Application of a maize transcription factor ZmERF1a and its encoding gene in regulating plant grain yield

Abstract

The present invention discloses the application of a maize transcription factor ZmERF1a and its coding gene in regulating plant grain yield, belonging to the technical fields of molecular biology and genetic engineering. The present invention screens, through DNA pull down and mass spectrometry cloning, a transcription factor ZmERF1a that regulates maize kernel weight and its coding gene ZmERF1a, the amino acid sequence of which is as shown in SEQ ID NO.2. The present invention verifies that the ZmERF1a protein can bind to the promoter of the ZmNAD gene and regulate the hundred-kernel weight of maize. The transgenic experimental results of the present invention show that in Arabidopsis thaliana plants overexpressing ZmERF1a, the overexpression of the ZmERF1a gene can increase the thousand-kernel weight of grains by 14.8% - 37.4%. The transcription factor ZmERF1a provided by the present invention has broad application prospects and huge economic benefits in regulating maize grain yield.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and genetic engineering, and in particular to application of a corn transcription factor ZmERF1a and a coding gene thereof in regulating plant grain yield. Background Art

[0002] Corn (Zea mays L.) is a major global food crop and a crucial source of human nutrition, animal feed, and bioenergy. According to statistics, my country's corn planting area reached 44,219,000 hectares in 2023, with a total output of 288.84 million tons, ranking first among food crops in both sown area and total output (National Bureau of Statistics, 2024). However, with socioeconomic development, market demand for corn continues to grow. Simultaneously, constraints such as population, land resources, and the environment pose greater pressure to further increase corn grain yield. Kernel weight is a key trait affecting corn yield, and conventional breeding to increase corn grain yield has currently reached a bottleneck.

[0003] Transcription factors are protein molecules that specifically bind to cis-acting elements in the promoter region of eukaryotic genes, thereby regulating the expression of target genes. Plant growth and development are generally regulated by multiple genes. Transcription factors are activated through a series of signaling pathways, binding to corresponding cis-acting elements and activating the RNA polymerase II transcription complex, thereby initiating the transcriptional expression of the corresponding functional gene. AP2 / ERF (APETALA2 / ethylene-responsive factor) is a family of transcription factors widely found in plants. Proteins in this family contain an AP2 / ERF domain, possess DNA-binding functions, and are closely related to plant growth and development. Because the functions of AP2 / ERF family transcription factors are complex and they participate in diverse signal transduction pathways, further research is needed to understand how genes in this family regulate maize kernel weight by participating in various signaling pathways and regulating the expression of downstream genes.

[0004] With the rapid development of molecular biology, technologies such as molecular marker-assisted selection breeding and genetic engineering breeding have been widely used in corn breeding research. Finding functional genes that control corn kernel weight is the key to carrying out molecular breeding and genetic engineering breeding for high-yield corn. Summary of the Invention

[0005] The purpose of the present invention is to provide a maize transcription factor ZmERF1a and its encoding gene for use in regulating plant grain yield to solve the problems existing in the above-mentioned prior art. The present invention screened out a transcription factor ZmERF1a that regulates maize grain weight through DNA pull-down and mass spectrometry identification. By introducing or improving this transcription factor, the expression of a series of grain weight-related genes can be regulated, thereby increasing maize yield and providing genetic resources for breeding high-yield maize varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of a maize transcription factor ZmERF1a in any one of the following:

[0008] (1) Application in regulating plant grain yield;

[0009] (2) Application in cultivating transgenic plants with high grain yield;

[0010] (3) Application in preparing products for increasing plant grain yield;

[0011] The amino acid sequence of the maize transcription factor ZmERF1a is shown in SEQ ID NO.2.

[0012] The present invention also provides an application of a coding gene of the maize transcription factor ZmERF1a in any one of the following:

[0013] (1) Application in regulating plant grain yield;

[0014] (2) Application in cultivating transgenic plants with high grain yield;

[0015] (3) Application in preparing products for increasing plant grain yield;

[0016] The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0017] The present invention also provides an application of a recombinant vector containing the coding gene in any one of the following:

[0018] (1) Application in regulating plant grain yield;

[0019] (2) Application in cultivating transgenic plants with high grain yield;

[0020] (3) Application in preparing products for increasing plant grain yield.

[0021] The present invention also provides an application of an engineered bacterium containing the recombinant vector in any one of the following:

[0022] (1) Application in regulating plant grain yield;

[0023] (2) Application in cultivating transgenic plants with high grain yield;

[0024] (3) Application in preparing products for increasing plant grain yield.

[0025] Preferably, increasing the expression level of the maize transcription factor ZmERF1a or its coding gene in a plant increases the grain yield of the plant;

[0026] The plant is Arabidopsis thaliana or maize.

[0027] Preferably, the method for increasing the expression level of the maize transcription factor ZmERF1a or its coding gene in a plant includes constructing an overexpression vector of the coding gene of the maize transcription factor ZmERF1a, transforming the overexpression vector into Agrobacterium, and then infecting the plant.

[0028] The present invention also provides a method for increasing the grain yield of a plant, including the step of increasing the expression level of the coding gene of the maize transcription factor ZmERF1a in the plant to increase the grain yield of the plant;

[0029] The nucleotide sequence of the coding gene is as shown in SEQ ID NO.1.

[0030] Preferably, the method for increasing the expression level of the coding gene of the maize transcription factor ZmERF1a includes the following steps:

[0031] Constructing an overexpression vector of the coding gene of the maize transcription factor ZmERF1a, transforming the overexpression vector into Agrobacterium, and then infecting the plant;

[0032] The plant is Arabidopsis thaliana or maize.

[0033] The present invention also provides a breeding method for transgenic plants with high grain yield, including the following steps:

[0034] Overexpressing the coding gene of the maize transcription factor ZmERF1a in plant cells, then cultivating the plant cells, and regenerating plants using the plant cells to obtain the transgenic plants with high grain yield;

[0035] The nucleotide sequence of the coding gene is as shown in SEQ ID NO.1.

[0036] Preferably, the plant is Arabidopsis thaliana or maize.

[0037] The present invention discloses the following technical effects:

[0038] The present invention cloned a transcription factor ZmERF1a that regulates corn kernel weight through DNA pull-down and mass spectrometry. Based on the association analysis of the gene ZmERF1a, significant SNPs related to 100-kernel weight were identified. Through yeast one-hybrid and electrophoretic mobility shift assays, it was verified that the ZmERF1a protein can bind to the promoter of the ZmNAD gene and regulate the 100-kernel weight of corn. In transgenic Arabidopsis plants overexpressing ZmERF1a, the overexpression of the ZmERF1a gene can increase the 1000-kernel weight of seeds by 14.8%-37.4%. The transcription factor ZmERF1a provided by the present invention has broad application prospects and great economic benefits in regulating corn grain yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1 It is a haplotype analysis result diagram based on the association analysis of ZmERF1a; wherein, A is the 100-kernel weight phenotypes of Hap1, Hap2, Hap3, and Hap4; different lowercase letters indicate statistically significant differences; B is the genotypes of Hap1, Hap2, Hap3, and Hap4;

[0041] Figure 2 It is an SDS-PAGE electrophoresis result diagram of the prokaryotic expression and purified protein of the transcription factor ZmERF1a; wherein, A is the electrophoresis result of the supernatant after prokaryotic expression and disruption, lane 1 represents the MBP prokaryotic expression protein, and lane 2 represents the MBP-ZmERF1a prokaryotic expression protein; B is the electrophoresis result of the purified protein; lane 1 represents the purified MBP protein, and lane 2 represents the purified MBP-ZmERF1a protein;

[0042] Figure 3 It is a yeast one-hybrid verification result diagram of the transcription factor ZmERF1a and the promoter of the ZmNAD gene;

[0043] Figure 4 It is an electrophoretic mobility shift assay result diagram of the transcription factor ZmERF1a and the promoter of the ZmNAD gene;

[0044] Figure 5 It is a positive detection result diagram of the transcription factor ZmERF1a gene in transgenic Arabidopsis lines; wherein, 1-16 represent the Arabidopsis individual numbers, and they are the positive homozygous lines of the T3 generation of transgenic Arabidopsis overexpressing ZmERF1a obtained after seed propagation

[0045] Figure 6 The phenotype diagram of 1000-grain weight of the transcription factor ZmERF1a gene in transgenic Arabidopsis thaliana lines; among them, A is the electrophoresis detection result of the expression level of the ZmERF1a gene; B is the statistical result of the expression level of the ZmERF1a gene; C is the grain observation diagram of transgenic Arabidopsis thaliana lines; D is the statistical result of 1000-grain weight of transgenic Arabidopsis thaliana lines; *** indicates P<0.001. Specific implementation manners

[0046] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0050] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0051] Example 1 Obtaining of maize transcription factor ZmERF1a and cloning of its encoding gene

[0052] The promoter of the maize ZmNAD gene was biotin-labeled at the 5'-end, and DNA pull-down was performed by the magnetic bead method to obtain a DNA-protein binding product. After SDS-PAGE silver staining identification, mass spectrometry identification was carried out, and the transcription factor ZmERF1a that interacts with the ZmNAD gene promoter fragment was screened. By designing gene primer pairs, the full-length CDS of the ZmERF1a gene was cloned. The sequences of the primer pairs are as follows:

[0053] Forward primer ZmERF1a-F: 5'-CCAGTTTCGTCAGCTCATCA-3' (SEQ ID NO.3);

[0054] Reverse primer ZmERF1a-R: 5'-TATCGCATATTCGCATTCCA-3' (SEQ ID NO.4).

[0055] The cloning of the ZmERF1a gene includes the following steps:

[0056] (1) Extraction of maize leaf RNA: Total RNA of maize leaves was extracted according to the instruction manual of the Trizol kit (Invitrogen);

[0057] (2) Reverse transcription to synthesize cDNA: According to the operation manual of the RT kit (TakaRa), reverse transcription was carried out to obtain cDNA;

[0058] (3) Cloning of the full-length CDS of the ZmERF1a gene: PCR amplification was carried out according to the instruction manual of the high-fidelity enzyme P505 (Vazyme); after detecting the ZmERF1a gene PCR product by 1% agarose gel electrophoresis, the target band was recovered according to the operation steps of the gel DNA recovery kit (Magen); according to the instruction manual of the pEASY-blunt gene cloning kit (TransGen), positive clones with correct sequencing were obtained. The CDS sequence of the obtained ZmERF1a gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0059] SEQ ID NO.1:

[0060] ATGCTGCTGAACCCGGCGTCAGAGGCGTCGGTGCTAGACACCATCCGGCAGCACCTCCTCGAGGAGCCAGCCGACGAGAGCTTCGGGAGCCTGGTGGCGGACCAGTGGAGCGGCTCGCTCCCGTTCCGCACCGACGACGCCGACGACATGGTGGTGTTCGGGGCGCTGCAGGACGCCTTCGCCTACGGCTGGCTGCCCGACGGCTCATTCGTGCACGTGAAGCCCGAGCCGGTGCGGTCCCCCGACTCGTCCTCCTACCCCTGCTCCTACGACGGCTCACCCTGCTTCGGCCTCCTGGACCCGGAGCCGCCGCTGACGCCCGGCACCACCACGCCCAGTAGTAGGGGGCAGGAGGAGGCCGCGGCGGCCATGGCCCGGGGCAAGCACTACAGGGGGGTGAGGCAGCGCCCGTGGGGCAAGTTCGCGGCGGAGATCAGGGACCCCGCCAGGAACGGCGCGCGCGTCTGGCTCGGCACGTACGACACCGCCGAGGACGCCGCGCTCGCCTACGACCGCGCCGCCTACCGCATGCGCGGCTCGCGCGCGCTCCTCAACTTCCCGCTCCGCATCGGCTCCGGGGACAAGCGCCCGTCGCCGGCGCCGCCCGAGCCCGCCACCTCCTCGGACTCCTCCTCGTCTTCGGCCAGCGGCTCGCACAAGAGGCGGAAGCGAGGCGAGGCCGCGGCTGCCAACATGGCCATGGCGCTGGTGCCCCCGCCCTCCCAGCTTAACCGGCCGGCCCAGCCGTGGTTCCCTGCCGCGCCGGTCGAGCAGGCGGCGATGGCTCCGCGCGTGGAGCAGATCGTCGTCTAG;

[0061] SEQ ID NO.2:

[0062] MLLNPASEASVLDTIRQHLLEEPADESFGSLVADQWSGSLPFRTDDADDMVVFGALQDAFAYGWLPDGSFVHVKPEPVRSPDSSSYPCSYDGSPCFGLLDPEPPLTPGTTTPSSRGQEEAAAAMARGKHYRGVRQRPWGKFAAEIRDPARNGARVWLGTYDTAEDAALAYDRAAYRMRGSRALLNFPLRIGSGDKRPSPAPPEPATSSDSSSSSASGSHKRRKRGEAAAANMAMALVPPPSQLNRPAQPWFPAAPVEQAAMAPRVEQIVV*。

[0063] Example 2 Association analysis and haplotype identification based on ZmERF1a

[0064] In 70 randomly selected maize inbred lines, the ZmERF1a gene body and upstream promoter region were amplified by PCR respectively, and a total of 51 variations (38 SNPs and 13 InDels) were identified. Combining the 51 variations and the 100-kernel weight phenotypic values of 70 maize inbred lines, a candidate gene association analysis was performed using the GLM model, and the significant variation detection threshold was set at P = 0.05 / N (N is the number of effective markers, N = 51). The results showed that three SNPs, SNP-10-140619436, SNP-10-140620265, and SNP-10-140620276, were significantly associated with the 100-kernel weight. Based on these significant SNPs, a total of four haplotypes were identified, namely Hap1 (ACC), Hap2 (ACG), Hap3 (CCG), and Hap4 (CTG), as Figure 1 shown. Among them, the phenotypic value of the 100-kernel weight of Hap4 was the largest and was significantly higher than that of Hap1 and Hap2. Therefore, Hap4 was determined as the excellent haplotype of ZmERF1a, and Hap1 with the smallest phenotypic value was the non-excellent haplotype.

[0065] Example 3 Construction of prokaryotic expression vector pC2X-MBP-ZmERF1a and protein expression

[0066] (1) Cloning of vector pC2X-MBP-ZmERF1a: The ZmERF1a gene obtained in Example 1 was used to obtain a positive clone with correct sequencing according to the instruction manual of the ClonExpress II cloning kit (Vazyme).

[0067] (2) Induced expression of ZmERF1a-MBP protein: The obtained pC2X-MBP-ZmERF1a prokaryotic expression vector was transformed into Rosetta strain for induced expression of the protein. The large amount of induced bacterial liquid was 400 mL, the final concentration of IPTG was 0.5 mM, the temperature was 16 °C, and the culture time was 12 - 16 h. After collecting the bacteria, they were resuspended with CB buffer (containing PMSF with a final concentration of 0.25 mM). The bacterial suspension was sonicated until the solution became clear. The supernatant after sonication was detected by SDS-PAGE electrophoresis, and the results were as shown in Figure 2 Figure A.

[0068] (3) Purification of ZmERF1a-MBP protein: Protein purification was carried out according to the operating steps of the Dextrin Beads gravity column product (Tian Di Ren He). The eluted protein after purification was detected by SDS-PAGE electrophoresis, and the results were as shown in Figure 2 Figure B.

[0069] Example 4 Verification of the binding of transcription factor ZmERF1a to the ZmNAD promoter

[0070] 1. Verification of the binding of ZmERF1a to the ZmNAD promoter by yeast one-hybrid

[0071] (1) Cloning of the Bait vector pAD-ZmERF1a: The ZmERF1a gene obtained in Example 1 was used to obtain a positive clone with correct sequencing according to the instruction manual of the ClonExpress II cloning kit (Vazyme).

[0072] (2) Transformation of pAD-ZmERF1a into the Prey yeast strain: According to the steps of the yeast transformation kit (Coolaber), the Bait vector pAD-ZmERF1a was transferred into the Prey yeast competent pAbAi-ZmNADproY1Hgold (containing the ZmNAD promoter) to obtain a yeast strain containing both Bait and Prey.

[0073] (3) One-to-one interaction verification: In the SD-Lue-Ura liquid medium, it was cultured until OD

[0072] , 600 ,

[0074] , Figure 3 , , , <00001​​​​​2. Gel retardation assay proves that ZmERF1a binds to the ZmNAD promoter

[0075] According to the steps of the EMSA / Gel-Shift kit (Beyotime), a gel retardation assay was performed using a biotin-labeled ZmNAD promoter probe, a mutant probe, and ZmERF1a protein. The probe sequences are shown as follows:

[0076]

[0077] The results are as Figure 4 shown, indicating that the transcription factor ZmERF1a can bind to the promoter of the ZmNAD gene in vitro experiments.

[0078] Example 5 Obtaining of transgenic ZmERF1a Arabidopsis plants

[0079] (1) Cloning of vector pRI-AN101-ZmERF1a: The ZmERF1a gene obtained in Example 1 was used to obtain a positive clone with correct sequencing according to the instruction manual of the ClonExpress II cloning kit (Vazyme).

[0080] (2) Obtaining of transgenic ZmERF1a Arabidopsis plants: Using the Agrobacterium-mediated floral dip method, the vector pRI-AN101-ZmERF1a was transferred into wild-type Arabidopsis. Positive identification of T0 generation Arabidopsis seeds was carried out by kanamycin resistance screening and PCR amplification of the ZmERF1a gene specific band. The results are as Figure 5 shown.

[0081] Example 6 Phenotypes of transgenic ZmERF1a Arabidopsis lines

[0082] (1) Analysis of the expression level of gene ZmERF1a in transgenic Arabidopsis: According to the methods in (1) and (2) of Example 1, cDNA of transgenic Arabidopsis plants was obtained. qRT-PCR was performed according to the instruction manual of the NovoScript quantitative kit (Novoprotein), using AtActin as the internal reference gene, setting 3 biological replicates, and designing 3 technical replicates for each sample to analyze the expression level of gene ZmERF1a in transgenic Arabidopsis plants. The results are as Figure 6 shown in A and B, indicating that the gene ZmERF1a was successfully integrated into the Arabidopsis genome and highly expressed.

[0083] (2) Thousand-seed weight phenotype of transgenic ZmERF1a Arabidopsis: The thousand-seed weight of three transgenic ZmERF1a Arabidopsis lines was measured, with 3 biological replicates and 3 technical replicates set for each line. The results are as Figure 6As shown in C and D: Compared with the wild type, the thousand-grain weight significantly increased after overexpression of the ZmERF1a gene, indicating that the transcription factor ZmERF1a can improve the seed yield of Arabidopsis thaliana.

[0084] The above results indicate that the present invention successfully cloned the full-length CDS of a maize AP2 / ERF family transcription factor ZmERF1a gene, verified that this transcription factor can bind to the promoter of the ZmNAD gene, regulate the kernel weight of maize, and successfully introduced it into Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana lines, providing gene resources for cultivating new high-yield maize varieties.

[0085] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a maize transcription factor ZmERF1a in any of the following: (1) Use in increasing the grain yield of plants; (2) Use in cultivating transgenic plants with high grain yield; (3) Use in preparing a product for increasing the grain yield of plants; Up-regulating the expression level of the maize transcription factor ZmERF1a or its coding gene in a plant to increase the grain yield of the plant; The plant is Arabidopsis thaliana or maize; The amino acid sequence of the maize transcription factor ZmERF1a is as shown in SEQ ID NO.

2.

2. Use of a coding gene of the maize transcription factor ZmERF1a as described in claim 1 in any of the following: (1) Use in increasing the grain yield of plants; (2) Use in cultivating transgenic plants with high grain yield; (3) Use in preparing a product for increasing the grain yield of plants; Up-regulating the expression level of the coding gene of the maize transcription factor ZmERF1a in a plant to increase the grain yield of the plant; The plant is Arabidopsis thaliana or maize; The nucleotide sequence of the coding gene is as shown in SEQ ID NO.

1.

3. Use of a recombinant vector containing the coding gene as described in claim 2 in any of the following: (1) Use in increasing the grain yield of plants; (2) Use in cultivating transgenic plants with high grain yield; (3) Use in preparing a product for increasing the grain yield of plants; Up-regulating the expression level of the coding gene in a plant to increase the grain yield of the plant; The plant is Arabidopsis thaliana or maize.

4. Use of an engineered bacterium containing the recombinant vector as described in claim 3 in any of the following: (1) Use in increasing the grain yield of plants; (2) Use in cultivating transgenic plants with high grain yield; (3) Use in preparing a product for increasing the grain yield of plants; ​ ​ 5. The application according to any one of claims 1-4, characterized in that, ​ 6. A method for increasing the grain yield of plants, characterized in that, ​ ​ ​ 7. The method according to claim 6, wherein ​ 8. A breeding method for transgenic plants with high grain yield, characterized in that, ​ ​ ​ The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

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