Application of corn transcription factor ZmERF1a and coding gene thereof in regulation and control of plant kernel yield

Abstract

The invention discloses application of a corn transcription factor ZmERF1a and a coding gene thereof in regulating and controlling the yield of plant kernels, and belongs to the technical field of molecular biology and genetic engineering. According to the invention, a transcription factor ZmERF1a with an amino acid sequence shown as SEQ ID NO.2 and a coding gene ZmERF1a of the transcription factor ZmERF1a, which is used for regulating and controlling the weight of corn kernels, are obtained through DNA (deoxyribonucleic acid) pull down and mass spectrum cloning and screening. The invention verifies that the ZmERF1a protein can be combined with the promoter of the ZmNAD gene to regulate and control the hundred-grain weight of corn. A transgenic experiment result shows that in an arabidopsis thaliana plant over-expressing the ZmERF1a, the thousand seed weight of grains can be increased by 14.8%-37.4% through the over-expression of the ZmERF1a gene. The transcription factor ZmERF1a provided by the invention has wide application prospects and economic benefits with huge potential in regulation and control of corn kernel yield.

Description

Technical Field

[0001] The 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 an important food crop in the world and an important source of human nutrition, animal feed and bioenergy. According to statistics, in 2023, my country's corn planting area will reach 44,219 thousand hectares, and the total output will reach 288.84 million tons, ranking first among food crops in both sown area and total output (National Bureau of Statistics, 2024). However, with the development of social economy, the total market demand for corn continues to grow. At the same time, there are constraints such as population, land resources and environment, which makes it more difficult to further increase corn grain yield. Grain weight is a key trait affecting corn yield. At present, conventional breeding to increase corn grain yield has encountered bottlenecks.

[0003] Transcription factors are protein molecules that can specifically bind to cis-acting elements in the promoter region of eukaryotic genes, thereby regulating the expression of target genes. The growth and development process of plants is generally regulated by multiple genes. Transcription factors are activated through a series of signal transductions, causing them to bind to the corresponding cis-acting elements and activate the RNA polymerase II transcription complex, thereby initiating the transcriptional expression of the corresponding functional genes. AP2 / ERF (APETALA2 / ethylene-responsive factor) is a family of transcription factors that are widely present in plants. The proteins in this family contain the AP2 / ERF domain, have DNA binding function, and are closely related to the growth and development of plants. Since the functions of AP2 / ERF family transcription factors are complex and they participate in different signal transduction pathways, further research is needed on how the genes in this family regulate corn grain weight by participating in various signal pathways and regulating downstream gene expression.

[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 the 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 corn 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 screens out a transcription factor ZmERF1a that regulates corn grain weight through DNA pull down and mass spectrometry identification. By introducing or improving the transcription factor, the expression of a series of grain weight-related genes can be regulated, thereby increasing corn yield and providing genetic resources for breeding high-yield corn 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 of the following:

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

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

[0010] (3) Application in the preparation of products for increasing plant seed 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 the coding gene of the maize transcription factor ZmERF1a in any of the following items:

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

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

[0015] (3) Application in the preparation of products for increasing plant seed 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 comprising the encoding gene in any of the following:

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

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

[0020] (3) Application in the preparation of products for increasing plant seed yield.

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

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

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

[0024] (3) Application in the preparation of products for increasing plant seed yield.

[0025] Preferably, the expression level of the maize transcription factor ZmERF1a or its encoding gene is up-regulated in the plant to increase the seed yield of the plant;

[0026] The plant is Arabidopsis thaliana or corn.

[0027] Preferably, the method for upregulating the expression level of the maize transcription factor ZmERF1a or its encoding gene in plants comprises the steps of constructing an overexpression vector of the encoding 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 plant seed yield, comprising the steps of increasing the expression level of the gene encoding the corn transcription factor ZmERF1a in the plant to increase the plant seed yield;

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

[0030] Preferably, the method for upregulating the expression level of the gene encoding the maize transcription factor ZmERF1a comprises the following steps:

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

[0032] The plant is Arabidopsis thaliana or corn.

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

[0034] Overexpressing a gene encoding a maize transcription factor ZmERF1a in a plant cell, then cultivating the plant cell, and using the plant cell to regenerate a plant, thereby obtaining the transgenic plant with high grain yield;

[0035] The nucleotide sequence of the coding gene is 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 clones a transcription factor ZmERF1a that regulates corn grain weight through DNA pull down and mass spectrometry. Based on the association analysis of the gene ZmERF1a, a significant SNP associated with 100-grain weight is identified. Through yeast single hybridization and gel retardation experiments, it is verified that the ZmERF1a protein can bind to the promoter of the ZmNAD gene and regulate the 100-grain weight of corn. In transgenic ZmERF1a Arabidopsis plants, overexpression of the ZmERF1a gene can increase the 1000-grain weight of grains by 14.8%-37.4%. The transcription factor ZmERF1a provided by the present invention has broad application prospects and huge potential economic benefits in regulating corn grain yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 Figure 1 is the result of haplotype analysis based on ZmERF1a association analysis; A is the 100-grain weight phenotype of Hap1, Hap2, Hap3 and Hap4; different lowercase letters indicate statistically significant differences; B is the genotype of Hap1, Hap2, Hap3 and Hap4;

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

[0042] Figure 3 This is the yeast one-hybrid validation result of transcription factor ZmERF1a and ZmNAD gene promoter;

[0043] Figure 4 This is the result of the gel retardation experiment of transcription factor ZmERF1a and ZmNAD gene promoter;

[0044] Figure 5 The positive detection result of transcription factor ZmERF1a gene in transgenic Arabidopsis strains; 1-16 represents the individual plant numbers of Arabidopsis, and the positive homozygous strains of transgenic ZmERF1a Arabidopsis T3 generation were obtained after seed propagation

[0045] Figure 6 Figure 1 is the 1000-grain weight phenotype of the transcription factor ZmERF1a gene in transgenic Arabidopsis thaliana lines; A is the electrophoresis detection result of ZmERF1a gene expression; B is the statistical result of ZmERF1a gene expression; 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. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Obtaining the Maize Transcription Factor ZmERF1a and Cloning the Encoding Gene

[0052] The promoter of the maize ZmNAD gene was labeled with biotin at the 5' end, and DNA was pulled down by magnetic beads to obtain DNA-protein binding products. After SDS-PAGE silver staining and mass spectrometry identification, the transcription factor ZmERF1a that interacts with the promoter fragment of the ZmNAD gene was screened. The full-length CDS of the ZmERF1a gene was cloned by designing a gene primer pair. The sequence of the primer pair is 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 ZmERF1a gene includes the following steps:

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

[0057] (2) Reverse transcription to synthesize cDNA: RT kit (TakaRa) operating instructions, reverse transcription to obtain cDNA;

[0058] (3) Cloning the full-length CDS of the ZmERF1a gene: PCR amplification was performed according to the instruction manual of the high-fidelity enzyme P505 (Vazyme); the PCR product of the ZmERF1a gene was detected by 1% agarose gel electrophoresis, and 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), a positive clone with correct sequencing was obtained. The obtained ZmERF1a gene CDS sequence 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] MLLNPASEASVLDTIRQHLLEEPADESFGSLVADQWSGSLPFRTDDADDMVVFGALQDAFAYGWLPDGSFVHVKPEPVRSPDSSSYPCSYDGSPCFGLLDPEPPLTPGTTTPSSRGQEEAAAAMARGKHYRGVRQR PWGKFAAEIRDPARNGARVWLGTYDTAEDAALAYDRAAYRMRGSRALLNFPLRIGSGDKRPSPAPPEPATSSDSSSSASGSHKRRKRGEAAAANMAMALVPPPSQLNRPAQPWFPAAPVEQAAMAPRVEQIVV*.

[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, and a total of 51 variants (38 SNPs and 13 InDels) were identified. Combining the 51 variants and the 100-grain weight phenotypic values ​​of 70 maize inbred lines, the GLM model was used for candidate gene association analysis, and the significant variant 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 correlated with 100-grain weight. Based on these significant SNPs, a total of four haplotypes were identified, namely Hap1 (ACC), Hap2 (ACG), Hap3 (CCG) and Hap4 (CTG), as shown in Figure 2. Figure 1 As shown. Among them, Hap4 has the largest phenotypic value of 100-grain weight, and is significantly higher than Hap1 and Hap2. Therefore, Hap4 was determined to be the superior haplotype of ZmERF1a, and Hap1 with the smallest phenotypic value was a non-superior 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 cloned according to the instruction manual of ClonExpress II cloning kit (Vazyme) to obtain a positive clone with correct sequencing.

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

[0068] (3) ZmERF1a-MBP protein purification: Protein purification was performed according to the operating procedures of the Dextrin Beads gravity column product (Tian Di Ren He). The purified eluted protein was detected by SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown in B.

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

[0070] 1. Yeast one-hybrid assay to verify the binding of ZmERF1a to the ZmNAD promoter

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

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

[0073] (3) One-to-one interaction verification: Culture in SD-Lue-Ura liquid medium until OD 600 =1, and the concentration gradient was diluted to 1×, 10×, 100× and 1000×. 2.5 μL of each bacterium was taken and cultured on the solid medium of SD-Lue-Ura+AbA (100 ng) for 60 h. The results are shown in Figure 3 As shown: ZmNADp1-ZmNADp4 are the promoters of the ZmNAD gene, indicating that the transcription factor ZmERF1a can bind to the promoter of the ZmNAD gene in yeast.

[0074] 2. Gel retardation assay proves that ZmERF1a binds to the ZmNAD promoter

[0075] According to the instructions of the EMSA / Ge1-Shift kit (Biyuntian), a gel retardation experiment was performed using a biotin-labeled ZmNAD promoter probe and a mutation probe and ZmERF1a protein. The probe sequences are as follows:

[0076]

[0077] The results are as follows Figure 4 As shown, it was shown that the transcription factor ZmERF1a could bind to the promoter of the ZmNAD gene in in vitro experiments.

[0078] Example 5 Obtaining transgenic ZmERF1a Arabidopsis plants

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

[0080] (2) Obtaining transgenic ZmERF1a Arabidopsis plants: The vector pRI-AN101-ZmERF1a was transferred into wild-type Arabidopsis by Agrobacterium-mediated inflorescence infection. The T0 generation Arabidopsis seeds were positively identified by kanamycin resistance screening and PCR amplification of ZmERF1a gene-specific bands. Figure 5 shown.

[0081] Example 6 Phenotype of transgenic ZmERF1a Arabidopsis lines

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

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

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

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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) Application in regulating plant seed yield; (2) Application in breeding transgenic plants with high grain yield; (3) Application in the preparation of products for increasing plant seed yield; The amino acid sequence of the maize transcription factor ZmERF1a is shown in SEQ ID NO.

2.

2. Use of the gene encoding the maize transcription factor ZmERF1a as claimed in claim 1 in any of the following: (1) Application in regulating plant seed yield; (2) Application in breeding transgenic plants with high grain yield; (3) Application in the preparation of products for increasing plant seed yield; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. Use of a recombinant vector comprising the coding gene according to claim 2 in any of the following: (1) Application in regulating plant seed yield; (2) Application in breeding transgenic plants with high grain yield; (3) Application in the preparation of products for increasing plant seed yield.

4. Use of an engineered bacterium comprising the recombinant vector according to claim 3 in any of the following: (1) Application in regulating plant seed yield; (2) Application in breeding transgenic plants with high grain yield; (3) Application in the preparation of products for increasing plant seed yield.

5. The use according to any one of claims 1 to 4, characterized in that: Up-regulating the expression of the corn transcription factor ZmERF1a or its encoding gene in the plant to increase the grain yield of the plant; The plant is Arabidopsis thaliana or corn.

6. The use according to claim 5, characterized in that The method for up-regulating the expression of the corn transcription factor ZmERF1a or its encoding gene in plants comprises the steps of constructing an overexpression vector of the encoding gene of the corn transcription factor ZmERF1a, transforming the overexpression vector into Agrobacterium, and then infecting the plant.

7. A method for increasing plant seed yield, characterized in that: The method comprises the steps of up-regulating the expression level of the gene encoding the corn transcription factor ZmERF1a in the plant to increase the seed yield of the plant; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The method for up-regulating the expression level of the gene encoding the maize transcription factor ZmERF1a comprises the following steps: Constructing an overexpression vector of the gene encoding the maize transcription factor ZmERF1a, transforming the overexpression vector into Agrobacterium, and then infecting plant plants; The plant is Arabidopsis thaliana or corn.

9. A method for breeding transgenic plants with high grain yield, characterized in that: The following steps are involved: Overexpressing a gene encoding a maize transcription factor ZmERF1a in a plant cell, then cultivating the plant cell, and using the plant cell to regenerate a plant, thereby obtaining the transgenic plant with high grain yield; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that The plant is Arabidopsis thaliana or corn.

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