Zmereb16 protein mutant for regulating plant type and cold tolerance of corn and application thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a ZmEREB16 protein mutant for regulating maize plant type and cold tolerance and application thereof. The ZmEREB16 protein mutant is obtained by introducing BTA transposon insertion, the mutant only affects the translation efficiency of ZmEREB16 protein, and does not affect the amino acid coding sequence of the protein conservative functional domain. Under the drive of a constitutive strong promoter, the protein expression level is lower than the expression intensity when the wild type ZmEREB16 is overexpressed, and the plant lethality problem is avoided. The ZmEREB16 protein mutant overexpression strain provided by the present application effectively maintains the stress resistance while reducing the negative impact on the growth state, whether applied to an inbred line or a hybrid background, the overall plant height is significantly reduced, and the normal growth and development process can be completed, which can provide ideal test materials for subsequent stress resistance trait improvement and plant type improvement research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a ZmEREB16 protein mutant that regulates maize plant architecture and cold tolerance, and its applications. Background Technology

[0002] Maize possesses multiple values, including food production, industrial processing, and feed applications. Therefore, conducting research on maize gene function analysis, trait improvement, and molecular breeding is crucial for enhancing maize yield, stress resistance, and quality. Current research indicates that plant resistance to low-temperature stress primarily relies on two core signaling pathways: the CBF (C-Repeat Binding Factor)-dependent signaling pathway and the CBF-independent signaling pathway. Among these, the CBF-dependent signaling pathway plays a vital and dominant role in regulating plant low-temperature tolerance and cold stress response, and is a core target for genetic improvement of crop cold tolerance.

[0003] In maize breeding, cultivating a high-light-efficiency plant type suitable for dense planting is a key strategy for increasing yield. Reducing plant height and ear height effectively enhances lodging resistance and adapts to mechanized operations; shortening the length of leaves above the ear and decreasing the leaf angle improves light penetration and ventilation in the lower and middle parts of the canopy, optimizing canopy light energy utilization efficiency. Therefore, synergistically regulating plant type traits such as plant height, ear height, leaf length above the ear, and leaf angle is of significant practical importance for breeding high-yielding maize varieties that tolerate dense planting. Furthermore, combining plant type optimization with improved cold tolerance helps achieve simultaneous breakthroughs in yield potential and yield stability.

[0004] However, while CBF transcription factors activate the expression of downstream cold-tolerant genes, they often have adverse effects on the normal growth and development of plants. Conventional CBF overexpression strategies usually lead to pleiotropic negative phenotypes such as severe dwarfing and slow growth, resulting in a difficult-to-break negative association between cold tolerance and ideal plant type, which seriously restricts the application of the CBF pathway in maize genetic improvement. Summary of the Invention

[0005] In view of this, the present invention proposes a ZmEREB16 protein mutant that regulates maize plant architecture and cold tolerance and its application.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides a ZmEREB16 protein mutant (named TE166), the amino acid sequence of which is shown in SEQ ID NO:4.

[0008] Secondly, the present invention provides a nucleic acid encoding the mutant of the said protein.

[0009] Thirdly, the present invention provides an expression cassette, vector, or host cell containing the nucleic acid.

[0010] Fourthly, this invention provides the application of biomaterials overexpressing the ZmEREB16 protein mutant in regulating maize plant architecture and / or enhancing cold tolerance, wherein the amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:4, and the biomaterial is any one of the following:

[0011] (a1) An expression cassette expressing the mutant of the protein;

[0012] (a2) An expression cassette containing the said nucleic acid;

[0013] (a3) The vector for expressing the protein mutant described in the study;

[0014] (a4) A vector containing the nucleic acid;

[0015] (a5) Host cells expressing the mutant of the protein;

[0016] (a6) A host cell containing the nucleic acid;

[0017] (a7) A host cell containing any of the biological materials (a1)-(a4).

[0018] Furthermore, the regulation of maize plant architecture includes any of the following:

[0019] (b1) Reduce plant height;

[0020] (b2) Reduce ear height;

[0021] (b3) Reduce the length of leaves above the spike;

[0022] (b4) Reduce the leaf angle.

[0023] Furthermore, the application is in the improvement of maize plant type and / or cold tolerance germplasm resources.

[0024] Fifthly, the present invention provides a method for regulating maize plant architecture and / or improving cold tolerance, comprising the following steps:

[0025] S1. Construct a recombinant vector overexpressing the ZmEREB16 protein mutant, and transfer the recombinant vector into wild-type maize plants to obtain transgenic plants;

[0026] S2. Select plants from the transgenic plants that have reduced plant height and / or increased cold tolerance compared with wild type;

[0027] The amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:4.

[0028] Furthermore, step S1 also includes: driving the overexpression of the ZmEREB16 protein mutant with the Ubi promoter.

[0029] Sixthly, the present invention provides a method for blocking the lethal effect caused by overexpression of wild-type ZmEREB16.

[0030] A recombinant vector overexpressing the ZmEREB16 protein mutant was constructed, wherein the recombinant vector contains a nucleic acid sequence encoding the ZmEREB16 protein mutant driven by the Ubi promoter;

[0031] The protein expression level of the mutant was lower than that of wild-type ZmEREB16 overexpression, thereby blocking the lethal effect caused by wild-type ZmEREB16 overexpression.

[0032] The amino acid sequence of the wild-type ZmEREB16 is shown in SEQ ID NO:2; the amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:4.

[0033] The beneficial effects of the present invention include at least the following:

[0034] This invention introduces a BTA fragment to obtain a mutated ZmEREB16 sequence, which is then operatively linked to the Ubi promoter. This results in a lower protein expression level driven by the promoter compared to wild-type ZmEREB16 overexpression. This strategy achieves constitutive expression while avoiding plant lethality, and still maintains a significantly higher expression level of the target protein than the wild type. Ultimately, this yields an overexpression line capable of normal growth and development, meeting the needs of breeding creation and molecular mechanism research.

[0035] From the perspective of plant growth, development, and stress tolerance, the ZmEREB16 protein mutant overexpression lines provided by this invention effectively balance stress resistance and growth status. This material retains the excellent stress-resistance physiological characteristics of the ZmEREB16 gene while successfully avoiding the lethal defects caused by high-dose expression. Whether applied to inbred lines or hybrids, the overall plant height is significantly reduced, and the plants exhibit robust growth, completing normal growth and development. This provides ideal experimental material for subsequent research on stress resistance and plant type improvement. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This study analyzed the sequence characteristics of TE166 (BTA transposon insertion ZmEREB16 mutant) and the expression levels of overexpression materials. Specifically, A shows the sequence alignment of wild-type ZmEREB16 CDS with the TE166 mutant CDS; B shows the amino acid sequence alignment of wild-type ZmEREB16 CDS with the TE166 mutant protein; C shows the effect of the BTA transposon insertion mutation on the function of ZmEREB16 protein using a dual-luciferase reporter assay; D shows the relative expression levels of ZmEREB16 mRNA in wild-type (WT) and two independent TE166 overexpression (OE) lines using quantitative real-time PCR (qPCR); and E shows the relative expression levels of ZmEREB16-Flag protein in wild-type and two independent TE166 overexpression lines using Western blot.

[0038] Figure 2 Plasmid map of the overexpression vector TE166-ZmEREB16-Flag;

[0039] Figure 3 Phenotypic analysis of the ZmEREB16 overexpressing line TE166 under cold treatment was performed. A shows the phenotypes of the ZmEREB16 overexpressing line and wild-type (WT) seedlings before and after cold treatment; each red seedling pot was sown with 9 corn kernels; the scale bar was 2 cm. B shows the statistical analysis of the survival rate of ZmEREB16 overexpressing line (OE) and wild-type (WT) seedlings after cold treatment. Differences were analyzed using t-tests, with a maximum value of **** and P < 0.0001. Error bars represent the standard error (SEM) of the mean from three pots (biological replicates).

[0040] Figure 4 This study analyzed the field phenotypic characteristics of the ZmEREB16 overexpressing line TE166 in Gansu Province. Figure A shows photographs of mature plants of the ZmEREB16 overexpressing line and the WT material; "25GS" indicates planting in Gansu Province, with a scale bar of 1 m. Figure B shows the statistical analysis of plant height, ear height, above-ear leaf length, leaf width, and leaf angle of the ZmEREB16 overexpressing line and the WT material. Differences were analyzed using t-tests: n = 15; *, P > 0.05; **, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

[0041] Figure 5This study analyzed the field phenotypic characteristics of the ZmEREB16 overexpressing line TE166 and its hybrid line (F1 generation obtained by crossing the ZmEREB16 overexpressing line with the B73 inbred line) in Hainan Province. In Figure A, mature plants of different materials are photographed; "25SY" represents plants grown in Sanya City, Hainan Province. The scale bar is 1 m. Figure B shows the statistical analysis of plant height, ear height, ear leaf length, leaf width, and leaf angle. Differences were analyzed using t-tests: n = 15; *, P > 0.05; **, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Reagents or instruments used, unless otherwise specified, are all commercially available products. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or according to the manufacturer's recommendations.

[0043] the term :

[0044] "Corn" refers to any corn plant and includes all plant varieties that can be bred with corn, including the whole plant, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which the plant can regenerate, plant callus, and complete plant cells in a plant or plant part, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stem, root, root tip, anther, etc.

[0045] "Plant height": refers to the height from the base of the plant stem to the tip of the tassel. In a specific implementation plan, the height from the base of the plant stem to the tip of the tassel is measured after pollination. Plant height can be determined using conventional methods known to those skilled in the art. "Reduced plant height": refers to the plant height of transgenic plants containing the target gene being lower than that of ordinary plants without the target gene, exhibiting a dwarfing phenotype.

[0046] "Ear height" refers to the height from the base of the peduncle of the first ear of the plant to the ground. In the specific implementation plan, the ear height of corn is measured after pollination. "Ear height reduction" means that the ear height of the transgenic plant containing the target gene is lower than that of the ordinary plant without the target gene.

[0047] "Leaf length above the ear": refers to the length of the first leaf above the node where the first ear of corn grows (usually measured as the distance from the leaf base to the leaf tip); "Leaf length above the ear reduced": refers to the length of the leaf above the ear of transgenic plants containing the target gene being lower than that of ordinary plants that have not been introduced with the target gene.

[0048] "Width of leaf above ear": refers to the width of the first leaf above the node where the first ear of corn grows (usually measured by the lateral width at the widest point of the leaf); "Reduced length of leaf above ear": refers to the length of the leaf above ear of transgenic plants containing the target gene being lower than that of ordinary plants that have not been introduced with the target gene.

[0049] "Leaf angle": refers to the angle between the midrib of the leaf on the corn ear and the stem; "Leaf angle reduced": refers to the leaf angle of the transgenic plant containing the target gene being smaller than that of the ordinary plant without the target gene.

[0050] "Nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides and hybridize with single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.

[0051] When the terms “encoded” or “encoded” are used in the context of a particular nucleic acid, it means that the nucleic acid contains the necessary information to guide the translation of that nucleotide sequence into a particular protein.

[0052] The terms “peptide” and “protein” are used interchangeably in this document to refer to polymers of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers.

[0053] The terms “vector” and “plasmid” are used interchangeably in this article: to refer to a nucleic acid molecule that is capable of autonomously replicating and / or expressing a target polynucleotide operatively linked to it in a suitable host cell.

[0054] The term "recombinant vector" refers to a recombinant nucleic acid construct obtained by inserting a foreign nucleic acid sequence (such as an expression cassette, target gene, etc.) into a vector backbone through genetic engineering, which can autonomously replicate and / or express the protein encoded by the foreign nucleic acid sequence in host cells.

[0055] "Expression cassette": refers to a nucleic acid construct containing a nucleic acid sequence encoding a target protein and expression regulatory elements necessary for transcription and / or translation of that nucleic acid sequence in a host cell. These expression regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyA signals. The expression cassette can be a separate linear nucleic acid fragment or integrated into a vector or the host cell genome.

[0056] The term "host cell" refers to a cell that has been introduced into or is capable of accepting exogenous nucleic acid molecules (such as recombinant vectors, expression cassettes, etc.), including prokaryotic and eukaryotic cells. The host cell includes, but is not limited to, Escherichia coli cells, Agrobacterium cells, yeast cells, and plant cells (such as corn cells). The term "containing" includes various forms of exogenous nucleic acid molecules, such as transient presence in the host cell, integration into the host cell genome, or presence in the host cell as episomes.

[0057] The term "overexpression" (OE) refers to the phenomenon where, after artificial modification of a target gene in a host cell or organism, its transcription and / or translation levels are higher than the normal expression levels of the gene in an unmodified control (such as wild-type). Overexpression can be achieved by operatively linking the target gene to a strong promoter, increasing the gene copy number, optimizing codons, using enhancers, or other means known in the art to improve gene expression levels. The terms "overexpression," "over-expression," and "excessive expression" are used interchangeably.

[0058] The term "expression" or "overexpression" of ZmEREB16 refers to the expression / overexpression of ZmEREB16, and / or the expression / overexpression of a ZmEREB16 protein mutant, with the specific meaning determined by the context. For example, in transgenic plants involving overexpression of a ZmEREB16 protein mutant, it should be understood that the transgenic plants contain both normal expression of the endogenous ZmEREB16 gene and overexpression of the exogenous gene encoding the ZmEREB16 protein mutant driven by the introduced recombinant vector. The expression of the endogenous gene is controlled by its own regulatory sequences and is not directly affected by the insertion of the exogenous vector.

[0059] Table 1 Sequence Information Table

[0060]

[0061]

[0062] 1. Obtaining the maize ZmEREB16 gene

[0063] Homology comparisons of the protein sequences of Arabidopsis AtCBFs (AtCBF1, AtCBF2, ATCBF3) in maize preliminarily identified members of the maize CBF homologous gene family, including ZmEREB16. Multiple sequence alignment and phylogenetic analysis using MEGA software revealed that although several candidate genes closely related to Arabidopsis and induced by low temperature existed, only ZmEREB16 (Zm00001eb250560) exhibited overexpression lethality among the three most homologous maize genes. The nucleotide sequence of its coding sequence (CDS) is shown in SEQ ID NO:1.

[0064] To analyze how ZmEREB16 gene overexpression affects plant growth and development, this invention creates ZmEREB16 overexpression materials.

[0065] 2. Obtaining ZmEREB16 overexpression materials

[0066] While previous studies have indicated that persistent high-intensity overexpression of CBF family genes can lead to pleiotropic adverse phenotypes such as dwarfing, delayed growth period, and malformed plant structure, the problem is more extreme in maize: although the maize ZmEREB16 gene is homologous to Arabidopsis CBF, it exhibits significant species-specific functional differences. In maize, the conventional Ubi constitutive strong promoter (pZmUbi1) exhibits a lethal effect, making it impossible to obtain positive transgenic materials. This renders the idea of ​​elucidating its function and breeding cold-resistant germplasm through traditional overexpression methods impossible.

[0067] Existing maize overexpression systems are highly dependent on a single pZmUbi1 promoter, whose expression activity is constant and its intensity is not adjustable. It only has a binary regulatory mode of "all on" or "all off", lacking a flexible and controllable expression regulation mechanism.

[0068] To obtain constitutively high expression plants, this invention attempted to insert a BTA transposon sequence into the 5' end of the ZmEREB16 gene. BTA stands for B73 active transposable element, a hAT family active transposon originating from the maize inbred line B73. The obtained BTA transposon insertion mutant sequence (as shown in SEQ ID NO:3, i.e., the TE166 sequence) was compared with the original ZmEREB16 gene sequence. The results showed that this newly inserted sequence introduced a new transcription start site within the gene, altering only the coding of the first 40 amino acids of the ZmEREB16 protein itself, without affecting the subsequent amino acid sequence, and without causing mutations in conserved domains. Figure 1 A and 1B).

[0069] (1) Verification of the inhibitory effect of ZmEREB16 expression

[0070] Two dual-luciferase reporter vectors were subsequently constructed using the pGreenII 0800-LUC vector. Both vectors used a 35S promoter to drive the target fragments: the control vector contained the original ZmEREB16 sequence (SEQ ID NO:1), which was fused with firefly luciferase (LUC) after removing the stop codon; the experimental vector contained the TE166 sequence at the same position (as shown in SEQ ID NO:3), which was also fused with firefly luciferase (LUC) after removing the stop codon. The plasmids were transformed into maize protoplasts and cultured overnight at 28°C for 14 h. Fluorescence values ​​were measured using the Novizan Dual Luciferase Reporter Assay Kit (DL101), and expression levels were quantitatively compared using the LUC / REN ratio.

[0071] The results showed that, driven by the 35s promoter, the insertion of the BTA fragment significantly suppressed the expression level of ZmEREB16 compared with the original ZmEREB16 sequence overexpression level. Figure 1 C).

[0072] (2) Construction, genetic transformation and strain identification of overexpression vector TE166-ZmEREB16-Flag

[0073] The plant expression vector WMV014-Flag, disclosed in patent CN119331070A, was used as the backbone. The TE166 sequence (SEQ ID NO:3) was modified by removing the terminator via BamHI restriction site and inserted downstream of the Ubi promoter to construct the overexpression vector TE166-ZmEREB16-Flag, which fused a 3×Flag tag to its C-terminus (plasmid map shown). Figure 2 (as shown), and transformed into the maize KN5585 inbred line. After resistance screening and molecular identification, two independent T2 generation overexpression lines were finally obtained, namely TE166#1 and TE166#2 (OE lines).

[0074] qPCR analysis showed that the relative expression levels of ZmEREB16 in TE166#1 and TE166#2 were significantly higher than those in the wild-type WT control. Figure 1 D). The sequences of ZmEREB16 and the internal control primers are shown in Table 1 as SEQ ID NO:5-8. Western blotting results showed that in the overexpression line, the expected molecular weight fusion protein band could be detected using the Flag antibody, while no such band was found in the WT control. Figure 1 E).

[0075] 3. Low-temperature tolerance test

[0076] The OE plants and their corresponding WT plants obtained above were planted in a PERCIVAL incubator (model LT-41VLC8). The incubator temperature was set at 24℃, the photoperiod was 12h light followed by 12h dark, and the light intensity was 100%. When the plants reached the two-leaf-one-heart stage, the incubator temperature was adjusted to 4℃ while keeping other parameters unchanged. Low-temperature treatment was performed under dark conditions for 5 days. Subsequently, the plants were transferred to 24℃ for recovery culture for 6 days under dark conditions, and the plant survival rate was measured. The plant survival rate was used to evaluate the plant's cold tolerance.

[0077] The results showed that almost all wild-type ZmEREB16 controls died, while the survival rate of the overexpressing lines was significantly improved. The survival rate of OE1 (TE166#1) was approximately 50%, and the survival rate of OE2 (TE166#2) was approximately 80%, with highly significant differences. Figure 3 This indicates that the ZmEREB16 gene plays a key positive regulatory role in the low-temperature response of maize.

[0078] 4. Phenotypic detection of plant growth and development

[0079] For field agronomic trait measurements, we selected two ZmEREB16 overexpression lines (TE166#1 and TE166#2, i.e., OE1 and OE2) and planted them in two different regions for phenotypic measurements. Once the plants reached flowering stage, we statistically analyzed plant height, ear height, leaf length, leaf width, and leaf angle.

[0080] (1) Planting location and time: Planted in the summer of 2025 at the Zhangye Experimental Farm of Gansu Academy of Agricultural Sciences, Ganzhou District, Zhangye City, Gansu Province.

[0081] The results are as follows Figure 4 As shown, in the two ZmEREB16 overexpression lines (i.e., OE1 and OE2):

[0082] The plant height is about 100-125 cm, which is significantly lower than the wild type's 200-215 cm by about 75-115 cm.

[0083] The ear height is about 40-60 cm, which is significantly lower than the wild type's 100-105 cm by about 40-65 cm;

[0084] The length of the spikelet leaves is about 50-60 cm, which is significantly reduced by about 10-20 cm compared to the 60-70 cm of the wild type.

[0085] There was no significant difference in leaf width in OE1, but in OE2, the leaf width was about 100 mm, which was significantly reduced by about 10-15 mm compared to the wild type's 110-115 mm.

[0086] The leaf angle is about 30°, which is significantly lower than the wild type's 32-34° by about 2-4°.

[0087] The above results indicate that ZmEREB16 overexpression leads to a significant reduction in plant height, and it plays an important role in maize growth and development.

[0088] (2) Planting location and time: Winter 2025 at the Nanfan Base in Sanya City, Hainan Province

[0089] The results are as follows Figure 5 As shown, in the two ZmEREB16 overexpression lines (i.e., OE1 and OE2):

[0090] The plant height is about 100 cm, which is significantly lower than the wild type's 200-220 cm by about 100-120 cm;

[0091] The ear height is about 30-40 cm, which is significantly lower than the wild type's 75-90 cm by about 35-50 cm;

[0092] The leaves are about 60 cm long, which is significantly shorter than the wild type's 70-80 cm by about 10-20 cm.

[0093] There was no significant difference in leaf width in the OE1 group; in the OE2 group, the leaf width was about 80 mm, which was significantly reduced by about 10-20 mm compared to the wild type's 90-100 mm.

[0094] The leaf angle is approximately 26-30°, which is significantly lower than the wild type's 36-38° by about 6-18°.

[0095] Comparative analysis revealed that the core regulatory effect of ZmEREB16 overexpression on maize plant architecture was highly conserved in both regions, but environmental dependence also existed. In Hainan, the plant height and ear height of the OE material were lower than in Gansu. Furthermore, the overexpressing line was crossed with the B73 inbred line to obtain the F1 generation, which was then planted in a 25SY experimental field. Phenotypic observations showed that ZmEREB16 also inhibited agronomic traits such as plant height in the hybrids. Plants carrying the OE allele were significantly shorter in height (approximately 30 cm) than the wild type, with a corresponding decrease in leaf angle of approximately 6°. Combined with the field agronomic traits observed at different planting sites and for different materials, it can be concluded that ZmEREB16 overexpression significantly reduces maize plant height, playing an important and stable role in the regulation of maize growth and development.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ZmEREB16 protein mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO:

4.

2. The nucleic acid encoding the protein mutant of claim 1.

3. An expression cassette, vector, or host cell comprising the nucleic acid of claim 2.

4. The application of biomaterials overexpressing the ZmEREB16 protein mutant in regulating maize plant architecture and / or enhancing cold tolerance, characterized in that... The amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:4, and the biological material is any one of the following: (a1) An expression cassette for expressing the protein mutant as described in claim 1; (a2) An expression cassette containing the nucleic acid as described in claim 2; (a3) A vector expressing the protein mutant as described in claim 1; (a4) A vector containing the nucleic acid as described in claim 2; (a5) Host cells expressing the protein mutant as described in claim 1; (a6) A host cell containing the nucleic acid as described in claim 2; (a7) A host cell containing any of the biological materials (a1)-(a4).

5. The application as described in claim 4, characterized in that, The regulation of maize plant architecture includes any of the following: (b1) Reduce plant height; (b2) Reduce ear height; (b3) Reduce the length of leaves above the spike; (b4) Reduce the leaf angle.

6. The application as described in claim 4, characterized in that, The application is in the improvement of maize plant type and / or cold tolerance germplasm resources.

7. A method for regulating maize plant architecture and / or improving cold tolerance, characterized in that, Includes the following steps: S1. Construct a recombinant vector overexpressing the ZmEREB16 protein mutant, and transfer the recombinant vector into wild-type maize plants to obtain transgenic plants; S2. Select plants from the transgenic plants that have reduced plant height and / or increased cold tolerance compared with wild type; The amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:

4.

8. The method as described in claim 7, characterized in that, Step S1 also includes: driving the overexpression of the ZmEREB16 protein mutant with the Ubi promoter.

9. A method for blocking the lethal effect induced by overexpression of wild-type ZmEREB16, characterized in that, A recombinant vector overexpressing the ZmEREB16 protein mutant was constructed, wherein the recombinant vector contains a nucleic acid sequence encoding the ZmEREB16 protein mutant driven by the Ubi promoter; The protein expression level of the mutant was lower than that of wild-type ZmEREB16 overexpression, thereby blocking the lethal effect caused by wild-type ZmEREB16 overexpression. The amino acid sequence of the wild-type ZmEREB16 is shown in SEQ ID NO:2; the amino acid sequence of the ZmEREB16 protein mutant is shown in SEQ ID NO:4.

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