CN117187270A - Corn gene ZmCPK28 and application thereof in regulating plant growth and development and environmental stress - Google Patents

Abstract

The invention discloses the corn gene ZmCPK28, its encoded protein, and its application in regulating plant growth and development and environmental stress. The present invention confirms that by using CRISPR/Cas9 gene editing technology to mutate the ZmCPK28 gene, corn plants that are drought-resistant and short-stemmed can be obtained; and by overexpressing the ZmCPK28 gene in corn, corn plants that utilize nitrogen nutrients efficiently and are resistant to high-light damage can be obtained.

Description

Maize gene ZmCPK28 and its role in regulating plant growth, development and environmental stress application

Technical field

The present invention relates to corn gene ZmCPK28, its encoded protein, and its application in regulating plant growth and development and environmental stress.

Background technique

Calcium ion (Ca ²⁺ ) plays an important role in plant growth and development, environmental stress response and complex signal transduction pathways, and is an indispensable second messenger in plants. In the signal transduction process involving Ca ²⁺ , in addition to relying on its own concentration changes, a variety of functional proteins are also required to participate, including calmodulin, calmodulin-like protein, calmodulin-like B subunit protein and calcium Calcium-dependent protein kinase (CPK). ^Among these calcium signal sensor proteins, CPK can not only sense Ca ²⁺ signals, but also directly convert Ca ²⁺ signals into phosphorylation reactions, thus having the dual functions of Ca ²⁺ sensors and responders.

CPK is a type of serine/threonine (Ser/Thr) protein kinase widely present in plants. As a Ca ²⁺ sensing protein in the cytoplasm, CPK phosphorylates various substrates, including ion channels, transcription factors and metabolic enzymes. , transmits calcium signals downstream and causes plant physiological responses, playing a key role in stem and root development, pollen tube growth, stomatal movement, hormone signal transduction, and stress tolerance. Currently, CPK genes in many plants have been cloned. In Arabidopsis thaliana, AtCPK10 was reported to be involved in the regulation of stomata by abscisic acid and Ca ²⁺ under drought conditions (Zou et al. 2010), and AtCPK23 plays an important role in plant responses to drought and salt stress (Ma and Wu 2007). Overexpression of AtCPK6 can enhance the salt and drought tolerance of plants (Xu et al. 2010). Overexpression of rice OsCPK7 can improve the cold resistance, salt tolerance and drought resistance of rice (Saijo et al. 2000), and overexpression of OsCPK12 can enhance the salt tolerance of plants (Asano et al. 2012). Low temperature can induce the expression of ZmCPK1 in maize (Berberich and Kusano 1997), and the ZmCPK11 gene is involved in tissue injury response (Szczegielniak et al. 2012). In tobacco, gibberellin or salt treatment can significantly induce the expression of NtCPK4 (Zhang et al. 2005). Overexpression of maize ZmCPK35 and ZmCPK37 can improve the drought tolerance of maize (Li et al. 2021). There are few reports on the specific functions of CPK genes in maize, and more related functions of CPK genes in maize need further study.

At present, there have been some reports on the role of CPK proteins in plants in nutrition, stress, growth and development, etc., but most of the research focuses on Arabidopsis thaliana. There are few relevant reports on corn, and its mechanism of action is not clear. Using corn ND101 as material, we use transgenic technology to increase the expression of the ZmCPK28 gene in corn or use CRISPR/Cas9 technology to knock out ZmCPK28 and regulate the growth and development, drought stress, nitrogen absorption and utilization, high light damage, etc. of corn through genetic modification. Research on this aspect is unclear.

Contents of the invention

The inventor unexpectedly discovered that by using CRISPR/Cas9 gene editing technology to mutate the ZmCPK28 gene, corn plants that are drought-resistant and dwarf-stem can be obtained; and by overexpressing the ZmCPK28 gene in corn, corn plants that utilize nitrogen nutrients efficiently and are resistant to high-light damage can be obtained .

Therefore, the purpose of the present invention is to provide the corn gene ZmCPK28, its encoded protein, and its application in regulating plant growth and development and environmental stress, especially related to the gene's role in regulating corn growth and development, drought stress, nitrogen absorption and utilization, and high light damage. etc. applications.

Therefore, according to a first aspect, the present invention relates to the maize gene ZmCPK28. The maize ZmCPK28 gene Genomic DNA involved in the present invention consists of 6948 bases and a total of 3 transcripts. This gene has 12 exons and 11 introns. The gene is derived from the ND101 inbred line. The Zea mays (B73_RefGen_v4) gene number of the maize ZmCPK28 involved in the present invention is Zm00001d015100, and the sequence is: Zea mays (B73_RefGen_v3) version code: GRMZM2G157068. The invention provides an isolated nucleic acid molecule comprising a sequence selected from:

1) The nucleotide sequence shown in SEQ ID NO:1;

2) At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or A nucleotide sequence with 99.5% identity, the polypeptide encoded by it has the function of regulating plant growth and development;

3) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 1 under stringent conditions;

4) A nucleotide sequence that expresses the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO:1;

5) Different transcripts produced from the nucleotide sequence shown in SEQ ID NO:1.

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

1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2;

2) Encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5 The nucleotide sequence of the amino acid sequence with % identity, the polypeptide encoded by it has the function of regulating plant growth and development;

3) A nucleotide sequence encoding an amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:2, and the encoded polypeptide has the ability to regulate plant growth. developmental functions.

In another aspect, the invention provides an isolated polypeptide transcribed and/or expressed from the nucleic acid molecule of the above aspect.

In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from:

1) The amino acid sequence shown in SEQ ID NO:2;

2) At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% with the amino acid sequence shown in SEQ ID NO:2 Identity of the amino acid sequence, the polypeptide has the function of regulating plant growth and development;

3) An amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and the polypeptide has the function of regulating plant growth and development.

In another aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the above aspect.

In another aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the above aspect, or the polypeptide described in the above aspect, or the recombinant vector described in the above aspect.

In another aspect, the present invention provides a transgenic plant comprising the nucleic acid molecule described in the above aspect, or comprising the recombinant vector described in the above aspect.

In some embodiments, the transgenic plant is a monocot or dicot, preferably a crop plant.

In some specific embodiments, the transgenic plant is selected from the group consisting of corn (Zea mays), sorghum (Sorghumbicolor, Sorghum vulgare), soybean (Glycine max), wheat (Triticum aestivum), rice (Oryza sativa), cotton (such as sea island Cotton (Gossypium barbadense), brassica (Brassica campestris), cabbage (Brassica oleracea), rape (Brassica napus), mustard (Brassica juncea), barley (Hordeum vulgare), rye (Secale cereale), oats (Avena sativa), Millet, millet (such as pearl millet (Pennisetum glaucum)), tomato (Lycopersicon esculentum), sunflower (Helianthusannuus), potato (Solanum tuberosum), peanut (Arachis hypogaea), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), tobacco (Nicotiana tabacum) or Arabidopsis thaliana (Arabidopsis thaliana); preferably Arabidopsis thaliana and corn; more preferably corn.

In another aspect, the present invention provides the application of the isolated nucleic acid molecule described in the above aspect or the isolated polypeptide described in the above aspect or the recombinant vector described in the above aspect in regulating plant growth and development, environmental stress and nitrogen absorption and utilization. .

In some embodiments, the application includes the steps of:

1) Introduce the nucleic acid molecules described in the above aspects or the recombinant vectors described in the above aspects into the target plants to obtain overexpression transgenic plants; and

2) Cultivate the plants so that the over-expression transgenic plants have the properties of tall stalks, high light tolerance, and high nitrogen utilization efficiency compared with control plants.

In some embodiments, the application includes the steps of:

1) Destroy the ZmCPK28 gene in plants or genes homologous to ZmCPK28 genes in other plants to obtain transgenic plants; and

2) Cultivate the plants so that the transgenic plants have drought resistance and short stem properties compared with control plants.

In some embodiments, the disruption can be achieved by knocking out or knocking down the ZmCPK28 gene.

In some embodiments, the disruption can be achieved by genome editing systems such as CRISP/Cas, TALENs, ZFNs, or other gene editing systems.

In some embodiments, the regulating plant growth and development includes, but is not limited to, regulating plant height; and the regulating plant environmental stress includes, but is not limited to, regulating plant tolerance to high light and drought.

In another aspect, the present invention provides the use of the nucleic acid molecule described in the above aspect or the polypeptide described in the above aspect or the recombinant vector described in the above aspect in the breeding of plants with altered traits, especially corn.

In some embodiments, the plant is a monocot or dicot, preferably a crop plant.

In some specific embodiments, the transgenic plant is selected from the group consisting of corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats, millet, millet, tomato, sunflower, One or more of potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco or Arabidopsis; Arabidopsis and corn are preferred; corn is more preferred.

In another aspect, the present invention provides a method for regulating plant growth and development, which includes:

1) Introduce the isolated nucleic acid molecule described in the above aspects or the recombinant vector described in claim 5 into a target plant to obtain a transgenic plant; and

2) Cultivate the plant, wherein compared with the control plant, the transgenic plant has the properties of tall stalk, high light tolerance, and high nitrogen utilization efficiency.

In another aspect, the present invention provides a method for regulating plant growth and development, which includes:

1) Destroy the ZmCPK28 gene in plants or genes homologous to ZmCPK28 genes in other plants to obtain transgenic plants; and

2) Cultivate the plant, wherein the transgenic plant has drought resistance and dwarf properties compared to the control plant.

In another aspect, the present invention provides a method for producing a transgenic plant, the transgenic plant comprising the introduced isolated nucleic acid molecule as described in the above aspect, or the recombinant vector as described in the above aspect, or its recombinant expression as described in the above aspect. The isolated polypeptide, the method includes the following steps:

1) Obtain the seeds of the transgenic plants;

2) Plant the seeds to obtain the transgenic plants whose traits can be stably inherited.

In another aspect, the invention provides a method for producing transgenic plants, the transgenic plants comprising disrupted ZmCPK28 genes in plants or genes homologous to ZmCPK28 genes in other plants, the method comprising the following steps:

1) Obtain the seeds of the transgenic plants;

2) Plant the seeds to obtain the transgenic plants whose traits can be stably inherited.

In some embodiments, the disruption is achieved by knocking out or knocking down the ZmCPK28 gene.

In some embodiments, the disruption is achieved by CRISP/Cas, TALEN, ZFN, or other gene editing systems.

In some specific embodiments, the transgenic plant is selected from the group consisting of corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats, millet, millet, tomato, sunflower, One or more of potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco or Arabidopsis; Arabidopsis and corn are preferred; corn is more preferred.

In another aspect, the present invention provides a CRISPR/Cas9 target gDNA, which includes the nucleotide sequence shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

Description of the drawings

Figure 1 shows the expression level detection results of ZmCPK28 transgenic overexpression plants.

Figure 2 is the sequence detection result of ZmCPK28 mutant.

Figure 3 shows the results of plant height determination of ZmCPK28 mutants and overexpression plants.

Figure 4 shows the phenotypes of ZmCPK28 mutants and overexpressed plants under drought treatment. The left picture shows conditions with sufficient water supply; the right picture shows drought conditions.

Figure 5 shows the relative water content of leaves of ZmCPK28 mutant and overexpression plants.

Figure 6 shows the water loss rate of detached leaves of ZmCPK28 mutant and overexpression plants.

Figure 7 shows the stomatal opening detection of ZmCPK28 mutants and overexpression plants.

Figure 8 shows the nitrogen nutrition hydroponic phenotype of ZmCPK28 overexpression plants, where:

A. Plant phenotype observation of ZmCPK28 overexpression plants under severe nitrogen deficiency (0.04mM NO ₃ ^- ) hydroponic conditions;

B. Observation of plant phenotypes of ZmCPK28 overexpression plants under nitrogen-limited (0.4mM NO ₃ ^- ) hydroponic conditions;

C. Observation of plant phenotypes of ZmCPK28 overexpression plants under hydroponic conditions with sufficient nitrogen nutrition (4mM NO ₃ ^- );

D. Observation of leaf phenotypes of ZmCPK28 overexpression plants under severe nitrogen deficiency (0.04mM NO ₃ ^- ) hydroponic conditions;

E. Observation of leaf phenotypes of ZmCPK28 overexpression plants under nitrogen-limited (0.4mM NO ₃ ^- ) hydroponic conditions;

F. Observation of leaf phenotypes of ZmCPK28 overexpression plants under hydroponic conditions with sufficient nitrogen nutrition (4mM NO ₃ ^- ).

Figure 9 is the determination of biomass and nitrogen content of ZmCPK28 related plants, where:

A. Biomass detection of ZmCPK28-related plants under different nitrogen treatment conditions;

B. Detection of nitrogen content of ZmCPK28-related plants under different nitrogen treatment conditions.

Figure 10 shows the instantaneous absorption of nitrogen nutrients by ZmCPK28 overexpressing plants, where:

A. Instantaneous absorption of nitrogen nutrition by ZmCPK28 overexpressing inbred plants;

B. Instantaneous absorption of nitrogen nutrients in hybrid plants overexpressing ZmCPK28.

Figure 11 is the analysis of panicle phenotype and seed test data of ZmCPK28 overexpression plants, where:

A. Observation of panicles of ZmCPK28 overexpression plants under different nitrogen fertilizer treatments;

B. Yield analysis of ZmCPK28 overexpression plants under different nitrogen fertilizer treatments;

C. Analysis of panicle length of ZmCPK28 overexpression plants under different nitrogen fertilizer treatments;

D. Analysis of panicle row number of ZmCPK28 overexpression plants under different nitrogen fertilizer treatments;

E. Analysis of row grain number of ZmCPK28 overexpression plants under different nitrogen fertilizer treatment conditions.

Figure 12 is an observation of the sunburn-sensitive phenotype of the ZmCPK28 mutant.

Detailed description of the invention

The following definitions and descriptions are provided to better define the present invention and to guide those skilled in the art in practicing the present invention. Unless defined otherwise, all techniques and terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise stated, the techniques employed and encompassed herein are standard methods well known to those skilled in the art to which this invention belongs. The materials, methods, and examples are provided for illustrative purposes only and do not limit the scope of the invention in any way.

As used herein, "plant" includes intact plants, transgenic plants, meristems, plant parts, plant cells and their progeny. Parts of plants include, but are not limited to, leaves, stems, tubers, roots, flowers (including, for example, bracts, sepals, petals, stamens, carpels, anthers, ovules, etc.), fruits, embryos, endosperms, seeds, pollen, meristems, Calli, protoplasts, microspores, etc.

As described herein, the available plant species generally cover higher plant species suitable for transgenic technology, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, gymnophytes, lycophytes Plants, bryophytes, and multicellular algae.

In some embodiments, plant parts or plant cells of the invention are regenerable. In other embodiments, the plant parts or plant cells of the invention are non-renewable.

In specific embodiments, plants suitable for the present invention are selected from the following: corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats, millet, millet, tomato , sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco, Arabidopsis, etc. Preferably, plants suitable for use in the present invention are maize or rice.

As used herein, the term "transgene" refers to a polynucleotide molecule artificially introduced into the genome of a host cell. The transgene can be heterologous to the host cell. The term "transgenic plant" refers to a plant containing a heterologous polynucleotide as described above, including plants regenerated from an initially transformed plant cell and progeny transgenic plants derived from subsequent generation or crosses of transgenic plants.

Any common method in the art known to those skilled in the art can be used to obtain transgenic plants, such as the introduction of heterologous nucleic acid sequences through electroporation or microinjection of plant cell protoplasts; or the introduction of heterologous nucleic acid sequences through DNA particle bombardment. The nucleic acid sequence is directly introduced into plant tissue cells; or the heterologous nucleic acid sequence is introduced into plant cells using Agrobacterium tumefaciens host cells.

As used herein, a "control plant" refers to a plant that does not contain recombinant DNA that confers enhanced traits. Control plants are used to identify and select transgenic plants with enhanced traits. Suitable control plants may be non-transgenic plants of the parental line used to generate the transgenic plants, for example, wild-type plants. Suitable control plants may also be transgenic plants containing recombinant DNA that confer other traits, for example, transgenic plants with enhanced herbicide tolerance.

As used herein, "trait" refers to the physiological, morphological, biochemical or physical characteristics of a plant or a specific plant material or cell. In some cases, the characteristic is visible to the human eye, such as seed or plant dimensions, or can be obtained through biochemical techniques (such as detection of protein, starch, certain metabolites, or oil content of seeds or leaves) or by observing metabolic or physiological processes. (e.g., by measuring tolerance to water deprivation or specific salt or sugar concentrations) or by measuring the expression level of one or more genes (e.g., using Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems) or measured through agricultural observations such as hyperosmotic stress tolerance and yield. Any technique can be used to measure the amount, comparative level, or difference of any selected chemical compound or macromolecule in a transgenic plant.

As used herein, "gene" or "gene sequence" refers to the partial or complete coding sequence of the gene, its complementary sequence, and its 5' and/or 3' untranslated region. A gene is also a functional unit of heredity and is physically a specific segment or sequence of nucleotides along the molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter can undergo subsequent processing, such as chemical modification or folding to obtain functional proteins or peptides. By way of example, a transcriptional regulatory gene encodes a transcriptional regulatory polypeptide, which may be functional or require processing to act as an initiator of transcription.

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 limitation. . As the nucleic acid used to construct the recombinant construct, DNA is preferred, as it is more stable and easier to handle than RNA.

The term “DNA” refers to a double-stranded DNA molecule of genomic or synthetic origin, a polymer or nucleotide molecule of deoxyribonucleotide bases, 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 typically shown from the 5' (upstream) end to the 3' (downstream) end.

Methods known to those skilled in the art can be used to isolate the DNA molecules or fragments thereof described herein. For example, PCR (polymerase chain reaction) technology can be used to amplify a specific starting DNA molecule and/or produce variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques, such as direct synthesis of fragments by chemical methods (such as automated oligonucleotide synthesizers).

The term "transcript" refers to the product of transcription of a DNA nucleotide sequence catalyzed by RNA polymerase. When an RNA transcript is a perfect complementary copy of a DNA sequence, it is called a primary transcript, or it can be RNA obtained by post-transcriptional processing of a primary transcript, called mature RNA.

The term "isolated" means that the molecule is at least partially separated from other molecules to which it is normally associated in its autologous or native state.

In some embodiments, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule that is at least partially separated from the nucleic acid to which it would normally flank the nucleic acid molecule in its autologous or native state. Accordingly, nucleic acid molecules are considered to be isolated herein if they are fused to regulatory or coding sequences to which they are not normally linked as a result of recombinant techniques. Such molecules are considered to be isolated even when integrated into the chromosome of the host cell or present in a nucleic acid solution with other nucleic acid molecules.

As used herein, a "polypeptide" includes a plurality of contiguous polymerized amino acid residues, for example, at least about 15 contiguous polymerized amino acid residues. Typically, a polypeptide contains a series of polymerized amino acid residues that are transcriptional regulators or domains or portions or fragments thereof. In addition, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) an inhibitory domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (v) a protein-protein interaction domain; vi) DNA binding domain; or other parts. The polypeptide optionally contains modified amino acid residues, naturally occurring amino acid residues not encoded by codons, non-naturally occurring amino acid residues.

As used herein, "protein" refers to a series of amino acids, oligopeptides, peptides, polypeptides or portions 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 extracellularly) in a greater amount than in its native state in a wild-type cell, e.g., in an amount greater than about 5 % or more than about 10% or more than about 20% or more than about 50% or more, which may alternatively be expressed as: 105%, 110%, 120% relative to the wild-type polypeptide normalized at 100%, 150% or more. This is not the result of a natural response in wild-type plants. Additionally, the isolated polypeptide is separated from other normally associated cellular components, for example, using various protein purification methods.

As used herein, "percent identity", "% identity" or "% identity" is a comparison between amino acid sequences or nucleotide sequences, based on the comparison of two sequences that are optimally aligned over a comparison window Determine (for example, gaps can be introduced into the first nucleotide sequence or amino acid sequence for optimal alignment with another nucleotide sequence or amino acid sequence). Those skilled in the art know how to calculate the percent identity between two sequences, for example, through Clustal, Bestfit, Blast, Fasta and other software. Percent identity is determined by counting the number of identical nucleotide or amino acid positions in the two sequences, dividing by the full length of the reference sequence (excluding gaps introduced into the reference sequence by the alignment process), and multiplying by 100%. The reference sequence may be, for example, SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. One of the other sequences may have one or more amino acid/nucleotide insertions, substitutions and deletions relative to the reference sequence.

In some embodiments, the nucleotide sequence of the invention is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% identical to the nucleotide sequence shown in SEQ ID NO:1 %, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% or 100% identity.

In some embodiments, the polypeptide sequence of the invention is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% identical to the amino acid sequence shown in SEQ ID NO:2 , 94%, 95%, 96%, 97%, 98%, 98%, 99% or 100% identity.

As used herein, the term "stringent conditions" or "stringent hybridization conditions" includes conditions under which a probe hybridizes to a target sequence to a higher detectable degree than other sequences (eg, at least two times greater than background). Stringent conditions vary depending on the sequence and environment. By adjusting the stringency of hybridization and/or wash conditioning, target sequences up to 100% complementary to the probe can be detected. Alternatively, the stringency can be adjusted so that some mismatches can exist in the sequence, thereby detecting less identical target sequences. Hybridization specificity depends on the posthybridization wash step, with key factors being the salt concentration and temperature of the wash solution. As desired, both the temperature and the salt concentration can be varied, or the temperature or the salt concentration can be held constant while the other variable is changed. Suitable stringent conditions to promote DNA hybridization are known to those skilled in the art. Examples of low stringency conditions are: hybridization in buffer containing 30-35% formamide, 1M NaCl, 1% SDS at 37°C and 1-2× SSC (Chloride) at 50°C-55°C. Sodium chloride/sodium citrate) washing. Examples of moderately stringent conditions include hybridization in a buffer containing 40-45% formamide, 1M NaCl, 1% SDS at 37°C and washing with 0.5-1×SSC at 55°C-60°C. Examples of highly stringent conditions include hybridization in a buffer containing 50% formamide, 1M NaCl, 1% SDS at 37°C and washing with 0.1×SSC at 60°C-65°C. Detailed descriptions of nucleic acid hybridization can be found in Haymes et al., Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, DC (1985); and Sambrook et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3 .6.

As used herein, "insertion," "deletion," or "substitution" of one or more nucleotides or amino acids does not impair the function of the original sequence (e.g., in this context Refers to the function of still retaining drought resistance and/or increasing crop yield under drought conditions). Those skilled in the art are aware of methods to accomplish 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, choosing to carry out such insertions, deletions and/or substitutions in non-conserved regions; or based on the degeneracy of the genetic code, modifying nucleotides through "silent mutation" without changing the polypeptide encoded by the nucleotides; or , through "conservative substitution", one amino acid in a protein is replaced by another amino acid with similar properties 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 non-polar (hydrophobic) amino acids , such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Conservative substitutions for amino acids within a native protein or polypeptide can be selected from other members of the group to which 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 chain is serine and threonine; with The group of amino acids with amide side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are Lysine, arginine, and histidine; and groups of amino acids with sulfur-containing side chains are cysteine and methionine. The natural conservative amino acid substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, tyrosine asparagine-glutamine and asparagine-glutamine.

As used herein, the term "recombinant" refers to forms of DNA and/or proteins and/or organisms that are not normally found in nature and are thus produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants.

In some embodiments, the recombinant vector further comprises a promoter, terminator, regulatory sequences, selectable markers, and/or any other sequences necessary for its expression in the host cell operably linked to the nucleotide sequence. In some specific embodiments, the recombinant vector is a plasmid.

As used herein, the term "promoter" generally refers to a DNA molecule involved 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; alternatively, the promoter can be a synthetically produced or manipulated DNA molecule. In some embodiments, a promoter may also be chimeric, ie, 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 (eg, Agrobacterium). In some embodiments, the promoter is a developmentally regulated, organelle-specific, tissue-specific, inducible, constitutive, or cell-specific promoter.

As used herein, "operably linked" can be used interchangeably with "operably linked" and refers to the function between a first sequence (such as a promoter) and a second sequence (such as a gene of interest). Sexual linkage, in which the promoter sequence initiates and mediates transcription of the second sequence. Typically, two sequences that are operably linked are contiguous. Those skilled in the art know how to select promoters, terminators and other sequences required for expression of genes in host cells.

In some specific embodiments, expression of a gene is controlled by a so-called "strong" promoter (ie, a promoter with high transcriptional potential such that the gene is strongly expressed).

As used herein, "host cell" refers to a cell that contains a recombinant vector and supports the replication and/or expression of the expression vector. The host cell may be a prokaryotic cell (eg, E. coli cells, Agrobacterium tumefaciens cells) or a eukaryotic cell (eg, yeast, insect, plant or animal cell).

In some embodiments, the host cell is preferably a monocotyledonous or dicot plant cell, including but not limited to, cells from corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats , millet, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco, Arabidopsis thaliana cells. Preferably, the host cell is a maize cell or a rice cell; more preferably, the host cell is a maize cell.

As used herein, "introducing" a nucleic acid molecule or an expression vector into a plant or plant cell means transfecting, transforming, transducing or incorporating the nucleic acid molecule or recombinant expression vector into a host cell such that the nucleic acid molecule can be expressed in the host cell. for autonomous reproduction or expression.

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

The CRISPR/Cas gene editing system described herein includes, for example, all components required for Cas9 or modified Cas9 enzyme, guide RNA and/or homology-directed repair template; including (a) CRISPR/Cas system nucleotide sequence or a nucleotide sequence encoding a CRISPR/Cas system nucleotide sequence and/or (b) a nucleotide sequence encoding a CRISPR/Cas enzyme, which sequences may be included in one or more recombinant viral vectors. The nucleotide sequence of (a) may be located on the same or different recombinant viral vector as the nucleotide sequence of (b).

In some embodiments, the viral vector can be a retroviral vector, optionally a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adeno-associated virus (AAV) vector (eg, an AAV8 vector), or a pox vector. Viruses (such as vaccinia virus).

In some embodiments, (a) a CRISPR/Cas system nucleotide sequence or a nucleotide sequence encoding a CRISPR/Cas system nucleotide sequence and/or (b) a nucleotide sequence encoding a CRISPR/Cas enzyme can be obtained by Liposomes, nanoparticles, exosomes, microvesicles or gene guns are delivered to the cells of an organism.

In some specific embodiments, a preferred CRISPR/Cas enzyme is a Type II CRISPR/Cas enzyme, preferably a Type II Cas9 enzyme or a biologically active fragment or derivative thereof.

The corn ZmCPK28 gene Genomic DNA involved in the present invention consists of 6948 bases and a total of 3 transcripts. The gene has 12 exons and 11 introns. The gene is derived from the ND101 inbred line, and its number in the maize genome database is GRMZM2G157068. Since the same DNA sequence in corn can produce different transcripts and translate into different proteins, the different transcripts produced by this sequence and the different proteins translated into each other are within the protection scope of this application.

The present invention uses gene editing methods to knock out ZmCPK28, and can obtain short-stem and drought-resistant corn plants; and overexpress the ZmCPK28 gene in corn, and can obtain high-stem corn plants that are resistant to high light and can efficiently utilize nitrogen nutrients. Therefore, the present invention has important theoretical guidance significance and production and application value for cultivating stress-resistant and high-yielding corn.

The technical effects of the present invention are as follows:

The present invention uses CRISPR/Cas9 gene editing technology to knock out the ZmCPK28 gene. After phenotypic testing and analysis of physiological traits such as plant drought resistance, it is found that the plant height of the zmcpk28 mutant is significantly lower than the control; the zmcpk28 mutant exhibits high-temperature sunburn injury traits. ; At the same time, the sensitivity to abscisic acid (ABA)-promoted stomatal closing process is increased, the water loss rate of detached leaves is reduced, and the drought resistance is significantly enhanced, indicating that the mutation of this gene can significantly reduce the plant height of corn, improve plant drought resistance, and improve Corn is sensitive to high light and high temperature; on the contrary, over-expression plants of the ZmCPK28 gene increase the plant height of corn, enhance the nitrogen use efficiency of corn, increase yield, and reduce the drought resistance of corn. In the embodiments of the present invention, gene editing technology is used to obtain drought-resistant dwarf plants. Compared with traditional breeding methods, the time is shorter and the purpose is stronger. It provides genetic resources for cultivating and improving corn and elucidates the role of ZmCPK28 in plant growth and development, drought and The important role of nitrogen absorption and utilization provides a theoretical basis.

Detailed ways

The corn receptor material inbred line used in the following examples is ND101; the Agrobacterium strain is EHA105; the cDNA of B73 as the PCR template and pBCXUN and pBUE411 as the vectors are provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

Example 1: Obtaining ZmCPK28 overexpressing corn plants

1.Construction and detection of ZmCPK28 gene overexpression vector

Extract total RNA from B73 maize (Zea mays L.), obtain cDNA by reverse transcription, use cDNA as a template, design primers (primers are as follows), use upstream and downstream primers and high-fidelity enzymes to amplify the CDS region of the target gene ZmCPK28, use Purify the PCR product using 1% agarose gel and gel recovery method. Connect the purified fragment to the constructed pBCXUN vector with Ubiquitin promoter through TA cloning method. Transform E. coli and screen positive clones. Complete the clone build. Sequence the constructed plasmid, compare and analyze the sequencing results and the target sequence, and select a vector containing a complete and correctly sequenced target gene sequence for subsequent experiments.

ZmCPK28-F primer:ATGGGCGCTTGCTTCTCCTCCGCCTCT

ZmCPK28-R primer:CGAGTCTTGCCTACCATTTTG

2. Identification of overexpressing transgenic corn plants

The constructed pBCXUN recombinant expression vector containing the target gene was transformed into Agrobacterium strain EHA105. The Agrobacterium-mediated transgenic operation was transferred into the young embryos of the corn inbred line ND101, dedifferentiated to form callus, and then differentiated to form tissues and organs through tissue culture to obtain the T ₀ generation transgenic plants.

The identified T ₀ generation plants were transplanted into the greenhouse of the West Campus of China Agricultural University, and the T ₁ generation transgenic plant offspring of the inbred line were inbred; the T ₁ generation transgenic plant offspring were selfed to obtain T ₂ generation transgenic seeds; T _{The second} -generation transgenic plants are then self-crossed to obtain T _-3 transgenic plant seeds; in each generation, PCR amplification is used to identify positive transgenic plants, and then self-crossing is performed.

In order to detect the expression efficiency of the ZmCPK28 gene in the T ₃ transgenic line, the total RNA from the seedling leaves of the recipient material ND101 and the four transgenic lines OE1, OE2, OE3, and OE4 were extracted and reverse transcribed into the corresponding cDNA. Fluorescence quantitative PCR experiment was performed to detect its expression level (Figure 1). And select 3 representative T ₃ -generation homozygous transgenic lines for subsequent experiments.

Example 2: Obtaining ZmCPK28 gene-edited corn plants

1.Construction and detection of ZmCPK28 gene editing vector

In order to study the function of ZmCPK28 in corn plants, the present invention uses CRISPR/Cas9 technology to edit the ZmCPK28 gene in the corn genome. ① Use a single target method to design the target: (C, 5'-ATAACACAGATATGTCGAC-3') and construct it into the pXUE411C-BG vector; ② Use a dual target method to design the target: (C1, 5'-ATAACACAGATATGTCGAC- 3';C2,5'-TTTCAAGCAATTCGCGCTT-3') and constructed into the pXUE411C-BG vector. Sequence the constructed plasmid and compare and analyze the sequencing results with the target sequence to ensure that the vector contains the complete target gene sequence (Figure 2).

2. Identification of gene-edited transgenic corn plants

The recombinant gene editing vector was introduced into Agrobacterium tumefaciens EHA105 strain, and the recombinant bacteria was introduced into the corn inbred line ND101 through Agrobacterium-mediated method. T ₀ generation transgenic seedlings were obtained by screening against Basta-resistant medium, and T ₀ generation transgenic plants were obtained. The seedling leaves of the T ₀ generation transgenic plants were taken to detect the copy number of the screening resistance gene Bar, and the transgenic positive plants were screened.

The identified T ₀ -generation transgenic plants are self-crossed to obtain T ₁ -generation gene-edited plants, and the T ₁ -generation gene-edited plants are self-crossed to obtain T ₂ -generation gene-edited plant offspring. Use target verification primers to detect gene editing of the target gene sequence to obtain the genotypes of each mutant. Sequence analysis revealed that the editing types of the three strains are d1+d28i3, d62, and d62 (Figure 3). The T ₂ generation plants were selfed to obtain T ₃ generation gene edited plants for subsequent experiments.

Example 3: Phenotypic detection of plant height of ZmCPK28 mutants and overexpression plants

Wild type, mutant and over-expression plants are germinated in a solar greenhouse at 28°C. The germinated seeds are sown on-demand in a matrix of imported soil: peat soil: vermiculite in equal proportions, 8kg per barrel, 1 seedling per pot. Culture under normal outdoor conditions. The 7 strains are completely randomly arranged, with at least 30 strains per strain, and are cultured normally until they become seedlings. After the powder is spun, the height from the base of the corn to the top of the tassel is measured. As shown in Figure 4, compared with the wild type, the plant height of the mutant line is significantly reduced, with the average plant height of the three lines reduced by 25%. The plant height of the overexpression lines increased significantly, with an average increase of 5% among the three lines.

Example 4: Application of ZmCPK28 mutants and overexpression plants in drought stress

1. Phenotypic detection of ZmCPK28 mutants and overexpressed plants under soil drought treatment

Sow the wild-type and ZmCPK28 gene mutant and over-expression seeds in a mixture of imported soil: peat soil: vermiculite in equal proportions, 140g per small pot, and 4 seeds per pot. When the seedlings grow to the three-leaf and one-heart stage, Carry out thinning, and keep 3 plants with relatively consistent growth size and status in each pot, and the seedlings will continue to grow in the greenhouse. One week later, add water to the tray. After the water is fully absorbed, transfer all the small pots to another dry tray. At this time, the planted wild-type, mutant, and overexpression plants are divided into two treatments. The control treatment continued to be cultured normally and watered on time. The drought-treated plants were no longer watered and subjected to drought stress treatment. They were observed regularly every day and took photos and records on 11-12 days. Figure 4 shows the phenotypic results under drought treatment conditions. The left side shows the normal watering situation, and the right side shows the drought treatment situation. From both, we can see that the ZmCPK28 gene mutant has obvious drought resistance compared with the control. Phenotype, its overexpression plants have drought-sensitive phenotype.

2. Detection of relative water content in leaves of ZmCPK28 mutants and overexpression plants

Observe the status of the seedlings in the pot. When a drought phenotype occurs, take 9 leaves each of the mutant, overexpression plant and control under normal watering and drought conditions, weigh the weight of the leaves and record them as W ₁ ; then cut the leaves. Put the section into a 50mL centrifuge tube, add distilled water, and allow the leaves to absorb water until they are saturated (about 24 hours). Then dry the water on the leaves with absorbent paper, reweigh it and record it as W ₂ ; after weighing, dry the leaves (80 ℃ for more than 8 hours), measure the dry weight and record it as W ₃ . Calculate the relative moisture content, relative moisture content = (W ₁ -W ₃ )/(W ₂ -W ₃ )×100% (Figure 5).

3. Detection of water loss rate in isolated leaves of ZmCPK28 mutants and overexpression plants

The wild type, ZmCPK28 mutant and over-expression plants were grown in the greenhouse for about 12 days. The uppermost unfolded leaf from the above ground was taken and the fresh weight was weighed with an electronic balance of 1/10000. The temperature was 27℃ and the humidity was 30%~ After being placed under 45% conditions for 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 hours, weigh. Calculate the water loss rate: water loss rate = (initial weight of the leaf - weight of the leaf at a certain time point) / initial weight of the leaf × 100%. Each experiment was repeated three times for wild type, mutant and overexpression plants, and the experiment was repeated three times independently. A water loss curve is made based on the water loss rate and time. Figure 6 shows that the water loss rates of the three ZmCPK28 mutant plants were significantly lower than those of the wild type, indicating that the three mutants are more drought-resistant than the control.

4. Detect the effect of exogenous ABA on stomatal opening of ZmCPK28 mutants and overexpression plants

This operation uses the leaves of wild type, ZmCPK28 mutant and overexpression plants as the operating objects, and sets up control groups and experimental groups (with ABA treatment) respectively. Apply transparent nail polish on the back of the corn leaves, peel off the epidermis, take photos with a microscope, and use Image J software to count the stomatal opening (expressed by the transverse diameter of the stomatal opening). The results of the stomatal movement experiment are shown in Figure 7. Under control conditions, there was no difference in stomatal opening between plants. However, under ABA treatment conditions, the stomatal opening of ZmCPK28 mutant plants was smaller than that of the wild type, indicating that they were sensitive to the action of ABA. ZmCPK28 is responsive to the regulation of plant stomatal movement and has great application value in plant response to drought stress.

Example 5: Application of ZmCPK28 mutants and overexpression plants in nitrogen absorption and utilization

This operation uses wild-type and ZmCPK28 overexpression strains as the operating subjects, and sets different concentrations of nitrogen nutrition to conduct nutrient solution culture experiments. Experimental results show that ZmCPK28 overexpression plants show a tolerance phenotype under nitrogen nutrition limitation (NL) and extreme nitrogen deficiency (ND) conditions. When the allele leaves of the wild type show typical nitrogen deficiency symptoms, overexpression The leaves of the expressing plants still remain green (Figure 8). The nitrogen content detection results showed that under low nitrogen and extreme nitrogen deficiency conditions, the nitrogen content and biomass of ZmCPK28 overexpression plants were significantly higher than those of the control (Figure 9).

The results of the nitrogen nutrient depletion experiment and the ¹⁵ N instantaneous absorption experiment showed that the nitrogen nutrient absorption rate of ZmCPK28 overexpression plants was significantly faster than that of the wild-type control (Figure 10). The results of field nitrogen fertilizer experiments show that ZmCPK28 overexpressing plants have very significant nitrogen nutrition advantages. Under low nitrogen treatment conditions, the corn ears of ZmCPK28 overexpressing plants are significantly larger than those of the wild type, and the yield and nitrogen content are significantly higher than those of the wild type. Statistics The data showed that the panicle length and row grain number of ZmCPK28 overexpression plants were significantly greater than those of the wild-type control (Figure 11).

Example 6: Application of ZmCPK28 in regulating sunburn resistance of corn

In field experiments in Hainan, where light intensity is relatively high, it was found that compared to the wild-type control, the zmcpk28 mutant exhibited a very severe sunburn injury phenotype. As shown in Figure 12, local leaves of the zmcpk28 mutant showed symptoms of chloroplasts caused by chloroplast destruction and degradation, and some areas even became transparent. This shows that ZmCPK28 plays a very important role in corn resistance to high light and sunburn stress.

The present invention has been described in detail with general descriptions and specific embodiments. On the basis of the present invention, some modifications or improvements can be made. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (29)

1. An isolated nucleic acid molecule, characterized in that it comprises a sequence selected from: 1) The nucleotide sequence shown in SEQ ID NO:1; 2) At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or A nucleotide sequence with 99.5% identity, the polypeptide encoded by it has the function of regulating plant growth and development; 3) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 1 under stringent conditions; 4) A nucleotide sequence that expresses the same or functionally deleted or mutated protein by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO:1; 5) Different transcripts produced from the nucleotide sequence shown in SEQ ID NO:1. 2. An isolated nucleic acid molecule, characterized in that it comprises a sequence selected from: 1) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2; 2) Encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5 The nucleotide sequence of the amino acid sequence with % identity, the polypeptide encoded by it has the function of regulating plant growth and development; 3) A nucleotide sequence encoding an amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:2, and the encoded polypeptide has the ability to regulate plant growth. developmental functions. 3. An isolated polypeptide, characterized in that it is transcribed and/or expressed from the nucleic acid molecule of claim 1 or 2. 4. An isolated polypeptide, characterized in that it contains an amino acid sequence selected from the following: 1) The amino acid sequence shown in SEQ ID NO:2; 2) At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% with the amino acid sequence shown in SEQ ID NO:2 Identity of the amino acid sequence, the polypeptide has the function of regulating plant growth and development; 3) An amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2. The polypeptide has the function of regulating plant growth and development. 5. A recombinant vector, characterized by comprising the nucleic acid molecule of claim 1 or 2. 6. A host cell, characterized by comprising the nucleic acid molecule of claim 1 or 2, or the polypeptide of claim 3 or 4, or the recombinant vector of claim 5. 7. A transgenic plant, characterized by comprising the nucleic acid molecule according to claim 1 or 2, or the recombinant vector according to claim 5. 8. The transgenic plant according to claim 7, wherein the transgenic plant is a monocotyledonous plant or a dicotyledonous plant, preferably a crop plant. 9. The transgenic plant according to claim 7 or 8, wherein the transgenic plant is selected from the group consisting of corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats, One or more of millet, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco or Arabidopsis; Arabidopsis and corn are preferred. 10. Use of the isolated nucleic acid molecule of claim 1 or 2, the isolated polypeptide of claim 3 or 4, or the recombinant vector of claim 5 in regulating plant growth and development. 11. Use of the isolated nucleic acid molecule of claim 1 or 2, the isolated polypeptide of claim 3 or 4, or the recombinant vector of claim 5 in regulating plants' response to environmental stress. 12. Use of the isolated nucleic acid molecule of claim 1 or 2, the isolated polypeptide of claim 3 or 4, or the recombinant vector of claim 5 in regulating nitrogen absorption and utilization of plants. 13. The application according to any one of claims 10-12, which includes the following steps: 1) Introducing the nucleic acid molecule of claim 1 or 2 or the recombinant vector of claim 5 into a target plant to obtain an overexpression transgenic plant; and 2) Cultivate the plants so that the over-expression transgenic plants have the properties of tall stalks, high light tolerance, and high nitrogen utilization efficiency compared with control plants. 14. The application according to any one of claims 10-12, comprising the following steps: 1) Destroy the ZmCPK28 gene in plants or genes homologous to ZmCPK28 genes in other plants to obtain transgenic plants; and 2) Cultivate the plants so that the transgenic plants have drought resistance and short stem properties compared with control plants. 15. The use according to claim 14, wherein the disruption is achieved by knocking out or knocking down the ZmCPK28 gene. 16. The application according to claim 14, wherein the destruction is achieved by a genome editing system of CRISP/Cas, TALEN, ZFN or other gene editing systems. 17. The application according to claim 10, wherein the regulating plant growth and development includes regulating the plant height. 18. The application according to claim 11, wherein said regulating plants to respond to environmental stress includes regulating plants' tolerance to high light and drought. 19. Use of the nucleic acid molecule of claim 1 or 2, the polypeptide of claim 3 or 4, or the recombinant vector of claim 5 in the breeding of plants with altered traits. 20. Use according to any one of claims 10 to 19, wherein the plant is a monocotyledonous or dicotyledonous plant, preferably a crop plant. 21. The application according to claim 20, wherein the plant is selected from the group consisting of corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats, millet, millet, One or more of tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco or Arabidopsis; Arabidopsis and corn are preferred. 22. A method for regulating plant growth and development, comprising: 1) Introducing the isolated nucleic acid molecule of claim 1 or 2 or the recombinant vector of claim 5 into a target plant to obtain a transgenic plant; and 2) Cultivate the plant, wherein compared with the control plant, the transgenic plant has the properties of tall stalk, high light tolerance, and high nitrogen utilization efficiency. 23. A method for regulating plant growth and development, comprising: 1) Destroy the ZmCPK28 gene in plants or genes homologous to ZmCPK28 genes in other plants to obtain transgenic plants; and 2) Cultivate the plant, wherein the transgenic plant has drought resistance and dwarf properties compared to the control plant. 24. A method for producing a transgenic plant, the transgenic plant comprising the introduced isolated nucleic acid molecule according to claim 1 or 2, or the recombinant vector according to claim 5, or its recombinant expression according to claim 3 or The isolated polypeptide described in 4, is characterized in that the method includes the following steps: 1) Obtain the seeds of the transgenic plants; 2) Plant the seeds to obtain the transgenic plants whose traits can be stably inherited. 25. A method for producing transgenic plants, the transgenic plants comprising disrupted ZmCPK28 genes in plants or genes homologous to ZmCPK28 genes in other plants, characterized in that the method includes the following steps: 1) Obtain the seeds of the transgenic plants; 2) Plant the seeds to obtain the transgenic plants whose traits can be stably inherited. 26. The method of claim 23 or 25, wherein said disruption is achieved by knocking out or knocking down the ZmCPK28 gene. 27. The method of claim 23 or 25, wherein the disruption is achieved by a genome editing system of CRISP/Cas, TALEN, ZFN or other gene editing systems. 28. The method according to any one of claims 22 to 27, wherein the plant is selected from the group consisting of corn, sorghum, soybean, wheat, rice, cotton, brassica, cabbage, rape, mustard, barley, rye, oats , millet, millet, tomato, sunflower, potato, peanut, sweet potato, cassava, sugar beet, sugar cane, tobacco or one or more of Arabidopsis thaliana; Arabidopsis thaliana and corn are preferred. 29. A CRISPR/Cas9 target gDNA, which comprises the nucleotide sequence shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.