Application of transcription factor ZmbZIP27 in regulation and control of corn plant height and ear position height

Abstract

The invention discloses application of a transcription factor ZmbZIP27 in regulation and control of corn plant height and ear height, and belongs to the technical field of plant genetic engineering and corn molecular breeding. The invention reveals that the bZIP transcription factor family member ZmbZIP27 has an important biological function of regulating and controlling the corn plant height and lodging resistance for the first time. A ZmbZIP27 knockout vector is constructed through a CRISPR / Cas9 gene editing technology, and an obtained homozygous mutant strain shows remarkable plant type improvement characteristics compared with a wild type: the plant height is reduced by 8-12%, and the ear height is reduced by 15-22%. And a new gene target and a breeding strategy are provided for creating a new high-yield lodging-resistant corn variety suitable for mechanical harvesting.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and maize molecular breeding technology, specifically to the application of transcription factor ZmbZIP27 in regulating maize plant height and ear height. Background Technology

[0002] corn( Zea mays Maize (L.) is an excellent food and cash crop, playing a vital role in grain and animal feed production due to its strong adaptability, high yield, and short growth cycle. Increasing planting density is a key method to improve maize yield; however, high planting density affects plant architecture, making maize more susceptible to lodging, which not only hinders yield increases but also impacts mechanized harvesting. Plant architecture-related traits, such as plant height (PH) and ear height (EH), directly influence the spatial structure of maize and are limiting factors for further increasing planting density. Plant height and ear height are closely related to maize's lodging resistance and also affect its suitability for mechanized harvesting. At higher planting densities, excessively high plant height and ear height, especially ear height, significantly affect the overall center of gravity of the maize plant, greatly increasing the probability of lodging. Appropriately reducing plant height and ear height can effectively improve maize's lodging resistance, enabling it to adapt to high-density planting conditions and accelerating the mechanized harvesting process. Therefore, exploring the genetic basis of maize plant height and ear height traits and identifying related genes for genetic improvement is of great significance for breeding maize varieties with moderately dwarfed plant height and ear height.

[0003] In recent years, based on methods such as mutant genetic analysis, map-based cloning, and association analysis, several genes regulating maize plant height and ear height have been cloned. Most of these genes are related to hormone synthesis, translocation, and signaling pathways. However, many of these genes are either closely linked to genes that are detrimental to high yield, or these genes themselves have some side effects that are detrimental to high yield. Currently, only a small number of these genes can be used in the breeding of dwarf maize varieties.

[0004] bZIP transcription factors (basic leucine zipper transcription factors) are an important class of DNA-binding proteins in eukaryotes. They recognize specific DNA sequences through their basic regions and form homodimers or heterodimers via leucine zipper structures. They are widely involved in processes such as plant growth and development, stress response, and animal immune defense. Arabidopsis bZIP family genes AtbZIP63 Activation via phosphorylation of SnRK1 kinase regulates energy stress response. Rice OsbZIP71Belonging to the A subgroup of the bZIP transcription factor family, these genes play a crucial role in plant abiotic stress responses by binding to the ABRE cis-elements and activating downstream stress-response genes. A total of 125 genes in the bZIP transcription factor family have been identified in maize, but their application in regulating traits such as plant height and ear height is rarely reported. Summary of the Invention

[0005] The purpose of this invention, in view of the aforementioned prior art, is to provide the application of the transcription factor ZmbZIP27 in regulating maize plant height and ear height. This invention uncovers a novel regulatory factor for maize plant height and ear height, providing a new genetic resource for breeding dwarf maize varieties suitable for high-density planting and mechanized harvesting.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the present invention, the transcription factor ZmbZIP27 is used in either (1) or (2) as follows: (1) Regulating maize plant height; (2) Regulate the height of corn ears; The amino acid sequence of the transcription factor ZmbZIP27 is shown in SEQ ID NO.2.

[0007] This invention has discovered that the transcription factor ZmbZIP27 is closely related to plant height and ear height traits in maize. Inactivating ZmbZIP27 in maize resulted in a slight reduction in plant height and ear height compared to the wild type. By appropriately reducing plant height and ear height, the overall center of gravity of the plant shifts downward, avoiding the problem of excessive torque on the stem due to the weight of the ear caused by a high ear position. This reduces the stem load, lowers the risk of breakage, and thus significantly improves the lodging resistance of maize.

[0008] A second aspect of the invention provides ZmbZIP27 The use of genes in at least one of the following (1)-(5): (1) Regulating maize plant height; (2) Regulate the height of corn ears; (3) Develop lodging-resistant maize varieties; (4) Develop maize varieties suitable for mechanized harvesting; (5) Develop maize varieties suitable for high-density planting; The ZmbZIP27 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] In the above applications, with ZmbZIP27 Genes can be used as targets to improve maize germplasm resources. For example, by... ZmbZIP27 Gene knockout or silencing can appropriately reduce the plant height and ear height of maize, thereby enabling the breeding of lodging-resistant maize varieties that are suitable for high-density planting. Furthermore, by appropriately reducing ear height, the probability of missed harvesting or ear detachment during mechanical harvesting can be decreased, improving the efficiency of mechanized harvesting.

[0010] A third aspect of the invention provides a target ZmbZIP27 The recombinant expression vector of the gene or the genetically engineered bacteria are used in at least one of the following (1)-(5): (1) Regulating maize plant height; (2) Regulate the height of corn ears; (3) Develop lodging-resistant maize varieties; (4) Develop maize varieties suitable for mechanized harvesting; (5) Develop corn varieties suitable for high-density planting.

[0011] In the above applications, the target ZmbZIP27 Recombinant gene expression vectors are targeted ZmbZIP27 Gene knockout vectors or silencing vectors.

[0012] Preferably, the knockout vector is a targeted vector. ZmbZIP27 CRISPR / Cas9 knockout vector for genes.

[0013] A fourth aspect of the present invention provides a method for reducing maize plant height and ear height, comprising the following steps: From corn plants ZmbZIP27 Gene knockout or silencing yields maize mutant lines with reduced plant height and ear height.

[0014] In the above methods, preferably, the CRISPR / Cas9 knockout vector is used to... ZmbZIP27 Gene knockout.

[0015] A fifth aspect of the present invention provides a method for improving the lodging resistance of corn, comprising the following steps: By inhibiting the corn plant ZmbZIP27 Improving maize lodging resistance can be achieved by reducing gene expression or decreasing the activity of transcription factor ZmbZIP27.

[0016] In the above methods, genetic engineering techniques can be used to... ZmbZIP27 Gene knockout or silencing to achieve inhibition ZmbZIP27 The purpose is to reduce gene expression or decrease the activity of transcription factor ZmbZIP27.

[0017] A sixth aspect of the present invention provides a method for breeding dwarf maize varieties suitable for high-density planting and mechanized harvesting, comprising the following steps: From corn plants ZmbZIP27 Gene knockout or silencing yields maize mutant lines with reduced plant height and ear height; By using maize mutant lines with reduced plant height and ear height as parents for self-pollination, or by hybridizing with other dwarf maize plants, dwarf maize varieties suitable for high-density planting and mechanized harvesting can be bred.

[0018] The beneficial effects of this invention are: This invention reveals for the first time members of the bZIP transcription factor family. ZmbZIP27 It possesses important biological functions in regulating maize plant height and lodging resistance. It was constructed using CRISPR / Cas9 gene editing technology. ZmbZIP27 The knockout vector yielded homozygous mutant lines that exhibited significant improvements in plant type compared to the wild type: plant height decreased by 8-12%, and ear height decreased by 15-22%. This provides new gene targets and breeding strategies for creating high-yielding, lodging-resistant maize varieties suitable for mechanized harvesting. Attached Figure Description

[0019] Picture 1 To utilize CRISPR / Cas9 to target genes specifically expressed in the intermeristor of the lower node of the spike. ZmbZIP27 Knockout; among which Picture 1 A in the middle is ZmbZIP27 A diagram illustrating gene structure and the location of knockout. The black bars in the diagram represent... ZmbZIP27 The exons of the gene are represented by black lines, introns by black lines, and a dual-target system is used. The red rectangles represent PAM sites. Picture 1 B is ZmbZIP27 Sequencing peak diagrams of three transgenic knockout lines obtained by three gene editing methods. ZmbZIP27-KO#1 A 1bp deletion occurs, causing a change in the protein sequence; ZmbZIP27-KO#2 A 1bp deletion causes a change in the protein sequence; ZmbZIP27-KO#3 A 4bp deletion occurs, causing a change in the protein sequence.

[0020] Picture 2 The plant height trait was compared between the wild-type (Z58) and knockout mutants. Picture 2 The diagram in center A shows the plant height phenotypes of four plants, from left to right: wild-type Z58, knockout mutant, and so on. ZmbZIP27-KO#1, ZmbZIP27-KO#2 , ZmbZIP27-KO# 3 ; Picture 2 B represents the plant height statistics of the four plant species in the bar chart. Picture 2 The bar chart in section C shows the ear height of the four plants.

[0021] Picture 3 Seed traits of wild-type (Z58) and knockout mutant, among which Picture 3 Image A shows a comparison of seed morphology, from top to bottom: wild-type Z58, knockout mutant. ZmbZIP27-KO#1, ZmbZIP27-KO#2 , ZmbZIP27-KO#3 ; Picture 3 B represents the statistical analysis of particle width; Picture 3 C represents the statistical analysis of grain length; Picture 3 D represents the statistical analysis of 100-grain weight. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] Terminology Explanation: Corn plant height: The vertical distance from the ground surface to the top of the plant (usually the top of the main stem).

[0024] Corn ear height: The height from the ground surface to the uppermost node on the stem where the ear of corn grows.

[0025] As mentioned earlier, maize plant type-related traits, such as plant height (PH) and ear height (EH), directly influence the spatial structure of maize and are limiting factors for further increasing maize planting density. Plant height and ear height are closely related to maize lodging resistance and also affect the suitability for mechanized harvesting. Currently reported genes regulating maize plant height and ear height are either closely linked to genes detrimental to high yield or have inherent side effects that discourage high yields. Only a small number of these genes are currently available for use in the breeding of dwarf maize varieties.

[0026] In view of this, this invention has conducted in-depth research on genes regulating maize plant height and ear height. Transcription factor ZmbZIP27 is a member of the bZIP transcription factor family. ZmbZIP27 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1; the amino acid sequence of the transcription factor ZmbZIP27 is shown in SEQ ID NO.2; as follows: ZmbZIP27 CDS region of a gene: ATGCTGATCTGCGATCTGCAGGGCGCGTACGCAAATGGGAGCATGGGAGACCCAGCTCTCGGATACACAGGGTCGATCGATCCAGGCGTCGCCGCGTTCGAGATCGAATACAGGCACTGGGTGGACGAGCAGAAGCGGCACACGGCGGAGCTGATGTCGGCGCTCCAGGGGCAGCAGACGTCGGAGCTGGAGCTCCGCTTGCTGGTGGAGACGGGGCTCAGCAACTACGAGCACCTCTTCAGGATCAAGGCGCTGGCCGCCAACGCGGACGTGTTCCACGTCATGTCGGGCGTGTGGAAGACCCCCGCCGAGAGGTTCTTCCTCTGGATCGGCGGGTTCAGGCCGTCGGAGGTCCTAAAGATTCTGAGCCCGCAGCTGGAGCCGCTGGCGGAGGCGCAGCGCATGCTCGTGGGCGGCCTCCAGCACACGTCAACGCAGGCGGAGGACGCGCTGTCGCAGGGCATGGAGAAGCTACAGCAGAACCTCGCCGAGATCCTGACCGCCGAGGCTGACCCCTTCGGGGCCCCCGACGCCTACATGCTGCAGATGGCCACTGCGGTGGAGAAGCTGAAAGAGCTCGTCAACTTCGTCACCCAGGTAACCGTCCATCCATCTCCTCCATGGATGGAACGCAAACGCAGGGCCTGGTCTGGAACGCACGCGCCGTGTCGTGTGGACGTGTGGTGTCCCTGTGGCTCACCCGTGCGGGTCTCTGCTCGGCTATGCTGTGCTGTGCAGGCGGACCATCTCCGGCTGATGACGCTGCAACAGATGCACAAGATCCTGACGACGCGGCAGGCGGCCAGGGGCCTGCTGGCGCTCGGCGACTACTTCCAGCGCCTCCGCACGCTGAGCTCGCTGTGGGCGGCGCGCCCGCGGGAGGCGGCGATTAGCTAG。

[0027] Transcription factor ZmbZIP27: MLICDLQGAYANGSMGDPALGYTGSIDPGVAAFEIEYRHWVDEQKRHTAELMSALQGQQTSELELRLLVETGLSNYEHLFRIKALAANADVFHVMSGVWKTPAERFFLWIGGFRPSEVLKILSPQLEPLAEAQRMLVGGLQHTSTQAED ALSQGMEKLQQNLAEILTAEADPFGAPDAYMLQMATAVEKLKELVNFVTQVTVHPSPPWMERKRRAWSGTHAPCRVDVWCPCGSPVRVSARLCCAVQADHLRLMTLQQMHKILTTRQAARGLLALGDYFQRLRTLSSLWAARPREAAIS.

[0028] The function of the transcription factor ZmbZIP27 is rarely reported. This invention constructs... ZmbZIP27 Gene knockout mutants, for ZmbZIP27 The function of the genes was studied, and the results showed that compared with the wild type, ZmbZIP27 The plant height and ear height of the gene knockout mutant are appropriately reduced. Therefore, ZmbZIP27 The gene can serve as a new regulatory gene for plant height and ear height, providing new genetic resources for creating dwarf maize varieties suitable for high-density planting and mechanized harvesting.

[0029] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0030] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, they are generally performed under conventional conditions, such as those described in J. Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual* (3rd Edition), Science Press, 2002; D.L. Spector et al., eds., *Cellular Laboratory Manual*, Science Press, 2001; or according to the conditions recommended by the manufacturer.

[0031] Example 1: ZmbZIP27 Construction of gene knockout mutants 1. Construction ZmbZIP27 CRISPR / Cas9 knockout vector: (1) Target design: Target gene ZmbZIP27The CDS sequence of the gene (shown in SEQ ID NO.1) was used to design knockout targets. Specifically, two 19bp specific sequences were selected from the CDS sequence of the target gene. The target was then predicted on the website (http: / / crispor.tefor.net / ) and a suitable target was selected (Table 1).

[0032] Table 1: Target Sequences (2) Primer design: Primers were designed based on the selected target sites, and the designed primer sequences are shown in Table 2.

[0033] Table 2: Primer sequences (3) PCR amplification: Using pBUE411 as a template, the target fragment was amplified using the designed primers (Table 2) and the PCR amplification system (Table 3). The PCR product was then recovered and purified. The pBUE411 empty vector plasmid was linearized using the KspAI restriction endonuclease from Thermo Scientific. Recombination ligation was then performed according to the recombination ligation reaction system prepared in Table 4, incubated at 50°C for 30 min, and then placed on ice.

[0034] Table 3: PCR amplification system Table 4: Recombinant Linkage Reaction System After ligation, all ligation products were transformed into *E. coli* TOP10 competent cells and incubated upside down at 37°C for 16 hours. Single colonies were then selected, positive clones were screened, and sent to the company for sequencing verification, thus constructing the desired product. ZmbZIP27 Gene CRISPR / Cas9 knockout vector.

[0035] 2. Agrobacterium-mediated genetic transformation: (1) Preparation of recombinant Agrobacterium: Remove 50 μL of Agrobacterium EHA105 competent cells from a -80°C freezer and thaw them in ice for 5 min. Add 1 μL of the constructed knockout vector to a centrifuge tube, gently tap to mix, and incubate on ice for 5 min. Quickly freeze in liquid nitrogen for 5 min, then incubate in a 37°C water bath for 5 min, followed by a rapid immersion in ice for 5 min. Transfer 1 mL of YEP to a centrifuge tube and incubate in a 28°C shaker at 180 rpm for 1 h. Then centrifuge at 5000 rpm for 1 min. In a clean bench, discard most of the supernatant, resuspend the culture, and transfer it to pre-prepared YEP medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin). Spread evenly, seal the plate, and invert it in a 28°C incubator for 48-72 h before identification to obtain recombinant Agrobacterium.

[0036] (2) Genetic transformation: Using maize inbred line Zheng 58 (Z58) as material, Agrobacterium-mediated genetic transformation was performed. The T0 generation was screened to identify CRISPR / Cas9-induced target gene mutations. Self-pollination was conducted from the T0 to T2 generations, followed by herbicide screening. DNA was extracted from leaves of transplanted plants and analyzed by PCR.

[0037] Ultimately, three different editing types were obtained, and CRISPR / Cas9 transgenic knockout lines that could stably inherit the Cas9 vector backbone were named respectively. ZmbZIP27-KO#1 , ZmbZIP27-KO#2 and ZmbZIP27-KO#3 Sequence alignment with the wild type was performed. ZmbZIP27-KO#1 A 1bp deletion was found at target 1; ZmbZIP27-KO#2 A 1bp deletion was found at target 1; ZmbZIP27-KO#3 A 4bp deletion occurred at target point 1, resulting in ZmbZIP27 Changes in protein structure lead to ZmbZIP27 Functional deficiency ( Picture 1 ).

[0038] Example 2: ZmbZIP27 Phenotypic assessment of gene knockout mutants 1. Test method: Wild-type maize (Z58) and the T2 generation obtained in Example 1 were planted in the field. ZmbZIP27-KO#1, ZmbZIP27- KO#2 as well as ZmbZIP27-KO#3 Seeds of the knockout mutant were removed, and plant height and ear height phenotypes were recorded at 70 days of growth. After ear maturity, grain traits such as grain length, grain width, and 100-grain weight were measured.

[0039] 2. Test Results: The results showed that, compared to WT, ZmbZIP27-KO#1 , ZmbZIP27-KO#2 as well as ZmbZIP27-KO#3 The plant height and ear height of mature plants from these three homozygous edited lines were significantly reduced. Picture 2 The results showed that by knocking out ZmbZIP27 Genes can reduce corn plant height and ear height, thereby improving corn's resistance to lodging.

[0040] Compared to wild type (WT), ZmbZIP27-KO#1 The grain width of homozygous edited lines was significantly increased, while ZmbZIP27- KO#2 The grain width of the strain was significantly reduced. ZmbZIP27-KO#3 The strains showed no significant changes. Regarding grain length, ZmbZIP27-KO#3 The number of strains increased significantly, while ZmbZIP27-KO#1 and ZmbZIP27-KO#2 No significant differences were observed between the strains. Furthermore, the 100-seed weight of all three edited strains was not significantly different from the wild type. Picture 3 ).

[0041] In summary, knockout ZmbZIP27 The gene can effectively reduce maize plant height and ear height, enhancing lodging resistance; while maintaining the stability of yield-related traits. This result indicates that... ZmbZIP27 Gene-edited lines have the potential to improve maize's tolerance to high-density planting, and are expected to increase yields through reasonable high-density planting.

[0042] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of transcription factor ZmbZIP27 in the following (1) or (2): (1) Regulating maize plant height; (2) Regulate the height of corn ears; The amino acid sequence of the transcription factor ZmbZIP27 is shown in SEQ ID NO.

2.

2. ZmbZIP27 The use of genes in at least one of the following (1)-(5): (1) Regulating maize plant height; (2) Regulate the height of corn ears; (3) Develop lodging-resistant maize varieties; (4) Develop maize varieties suitable for mechanized harvesting; (5) Develop maize varieties suitable for high-density planting; The ZmbZIP27 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.

2.

3. Targeted ZmbZIP27 The recombinant expression vector of the gene or the genetically engineered bacteria are used in at least one of the following (1)-(5): (1) Regulating maize plant height; (2) Regulate the height of corn ears; (3) Develop lodging-resistant maize varieties; (4) Develop maize varieties suitable for mechanized harvesting; (5) Develop corn varieties suitable for high-density planting.

4. The application according to claim 3, characterized in that, The target ZmbZIP27 Recombinant gene expression vectors are targeted ZmbZIP27 Gene knockout vectors or silencing vectors.

5. The application according to claim 4, characterized in that, The knockout vector is a targeted ZmbZIP27 CRISPR / Cas9 knockout vector for genes.

6. A method for reducing maize plant height and ear height, characterized in that, Includes the following steps: From corn plants ZmbZIP27 Gene knockout or silencing can produce maize mutant lines with reduced plant height and ear height.

7. The method according to claim 6, characterized in that, Using CRISPR / Cas9 knockout vectors to ZmbZIP27 Gene knockout.

8. A method for improving the lodging resistance of corn, characterized in that, Includes the following steps: By inhibiting the corn plant ZmbZIP27 Improving maize lodging resistance can be achieved by reducing gene expression or decreasing the activity of transcription factor ZmbZIP27.

9. A method for breeding dwarf maize varieties suitable for high-density planting and mechanized harvesting, characterized in that, Includes the following steps: From corn plants ZmbZIP27 Gene knockout or silencing yields maize mutant lines with reduced plant height and ear height; By using maize mutant lines with reduced plant height and ear height as parents for self-pollination, or by hybridizing with other dwarf maize plants, dwarf maize varieties suitable for high-density planting and mechanized harvesting can be bred.

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