A method for enhancing the fragrance of plants

Abstract

The present invention provides a method for enhancing plant fragrance, specifically, the present invention provides the use of an inhibitor of BADH2 gene or its encoded protein for enhancing plant fragrance; or preparing a composition or preparation for enhancing plant fragrance, wherein the BADH2 gene includes BADH2b gene. The present invention first discovered that inhibiting the expression of BADH2 gene or its encoded protein can significantly enhance the fragrance of plants.

Description

Technical Field

[0001] The invention relates to the field of agronomy, and in particular to a method for enhancing plant fragrance. Background Art

[0002] Fragrant rice is favored by consumers for its unique and rich aroma, so aroma has become an important quality trait indicator of rice. The price of fragrant rice is 2-3 times that of ordinary rice, and the price of BASMATI fragrant rice produced in India and Pakistan is much higher. The volatile aroma substances of fragrant rice contain more than 100 compounds, among which 2-acetyl-1-pyrroline (2AP) is the most important aroma substance.

[0003] my country has more than 600 million mu of corn planting area each year, of which about 20 million mu is for fresh corn, and the market value of corn seeds is about 20 billion yuan each year. In various reports, no fragrant corn germplasm has been found in either ordinary grain corn or fresh corn. Therefore, combining the existing reported biotechnology, improving corn fragrance and increasing corn added value are of great significance for enriching corn germplasm resources, expanding corn planting area, and increasing the scope of corn application.

[0004] Therefore, there is an urgent need in the art to develop a new method for enhancing plant fragrance. Summary of the invention

[0005] The object of the present invention is to provide a new method for enhancing plant fragrance.

[0006] The first aspect of the present invention provides a method for enhancing plant fragrance, comprising the steps of:

[0007] The expression level and / or activity of the BADH2 gene or its encoded protein in the plant is reduced or inhibited, thereby enhancing the fragrance of the plant, wherein the BADH2 gene includes the BADH2b gene.

[0008] In another preferred embodiment, the BADH2 gene also includes the BADH2a gene.

[0009] In another preferred embodiment, the BADH2 gene includes BADH2b and BADH2a genes.

[0010] In another preferred embodiment, the method comprises administering an inhibitor of the plant BADH2 gene or its encoded protein.

[0011] In another preferred example, the enhancing the fragrance of the plant comprises increasing the content of 4-aminobutyraldehyde or 2-acetyl-1-pyrroline (2AP) in the plant.

[0012] In another preferred embodiment, the enhancing the fragrance of the plant is to increase the content of 4-aminobutyraldehyde or 2-acetyl-1-pyrroline (2AP) in the plant.

[0013] In another preferred embodiment, the plants include crops, forestry plants, vegetables, fruits, flowers, and forage grasses (including lawn grasses).

[0014] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0015] In another preferred embodiment, the plant is selected from the following group: Poaceae, Leguminosae, Chenopodiaceae, Cruciferae, or a combination thereof.

[0016] In another preferred embodiment, the plant is selected from the group consisting of Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, strawberry, or a combination thereof.

[0017] In another preferred example, the plant is selected from corn.

[0018] In another preferred embodiment, the expression level or activity of the BADH2 gene or its encoded protein in the plant tissue or plant cell is reduced by ≥30%, ≥50%, ≥70%, and more preferably, ≥80%.

[0019] In another preferred embodiment, the “reduction or inhibition” refers to that the expression or activity of the BADH2 gene or its encoded protein is reduced to meet the following conditions:

[0020] The BADH2 gene or its encoded protein in the plant is completely or partially inactive, or the ratio of A1 / A0 is ≤80%, preferably ≤60%, more preferably ≤40%, and most preferably 0-30%; wherein A1 is the expression or activity of the BADH2 gene or its encoded protein in the plant; and A0 is the expression or activity of the same BADH2 gene or its encoded protein in a wild-type plant of the same type.

[0021] In another preferred embodiment, the reduction or inhibition refers to that compared with the expression level E0 of the BADH2 gene or its encoded protein in the wild-type plant, the expression level E1 of the BADH2 gene or its encoded protein in the plant is 0-80% of the wild-type, preferably 0-60%, more preferably 0-40%, and more preferably 0-30%.

[0022] In another preferred embodiment, the reduction or inhibition of the expression and / or activity of the BADH2 gene or its protein is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of gene or protein inhibitors, or a combination thereof.

[0023] In another preferred embodiment, the gene mutation is obtained by one or more of the following methods: natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus or bacteria-mediated mutagenesis), gene editing or biosynthesis.

[0024] In another preferred embodiment, the mutation region includes exon and / or intron regions.

[0025] In another preferred embodiment, the inhibitor is selected from the following group: antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, small molecule ligand, or a combination thereof.

[0026] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

[0027] In another preferred embodiment, the method comprises the steps of:

[0028] (i) providing a plant or plant cell; and

[0029] (ii) introducing the BADH2 gene or an inhibitor of its encoded protein into the plant or plant cell, thereby obtaining a modified plant or plant cell.

[0030] In another preferred embodiment, the method comprises the steps of:

[0031] (i) providing a plant or plant cell; and

[0032] (ii) using a gRNA targeting the BADH2 gene and a corresponding Cas protein to introduce into the plant or plant cell; in a preferred embodiment, an expression vector containing the gRNA and Cas protein is introduced into the plant or plant cell.

[0033] In another preferred embodiment, the BADH2 gene includes a wild-type BADH2 gene and a mutant BADH2 gene.

[0034] In another preferred embodiment, the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (ie, the function is the same or substantially the same as that of the wild-type encoded protein).

[0035] In another preferred embodiment, the polypeptide encoded by the mutant BADH2 gene is identical or substantially identical to the polypeptide encoded by the wild-type BADH2 gene.

[0036] In another preferred embodiment, the mutant BADH2 gene comprises a polynucleotide having a homology ≥80% (preferably ≥90%, more preferably ≥95%, more preferably ≥98% or 99%) compared to the wild-type BADH2 gene.

[0037] In another preferred embodiment, the mutant BADH2 gene comprises a polynucleotide having 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' end and / or 3' end of the wild-type BADH2 gene.

[0038] In another preferred embodiment, the BADH2 gene includes a cDNA sequence, a CDS sequence, a genomic sequence, or a combination thereof.

[0039] In another preferred embodiment, the BADH2 gene is derived from one or more plants selected from the following group: Gramineae, Leguminosae, Chenopodiaceae, and Cruciferae.

[0040] In another preferred embodiment, the BADH2 gene is derived from one or more plants selected from the following group: Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, and strawberry.

[0041] In another preferred embodiment, the BADH2 gene is derived from corn.

[0042] In another preferred embodiment, the amino acid sequence of the protein encoded by the BADH2a gene is selected from the following group:

[0043] (i) a polypeptide having an amino acid sequence as shown in SEQ ID NO.: 1;

[0044] (ii) a polypeptide derived from (i) having the same or similar function (betaine aldehyde dehydrogenase activity) formed by replacing, deleting or adding one or several (e.g., 1-10) amino acid residues in the amino acid sequence as shown in SEQ ID NO.: 1;

[0045] or (iii) a polypeptide having an amino acid sequence homology ≥90% (preferably ≥95%, more preferably ≥98%) to the amino acid sequence of SEQ ID NO.: 1 and having the same or similar functions.

[0046] In another preferred embodiment, the amino acid sequence of the protein encoded by the BADH2b gene is selected from the following group:

[0047] (i) a polypeptide having an amino acid sequence as shown in SEQ ID NO.: 2;

[0048] (ii) a polypeptide derived from (i) having the same or similar function (betaine aldehyde dehydrogenase activity) formed by replacing, deleting or adding one or several (e.g., 1-10) amino acid residues in the amino acid sequence as shown in SEQ ID NO.: 2;

[0049] or (iii) a polypeptide having an amino acid sequence homology ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%) to the amino acid sequence of SEQ ID NO.: 2 and having the same or similar functions

[0050] In another preferred embodiment, the nucleotide sequence of the BADH2a gene is selected from the following group:

[0051] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 1;

[0052] (b) a polynucleotide whose sequence is shown in SEQ ID NO.: 3;

[0053] (c) a polynucleotide having a nucleotide sequence homology ≥ 95% (preferably ≥ 98%, more preferably ≥ 99%) to the sequence shown in SEQ ID NO.: 3;

[0054] (d) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.: 3;

[0055] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0056] In another preferred embodiment, the nucleotide sequence of the BADH2b gene is selected from the following group:

[0057] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 2;

[0058] (b) a polynucleotide whose sequence is shown in SEQ ID NO.: 4;

[0059] (c) a polynucleotide having a nucleotide sequence homology ≥95% (preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID NO.: 4;

[0060] (d) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.: 4;

[0061] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0062] In another preferred embodiment, the reducing or inhibiting the expression level and / or activity of the BADH2b gene or its encoded protein is achieved by mutating the BADH2b gene.

[0063] In another preferred embodiment, the reduction or inhibition of the expression level and / or activity of the BADH2b gene and the BADH2a gene or the proteins encoded therein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene.

[0064] In another preferred embodiment, the mutation includes insertion mutation, deletion mutation, frameshift mutation and substitution mutation.

[0065] In another preferred embodiment, the method further comprises the step of testing the increase in the content of aroma substances in the plant.

[0066] In another preferred embodiment, the content of the aromatic substance in the plant is 0.01-20 mg / kg, preferably, 0.05-10 mg / kg, more preferably, 0.1-5 mg / kg, more preferably, 0.1-2 mg / kg, more preferably, 0.2-1 mg / kg.

[0067] In another preferred example, the aroma substance includes 2AP.

[0068] The second aspect of the present invention provides a use of an inhibitor of a BADH2 gene or a protein encoded therein for enhancing the fragrance of a plant; or for preparing a composition or preparation for enhancing the fragrance of a plant, wherein the BADH2 gene includes a BADH2b gene.

[0069] In another preferred embodiment, the BADH2 gene also includes the BADH2a gene.

[0070] In another preferred embodiment, the BADH2 gene includes BADH2b and BADH2a genes.

[0071] In another preferred embodiment, the composition comprises an agricultural composition.

[0072] In another preferred embodiment, the preparation includes an agricultural preparation.

[0073] In another preferred embodiment, said enhancing the aroma of the plant comprises increasing the content of aroma substances in the plant.

[0074] In another preferred example, the aroma substance includes 2AP.

[0075] In another preferred embodiment, the composition comprises (a) an inhibitor of BADH2 gene or its encoded protein, wherein the BADH2 gene includes BADH2b gene; and (b) an agronomically acceptable carrier.

[0076] In another preferred embodiment, the composition further comprises BADH2a gene.

[0077] In another preferred embodiment, the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.

[0078] In another preferred embodiment, the inhibitor is selected from the following group: antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, small molecule ligand, or a combination thereof.

[0079] In another preferred embodiment, the antisense nucleic acid is selected from the following group: antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.

[0080] In another preferred embodiment, the interfering RNA is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.

[0081] In another preferred embodiment, the composition further comprises other substances that enhance the aroma of crops.

[0082] The third aspect of the present invention provides a composition for enhancing plant fragrance, comprising:

[0083] (a) an inhibitor of BADH2 gene or a protein encoded thereof, wherein the BADH2 gene includes the BADH2b gene; and

[0084] (b) an agriculturally acceptable carrier.

[0085] In another preferred embodiment, the composition further comprises BADH2a gene.

[0086] In another preferred embodiment, the composition comprises an agricultural composition.

[0087] In another preferred embodiment, the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.

[0088] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt% of component (a), based on the total weight of the composition.

[0089] In another preferred embodiment, the inhibitor is selected from the following group: antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, small molecule ligand, or a combination thereof.

[0090] In another preferred embodiment, the antisense nucleic acid is selected from the following group: antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.

[0091] In another preferred embodiment, the interfering RNA is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.

[0092] In another preferred example, the composition further comprises other substances that enhance the fragrance of plants.

[0093] The fourth aspect of the present invention provides a use of the composition according to the third aspect of the present invention for enhancing the fragrance of plants.

[0094] A fifth aspect of the present invention provides a method for preparing genetically engineered plant tissues or plant cells, comprising the steps of:

[0095] The expression and / or activity of the BADH2 gene or its encoded protein in plant tissues or plant cells is reduced or inhibited, thereby obtaining genetically engineered plant tissues or plant cells.

[0096] In another preferred embodiment, the reduction of the expression and / or activity of the BADH2 gene or its protein is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of gene or protein inhibitors, or a combination thereof.

[0097] A sixth aspect of the present invention provides a method for preparing a genetically engineered plant, comprising the steps of:

[0098] The genetically engineered plant tissue or plant cell prepared by the method described in the fifth aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.

[0099] A seventh aspect of the present invention provides a method for improving plants, the method comprising the steps of:

[0100] (a) providing a plant cell, plant tissue or plant part, and introducing a BADH2 gene or an inhibitor of its encoded protein into the plant cell, plant tissue or plant part; wherein the BADH2 gene includes a BADH2b gene;

[0101] (b) regenerating the plant cells, plant tissues, or plant parts in step (a) into plants.

[0102] In another preferred embodiment, in step (a), the plant cells, plant tissues, and plant parts are modified using gene editing technology, so that the expression or activity of the BADH2 gene or its encoded protein in the plant cells, plant tissues, and plant parts is reduced.

[0103] In another preferred embodiment, the BADH2 gene also includes the BADH2a gene.

[0104] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.

[0105] In another preferred embodiment, the method is used to enhance the fragrance of plants.

[0106] In another preferred embodiment, the method is used to increase the content of flavor substances.

[0107] In another preferred embodiment, the method further comprises the step of testing the increase in the content of aroma substances in the plant cells, plant tissues, plant parts or plants.

[0108] The eighth aspect of the present invention provides a genetically engineered plant, wherein the plant is prepared by the method described in the seventh aspect of the present invention.

[0109] The ninth aspect of the present invention provides a method for screening or identifying fragrant plants, which detects the expression level of BADH2 gene and / or its protein in the plant, wherein the BADH2 gene includes BADH2b gene.

[0110] In another preferred embodiment, the BADH2 gene also includes the BADH2a gene.

[0111] In another preferred embodiment, the detected parts of the plant include callus, fruit, seed, flower, stem, leaf, ear and root of the plant.

[0112] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 The editing mode of the aroma gene of the C43 plant in the Zheng 58 background is shown, where A, the editing form of the two genes ZmBADH2a and ZmBADH2b in the DNA sequence; B, the editing form of the two genes ZmBADH2a and ZmBADH2b in the cDNA sequence; C, the deduced protein sequence of the two proteins ZmBADH2a and ZmBADH2b after editing. The blue markers are the same amino acid sequences as Zheng 58, and the red markers are the amino acid sequences of frameshift mutations. Zheng 58: maize inbred line Zheng 58; C43 / Zheng 58: gene-edited plant C43 in the Zheng 58 background.

[0114] Figure 2A, shows the editing mode of the fragrance gene of the P64 plant in the Zheng 58 background. A, the editing form of the ZmBADH2a gene in the DNA sequence; B, the editing form of the ZmBADH2a gene in the cDNA sequence; C, the deduced protein sequence of the edited ZmBADH2a. The blue markers are the same amino acid sequences as Zheng 58, and the red markers are the amino acid sequences of frameshift mutations. Zheng 58: maize inbred line Zheng 58; P64 / Zheng 58: gene-edited plant P64 in the Zheng 58 background. .

[0115] Figure 3 The content of 2AP in C43 and P64 seeds under the background of Zheng 58 is shown; among them, 1 is C43 / Zheng 58, 2 is P64 / Zheng 58, and 3 is unedited Zheng 58. The gray dotted line in the figure indicates the peak time of 2AP.

[0116] Figure 4 The editing mode of the fragrance gene of the P302 plant in the XCW175 background is shown, where A, the edited form of the two genes ZmBADH2a and ZmBADH2b in the DNA sequence; B, the edited form of the two genes ZmBADH2a and ZmBADH2b in the cDNA sequence; C, the deduced protein sequence of the two proteins ZmBADH2a and ZmBADH2b after editing. The blue marker is the same amino acid sequence as Zheng 58, and the red marker is the amino acid sequence of the frameshift mutation. XCW175: maize inbred line XCW175; P302 / XCW175: gene-edited plant P302 in the XCW175 background.

[0117] Figure 5 The figure shows the 2AP content of P302 plants in the XCW175 background. The gray dotted line in the figure indicates the peak time of 2AP, where 1 is P302, 2 is XCW175. DETAILED DESCRIPTION

[0118] After extensive and in-depth research, the inventors unexpectedly discovered for the first time through the research and screening of a large number of plant trait loci that when the expression of BADH2a and BADH2b genes or their encoded proteins is simultaneously inhibited, the fragrance of the plant can be significantly enhanced. The inventors completed the present invention on this basis.

[0119] Unless defined in this application, the scientific terms or professional terms used in the present invention have the meanings understood by those skilled in the art. When the meanings understood by those skilled in the art conflict with the meanings defined in this application, the meanings defined in this application shall prevail.

[0120] As used herein, the term "enhancing the aroma of a plant" includes imparting aroma to a plant without aroma, and also includes improving the aroma of a fragrant plant.

[0121] As used herein, the "Cripsr preparation" refers to a combination of effective ingredients that can achieve gene editing effects, including gRNA (guide RNA) or its coding sequence and Cas protein or its coding sequence, and may further include a vector, as well as elements that are conducive to homologous recombination or gene expression.

[0122] The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. Between two sequences. Usually, the comparison is made when the two sequences are aligned to produce maximum identity. Alignment methods are conventional methods known to those skilled in the art, such as the BLAST algorithm.

[0123] The term "genetic engineering" refers to the technology of modifying and utilizing nucleotides that control biological genetic information through artificial intervention to obtain new genetic characteristics, new varieties, or new products, including all genetic modification technologies disclosed in the art, such as gene mutagenesis, transgenic or gene editing. Methods of gene mutagenesis include but are not limited to physical mutagenesis (such as ultraviolet mutagenesis), chemical mutagenesis (such as acridine dyes), biological mutagenesis (such as virus, phage mutagenesis), etc. In a preferred embodiment, the genetic engineering modification of the present invention includes gene editing of members of the BADH2 gene family using one or more sgRNA-mediated Cas nucleases.

[0124] BADH2 gene

[0125] Badh2 is a gene encoding betaine aldehyde dehydrogenase, which has aldehyde dehydrogenase activity and may catalyze the oxidation of betaine aldehyde, 4-aminobutyraldehyde (AB-ald) and 3-aminopropanal, while 4-aminobutyraldehyde is a synthetic precursor of 2-acetyl-1-pyrroline (2AP), which is closely related to plant fragrance. In the present invention, the BADH2 gene family includes BADH2a and BADH2b.

[0126] As used herein, the term "BADH2 gene of the present invention" includes BADH2 genes or variants thereof in monocotyledonous or dicotyledonous plants, such as corn and rice. In a preferred embodiment, the nucleotide sequence of the BADH2 gene of the present invention is shown in SEQ ID NO.: 3 or 4.

[0127] >Zm00001d050339 (ZmBADH2a) genomic DNA sequence:

[0128]

[0129] >Zm00001d032257 (ZmBADH2b) genomic DNA sequence:

[0130]

[0131] The present invention also includes nucleic acids having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%, or 100%) homology with the preferred gene sequence of the present invention (SEQ ID NO.: 3 or 4), which can also effectively enhance the fragrance of plants. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids according to the percentage of identical positions. In this article, variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, etc.

[0132] In the present invention, the nucleotide sequence in SEQ ID NO.: 3 or 4 may be substituted, deleted or added with one or more nucleotides to generate a derivative sequence of SEQ ID NO.: 3 or 4. Due to the degeneracy of codons, even if the homology with SEQ ID NO.: 3 or 4 is low, it can still basically encode the amino acid sequence shown in SEQ ID NO.: 1 or 2. In addition, the meaning of "the nucleotide sequence in SEQ ID NO.: 3 or 4 is substituted, deleted or added with at least one nucleotide derivative sequence" also includes nucleotide sequences that can hybridize with the nucleotide sequence shown in SEQ ID NO.: 3 or 4 under moderate stringency conditions, more preferably under highly stringent conditions. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) nucleotides at the 5' and / or 3' ends.

[0133] It should be understood that although the gene provided in the examples of the present invention is derived from corn, the gene sequence of BADH2 derived from other similar plants and having a certain homology (such as having more than 80%, such as 85%, 90%, 95% or even 98%, 99%, or 100% sequence identity) with the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO.: 3 or 4) is also included in the scope of the present invention, as long as those skilled in the art can easily separate and obtain the sequence from other plants according to the information provided in the present application after reading the present application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0134] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or artificially synthesized DNA, and the DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO.: 3 or 4 or a degenerate variant.

[0135] The polynucleotide encoding the mature polypeptide includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0136] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and / or non-coding sequences. The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0137] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.

[0138] The full-length sequence of BADH2 nucleotide of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify and obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. Usually it is cloned into a vector, then transferred into a cell, and then the relevant sequence is isolated from the host cell after the proliferation by conventional methods.

[0139] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, very long fragments of the sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0140] BADH2 gene encoded polypeptide

[0141] As used herein, the terms "polypeptide of the present invention" and "protein encoded by BADH2 gene" can be used interchangeably and refer to polypeptides and variants of BADH2 derived from plants (such as corn). In a preferred embodiment, a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.: 1 or 2.

[0142] >Zm00001d050339 (ZmBADH2a) protein sequence:

[0143] MASPAMVPLRQLFVDGEWRPPAQGRRLPVVNPTTEAHIGEIPAGTAEDVDAAVAAARAALKRNRGRDWARAPGAVRAKYLRAIAAKVIERKPELAKLEALDCGKPYDEAAWDMDDVAGCFEYFADQAEALDKRQNSPVSLPMETFKCHLRREPIGVVGLITPWNYPLLMATWKIAPALAAGCTAVLKPSELASVTCLELADICKEVGLPSGVLNIVTGLGPDAGAPLSAHPDVDKVAFTGSFETGKKIMASAAPMVKPVTLELGGKSPIVVFDDVDIDKAVEWTLFGCFWTNGQICSATSRLLIHTKIAKKFNERMVAWAKNIKVSDPLEEGCRLGPVVSEGQYEKIKKFISNAKSQGATILTGGVRPAHLEKGFFIEPTIITDITTSMEIWREEVFGPVLCVKEFSTEDEAIELANDTQYGLAGAVISGDRERCQRLSEEIDAGCIWVNCSQPCFCQAPWGGNKRSGFGRELGEGGIDNYLSVKQVTEYISDEPWGWYQSPSKL(SEQ ID NO.:1)

[0144] >Zm00001d032257((ZmBADH2b) protein sequence:

[0145] MMASQAMVPLRQLFVDGEWRPPAQGRRLPVVNPTTEAHIGEIPAGTAEDVDAAVAAARAALKRRNRGRDWARAPGAVRAKYLRAIAAKVIERKQELAKLEALDCGKPYDEAAWDMDDVAGCFEYFADQ AEALDKRQNSPVSLPMETFKCHLRREPIGVVGLITPWNYPLLMATWKVAPALAAGCAAVLKPSELASVTTCLELADICKEVGLPPGVLNIVTGLGPDAGAPLSAHPDVDKVAFTGSFETGKKIMAAAAP MVKPVTLELGGKSPIVVFDDVDIDKAVEWTLFGCFWTNGQICSATSRLLVHTKIAKEFNEKMVAWAKNIKVSDPLEEGCRLGPVVSEGQYEKIKKFILNAKSEGATILTGGVRPAHLEKGFFIEPTI ITDITTSMEIWREEVFGPVLCVKEFSTEDEAIELANDTQYGLAGAVISGDRERCQRLSEEIDAGIIWVNCSQPCFCQAPWGGNKRSGFGRELGEGGIDNYLSVKQVTEYISDEPWGWYRSPSKL(SEQ ID NO.:2)

[0146] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99% or 100%) homology with the sequence shown in SEQ ID NO.: 1 or 2 of the present invention.

[0147] The "same or similar functions" mainly refers to having betaine aldehyde dehydrogenase activity.

[0148] The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production scheme, the polypeptide of the present invention can be glycosylated, or can be non-glycosylated. The polypeptide of the present invention may also include or not include an initial methionine residue.

[0149] The present invention also includes BADH2 protein fragments and analogs having BADH2 protein activity. As used herein, the terms "fragment" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the natural BADH2 protein of the present invention.

[0150] The polypeptide fragments, derivatives or analogs of the present invention may be: (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having a substitution group in one or more amino acid residues; or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0151] In the present invention, the polypeptide variant is an amino acid sequence as shown in SEQ ID NO.: 1 or 2, a derivative sequence obtained by replacing, deleting or adding at least one amino acid, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the protein, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and adding one or more amino acids to the C-terminus and / or the end is usually not changed. These conservative variations are preferably generated by substitution according to Table 1.

[0152] Table 1

[0153]

[0154]

[0155] The present invention also includes analogs of the claimed proteins. The difference between these analogs and the natural SEQ ID NO.: 1 or 2 may be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. The analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known biological techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

[0156] Modifications (usually without altering the primary structure) include: chemical derivatization of proteins in vivo or in vitro such as acetylating or carboxylating. Modifications also include glycosylation, such as those carried out during protein synthesis and processing. Such modifications can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylases or deglycosylases). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0157] Agricultural preparations

[0158] The active substances of the present invention (such as inhibitors of BADH2 gene or its encoded protein) can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, and coatings for seeds.

[0159] These preparations can be produced in a known manner, for example by mixing the active substance with an extender, i.e. a liquid, liquefied gas or solid diluent or carrier and optionally a surfactant, i.e. an emulsifier and / or dispersant and / or foam former. For example, when water is used as the extender, an organic solvent can also be used as an auxiliary agent.

[0160] When a liquid solvent is used as a diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons, for example xylene, toluene or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, for example chlorobenzene, vinyl chloride or methylene chloride; aliphatic hydrocarbons, for example cyclohexane or paraffin, for example mineral oil fractions; alcohols, for example ethanol or ethylene glycol and their ethers and lipids; ketones, for example acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; or unusual polar solvents, for example dimethylformamide and dimethyl sulfoxide, and water.

[0161] As for the diluent or carrier of liquefied gas, it refers to the liquid that will become gas at normal temperature and pressure, such as aerosol propellants, such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.

[0162] Solid carriers can be ground natural minerals, such as kaolin, clay, talc, quartz, atavistic clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals, such as highly dispersed silicic acid, alumina and silicates. Solid carriers for particles are crushed and classified natural zircons, such as calcite, marble, pumice, sepiolite and dolomite, and inorganic and organic coarse powders and synthetic particles, and organic materials such as sawdust, coconut shells, corn cobs and tobacco stem particles, etc.

[0163] Nonionic and anionic emulsifiers can be used as emulsifiers and / or foam formers. For example, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, such as alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and albumin hydrolysates. Dispersants include, for example, lignin sulfite waste liquor and methylcellulose.

[0164] Binders such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or emulsions, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, may be used in the formulations.

[0165] Colorants such as inorganic dyes, such as iron oxide, cobalt oxide and Prussian blue; organic dyes, such as azo dyes or metal phthalocyanine dyes; and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, aluminum and zinc, etc. can be used.

[0166] In the present invention, the "agricultural preparation" is generally an agricultural plant growth regulator, which contains an inhibitor of BADH2 gene or its encoded protein as an active ingredient for improving plant traits (eg, enhancing plant fragrance); and an agriculturally acceptable carrier.

[0167] As used herein, the "agriculturally acceptable carrier" is a pesticide-acceptable solvent, suspending agent or excipient for delivering the active substance of the present invention to plants. The carrier can be a liquid or a solid. Suitable agriculturally acceptable carriers for the present invention are selected from the group consisting of water, buffer, DMSO, surfactants such as Tween-20 or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.

[0168] The agricultural formulation of the present invention may include an agricultural composition.

[0169] The agricultural preparation of the present invention can be used in combination with other substances that enhance the aroma of plants. The other substances that enhance the aroma can be plant growth regulators known to those skilled in the art.

[0170] The agricultural formulation of the present invention can be in various forms, as long as the active ingredients can be effectively delivered to the plant body. From the standpoint of ease of preparation and application, the preferred agricultural formulation is a spray or solution formulation.

[0171] The agricultural formulations of the present invention generally contain 0.0001-99 wt%, preferably 0.1-90 wt% of the active ingredient of the present invention, based on the total weight of the agricultural formulation. The concentration of the active ingredient of the present invention in the commercial formulation or dosage form may vary over a wide range. The concentration of the active ingredient of the present invention in the commercial formulation or dosage form may be from 0.0000001-100% (g / v), preferably between 0.0001 and 50% (g / v).

[0172] Improvement of plant traits

[0173] The present invention also provides a method for improving plant traits, wherein the improvement comprises: enhancing the fragrance of the plant, comprising the steps of: reducing the expression level and / or activity of the BADH2 gene or its encoded protein in the plant, or adding an inhibitor of the BADH2 gene or its encoded protein.

[0174] In the present invention, other substances that can enhance the aroma of plants can be further used to treat plants or plant seeds by conventional methods, thereby improving the properties of the corresponding plants.

[0175] The main advantages of the present invention include:

[0176] (1) The present invention first discovered that plant fragrance can be significantly enhanced by inhibiting BADH2b, or inhibiting the expression or activity of BADH2a and BADH2b genes or their encoded proteins.

[0177] (2) The present invention first discovered that the content of flavor substances (such as 2AP) can be increased by inhibiting BADH2b or inhibiting the expression or activity of BADH2a and BADH2b genes or their encoded proteins.

[0178] (3) The present invention provides a method for rapidly improving the aroma of corn, and the improved corn aroma (2AP content) is significantly increased compared to the wild type.

[0179] (4) This invention creates a new corn germplasm resource, which has important theoretical and practical application significance.

[0180] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

[0181] Example

[0182] 1. Target design and vector construction

[0183] The protein sequence of the OsBADH2 gene was obtained through the NCBI (https: / / www.ncbi.nlm.nih.gov) website, and then two homologous genes were found after Blast with the corn protein database, namely Zm00001d032257 (ZmBADH2b) located on chromosome 1 and Zm00001d050339 (ZmBADH2a) located on chromosome 4.

[0184] gRNA was designed using target Design (http: / / skl.scau.edu.cn / targetdesign / ), in which ZmBADH2-T1 (CGCCAGCGATGGTCCCGCTG (SEQ ID NO.: 5)) located in the first exon region, ZmBADH2-T2 (AGTCGCGGCCACGGTTCCTC (SEQ ID NO.: 6)) located in the second exon region, and ZmBADH2-T3 (AATTAGGCTAGAGCAAAGAG (SEQ ID NO.: 7)) located in the second intron region are all sequences specific to the ZmBADH2a gene, in which ZmBADH2-T1 has 2 base mismatches with the corresponding sequence of ZmBADH2b, ZmBADH2-T2 has 2 base mismatches with the corresponding sequence of ZmBADH2b, and ZmBADH2-T3 has 3 base mismatches with the corresponding sequence of ZmBADH2b. ZmBADH2-T1 was combined with ZmBADH2-T2 and ZmBADH2-T3 to construct into gene editing vectors initiated by U6 promoter and spaced by glycine tRNA, respectively, to construct vectors P0195 and P0196. In addition, ZmBADH2-T1 and ZmBADH2-T2 were constructed into vector P0077, and the two gRNAs were spaced by glycine tRNA.

[0185] ZmBADH2-T4 (GGTTGACGACGGGGAGGCGG (SEQ ID NO.: 8)) located in the first exon region and ZmBADH2-T5 (GCGGCGCTCAAGAGGAACCG (SEQ ID NO.: 9)) located in the second exon region are the common sequences of the two genes. These two gRNAs were constructed into a gene editing vector using glycine tRNA spacer initiated by the U6 promoter to construct vector P0593.

[0186] The specific construction method is as follows:

[0187] 1) Use primer pairs

[0188] ZmBADH2-T1F (TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT (SEQ ID NO.: 10)) and

[0189] ZmBADH2-T2R

[0190] (TAGGTCTCTAAACAGTCGCGGCCACGGTTCCTCTGCACCAGCCGGGAATCG (SEQ ID NO.:11)), ZmBADH2-T1F (TAGGTCTCTTGCACGCCAGCGATGGTCCCGCTGGTTTTAGAGCTAGAAATAGCAAGT (SEQ ID NO.: 12)) and ZmBADH2-T3R (TAGGTCTCTAAACAATTAGGCTAGAGCAAAGAGTGCACCAGCCGGGAATCG (SEQ ID NO.: 13)), ZmBADH2-T4F (TAGGTCTCTTGCAGGTTGACGACGGGGAGGCGGGTTTTAGAGCTAGAAATAGCAAGT (SEQ ID NO.:14)) and ZmBADH2-T5R(TAGGTCTCTAAACGGCGGCGCTCAAGAGGAACCGTGGTGCACCAGCCGGGAATCG (SEQ ID NO.:15))

[0191] Plasmid P0055 was used as template for amplification, and fragments of about 200 bp were recovered by tapping, namely ZmBADH2-T1&2, ZmBADH2-T1&3 and ZmBADH2-T4&5. The recovered fragments were digested with BsaI and recovered using a kit;

[0192] 2) The backbone vectors P0174 and P0522 were digested with BsaI, and 23Kb fragments were recovered respectively;

[0193] 3) ZmBADH2-T1&2, ZmBADH2-T1&3 and P0174 were respectively connected with T4 ligase to construct the final vectors P0195 and P0196; ZmBADH2-T4&5 was connected with P0522 to construct the final vector P0593;

[0194] 4) The plasmid P0055 was digested with BsaI, and the liquid was recovered by the kit, and connected with ZmBADH2-T1&3 digested with BsaI. After plasmid amplification, the plasmid was double-digested with EcoRV and PstI, and the 1020 bp fragment was recovered by cutting the gel; the P0185 plasmid was double-digested with SmaI and PstI to recover the vector fragment; the 1020 bp DNA fragment was connected to the vector of P0185 with T4 DNA ligase to construct the final vector P0077.

[0195] 5) The ligation products in 3) and 4) above were transformed into competent E. coli Trans-T1, spread on Kan plate for culture, pick 8 colonies for liquid culture for 2 hours, perform PCR bacterial liquid detection, select 2 correct monoclones, and send the bacterial liquid for testing.

[0196] 6) Select the single clone with correct sequencing for expansion, bacterial preservation, plasmid extraction, and transformation of Agrobacterium EHA105. Pick 5 single colonies for culture. After the PCR test is correct, preserve the bacteria for use.

[0197] 2. Genetic transformation

[0198] 2.1 Transformation of Agrobacterium

[0199] The vector in 1 was transferred into the Agrobacterium strain EHA105 by heat shock method, and a single clone was picked out for liquid culture and PCR identification and stored in a -80°C refrigerator for later use.

[0200] 2.2 Bacteria Activation

[0201] Take out the bacteria from the -80 refrigerator, streak them on YEP solid culture medium, and culture them in the dark at 28℃. Usually colonies will grow in 1 to 2 days. Take the colonies and spread them on new YEP plates and culture them in the dark at 28℃. The bacteria on the plates will grow well in 12 to 24 hours.

[0202] 2.3 Preparation of Agrobacterium infection medium

[0203] Scrape fresh bacteria from the newly activated bacterial plate, resuspend them in the infection solution to which acetosyringone (AS) has been added in advance, use a 1 ml pipette to break up the bacteria until no granular bacteria are visible, adjust OD550 to 0.3-0.4, and culture on a shaker at room temperature with low speed for 2-4 hours.

[0204] 2.4 Taking corn embryos

[0205] Take corn ears about 10 days after pollination, remove the bracts and filaments, immerse them in 75% alcohol for disinfection for 5 to 10 minutes, shake them 2 to 3 times during the period, pick out 1.5 to 2 mm young embryos, and put them into the infection medium (without Agrobacterium) containing AS, with 50 to 70 young embryos in each tube.

[0206] 2.5 Infection

[0207] Wash the young embryos to be transformed with the infection solution three times until the infection solution is clear, pour out the infection solution and add 1 ml of bacterial solution, gently invert 10 times, and let it stand for 5 to 10 minutes. Take a clean culture dish and put 3 sterilized filter papers. After the infection is completed, invert it several times and quickly pour the bacterial solution on the filter paper. Hold the culture dish and change the direction to evenly distribute the bacterial solution carrying the young embryos on the filter paper.

[0208] 2.6 Co-culture

[0209] When the bacterial liquid can no longer be seen on the top layer of filter paper, use tweezers to pick up the upper layer of filter paper, and stick the side stained with immature embryos on the co-culture medium. Use tweezers to drive out the bubbles between the filter paper and the medium, then use tweezers to pinch a corner of the filter paper and quickly peel it off. Use an embryo peeling knife to transfer the immature embryos remaining on the filter paper to the medium, with the shield side of the immature embryo facing up, and culture in the dark at 22°C for 3 days.

[0210] 2.7 Recovery culture

[0211] After 3 days of co-culture, the embryos were transferred to recovery medium, 30 to 40 embryos per dish, and cultured in the dark at 25°C for 10 to 14 days. The embryos transformed with the P0077 vector alone were recovered for 7 days.

[0212] 2.8 Screening

[0213] After recovery, the young embryos transformed with the P0077 vector were transferred to a screening medium with the corresponding screening pressure of dipropyl ammonium phosphate, cultured in the dark at 25-28°C for 14 days, and then transferred to a screening medium with a higher screening pressure and cultured in the dark at 25-28°C for 14 days.

[0214] The immature embryos transformed with the four vectors P0195, P0196, P0593, and P0594 do not require the callus screening stage.

[0215] 2.9 Differentiation

[0216] The resistant calli obtained by transferring the P0077 vector were placed on a differentiation medium for differentiation, clipped into small pieces and spread flat on the medium, cultured in the dark at 25-28°C, and the medium was changed every 14 days.

[0217] The immature embryos transformed with P0195, P0196, P0593, and P0594 vectors were directly differentiated in differentiation medium.

[0218] 2.10 Rooting

[0219] Transfer the differentiated seedlings to the rooting medium, with 3 to 4 seedlings in each bottle, and culture them under light at 25 to 28°C until they grow into complete plants. After 7 days of rooting culture, small white roots will grow and can be sampled for testing.

[0220] 3. Screening of positive seedlings

[0221] For the regenerated seedlings, a small amount of leaves were taken to extract genomic DNA using the CTAB method.

[0222] For plants transformed with the P0077 vector, three pairs of primers, namely, ZmCas9-jc-F2 (TCACCGACGAGTACAAGGTC (SEQ ID NO.: 16)) and ZmCas9-jc-R1 (GTCTGGACGAGCTGGATGAA (SEQ ID NO.: 17)), ZmCas9-jc-F5 (TTCAAGGAGGACATCCAGAAG (SEQ ID NO.: 18)) and ZmCas9-jc-R5 (TCTCGTCGTACTTGGTGTTCAT (SEQ ID NO.: 19)), 35S-F2 (TCATTTGGAGAGGACACGCT (SEQ ID NO.: 20)) and sp110 (GGAGAAACTCGAGTCAAATCTCG (SEQ ID NO.: 21)), were used for detection respectively. If any pair of primers can amplify the target product, the regenerated seedling is a transgenic positive seedling.

[0223] For the transgenic plants of P0195 and P0196, three pairs of primers, Cas9-jc-F1 (AAGAAGCGGAAGGTCGGTAT (SEQ ID NO.: 22)) and Cas9-jc-R1 (CTCAGGTGGTAGATGGTGGG (SEQ ID NO.: 23)), Cas9-jc-F3 (CAGAAAGAGCGAGGAAACCA (SEQ ID NO.: 24)) and Cas9-jc-R3 (CCTCAAACAGTGTCAGGGTCA (SEQ ID NO.: 25)), SIE1-jc-F1 (CCCGAGGATAATGAGCAGAA (SEQ ID NO.: 26)) and NOS-jc-R1 (CCGATCTAGTAACATAGATGACA (SEQ ID NO.: 27)), were used for detection respectively. If any pair of primers can amplify the target product, the regenerated seedling is a transgenic positive seedling.

[0224] For the plants transfected with P0593, three pairs of primers, Cas9-jc-F1 (AAGAAGCGGAAGGTCGGTAT (SEQ ID NO.: 28)) and Cas9-jc-R1 (CTCAGGTGGTAGATGGTGGG (SEQ ID NO.: 29)), Cas9-jc-F3 (CAGAAAGAGCGAGGAAACCA (SEQ ID NO.: 30)) and Cas9-jc-R3 (CCTCAAACAGTGTCAGGGTCA (SEQ ID NO.: 31)), 35S-F2 (TCATTTGGAGAGGACACGCT (SEQ ID NO.: 32)) and sp110 (GGAGAAACTCGAGTCAAATCTCG (SEQ ID NO.: 33)), were used for detection respectively. If any pair of primers can amplify the target product, the regenerated seedling is a transgenic positive seedling.

[0225] 4. Genetic testing

[0226] For transgenic positive seedlings, primer pairs ZmBADH2a-73F (GAGACGTCCTCGCTTTCCAC (SEQ ID NO.: 34)) and ZmBADH2a-904R (ATGTGCACGCTGCGTTTTAC (SEQ ID NO.: 35)) were used to amplify the corresponding fragment of the ZmBADH2a gene, and primer pairs BADH2b-jc-F1 (GAAGTCCACTGCCGAGTTGC (SEQ ID NO.: 36)) and BADH2b-jc-R1 (CGACTGAGTTGTCTCACACTGA (SEQ ID NO.: 37)) were used to amplify the corresponding fragment of the ZmBADH2b gene. The amplified products were sequenced by the Sanger method to confirm the editing form. If the sequencing results showed double peaks, the PCR product was connected to the T vector, and 5 clones were selected for sequencing to confirm the editing form.

[0227] 5. Experimental results

[0228] The young embryos of Zheng 58 were transformed with P0077 to obtain the edited plant C43. The editing result was that an A was inserted after the +24nt of the CDS of ZmBADH2a, resulting in a frameshift mutation in the subsequent coding region and a stop codon TGA formed at 265-267nt of the new CDS, which would lead to the early termination of protein translation; a base G was deleted at the +27nt of the CDS of ZmBADH2b, resulting in a frameshift mutation, and a stop codon TAA formed at 262-264nt of the new CDS, which would lead to the early termination of protein translation. For specific editing methods, see Figure 1 .

[0229] P0077 was used to transform the young embryos of Zheng 58 to obtain the edited plant P64. The editing of the edited plant P64 resulted in the insertion of a C after the +24nt of the CDS of ZmBADH2a, which caused a frameshift mutation in the subsequent coding region ( Figure 2 ), and a termination codon TGA is formed at 265-267nt of the new CDS, which will lead to premature termination of protein translation; while the ZmBADH2b gene in the P64 plant did not undergo editing. For specific editing methods, see Figure 2 .

[0230] In summary, the C43 plant was edited in both ZmBADH2a and ZmBADH2b genes, while the P64 plant was edited only in ZmBADH2a. The seed extracts of C43 and P64 plants were analyzed by mass spectrometry for the content of the aroma substance 2AP; Figure 3As shown, the content of 2AP in the seeds of C43 plants was 0.648 mg / kg, while no 2AP was detected in the seeds of Zheng 58 wild type and P64 plants ( Figure 3 ).

[0231] P0593 was used to transform the young embryos of waxy corn inbred line XCW175 to obtain the edited plant P302. The edited plant was edited at two positions in ZmBADH2a and ZmBADH2b, and the editing results were a C inserted after +77nt and an A inserted after +188nt of the CDS of ZmBADH2a. The protein deduced from the edited CDS began to undergo a frameshift mutation at the 27th amino acid, and a stop codon TGA was formed at 262-264nt of the new CDS, which would lead to the early termination of protein translation; AA was inserted at +80nt of the CDS of ZmBADH2b and an A was deleted at +191nt. The protein deduced from the edited CDS began to undergo a frameshift mutation at the 27th amino acid, and a stop codon TAA was formed at 265-267nt of the new CDS, which would lead to the early termination of protein translation. For specific editing methods, see Figure 4 .

[0232] The content of 2AP, an aromatic substance, in the seed extract of P302 was 0.266 mg / kg by mass spectrometry analysis. However, no 2AP was detected in the wild type of XCW175 ( Figure 5 ).

[0233] In addition, P0196 was used to transform the young embryos of the waxy corn inbred line N355 to obtain the edited plant 282, which was self-pollinated and three edited plants were identified in its offspring plants, namely 288-4, 288-5 and 288-6. These three plants were homozygous edited in ZmBADH2a and ZmBADH2b, and the editing method was the same. The editing method was: an A was inserted after the +24nt of the CDS of ZmBADH2a, resulting in a frameshift mutation in the subsequent coding region, and a stop codon TGA was formed at 265-267nt of the new CDS, which would lead to the premature termination of protein translation; a base G was deleted at the +27nt of the CDS of ZmBADH2b, resulting in a frameshift mutation, and a stop codon TAA was formed at 262-264nt of the new CDS, which would lead to the premature termination of protein translation. Three plants were self-pollinated, and immature grains of waxy corn were taken 27 days after pollination. The content of 2AP aroma substance was analyzed by mass spectrometry. The content of 2AP in the three plants was 0.774 mg / kg, 0.826 mg / kg, and 0.503 mg / kg respectively. No 2AP was detected in the grains of the control N355 inbred line at the same time.

[0234] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Shandong Shunfeng Biotechnology Co., Ltd. <120> A method for enhancing the fragrance of plants <130> P2020-0635 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 505 <212> PRT <213> Corn (Zea mays) <400> 1 Met Ala Ser Pro Ala Met Val Pro Leu Arg Gln Leu Phe Val Asp Gly 1 5 10 15 Glu Trp Arg Pro Pro Ala Gln Gly Arg Arg Leu Pro Val Val Asn Pro 20 25 30 Thr Thr Glu Ala His Ile Gly Glu Ile Pro Ala Gly Thr Ala Glu Asp 35 40 45 Val Asp Ala Ala Val Ala Ala Ala Arg Ala Ala Leu Lys Arg Asn Arg 50 55 60 Gly Arg Asp Trp Ala Arg Ala Pro Gly Ala Val Arg Ala Lys Tyr Leu 65 70 75 80 Arg Ala Ile Ala Ala Lys Val Ile Glu Arg Lys Pro Glu Leu Ala Lys 85 90 95 Leu Glu Ala Leu Asp Cys Gly Lys Pro Tyr Asp Glu Ala Ala Trp Asp 100 105 110 Met Asp Asp Val Ala Gly Cys Phe Glu Tyr Phe Ala Asp Gln Ala Glu 115 120 125 Ala Leu Asp Lys Arg Gln Asn Ser Pro Val Ser Leu Pro Met Glu Thr 130 135 140 Phe Lys Cys His Leu Arg Arg Glu Pro Ile Gly Val Val Gly Leu Ile 145 150 155 160 Thr Pro Trp Asn Tyr Pro Leu Leu Met Ala Thr Trp Lys Ile Ala Pro 165 170 175 Ala Leu Ala Ala Gly Cys Thr Ala Val Leu Lys Pro Ser Glu Leu Ala 180 185 190 Ser Val Thr Cys Leu Glu Leu Ala Asp Ile Cys Lys Glu Val Gly Leu 195 200 205 Pro Ser Gly Val Leu Asn Ile Val Thr Gly Leu Gly Pro Asp Ala Gly 210 215 220 Ala Pro Leu Ser Ala His Pro Asp Val Asp Lys Val Ala Phe Thr Gly 225 230 235 240 Ser Phe Glu Thr Gly Lys Lys Ile Met Ala Ser Ala Ala Pro Met Val 245 250 255 Lys Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Ile Val Val Phe 260 265 270 Asp Asp Val Asp Ile Asp Lys Ala Val Glu Trp Thr Leu Phe Gly Cys 275 280 285 Phe Trp Thr Asn Gly Gln Ile Cys Ser Ala Thr Ser Arg Leu Leu Ile 290 295 300 His Thr Lys Ile Ala Lys Lys Phe Asn Glu Arg Met Val Ala Trp Ala 305 310 315 320 Lys Asn Ile Lys Val Ser Asp Pro Leu Glu Glu Gly Cys Arg Leu Gly 325 330 335 Pro Val Val Ser Glu Gly Gln Tyr Glu Lys Ile Lys Lys Phe Ile Ser 340 345 350 Asn Ala Lys Ser Gln Gly Ala Thr Ile Leu Thr Gly Gly Val Arg Pro 355 360 365 Ala His Leu Glu Lys Gly Phe Phe Ile Glu Pro Thr Ile Ile Thr Asp 370 375 380 Ile Thr Thr Ser Met Glu Ile Trp Arg Glu Glu Val Phe Gly Pro Val 385 390 395 400 Leu Cys Val Lys Glu Phe Ser Thr Glu Asp Glu Ala Ile Glu Leu Ala 405 410 415 Asn Asp Thr Gln Tyr Gly Leu Ala Gly Ala Val Ile Ser Gly Asp Arg 420 425 430 Glu Arg Cys Gln Arg Leu Ser Glu Glu Ile Asp Ala Gly Cys Ile Trp 435 440 445 Val Asn Cys Ser Gln Pro Cys Phe Cys Gln Ala Pro Trp Gly Gly Asn 450 455 460 Lys Arg Ser Gly Phe Gly Arg Glu Leu Gly Glu Gly Gly Ile Asp Asn 465 470 475 480 Tyr Leu Ser Val Lys Gln Val Thr Glu Tyr Ile Ser Asp Glu Pro Trp 485 490 495 Gly Trp Tyr Gln Ser Pro Ser Lys Leu 500 505 <210> 2 <211> 506 <212> PRT <213> Zea mays <400> 2 Met Met Ala Ser Gln Ala Met Val Pro Leu Arg Gln Leu Phe Val Asp 1 5 10 15 Gly Glu Trp Arg Pro Pro Ala Gln Gly Arg Arg Leu Pro Val Val Asn 20 25 30 Pro Thr Thr Glu Ala His Ile Gly Glu Ile Pro Ala Gly Thr Ala Glu 35 40 45 Asp Val Asp Ala Ala Val Ala Ala Ala Arg Ala Ala Leu Lys Arg Asn 50 55 60 Arg Gly Arg Asp Trp Ala Arg Ala Pro Gly Ala Val Arg Ala Lys Tyr 65 70 75 80 Leu Arg Ala Ile Ala Ala Lys Val Ile Glu Arg Lys Gln Glu Leu Ala 85 90 95 Lys Leu Glu Ala Leu Asp Cys Gly Lys Pro Tyr Asp Glu Ala Ala Trp 100 105 110 Asp Met Asp Asp Val Ala Gly Cys Phe Glu Tyr Phe Ala Asp Gln Ala 115 120 125 Glu Ala Leu Asp Lys Arg Gln Asn Ser Pro Val Ser Leu Pro Met Glu 130 135 140 Thr Phe Lys Cys His Leu Arg Arg Glu Pro Ile Gly Val Val Gly Leu 145 150 155 160 Ile Thr Pro Trp Asn Tyr Pro Leu Leu Met Ala Thr Trp Lys Val Ala 165 170 175 Pro Ala Leu Ala Ala Gly Cys Ala Ala Val Leu Lys Pro Ser Glu Leu 180 185 190 Ala Ser Val Thr Cys Leu Glu Leu Ala Asp Ile Cys Lys Glu Val Gly 195 200 205 Leu Pro Pro Gly Val Leu Asn Ile Val Thr Gly Leu Gly Pro Asp Ala 210 215 220 Gly Ala Pro Leu Ser Ala His Pro Asp Val Asp Lys Val Ala Phe Thr 225 230 235 240 Gly Ser Phe Glu Thr Gly Lys Lys Ile Met Ala Ala Ala Ala Pro Met 245 250 255 Val Lys Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Ile Val Val 260 265 270 Phe Asp Asp Val Asp Ile Asp Lys Ala Val Glu Trp Thr Leu Phe Gly 275 280 285 Cys Phe Trp Thr Asn Gly Gln Ile Cys Ser Ala Thr Ser Arg Leu Leu 290 295 300 Val His Thr Lys Ile Ala Lys Glu Phe Asn Glu Lys Met Val Ala Trp 305 310 315 320 Ala Lys Asn Ile Lys Val Ser Asp Pro Leu Glu Glu Gly Cys Arg Leu 325 330 335 Gly Pro Val Val Ser Glu Gly Gln Tyr Glu Lys Ile Lys Lys Phe Ile 340 345 350 Leu Asn Ala Lys Ser Glu Gly Ala Thr Ile Leu Thr Gly Gly Val Arg 355 360 365 Pro Ala His Leu Glu Lys Gly Phe Phe Ile Glu Pro Thr Ile Ile Thr 370 375 380 Asp Ile Thr Thr Ser Met Glu Ile Trp Arg Glu Glu Val Phe Gly Pro 385 390 395 400 Val Leu Cys Val Lys Glu Phe Ser Thr Glu Asp Glu Ala Ile Glu Leu 405 410 415 Ala Asn Asp Thr Gln Tyr Gly Leu Ala Gly Ala Val Ile Ser Gly Asp 420 425 430 Arg Glu Arg Cys Gln Arg Leu Ser Glu Glu Ile Asp Ala Gly Ile Ile 435 440 445 Trp Val Asn Cys Ser Gln Pro Cys Phe Cys Gln Ala Pro Trp Gly Gly 450 455 460 Asn Lys Arg Ser Gly Phe Gly Arg Glu Leu Gly Glu Gly Gly Ile Asp 465 470 475 480 Asn Tyr Leu Ser Val Lys Gln Val Thr Glu Tyr Ile Ser Asp Glu Pro 485 490 495 Trp Gly Trp Tyr Arg Ser Pro Ser Lys Leu 500 505 <210> 3 <211> 5933 <212> DNA <213> Zea mays <400> 3 aaccgccacg tgctgaaccg cctgccgcac cgcactagcg tccgcaacag caggagacgt 60 cctcgctttc cacctgcaca gagcgccagg gccgtagcgt gccgatcgct cgatcccacc 120 aactgaggcc actgtctagt tgccgactct cctcgcaggc cgcaatctcg cagacacgca 180 ccgtcgcggt atccgtatcg ccagagtcct cacggatggc ctcgccagcg atggtcccgc 240 tgcggcagct cttcgtcgac ggcgagtggc gcccgcccgc gcagggccgc cgcctccccg 300 tcgtcaaccc caccaccgag gctcacatcg gtgagtaagc tccccccgatc actgtcgcct 360 tctattatta tacttacta gctttttact tggcctggcg ctgatgcgct gggtttcgcg 420 tgcgcgcgca ggcgagatcc ctgcgggcac agcggaggac gtggacgccg cggtggcggc 480 ggcgcgggcg gcgctcaaga ggaaccgtgg ccgcgactgg gcgcgcgccc cggggccgt 540 ccgggccaag tacctccgcg ctatcgccgc caaggtacgc tggtcctctt tgctctagcc 600 taattgggcc agaaaccact cctgtgctgt gcctctgctt ccaatggttg gtacctacta 660 gcccctaagc tagtaagtta ttcagagtgg attgaatgtg tgacaactaa gtaggggtg 720 tttgggagtg aagtttttc aaagttttag aagaatactg cagatttctc aaaaaactata 780 gtattatata ctaaaaaggt gtttggcaag ccagctaaaa tctctgtttt caaaactaaa 840 gtattacaaa tactgcagct cttttgaagt attctaaact tatgtttata cctcagtttt 900 ctctgtaaaa cgcagcgtgc acatctctcc cacgtgcaaa aaaatacttt gcttccaaac 960 acctctatgt attcaatact gtggtattct aaaactgtag tattttaaac tgtggtattg 1020 tcatgactaa aatataccgt agtatttcaa aaaccgtggt ttccaaaaac tttgttccca 1080 aacaggcccg tacttgtgaa atatacgttg gtttccgtac actccattac tgtttctgcg 1140 ttacatgagc agaggcacag aaagtacaaa ttaaactcct ccaattgtaa accaatctat 1200 tcagggaaa tccttatgtt ctgcttttgg tgtgaacaaa ctattgcact tcccatcaat 1260 acagggtgtg ttagctttat tatcaactcc aatttcaaaa cacctcaaaa gatctttctc 1320 tttaaacttc aattgagaaa gtcttgtaac atttggctgg gccatcaaag ttttgttcaa 1380 atatattcac atatgtcaca ttttgtgtat aagaatgtct tcttattcag gtgattgaga 1440 ggaaacctga gctggctaag ctagaggcac ttgattgtgg gaagccttat gatgaagcag 1500 catgggacat ggtatgtgac accttatgga agtgcaattt attttcacta gcttgcaatt 1560 acaatctgtt tggtattcca tttatcagga tgatgttgct gggtgctttg agtactttgc 1620 ggatcaggca gaagccttgg ataaaaggca aaattcccca gtttctcttc caatggaaac 1680 ttttaaatgt catctccgaa gagagcctat tggggtagtt gggctgataa ctccttggta 1740 ttttcatttc cccctcactt agctgttact tgtatttatg accaaaactc acaggcgagt 1800 gccttattca gtaatacaac ttcatttctc atagatctca gggttgtata aatgtatagc 1860 gaataaactg catattctct aaccacacac agtcttttaa acctaatttc gataactttc 1920 ctcatcacca ttctgaaata tttccagtat tcagatttgt acttcctatc cagtgttgaa 1980 tctcacctat atatccaagt taatgtcaat tttttttgct tgtgtcaatg tttggccaat 2040 atcaagtatt ctttctatgt tgatatgaca gtgagctttg tcctagtata agtgtacgct 2100 tcttttctag aaactcacta ttgtgttact atctttgttc acataattgt tgtgcgggcc 2160 atttcagact attatgtcta tatataagat gctgttgact aagtaattat ttacttatat 2220 ttatgttttc ttcatatgtt cttgttttac ttatctgat tctgactcta aagcattata 2280 ctttgtactt gtatttgtgt tgcaggaact atcctctact gatggctaca tggaagatag 2340 cccctgcatt ggctgctggt tgtacagctg tgctgaagcc atctgaactg gcttctgtgt 2400 aagtattagc atgttatatc atgttcatgc tacaagcaac agcattttat gtgcctacgt 2460 tttgaattac caggacttgc ttagagcttg ctgatatctg taaagaagtc ggtctccctt 2520 ctggtgtctt gaacattgtg acaggattag gtcctgatgc tggcgctcct ttgtcagcac 2580 acccagatgt tgacaaggta aactattctg catataatga cataatcatg catgagcact 2640 cctcatagca tgggttttct gtttatttag gtcgctttta ctgggagttt tgaaactggc 2700 aagaaaatta tggcatcggc agctcctatg gtcaaggttc gtctgtgaat tatgttttat 2760 ttatagtaat gtgctgtgcc gtgttgtttt gctctcttct agttgataaa ttatcagaaa 2820 gtatgatttg agtgccctat tttaagagga aattctgttg cattccttaa cattgcattt 2880 ttcttgacag cctgttacac tggaacttgg tggaaaaagt cctatagtag tatttgatga 2940 tgttgacatt gacaaaggta cgtttatatt ctaaggatta caaaattcct gaagttcatt 3000 ggtatttgtg gattgttgca atttgcagag aatttaactg atgagaatgc taaatgccta 3060 aacccttcaa agcccttcat cggtttctgg gattttcata gaaatactca gctgagcctg 3120 atcaggctac tacaataaga attatttgg tttgaaaatt gattgttagg atttgtaaaa 3180 tacagtccat aaagttgtgg ggaaaaccca catgcttgct atagtttgga cttttgacca 3240 cagcaccaga ctttttatta gctgcatttt gcacaaaatt tagagatgca gcatgccagt 3300 tgaataaaaa gatgaaacag aaagtatttg atgcattatg tatatgttac atgatttgat 3360 ttcatcatag accttgagaa atataggct gaacttcttt gtagaacata agttttagtt 3420 ccaagaaagg gaacttttac aaattcactg accaacttac tttagttatg ctgatacttt 3480 gctcattgtg gaattatgga ttatgctata cttccatcat tgatcattcg ttctaaatat 3540 aaaacagctg tcgagtggac tctgtttggg tgcttttgga ccaatggtca gatttgcagc 3600 gcaacatctc gtttcttat ccatgtaagt ttcatatgtc attgccagta gcagttggga 3660 tacttttcac acataatagc tgtcaaccat aatatatttc cttgagtggt gttgctttag 3720 ctttaccccg ccgtgccgtt aacttctgat tcgagaaggc taccatttca aaatcgctgt 3780 actgaatata tagcagtata ctatgaggtt tacacctata ttgaacaata tatgtgcccc 3840 attgcctaag gttcacatc ttcaatttgt tgtgaactgt gcactttggt ttggtgtata 3900 tttagaagta tatattctca atgacttgat tgtttttact ccctctgata ccaaatgcaa 3960 gtagtttagg acatagtcct gatctccaat gtgacactgt ttgaccgact tgaaaaaaaa 4020 aaacctaac aattgggatt ggaggaata ggttaacaat ggctatttac tgctatatat 4080 tactctttca aatcagatct caattatgaa tcagttatgt tatgtactta tgttattgtt 4140 gtcagtaaca cattagcaccaa gttctgcctc caatgaaacc gttgcagaca aaaattgcta 4200 aaaaatttaa tgagaggatg gttgcatggg ccaaaaatat taaggtttcg gatccacttg 4260 aagagggtttg caggcttggg ccagttgtta gtgaaggaca ggtactacat gtcaactttg 4320 tctaaatgta aattcaccat gccaatgcta tgatttgtag cttctttctt atttgacaat 4380 ttggtacttc tgatatagta gctgtaaact ttgtctaaat gtaaattcac catgcgaatg 4440 ctataatttg tagcttcttt ctgatttgac aatttggtac ttctgattta gtagttgtac 4500 actcttgttt ttgctagttt ctacaggatg tagcttcctc tgattttgag aggatctgtg 4560 ttcagatgca ctgaccaaca ccacctttag atttactttg ttatatttta gtatgttttt 4620 acagcaagag ctatggtatg gattaatgtt tatggtcata ctggattatg agaaaatgac 4680 accctaatca ccaccatatt ggtaaccata cacatagtttt cagattacct tgtttcatct 4740 atagttgttc ataggagcct tatatatatt tttgtgttct cagtatgaga agattaagaa 4800 gttcatatcg aatgccaaaa gccaaggtgc tactattctg actggaggtg ttagacctgc 4860 ggtaaggcct gcatttggat ttacaaagat ccacatgcta aaagcatgct atagagagat 4920 agaactaatg tttcatgttt tacagcatct tgagaagggg ttctttattg aaccaactat 4980 tattactgat atcaccacat caatggaaat ctggagggag gaagtctttg gtccagtcct 5040 gtgcgttaaa gaatttagca ctgaagatga agccatcgaa cttgccaacg atacacagtg 5100 agctgattct ttttagcgca gtttcgatgt cttttttgca ggcttcacag gtcatgtgta 5160 gtccatagct tatgaattta tgccagtatc cttcttttga atttttgata ggtatggttt 5220 ggctggtgct gtaatttctg gtgatcggga gcgctgccag aggttatctg aggtacgtat 5280 aagtgaagag gtccacagta tttggttggc aaattgactg catcagactg atactcagag 5340 tgcacttttg tgtgtgtgtg tgtgtgtatg tggggtgggg ggggataagg ggtcaccatt 5400 atagctctgt gcttctgtcc atgctatcac tcgtttgaag ggttcaattt ggtacttttg 5460 ataccatacc actcaattcg attttcgtgt tgcaacatgg aattcgtggt gcacttgctc 5520 tcagaactgg ttttgggtat ttctactaca catctctaat gaaacttgcg caacaggaga 5580 ttgacgctgg atgcatctgg gtaaactgct cgcaaccctg cttctgccaa gctccctggg 5640 gcgggaacaa gcgcagtgga tttggacgtg agcttggaga agggtgggta acatggaacg 5700 acaacatttc taacagatat tgctatggga tctgaatgac actggctgtt tatctgtagg 5760 ggcattgata actacctgag cgtcaagcaa gtcacggagt acatctctga tgagccgtgg 5820 ggatggtacc aatccccctc caagctgtaa actggcaaga cgaaaatttg tctgtttcgg 5880 tagaataaat atatctcgat gctatacaga acgaacccgt gtatcacatt gaa 5933 <210> 4 <211> 5807 <212> DNA <213> Zea mays <400> 4 atctgtgtag tatttccgtt aagcctgaat ggagtcgcgg acagcgtgcc gataaagcgt 60 tccagttata tctctatctg tatagcctac actacacagg gccgcagctt gccgatccct 120 agcgacgaag tccactgccg agttgccgac tcaccgactc ccctcgcaag tcgcaatctc 180 cactctccag agtccaaact ccactcctca ccgatgatgg cctcgcaagc gatggtaccg 240 ctgcggcagc tctttgtcga cggcgagtgg cgcccgcccg cgcagggccg ccgcctcccc 300 gtcgtcaacc ctaccactga ggctcacatc ggtgagtgac cgccccccga tcgcaatcat 360 ccctgttttc ttctattata cttgttgctt ggcctggcgc tgatgcgcta ggtctcgcgt 420 gcacgcgcag gcgagatccc ggcgggcacg gcggaggacg tggacgccgc ggtggcggcg 480 gcgcgcgcgg cgctcaagag gaaccgcggc cgcgattggg cgcgcgcc gggggccgtt 540 cgggccaagt acctccgcgc catcgccgcc aaggtacgct ggtcctcttt gctctagcta 600. atttaattgg gtcaaaaacg actccagtgc tgtgcttctg cttccaagca ttggtatcta ctccctccgc cccaata atgtacatct cgcttaggaa tactaccata attcggatgt gaattcagtg tgagacaact cagtcgcggc atctattttt tgtgctttga tgggtatgat 780 tggtgactgg atctgtactt ggggatgcta ggagtgaact tctcagcacc tttgctatat 840 gcgttttcct cacttcaa cactgttact gttttacatg cacagaggca cagaaagtac aaattaagtt ccttctattc aggagaagtc cttatgttcc gcttttgttg tgaacaagct attgctttct gccaatacag ggtgttttga cctctagtat caactccaaa ttcaaaacac cttaaagat ctttctcttt aaacttccat ttgtaaagtc ttgtaacatt tggttgggtc atcaaagttt tgttgaaata tatttacaca tgtcacattt tgcatgtaaa aatgtcttct tattcaggta attgagga aacaagagct agctaagcta gaggcacttg attgtgggaa gccttatgat gaagctgcat gggacatggt atgtgacaca ccttatggaa gtgtgcaatt 1260 tttttttcca gtttgcaatg acaatctgtc tggtattcca tttatcagga tgatgttgct 1320 gggtgctttg agtacttcgc agatcaggca gaagctttgg acaaaagaca aaattcccca 1380 gtttctcttc cgatggaaac ttttaaatgc catctccgaa gagagcctat tggggtagtt 1440 gggctgataa ctccttggta ttttcatacc tacctcacct tgctgttatt cgtatttatg 1500 atcaaagctc acaggcaagc gacctgtatt cagtagtaca gcttcgtttc ctgtaagacc 1560 tcagagttct atagcaaaaa aatctgcata ttctctaacc acacaatttt cttaaactat 1620 tttttgacaa ctttcctcat catcgtcctg aaatatttcc agtgttcata tttgtacttc 1680 ctatccagtt tttaatctca cctatgatct atatatccaa gttaatgtca aattatttgc 1740 ttgtgtgaat gtttggccaa tgtcaatatg ctatcttggc acaacgttgt gctttgtccc 1800 agtacaactg ttgcttctgc tccataagct cactattctg ctattatcct ttttcacgta 1860 tttgttgggc cggggcattt cactttattg tgtctatata taaaatgttg ttgactaagt 1920 aactgattta tctatttttt catatgtttt tgttttactt attctgattc taaagtatta 1980 tgctttgtac ttgtatttgt attgcaggaa ctatcctctc ctgatggcta catggaaggt 2040 agctcctgct ctggctgctg gttgtgcagc tgtgctaaag ccatctgaat tggcttctgt 2100 gtaagtatta gcattctata ttctcatgct agaagcaatg gcatttgaag tgcctacctt 2160 ttgaattccc aggacttgct tagagcttgc tgatatctgt aaagaagtcg gtttctccc 2220 cggtgtctta aacattgtga caggattagg tcctgatgct ggtgctcctt tgtcagcaca 2280 cccagatgtt gaagaggtaa actgttctgc atataatgac ataatcatgc atgtgcatga 2340 cacatcaatg aaaacattat ttctcatatc atgtgttttc tgtgtttgta ggtcgctttt 2400 actgggagtt ttgaaactgg taagaagatt atggcggctg cagctcctat ggttaaggtt 2460 tgtctgtgaa tttatgtttt attcaagta atgaactgtg cattattcat tttgctctct 2520 2580 ttgcattctt taacattgca tttttcttga cagcctgtta cactggaact tggtggaaaa 2640 agtcctatag tagtatttga tgatgttgac attgacaaag gtacatgtat attctaaggc 2700 ttactaaatt cctaaatttc attgatattt gtggattgtt gcaaattgca aagaattgaa 2760 ctgatcacaa tgctaaacgc ctaaaactcc tcaaagccct tcttggtatc tgagcttttc 2820 gtagaaatac tcaactgagc aggctactac aacaagaatt gctttggtat tgaaaattga 2880 ttgttaggat ttgtaaaata cagtacataa aattgcgggg gaaactacat gcttgctatg 2940 gttttgacca gagtaccaga ctttgtgttg gttgtctgca tctgacacaa aatttggaga 3000 tgcattatgc cagttgaata aaaagatgaa atcgaaagta ttttatgcat tatgtacatg 3060 ttatatgatt tcatatttca tcattgtttt tgagaaatat agggctgaac ttctgtggag 3120 aacataagct ttagttccaa gaaaggggat tttataaatt tactgattct gacttgcttc 3180 agttatgctg atactttgct caacatggaa ttgtggattc tgatatgctt ccatcgttga 3240 tccttctaaa taacagctgt tgagtggact ctgtttgggt gcttttggac caatggtcag 3300 atttgcagcg caacatctcg tcttcttgtc catgtaagtg ccacatgtta ttgcttgtat 3360 tgtcacttgg aatacttctc acacttaata gttgtcaacc atgatctatt tcttggagtg 3420 gtgtcactta gcttgtcctt gccctgatgt taacttctga taatcttctg cgggagtgat 3480 tttaaaattg cctcacagaa taaatagcag catatatgtg cttacgcct gttttgaata 3540 atagctggt gtgtgtgttg tgtttcctag tgttgttttt tccttacctt tgctacctta 3600 atgaaatgac atgcaactct tgcattgttt gagaaaaaaa aacaatatat gcgttccatt 3660 gcttaaggtc tgggtttgaa ctttgaactc ttcaatttgc gcacttaaaa aaaaagtctt 3720 ctatttgctg tgaagtgtgt gcttccatct ggtgcatatt tagaaatata atctcaatga 3780 cttgattgtt ttttactcat ctgatacgaa acctaagatg tttaggacat tgccacggtc 3840 tccaatgtaa cactttgact ttagttttgt aagttatatt attaaaagg gacacaccta 3900 tatatttat gaaattattt ttcatgatga actagtaaca ttggtcatgc tttactaatc 3960 tatgcaactt gacatatatt gatgagtcaa gaaagaagtt tgaccgactt gaatcgaaaa 4020 atgacctaca attgggacca aaaggagtag gttaaccatg gctatttatc agtctatatt 4080 actatttgtc tcttccaaat cacattcag ttatgaatca gttatgcact atgttattgt 4140 tgtcagtaac atagtaatca agttttgcct tcaatgaac tgttgcagac aaaaattgct 4200 aaagaattta atgagagat gttgcatgg gccaagaata ttaggttc tgatccgctt 4260 gaagaggggtt gcagactttgg gccagttgtt agtgaagggc agttattaca tgttaacttc 4320 atctaaatct aacgtcacca ttgacaatgc tatgatttt gtctcctttc tgatttgaca 4380 gttcggtact tcctatttag ttgctataca ctcttgttct tcttacttc tacgggctac 4440 aggaccagga tgtagctccc gccgattttg agaggatcta tttgatatg cactgactga 4500 caccaccttc tttatac ttgttatat tacagtattt ttgttacaga aagagctatg 4560 gtatagatta atgtttatgg tcatatat aacacaagac tgagaaagtg gcaccctataat 4620 cgccactatc ttggcaacca tatacatagt ttcagattac cttgttgctt tatagttgtt 4680 cacgagtctt attcatgttt tgcgttctc agtatgagaa gattaaaag ttcatattga 4740 atgccaaaag cgaaggtgct actattctga ctggaggtgt tagactgcg gtaggcctg 4800 catttggatt tacatggacc acatgttata gtgagacata actaatgttt tatattttac 4860 agcatcttga gaaggggttc tttattgaac caacaatcat tactgatatc accacatcaa 4920 tggaaatttg gagggaggaa gtcttcggtc cagtcctgtg tgttaaagaa tttagcactg 4980 aagatgaagc cattgaactg gccaatgata cacagtgagc tacttatttt tagtacaact 5040 tcaatgctct tttgcaggct ttgcatctca tgcgtagtca gtagcttaag aattcatgtc 5100 actatccttt cttttatagg tatggtttag ctggtgctgt aatttctggt gatcgtgagc 5160 gctgccagag attatctgag gtatgtttag tgaaggggtt cacagtattt ggctcgtggt 5220 ttgatgactg aatcagactg atactcggag tctctctctc tctctctctc tcttgctctg 5280 tccatgttgc cacattgact tgctctcaga actgtttttg ggtactccta ctagacatct 5340 ctaatgaaaa cttgcgcaac aggagattga tgctggaatt atctgggtaa actgctcaca 5400 gccctgcttc tgccaggctc cctggggcgg taacaagcgt agtggatttg gacgtgagct 5460 tggagaaggg tgggtaacat ggaacgacaa cattttaaga cgtctaacag ctatatttgg 5520 gatctgactg acactgcctg tttacctgta ggggcattga taactacctg agcgtcaagc 5580 aagtcacgga gtacatctct gatgagccgt ggggatggta tcgatccccc tccaagctgt 5640 aaactggcat gaaattttgt ttgttccatt agaataaata tatctcgatg ctatacagaa 5700 cgaacccgtg tatcaaatgg agtatgacat tgtgaaaaag gcacatcagc ggaacaaatg 5760 tcagagttga caattccctc taatgcaaat agttaactcg cccttga 5807 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 cgccagcgat ggtcccgctg 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 agtcgcggcc acggttcctc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 aattaggcta gagcaaagag 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8 ggttgacgac ggggaggcgg 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 gcggcgctca agaggaaccg 20 <210> 10 <211> 57 <212> DNA <213> Artificial sequence <400> 10 taggtctctt gcacgccagc gatggtcccg ctggttttag agctagaaat agcaagt 57 <210> 11 <211> 51 <212> DNA <213> Artificial sequence <400> 11 taggtctcta aacagtcgcg gccacggttc ctctgcacca gccgggaatc g 51 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <400> 12 taggtctctt gcacgccagc gatggtcccg ctggttttag agctagaaat agcaagt 57 <210> 13 <211> 51 <212> DNA <213> Artificial sequence <400> 13 taggtctcta aacaattagg ctagagcaaa gagtgcacca gccgggaatc g 51 <210> 14 <211> 57 <212> DNA <213> Artificial sequence <400> 14 taggtctctt gcaggttgac gacggggagg cgggttttag agctagaaat agcaagt 57 <210> 15 <211> 54 <212> DNA <213> Artificial sequence <400> 15 taggtctcta aacgcggcgc tcaagaggaa ccgtggtgca ccagccggga atcg 54 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 tcaccgacga gtacaaggtc 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 gtctggacga gctggatgaa 20 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <400> 18 ttcaaggagg acatccagaa g 21 <210> 19 <211> twenty two <212> DNA <213> Artificial sequence <400> 19 tctcgtcgta cttggtgttc at 22 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <400> 20 tcatttggag aggacacgct 20 <210> twenty one <211> twenty three <212> DNA <213> Artificial sequence <400> twenty one ggagaaactc gagtcaaatc tcg 23 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two aagaagcgga aggtcggtat 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <400> twenty three ctcaggtggt agatggtggg 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four cagaaagagc gaggaaacca 20 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <400> 25 cctcaaacag tgtcagggtc a 21 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <400> 26 cccgaggata atgagcagaa 20 <210> 27 <211> twenty three <212> DNA <213> Artificial sequence <400> 27 ccgatctagt aacatagatg aca 23 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <400> 28 aagaagcgga aggtcggtat 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <400> 29 ctcaggtggt agatggtggg 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <400> 30 cagaaagagc gaggaaacca 20 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <400> 31 cctcaaacag tgtcagggtc a 21 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <400> 32 tcatttggag aggacacgct 20 <210> 33 <211> twenty three <212> DNA <213> Artificial sequence <400> 33 ggagaaactc gagtcaaatc tcg 23 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <400> 34 gagacgtcct cgctttccac 20 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <400> 35 atgtgcacgc tgcgttttac 20 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <400> 36 gaagtccact gccgagttgc 20 <210> 37 <211> twenty two <212> DNA <213> Artificial sequence <400> 37 cgactgagtt gtctcacact ga 22

Claims

1. A method for enhancing plant fragrance, characterized in that: Includes steps: Reducing or inhibiting the expression amount and / or activity of the BADH2 gene or its encoded protein in the plant, thereby enhancing the fragrance of the plant, wherein the BADH2 gene includes a BADH2a gene and a BADH2b gene; the amino acid sequence of the protein encoded by the BADH2a gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the BADH2b gene is shown in SEQ ID NO.2; The method for enhancing the aroma of the plant comprises increasing the content of 2-acetyl-1-pyrroline (2AP) in the plant; the plant is corn; The reduction or inhibition of the expression level and / or activity of the BADH2 gene or its encoded protein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene, wherein the mutation is selected from any one of the following (i)-(ii): (i) an A is inserted after +24nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and a base G is deleted at +27nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene; (ii) A C is inserted after +77nt and an A is inserted after +188nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and an AA is inserted at +80nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene and an A is deleted at +191nt.

2. A composition for enhancing plant fragrance, characterized in that: include: (a) an inhibitor of BADH2 gene or its encoded protein, which reduces the expression level and / or activity of BADH2 gene or its encoded protein in the plant, wherein the BADH2 gene includes BADH2a gene and BADH2b gene; the amino acid sequence of the protein encoded by the BADH2a gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the BADH2b gene is shown in SEQ ID NO.2; and (b) an agriculturally acceptable carrier; The plant is corn; The reduction or inhibition of the expression level and / or activity of the BADH2 gene or its encoded protein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene, wherein the mutation is selected from any one of the following (i)-(ii): (i) an A is inserted after +24nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and a base G is deleted at +27nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene; (ii) A C is inserted after +77nt and an A is inserted after +188nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and an AA is inserted at +80nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene and an A is deleted at +191nt.

3. Use of the composition according to claim 2, characterized in that: Used to enhance the aroma of plants; the plant is corn.

4. A method for preparing genetically engineered plant tissue or plant cells, characterized in that: Includes steps: Reducing or inhibiting the expression and / or activity of the BADH2 gene or its encoded protein in plant tissues or plant cells, thereby obtaining genetically engineered plant tissues or plant cells; The BADH2 gene includes a BADH2a gene and a BADH2b gene; the amino acid sequence of the protein encoded by the BADH2a gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the BADH2b gene is shown in SEQ ID NO.2; The plant is corn; The reduction or inhibition of the expression level and / or activity of the BADH2 gene or its encoded protein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene, wherein the mutation is selected from any one of the following (i)-(ii): (i) an A is inserted after +24nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and a base G is deleted at +27nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene; (ii) A C is inserted after +77nt and an A is inserted after +188nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and an AA is inserted at +80nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene and an A is deleted at +191nt.

5. A method for preparing a genetically engineered plant, characterized in that: Includes steps: The genetically engineered plant tissue or plant cell prepared by the method of claim 4 is regenerated into a plant body, thereby obtaining a genetically engineered plant; the plant is corn.

6. A method for improving plants, characterized in that: The method comprises the steps of: (a) providing a plant cell, plant tissue, or plant part, and introducing an inhibitor of a BADH2 gene or a protein encoded therein into the plant cell, plant tissue, or plant part to reduce the expression level and / or activity of the BADH2 gene or a protein encoded therein in the plant; wherein the BADH2 gene comprises a BADH2a gene and a BADH2b gene; the amino acid sequence of the protein encoded by the BADH2a gene is shown in SEQ ID NO.1; and the amino acid sequence of the protein encoded by the BADH2b gene is shown in SEQ ID NO.2; (b) regenerating the plant cell, plant tissue or plant part in step (a) into a plant; The method is used to enhance the aroma of a plant; the plant is corn; The reduction or inhibition of the expression level and / or activity of the BADH2 gene or its encoded protein is achieved by simultaneously mutating the BADH2b gene and the BADH2a gene, wherein the mutation is selected from any one of the following (i)-(ii): (i) an A is inserted after +24nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and a base G is deleted at +27nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene; (ii) A C is inserted after +77nt and an A is inserted after +188nt of the CDS (coding region) of the nucleotide sequence of the BADH2a gene, and an AA is inserted at +80nt of the CDS (coding region) of the nucleotide sequence of the BADH2b gene and an A is deleted at +191nt.

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