lines OE3 and OE21 were planted in an experimental field at the Nankou Pilot Base of the Chinese Academy of Agricultural Sciences in Changping District, Beijing. Phenotypic development at the silking stage was observed under normal nitrogen application levels. Fresh weight of different parts of the maize plant at different grain-filling stages was also recorded. Phenotypic comparisons at the silking stage were compared. Picture 2 As shown. Picture 3 and Picture 4 Statistical results show that during the grain-filling stage, corn ZmHY5.1 The fresh weight of the aboveground parts (including stems and leaves) of the overexpressing plants was significantly higher than that of the wild type. The total fresh weight of the stems and leaves was also significantly higher than that of the wild type, especially the fresh weight of the stems and leaves in the lower part of the ear. This indicates that... ZmHY5.1 Genes can regulate the biomass of maize stalks and leaves, and can increase the biomass of maize stalks and leaves. ZmHY5.1 Genes are expressed in order to increase maize biomass.

[0045] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. At least one of the following applications of a protein or a substance that regulates the content or activity of said protein: D1) Regulates plant biomass; D2) Prepare products that regulate plant biomass; D3) Cultivating plants with altered biomass; D4) Preparation of products from plants with altered biomass; The protein is either A1), A2), or A3). A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has more than 98% identity with A1) and has the same function as the amino acid sequence shown in SEQ ID No. 1 in the sequence listing, after substitution and / or deletion and / or addition of amino acid residues; A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The application according to claim 1, characterized in that: The substance is any one of B1) to B7) below: B1) The nucleic acid molecule that encodes the protein; 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), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

3. The application according to claim 2, characterized in that: B1) The nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No. 3 in the sequence listing, positions 110-622.

4. The application according to any one of claims 1-3, characterized in that: The substance that regulates the protein content or activity is a substance that increases the protein content or activity; the substance that regulates plant biomass is a substance that increases plant biomass; and the plant whose biomass is altered is a plant whose biomass is increased.

5. The application according to any one of claims 1-4, characterized in that: The plant is M1, M2, or M3. M1) Monocotyledons; M2) Gramineae plants; M3) corn.

6. Any of the following methods: X1) Methods to increase plant biomass include: To enable plants to express the protein described in claim 1, or to increase the content or activity of the protein described in claim 1 in plants, thereby increasing plant biomass; X2) A method for cultivating plants with increased biomass, comprising: expressing the protein of claim 1 in the plant, or increasing the content or activity of the protein of claim 1 in the plant, to obtain a target plant with increased biomass.

7. The method according to claim 6, characterized in that: The methods described in X1) and X2) are achieved by introducing the gene encoding the protein into the plant.

8. The method according to claim 6 or 7, characterized in that: The plant is M1, M2, or M3. M1) Monocotyledons; M2) Gramineae plants; M3) corn.

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