CN115125259B - Dwarf 20 protein, its encoding gene and application - Google Patents

Abstract

The invention relates to a maize gene 'short 20' and a coded protein thereof. The invention also researches the application of the gene.

Description

Dwarf 20 protein, its encoding gene and application

Technical Field

The present invention relates to a corn gene "Ai 20" and its encoded protein, a method for obtaining "Ai 20" transgenic plants, and the role of the above gene and protein in plant growth and development.

Background technique

Ai 20 encodes an IRE/NPH/phosphoinositide-dependent/ribosomal protein S6 kinase. This gene is specifically expressed in maize anthers. So far, no relevant research reports on this gene in maize have been found.

The Arabidopsis homologous gene AT1G51170.1 encodes an active AGC-VIII protein kinase that interacts with the putative transcription factor ATS, AGC2-3, to regulate planar growth during ovule integument development. Mutants exhibit ectopic growth of filaments and petals, as well as abnormal embryogenesis. Balaji et al. ^[1] have demonstrated that the Arabidopsis AGC VIII protein kinase UNICORN (UCN) maintains planar growth by inhibiting the formation of ectopic multicellular protrusions in several floral tissues, including the integument. UCN encodes an AGC-active kinase that controls this process by directly interacting with ABERRANT TESTA SHAPE (ATS), a member of the KANADI (KAN) family of transcription factors, thereby inhibiting its activity (Balaji et al. ^[2] , 2013).

The rice homologous gene LOC_Os08g39460.1 encodes an AGC kinase. Ana et al. found that AGC kinase plays an important role in regulating plant growth, immunity and cell death, as well as its connection with the stress-induced mitogen-activated protein kinase signaling cascade (Ana et al. ^[3] , 2012). For example, Matsui et al. studied the interaction between AGC kinase OsOxi1 and OsPti1a, and the results showed that the interaction between the two can positively regulate the basic disease resistance of rice. In eukaryotes, AGC kinase family proteins are regulated by 3-phosphoinositide-dependent protein kinase 1 (Pdk1). Therefore, the study showed that OsPdk1 positively regulates the basic disease resistance of rice through the OsOxi1-OsPti1a cascade phosphorylation (Matsui et al. ^[4] , 2010).

It can be inferred that this gene is likely related to the growth and development of plant reproductive organs and is involved in plant stress resistance pathways, such as disease resistance. Further research and confirmation is needed on the specific function of this gene in corn.

Summary of the invention

The following definitions and methods are used to better define the present invention and guide those skilled in the art to implement the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the art.

The invention relates to a corn gene "Dwarf 20", the gene sequence of which is derived from B73, and in the corn genome database, the B73_RefGen_v4 version is coded as Zm00001d031426, and the B73_RefGen_v3 version is coded as GRMZM2G050427.

The "Dwarf 20" gene contains one exon and one transcript, which translates 442 amino acids and encodes a kinase protein with two functional domains: Protein kinase (8-381) and AGC-kinase C-terminal (382-442).

The amino acid sequence of the "Dwarf 20" protein (SEQ ID NO: 1) consists of 442 amino acid residues. The coding region CDS (Coding Sequence) of the "Dwarf 20" gene is shown in the nucleotide sequence SEQ ID NO: 2. The DNA sequence of the "Dwarf 20" gene is shown in SEQ ID NO: 3.

First, in the first aspect, the present invention relates to an isolated nucleic acid molecule, characterized in that the nucleic acid molecule comprises a sequence selected from the following:

1) the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence;

2) a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence shown in SEQ ID NO: 3;

3) a nucleotide sequence that hybridizes to SEQ ID NO: 3 under stringent conditions; or

4) Variants derived from the nucleotide sequence shown in SEQ ID NO: 3 by deletion, substitution, insertion and/or addition of one or more nucleotides.

In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:3; in specific embodiments, the nucleotide sequence of the isolated nucleic acid molecule is shown in SEQ ID NO:3.

In some embodiments, the CDS sequence corresponding to the nucleic acid molecule is shown in SEQ ID NO:2.

In another aspect, the present invention also provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of:

1) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1;

2) a nucleotide sequence encoding an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 1; or

3) A nucleotide sequence encoding a variant derived from the sequence shown in SEQ ID NO: 1 by deletion, substitution, insertion and/or addition of one or more amino acid residues.

On the other hand, the present invention also provides an isolated polypeptide, characterized in that it comprises an amino acid sequence selected from the following:

1) the amino acid sequence shown in SEQ ID NO: 1;

2) an amino acid sequence encoded by the nucleic acid molecule described above;

3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 1; or

4) An amino acid sequence derived from the sequence shown in SEQ ID NO: 1 by deleting, substituting, inserting and/or adding one or more amino acid residues.

In some embodiments, the polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO:1; in specific embodiments, the polypeptide of the present invention has the amino acid sequence shown in SEQ ID NO:1.

In another aspect, the present invention also provides a recombinant vector comprising the isolated nucleic acid molecule defined above.

On the other hand, the present invention also provides a host cell, which comprises the isolated nucleic acid molecule defined above, or comprises the isolated polypeptide defined above, or comprises the recombinant vector defined above.

In another aspect, the present invention also provides a transgenic plant comprising the isolated nucleic acid molecule defined above, or the isolated polypeptide defined above, or the recombinant vector defined above.

In some embodiments, the transgenic plant is a monocot or a dicot; in a specific embodiment, the transgenic plant is preferably a crop plant.

In some specific embodiments, the plant is selected from the group consisting of corn (Zea mays), rapeseed (Brassicanapus), turnip (Brassica rapa), mustard (Brassica juncea), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), yellow rice (Panicum miliaceum), millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), beet (Betavulgaris), sugarcane (Saccharum spp.), oat (Avena sativa), barley (Hordeum vulgare) and Arabidopsis thaliana; in a preferred embodiment, the plant of the present invention is selected from Arabidopsis thaliana or maize.

In some embodiments, the transgenic plants of the present invention have altered traits compared to plants that have not undergone corresponding genetic modification or wild-type plants, wherein the altered traits are selected from yield, plant height, ear length, ear thickness, bald tip length, number of ear rows, number of grains per row, normal number of grains per ear, grain width, grain thickness, grain weight in a plot, grain moisture, 100-grain weight, internode spacing, etc.; for example, increased yield, decreased plant height, increased 100-grain weight, decreased internode spacing, etc.

On the other hand, the present invention also provides a method for producing a transgenic plant with altered traits, the method comprising introducing a nucleic acid molecule as defined above, or a recombinant vector as defined above, or a host cell as defined above into a plant to obtain a corresponding transgenic plant; the transgenic plant has altered traits compared to a plant that has not undergone corresponding transformation or a wild-type plant, wherein the altered traits are selected from yield, plant height, ear length, ear thickness, bald tip length, number of ear rows, number of grains per row, normal number of grains per ear, grain width, grain thickness, grain weight per plot, grain moisture, 100-grain weight, and internode distance; wherein the changes in the traits include, for example, increased yield, decreased plant height, increased 100-grain weight, decreased internode distance, etc.

On the other hand, the present invention also provides a method for regulating plant growth and development or plant stress resistance, the method comprising transforming a plant with the recombinant vector defined above, or transfecting a plant with the host cell defined above; wherein, compared with a plant that has not been transformed/transfected accordingly, or compared with a wild-type plant, the transgenic plant has an increased yield.

On the other hand, the present invention relates to the use of the nucleic acid molecule as defined above, the polypeptide as defined above, or the recombinant vector as defined above in regulating plant growth and development or plant stress resistance.

In some embodiments, the above-mentioned plant or transgenic plant is a monocotyledonous plant or a dicotyledonous plant; in a specific embodiment, the plant is preferably a crop plant.

In a preferred embodiment, the plant is selected from the group consisting of corn (Zea mays), rapeseed (Brassica napus), turnip (Brassica rapa), mustard (Brassica juncea), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), yellow rice (Panicum miliaceum), millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot spp. esculenta), beet (Betavulgaris), sugarcane (Saccharum spp.), oat (Avena sativa), barley (Hordeum vulgare) and Arabidopsis thaliana; more preferably, the plant of the present invention is selected from Arabidopsis thaliana or corn.

As used herein, the term "plant" includes whole plants, transgenic plants, meristems, shoot organs/structures (such as leaves, stems and tubers), roots, flowers and flower organs/structures (such as bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryos, endosperms and seed coats) and fruits (mature ovaries), plant tissues (such as vascular tissues, ground tissues, etc.) and cells (such as guard cells, egg cells, pollen, mesophyll cells, etc.) and their progeny. The plant species that can be used in the present invention are generally as broad as the species of higher or lower plants that can be treated with transformation and breeding techniques, including angiosperms (monocotyledons and dicotyledons), gymnosperms, ferns, horsetails, gymnophytes, lycophytes, mosses and multicellular algae.

As used herein, the term "transgenic" refers to a nucleotide molecule artificially introduced into the genome of a host cell. Such a transgene may be heterologous to the host cell. As used herein, a "transgenic plant" refers to a plant whose genome has been altered by the stable integration of recombinant DNA. Transgenic plants include plants regenerated from initially transformed plant cells and subsequent generation or hybridization of transgenic plants.

As used herein, "control plants" refer to plants that do not contain recombinant DNA that changes a trait. Control plants are used to identify and screen transgenic plants with altered traits. Suitable control plants can be non-transgenic plants of the parental line used to generate the transgenic plant, for example, wild-type plants that do not contain the corresponding recombinant DNA. Suitable control plants can also be transgenic plants that contain recombinant DNA that imparts other traits, for example, transgenic plants with enhanced herbicide tolerance.

As described herein, " traits " are the physiology, morphology, biochemistry or physical characteristics of plant or specific plant material or cell. In some cases, this feature is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques (such as detecting the oil content of protein, starch, metabolite or seed or leaf) or by observing metabolism or physiological processes (such as by measuring the tolerance to water deprivation or specific salt or sugar concentration) or by measuring the expression level of one or more genes (by utilizing Northern Blotting, Western Blotting, RT-PCR, microarray gene expression determination method or reporter gene expression system) or by agricultural observation (such as hyperosmotic stress tolerance and yield). Any technology can be used to measure the amount, comparative level or difference of any selected chemical compound or macromolecule in transgenic plants.

The proterties with special economic benefits are the yield of increase. Yield is usually defined as the measurable generation of the economic value from crops. This can be defined in terms of quantity and/or quality. Yield directly depends on several factors, such as, the number and size of organs, plant structure (such as the number of branches), seed production, leaf senescence, etc. And root development, nutrient uptake, stress tolerance, etc. can also be the important factors determining yield. With regard to crop plants, such as corn, rice, etc., yield means the amount of harvested grain, and particle diameter and grain weight proterties are critical in determining yield. Therefore, increasing particle diameter and grain weight is important for increasing the yield of crops.

As used herein, the term "nucleic acid" refers to any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and its length is not subject to any particular restrictions. For nucleic acids used to construct recombinant constructs, DNA is preferred, which is more stable and easy to handle than RNA.

The term "DNA" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases or a nucleotide molecule, read from the 5' end (upstream) to the 3' end (downstream). The term "nucleotide sequence" refers to the sequence of nucleotides of a DNA or RNA molecule, usually displayed from the 5' (upstream) end to the 3' (downstream) end.

Methods known to those skilled in the art can be used for separation and DNA molecules as herein described or its fragment.For example, PCR (polymerase chain reaction) technology can be used for amplification of specific initial DNA molecules and/or generation of variants of the original molecule.DNA molecules or its fragments can also be obtained by other technologies, such as by chemical method direct synthesis fragments (such as automated oligonucleotide synthesizer).

As described herein, the term "isolated" refers to at least partially separating a molecule from other molecules that are usually associated with it in its natural or natural state. In some embodiments, the term "isolated nucleic acid molecule" or "isolated DNA molecule" refers to a nucleic acid molecule (such as a DNA molecule) that is at least partially separated from the nucleic acid that is usually flanked by a gene or DNA molecule in its natural or natural state. Therefore, nucleic acid molecules that are fused to a regulatory or coding sequence that is usually unrelated to it by recombinant technology are considered to be isolated in this article. Even when integrated into the chromosome of a host cell or present in a nucleic acid solution together with other DNA molecules, the molecule is also considered to be isolated.

As used herein, a "polypeptide" comprises a plurality of consecutive polymerized amino acid residues, such as at least about 15 consecutive polymerized amino acid residues. Typically, a polypeptide comprises a series of polymerized amino acid residues, which is a transcriptional regulator or a domain or part or fragment thereof. In addition, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) an inhibition domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA binding domain; or other parts. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by codons, and non-naturally occurring amino acid residues. As used herein, a "protein" refers to a series of amino acids, oligopeptides, peptides, polypeptides, or parts thereof, whether naturally occurring or synthetic.

The term "isolated polypeptide" refers to a polypeptide, whether naturally occurring or recombinant, that is present in a cell (or outside a cell) at a higher level than the polypeptide in its native state in a wild-type cell, for example, at a level greater than about 5%, greater than about 10%, greater than about 20%, greater than about 50%, or more, which can alternatively be expressed as: 105%, 110%, 120%, 150% or more relative to the wild-type polypeptide normalized to 100%. This is not the result of a natural reaction of a wild-type plant. In addition, the isolated polypeptide is separated from other normally associated cellular components, for example, using various protein purification methods.

As used herein, the term "stringent conditions" are those described by Sambrook et al. (1989) and Haymes et al.: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Appropriate stringent conditions that promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC), about 45°C, followed by washing with 2.0×SSC at 50°C, are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0×SSC at 50°C to a high stringency of about 0.2×SSC at 50°C. In addition, the temperature in the washing step can be increased from a low stringency condition of about 22°C at room temperature to a high stringency condition of about 65°C. Both temperature and salt can be varied, or the temperature or salt concentration remains constant while the other variable is changed. For example, moderately stringent conditions are at about 2.0X SSC and about 65°C.

In one aspect of the invention, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 3. In another aspect of the invention, the isolated nucleic acid molecule of the invention has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% and 99.5% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the isolated nucleic acid molecule of the invention has 95% 96%, 97%, 98%, 98.5%, 99% and 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 3.

The term "percent identity" or "% identity" is a comparison between amino acid sequences or nucleotides, determined by comparing the two sequences of the best pairing over a comparison window. Those skilled in the art know how to calculate the percent identity between two sequences, and there are many tools available for use (e.g., Clustal, Bestfit, Blast, Fasta, etc.). One of the two sequences may have one or more insertions, substitutions, and/or deletions of amino acids or nucleotides relative to the other sequence.

In the vector, the gene of the present invention is usually operably linked to a promoter, a terminator and/or any other sequences necessary for its expression in yeast.

The terms "operably linked" and "operably linked" are used interchangeably and refer to the functional connection between elements that enable gene expression, and optionally, the regulation (5' and 3' regulatory sequences) and sequences of reporter genes controlled by these elements. Those skilled in the art know how to select promoters, terminators and other regulatory sequences required for expressing a gene.

As described herein, the term "promoter" generally refers to a DNA molecule that participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. In some embodiments, the promoter can be initially isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene; or, the promoter can be a synthetically produced or manipulated DNA molecule. In some embodiments, the promoter can also be chimeric, i.e., a promoter produced by the fusion of two or more heterologous DNA molecules. Plant promoters include promoter DNA obtained from plants, plant viruses, fungi and bacteria (such as Agrobacterium). In some embodiments, the promoter is a developmentally regulated, organelle-specific, tissue-specific, inducible, constitutive or cell-specific promoter. In some specific embodiments, the expression of a gene is controlled by a so-called "strong" promoter (i.e., a promoter with high transcriptional potential so that the gene is strongly expressed).

The isolated nucleic acid molecules of the present invention also include variant sequences derived from the sequence shown in SEQ ID NO: 3 by deletion, substitution, insertion and/or addition of one or more nucleotides.

As mentioned above, "insertion", "deletion", "substitution" or "addition" of one or more nucleotides or amino acids, the insertion, deletion, substitution and/or addition here will not damage the function of the original sequence (for example, in this article it means that the function of regulating plant growth and development or regulating plant stress resistance is still retained). Those skilled in the art are aware of methods for completing the insertion, deletion and/or substitution of one or more nucleotides/amino acids in the original sequence while retaining the biological function of the original sequence. For example, such insertion, deletion, addition, substitution and/or addition are selected in non-conserved regions; or based on the degeneracy of the genetic code, nucleotides are modified by "silent variation" without changing the polypeptide encoded by the nucleotide; or, an amino acid in a protein is replaced by another amino acid with similar properties by "conservative substitution" without affecting the biological function of the protein.

Conservative substitutions can occur within the following groups: 1) acidic (negatively charged) amino acids, such as aspartic acid and glutamic acid; 2) basic (positively charged) amino acids, such as arginine, histidine and lysine; 3) neutral polar amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; and 4) neutral nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Conservative substitutions for amino acids within natural proteins or polypeptides can be selected from other members of the group to which the naturally occurring amino acids belong. For example, the amino acid group with aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; the amino acid group with aliphatic-hydroxyl side chains is serine and threonine; the amino acid group with amide-containing side chains is asparagine and glutamine; the amino acid group with aromatic side chains is phenylalanine, tyrosine and tryptophan; the amino acid group with basic side chains is lysine, arginine and histidine; and the amino acid group with sulfur-containing side chains is cysteine and methionine. Natural conservative amino acid substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, aspartic acid-glutamic acid and asparagine-glutamine.

"Mutation" as described herein refers to the sudden and heritable variation of genomic DNA molecules. At the molecular level, gene mutation means the change of the arrangement sequence of base pair composition or structure. The generation of gene mutation can be spontaneous or induced. Methods for artificially inducing gene mutations include physical factors (such as gamma rays, x-rays, UV, neutron beams, etc.), chemical factors (such as alkylating agents, base analogs, antibiotics, etc.) and biological factors (such as certain viruses, bacteria, etc.). In addition, recombinant DNA technology can be used to introduce specific mutations at designated sites in DNA molecules in order to perform site-directed mutagenesis. Those skilled in the art can use any of these well-known mutagenesis methods to obtain variant sequences of the sequence shown in SEQ ID NO:3 comprising the deletion, substitution, insertion and/or addition of one or more nucleotides.

As described herein, the term "recombination" refers to the form of DNA and/or protein and/or organism that is usually not present in nature and is therefore generated by human intervention. This human intervention can produce recombinant DNA molecules and/or recombinant plants. As used herein, "recombinant DNA molecules" are DNA molecules comprising a combination of DNA molecules that are non-natural and exist together and are the result of human intervention, for example, DNA molecules comprising the following combination: at least two DNA molecules that are heterologous to each other, and/or artificially synthesized and comprising a DNA molecule of a polynucleotide sequence derived from a polynucleotide sequence that usually exists in nature, and/or comprising a transgenic DNA of the genomic DNA of an artificially introduced host cell and a DNA molecule of the relevant flanking DNA of the host cell genome. The example of a recombinant DNA molecule is derived from inserting a gene into the corn genome described herein, which can ultimately cause recombinant RNA to be transcribed in an organism and/or a DNA molecule that a polypeptide or protein is expressed in the organism.

As used herein, "host cell" refers to a cell that contains a recombinant vector and supports the expression vector for replication and/or expression. The host cell can be a prokaryotic cell (e.g., an Escherichia coli cell, an Agrobacterium tumefaciens cell) or a eukaryotic cell (e.g., a yeast, insect, plant, or animal cell).

In some embodiments, the host cell is preferably a monocotyledonous or dicotyledonous plant cell, including but not limited to cells from corn, rapeseed, turnip, mustard, alfalfa, rice, rye, sorghum, pearl millet, yellow rice, millet, finger millet, sunflower, safflower, wheat, soybean, tobacco, potato, peanut, cotton (sea island cotton, upland cotton), sweet potato, cassava, sugar beet, sugar cane, oat, barley and Arabidopsis. Preferably, the host cell is a corn cell or a rice cell; more preferably, the host cell is a corn cell.

As described herein, "introducing" a nucleic acid molecule or an expression vector into a plant or a plant cell means transfecting, transforming, transducing or incorporating the nucleic acid molecule or the recombinant expression vector into a host cell, so that the nucleic acid molecule can autonomously replicate or express in the host cell.

In some embodiments, the introduced nucleic acid molecule is integrated into the host cell genomic DNA (e.g., chromosome, plasmid, plastid or mitochondrial DNA), and the expression of the nucleotide sequence is controlled by a regulatable promoter region. In other embodiments, the introduced nucleic acid molecule is not integrated into the cell genome.

The present invention obtains plants with significant changes in traits such as ear length, ear thickness, bald tip length, number of ear rows, number of grains per row, number of normal grains per ear, grain width, grain thickness, grain weight in a plot, grain moisture, and 100-grain weight by overexpressing the gene "Dwarf 20" in corn; this suggests that the gene may be related to plant growth and development and participate in the plant stress resistance pathway.

A more detailed description of some preferred embodiments of the invention follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: “Ai 20” gene structure and protein functional domains

Figure 2: Schematic diagram of the pBCXUN vector structure

Figure 3: Schematic diagram of gene overexpression vector structure

Figure 4: Expression analysis of Ai 20 in leaves of maize inbred line B73-329 (WT) and T3 transgenic pure lines

Figure 5: Comparison of seedling vigor of transgenic plants before harvest in 2018

Figure 6: Comparison of SPAD (chlorophyll content) values of transgenic plants before harvest in 2018

Figure 7: Comparison of silking days before harvest of transgenic plants in 2018

Figure 8: Comparison of the number of days before the harvest of transgenic plants in 2018

Figure 9: Comparison of plant height data of transgenic plants before harvest in 2018

Figure 10: Comparison of grain weights of transgenic plants in different plots after harvest in 2018

Figure 11: Comparison of ear length data of transgenic plants after harvest in 2018

Figure 12: Comparison of ear width data of transgenic plants after harvest in 2018

Figure 13: Comparison of ear-row numbers of transgenic plants after harvest in 2018

Figure 14: Comparison of the number of grains per row of transgenic plants after harvest in 2018

Figure 15: Comparison of normal grain number per ear of transgenic plants after harvest in 2018

Figure 16: Comparison of grain width of transgenic plants after harvest in 2018

Figure 17: Comparison of grain thickness of transgenic plants after harvest in 2018

Figure 18: Comparison of 100-grain weight of transgenic plants after harvest in 2018

Figure 19: Comparison of silking days before harvest of GM plants in 2020

Figure 20: Comparison of the number of days before the GM plants shed pollen in 2020

Figure 21: Comparison of plant height of GM plants before harvest in 2020

Figure 22: Comparison of ear height of transgenic plants before harvest in 2020

Figure 23: Comparison of internode spacing of transgenic plants before harvest in 2020

Figure 24: Comparison of plant height between control plants and transgenic plants

Figure 25: Comparison of internode distance between control plants and transgenic plants (left is transgenic plant, right is control plant)

Specific embodiment

The following examples describe some specific embodiments of the present invention. However, it should be understood that the examples and drawings are given by way of illustration only and do not limit the scope of the present invention.

pBCXUN: Based on the vector pCXUN (NCBI GenBank: FJ905215), the HYG gene was replaced with Bar through the XhoⅠ site; the Bar gene came from pCAMBIA3301, the promoter for initiating the expression of the target gene in the pCXUN vector was maize Ubiquitin-1, and the terminator was T-nos.

Example 1: Construction of an overexpression vector for the “Dwarf 20” gene

1. Obtaining the "Dwarf 20" gene in corn

According to the manufacturer's instructions, RNA was extracted from anthers of corn variety B73 using a magnetic bead plant total RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd., catalog number AU3402), and then reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific, catalog number 4368814) to create a cDNA template.

Using maize cDNA as a template, primers were designed based on the sequence analysis of the coding region of Ai 20, and the coding region of the gene was amplified using the upstream primer "Ai 20"-F, the downstream primer "Ai 20"-R and a high-fidelity enzyme to obtain the target gene sequence, which was then connected to the pBCXUN vector with Ubiquitin as the promoter. The primer sequences are shown in Table 1:

Table 1 Primers for amplification of “Dwarf 20”

Primer number Primer sequences "Dwarf 20"-F (SEQ ID NO: 4) 5’-GCCAGCAGACATGGACATCGAC-3’ "Dwarf 20"-R (SEQ ID NO: 5) 5’-TAGCCCGAGCTCAGAGTCAGAAC-3’

2. Preparation of overexpression vector

The DNA molecule of the "Dwarf 20" insertion sequence shown in SEQ ID NO: 6 was connected to the pBCXUN vector by TA cloning method to obtain the recombinant expression vector pBCXUN-"Dwarf 20" (sequencing verified), specifically:

The PCR product was purified using a 1% agarose gel and a gel recovery kit, and the purified "Dwarf 20" cDNA sequence was ligated with the vector pBCXUN (NCBI GenBank: FJ905215, see Plant Physiol. 150 (3), 1111-1121 (2009)) digested with XcmI by TA cloning to obtain a recombinant expression vector pBCXUN-"Dwarf 20" (Figure 3). The recombinant expression vector was used to transform Escherichia coli, and positive clones were screened, and the cloning construction was completed in one step.

The obtained plasmid was sent to a sequencing company for sequencing, and the sequencing results were compared with the target sequence to ensure that the vector contained a complete target gene sequence, indicating that the target gene had been transferred into pBCXUN and the cloning construction was completed.

Example 2: Preparation of "Ai 20" overexpression transgenic corn

The recombinant expression vector pBCXUN-"Dwarf 20" was introduced into the Agrobacterium EHA105 strain to obtain the recombinant bacteria. Then, the recombinant bacteria were introduced into the corn inbred line B73-329 through the Agrobacterium-mediated method to obtain T0 generation transgenic plants.

The seedling leaves of the T0 transgenic plants were taken to extract genomic DNA. Using the genomic DNA as a template, PCR amplification was performed using primers Ubip-F (targeting the 5' end of the Ubi1P promoter of the recombinant expression vector pBCXUN-"Dwarf 20") and primer Nos-R (targeting the 3' end of the Nos terminator of the recombinant expression vector pBCXUN-Dwarf 20) and the target gene's own primers "Dwarf 20"-F and "Dwarf 20"-R combination.

The genomic DNA of the seedling leaves of the maize inbred line B73-329 was used as a negative control, and the plasmid of the recombinant expression vector pBCXUN-"Ai 20" was used as a positive control.

Table 2: Primers for identification of “Ai 20” T0 transgenic materials

The PCR amplification products were detected by agarose gel electrophoresis. The results showed that both the transgenic plants and plasmids could amplify a single band of the target gene: the combined amplification fragment size of Ubip-F (SEQ ID NO: 7) and "Dwarf 20"-R (SEQ ID NO: 5) was 1464bp; the combined amplification fragment size of "Dwarf 20"-F (SEQ ID NO: 4) and Nos-R (SEQ ID NO: 8) was 1436bp, while the parent B73-329 did not amplify the corresponding band, indicating that the "Dwarf 20" gene was successfully transferred into the transgenic plant.

The identified T0 transgenic plants were self-pollinated to obtain T1 transgenic plant progeny; the T1 transgenic plant progeny were self-pollinated again to obtain T2 transgenic plant progeny; the T2 transgenic plant progeny were self-pollinated again to obtain T3 transgenic plant progeny; in each generation, the above-mentioned PCR amplification method was used to identify positive transgenic plants, and then self-pollinated again. Five representative T3 homozygous transgenic lines (i.e., Ai20-1, Ai20-2, Ai20-3, Ai20-4, Ai20-5) were selected for subsequent functional analysis experiments.

Example 3: Detection of gene expression level of Ai20

In this example, five representative T3 homozygous transgenic lines (i.e., Ai20-1, Ai20-2, Ai20-3, Ai20-4, Ai20-5) and the maize inbred line B73-329 (WT) were used as test plants.

According to the manufacturer's instructions, the RNA of the V4 leaves of the tested plants was extracted using a magnetic bead plant total RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd., catalog number AU3402), and then the RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific, catalog number 4368814).

The SYBR Premix Ex TaqTM II (Tli RNaseH Plus) kit (Takara, catalog number RR820A) was used, cDNA was used as a template, and GTY-rF (SEQ ID NO: 9: AGTATTGGGGATCCGAATTTC) and GTY-rR (SEQ ID NO: 10: TAATCATAAAAACCCATCTCATAA) were used for real-time fluorescence quantitative PCR amplification to detect the expression level of the transferred "Dwarf 20" gene (refer to the patent of Wang Xiqing et al. from China Agricultural University, application number: CN201810636371.7, announcement number: CN108624709B). The cDNA of maize inbred line B73-329 (WT) was used as a control, and the maize Actin gene was used as an internal reference gene, and the detection primers were: ZmActin-rF (SEQ ID NO: 11: GAGCTCCGTGTTTCGCCTGA) and ZmActin-rR (SEQ ID NO: 12: CAGTTGTTCGCCCACTAGCG). The reaction procedure of fluorescence quantitative PCR amplification is shown in Table 3 below.

Table 3: Reaction procedure of fluorescence quantitative PCR amplification

The results of fluorescence quantitative PCR are shown in Figure 4. As can be seen from Figure 4, the expression levels of "Ai 20" in the transgenic lines Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5 were significantly higher than those in the maize inbred line B73-329 (WT). These results indicate that the "Ai 20" gene was successfully overexpressed in the T3 generation homozygous transgenic lines Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5.

Example 4: Field performance

The five overexpression transgenic lines were tested with control plants for multiple years at multiple sites. Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, Ai 20-5 and WT (wild type) were planted in two pilot sites (Gongzhuling and Zhuozhou, 6 replicates per site) in 2018 and in two pilot sites (Gongzhuling and Kaifeng, 3 replicates per site) in 2020.

From the seedling stage to the milky stage, the field characteristics of each strain were investigated, including seedling vigor, SPAD (chlorophyll content) value, silking stage, pollen shedding stage, plant height, and ear height. After harvest, the field characteristics of each strain were measured, including yield, ear length, ear diameter, bald tip length, number of ear rows, number of grains per row, number of normal grains per ear, grain width, grain thickness, grain weight in a plot, grain moisture, and 100-grain weight.

The results of the comparison of field traits between transgenic corn and control plants measured before harvest in 2018 showed that the seedling vigor of the five transgenic lines, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, decreased by 15.4%, 25.4%, 25.4%, 20.4%, and 17.9%, respectively, reaching an extremely significant level (Figure 5); the SPAD values of the three transgenic lines, Ai 20-1, Ai 20-3, and Ai 20-5, decreased by 6.1%, 11.3%, and 13.2%, respectively, reaching an extremely significant level (Figure 6); the silking period of one transgenic line, Ai 20-3, was delayed by 5%, reaching a significant level The pollen shedding period of one transgenic line, Ai 20-3, was delayed by 5.7%, reaching a significant level (Figure 8); the plant heights of five transgenic lines, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, were reduced by 43.9%, 42.0%, 42.6%, 34.1%, and 41.6%, respectively, reaching an extremely significant level (Figure 9); the ear heights of five transgenic lines, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, were reduced by 70.8%, 66.2%, 63.1%, 57.6%, and 63.1%, respectively, reaching an extremely significant level.

The results of the comparison of field traits between transgenic corn and control plants measured after harvest in 2018 showed that the yield of the five transgenic lines, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, decreased by 50.6%, 40.7%, 72.4%, 35.2%, and 36.5%, respectively, reaching an extremely significant level (Figure 10); the ear length of the four transgenic lines, Ai 20-1, Ai 20-3, Ai 20-4, and Ai 20-5, decreased by 16.5%, 7.3%, 8.3%, and 13.4%, respectively, reaching an extremely significant level (Figure 11); The ear diameter of the five transgenic lines, Ai20-1, Ai20-2, Ai20-3, Ai20-4, and Ai20-5, decreased by 3.9%, increased by 2.4%, increased by 0.7%, decreased by 0.4%, and decreased by 3.1%, respectively, and the difference was not significant (Figure 12); the bald tip length of the five transgenic lines, Ai20-1, Ai20-2, Ai20-3, Ai20-4, and Ai20-5, increased by 25.1%, increased by 6.6%, increased by 20.7%, decreased by 15.6%, and decreased by 23.4%, respectively, reaching an extremely significant level; the number of ear rows of one transgenic line, Ai20-5, The difference in the other strains was not significant (Figure 13); the number of grains per row of the four transgenic strains, Ai 20-1, Ai 20-3, Ai 20-4, and Ai 20-5, decreased by 24.3%, 12.3%, 13.5%, and 12.7%, respectively, reaching an extremely significant level (Figure 14); the number of normal grains per ear of the five transgenic strains, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, decreased by 29.2%, 6.4%, 6.5%, 15.5%, and 20.5%, respectively, reaching an extremely significant level (Figure 15); The grain width of one transgenic line, Ai20-2, increased by 5.4%, reaching an extremely significant level, while the differences in other transgenic lines were not significant (Figure 16); the grain thickness of two transgenic lines, Ai20-1 and Ai20-3, decreased by 6.4% and increased by 5.6%, respectively, reaching an extremely significant level, while the differences in other transgenic lines were not significant (Figure 17); the 100-grain weight of five transgenic lines, Ai20-1, Ai20-2, Ai20-3, Ai20-4, and Ai20-5, decreased by 6.1%, 4.2%, 7.6%, 6.0%, and 6.7%, respectively, reaching a significant level (Figure 18).

The results of the comparison of field traits between transgenic corn and control plants measured before harvest in 2020 showed that among the five transgenic lines, Ai 20-1, Ai 20-2, Ai 20-3, Ai 20-4, and Ai 20-5, the silking period of one transgenic line, Ai 20-3, was delayed by 2.7%, reaching a significant level (Figure 19); the pollination period of one transgenic line, Ai 20-3, was delayed by 1.6%, reaching a significant level (Figure 20); 2. The plant heights of Ai20-3, Ai20-4 and Ai20-5 decreased by 42.8%, 33.8%, 40.6%, 39.5% and 43.2%, respectively, reaching an extremely significant level (Figure 21); the ear heights of the five transgenic lines Ai20-1, Ai20-2, Ai20-3, Ai20-4 and Ai20-5 decreased by 52.9%, 47.3%, 56.8%, 57.0% and 55.8%, respectively, reaching an extremely significant level (Figure 22). The internodes of the first node under the ear of the five transgenic lines Ai20-1, Ai20-2, Ai20-3, Ai20-4, and Ai20-5 decreased by 69.6%, 66.7%, 71.2%, 70.6%, and 65.3%, respectively, reaching an extremely significant level; the internodes of the second node under the ear decreased by 64.5%, 63.7%, 69.4%, 68.7%, and 62.0%, respectively, reaching an extremely significant level; the internodes of the third node under the ear decreased by 58.3%, 59.1%, 66.5%, 64.1%, and 54.6%, respectively, reaching an extremely significant level The internodes of the 4th node under the ear decreased by 52.1%, 54.0%, 63.4%, 57.8% and 46.8%, respectively, reaching an extremely significant level; the internodes of the 5th node under the ear decreased by 51.7%, 53.3%, 65.1%, 58.4% and 42.4%, respectively, reaching an extremely significant level; the internodes of the 6th node under the ear decreased by 53.6%, 58.4%, 68.5%, 62.7% and 44.2%, respectively, reaching an extremely significant level; the internodes of the 7th node under the ear decreased by 56.5%, 57.7%, 68.0% and 65. 5%, 48.1%, reaching an extremely significant level; the internode distance at ear position decreased by 67.0%, 65.1%, 70.3%, 67.9%, 63.5%, respectively, reaching an extremely significant level; the internode distance at the first node on the ear decreased by 47.9%, 44.9%, 47.1%, 48.6%, 34.7%, respectively, reaching an extremely significant level; the internode distance at the second node on the ear decreased by 43.7%, 39.6%, 34.2%, 41.2%, 27.9%, respectively, reaching an extremely significant level; the internode distance at the third node on the ear decreased by 46.1%, 41.6%, 43.9%, 39.6%, 34.2%, 41.2%, 27.9%, respectively, reaching an extremely significant level; .5%, 37.2%, 42.1% and 30.2%, respectively, reaching an extremely significant level; the internodes of the 4th node on the ear decreased by 48.7%, 44.4%, 42.5%, 43.9% and 33.6%, respectively, reaching an extremely significant level; the internodes of the 5th node on the ear decreased by 47.3%, 42.2%, 42.4%, 41.7% and 33.1%, respectively, reaching an extremely significant level; the internodes of the 6th node on the ear decreased by 40.3%, 36.5%, 34.8%, 35.8% and 29.2%, respectively, reaching an extremely significant level (Figure 23).

summary

The "Dwarf 20" gene can significantly reduce the plant height and ear position of corn, and can keep the number of leaves and flowering period unchanged when the plant height is reduced. This performance ensures that the photosynthesis efficiency of corn does not decrease when the plant height is reduced, ensuring the accumulation requirements for grain filling, thereby achieving the goal of not significantly reducing yield under normal density.

"Dwarf 20" can significantly shorten the internode spacing, and because the internode spacing below the ear is shortened more significantly than the internode spacing above the ear, the reduction in ear position is more significant than the reduction in plant height. This plant type has a strong lodging resistance, which can increase the planting density of corn without causing lodging, thereby increasing yield by increasing density. Dwarfed corn can have a lower lodging rate than tall corn when infected with stalk rot, thereby avoiding yield losses and reducing harvest costs.

The "Dwarf 20" gene dwarfs corn, allowing farmers to access corn fields throughout the growing season, expanding the time window for fertilization and pest control, thereby increasing yields and reducing the capital investment in large overhead sprayers. The "Dwarf 20" gene dwarfs corn, making it easier for seed companies to "emasculate" large-scale seed production, significantly reducing the labor costs of seed production.

The present invention has been described in detail above with general description and specific embodiments, and some modifications or improvements may be made to it on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

references :

[1]Balaji Enugutti,Charlotte Kirchhelle,Maxi Oelschner,Ramón AngelTorres Ruiz,Ivo Schliebner,Dario Leister,Kay Schneitz.Regulation of planargrowth by the Arabidopsis AGC protein kinase UNICORN[J].Proceedings of theNational Academy of Sciences of the United States of America,2012,109(37).

[2]Balaji Enugutti,Kay Schneitz.Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN[J].Kay Schneitz,2013,13(1).

[3]Ana Victoria Garcia, Mohamed Al-Yousif, Heribert Hirt. Role of AGCkinases in plant growth and stress responses[J]. Cellular and Molecular Life Sciences, 2012, 69(19).

[4] Matsui Hidenori, Miyao Akio, Takahashi Akira, Hirochika Hirohiko. Pdk1 kinase regulates basal disease resistance through the OsOxi1-OsPti1aphosphorylation cascade in rice[J]. Plant & cell physiology, 2010, 51(12).

Claims (5)

1. A method for producing a transgenic plant with an altered trait, characterized in that the method comprises introducing a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 1, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the recombinant vector into a plant to obtain a corresponding transgenic plant; the transgenic plant has an altered trait compared to a wild-type plant, wherein the altered trait is a reduced plant height, a shortened internode distance, and an enhanced lodging resistance; The shortening of the internodes below the ear was more significant than that above the ear, and thus the reduction of ear position was more significant than the reduction of plant height. The plant is corn. 2. A method for regulating plant growth and development or plant stress resistance, wherein the method comprises transforming/transfecting a plant with a recombinant vector comprising a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 1 or a host cell comprising the nucleic acid molecule or the recombinant vector to obtain a transgenic plant with shortened internode distance, reduced plant height and enhanced lodging resistance compared with a wild-type plant; The shortening of the internodes below the ear was more significant than that above the ear, and thus the reduction of ear position was more significant than the reduction of plant height. The plant is corn. 3. Use of a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 1 in regulating plant growth and development or plant stress resistance, wherein the transgenic plants obtained have shortened internodes, reduced plant height and enhanced lodging resistance compared with wild-type plants; The shortening of the internodes below the ear was more significant than that above the ear, and thus the reduction of ear position was more significant than the reduction of plant height. The plant is corn. 4. Use of a recombinant vector containing the nucleic acid molecule in regulating plant growth and development or plant stress resistance, wherein the transgenic plants obtained have shortened internodes, reduced plant height and enhanced lodging resistance compared with wild-type plants; The shortening of the internodes below the ear was more significant than that above the ear, and thus the reduction of ear position was more significant than the reduction of plant height. The plant is corn. 5. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in regulating plant growth and development or plant stress resistance, wherein the transgenic plants obtained have shortened internode distance, reduced plant height and enhanced lodging resistance compared with wild-type plants; The shortening of the internodes below the ear was more significant than that above the ear, and thus the reduction of ear position was more significant than the reduction of plant height. The plant is corn.