Use of a maize ZmMKK5 gene in regulating maize kernel development

Abstract

The application discloses application of a corn ZmMKK5 gene in regulation of corn kernel development and belongs to the technical field of genetic engineering. The nucleotide sequence of the ZmMKK5 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the ZmMKK5 gene coding protein is shown as SEQ ID NO. 2. The application finds for the first time that by inhibiting the expression of the coding gene ZmMKK5 of the ZmMKK5 protein, the length, width, hundred-grain weight of the corn kernel and the diameter of the corn kernel starch granule can be significantly reduced, a scientific basis is provided for further fully tapping the production potential of corn and improving the corn yield, the application prospect is good, and the ZmMKK5 gene has important guiding significance for improving the corn yield and high-yield and stable-yield breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the maize ZmMKK5 gene in regulating maize kernel development. Background Technology

[0002] Maize (Zea mays L.), as my country's largest grain crop, is widely planted throughout the country and plays a vital role in local agricultural production and the economy. Understanding its grain development mechanism is crucial for ensuring food security. Grain size, as a core constituent trait of the three yield factors (ear number, grain number, and grain weight), is influenced by the synergistic effects of genetic regulatory networks and the external environment. Many scholars both domestically and internationally have studied this trait, and after years of research, some progress has been made in understanding the molecular genetic mechanisms of maize grain size. However, gene resources are relatively scarce, and there is an urgent need to discover and utilize more superior alleles. Therefore, using genetic engineering technology to select genes suitable for breeding is a key research focus.

[0003] Mitogen-activated protein kinase kinase (MAPKK) is a key member of the MAPK cascade in plants. The MAPK cascade pathway is a highly conserved signal transduction system composed of MAPKKK-MAPKK-MAPK, which regulates cell proliferation and differentiation through stepwise phosphorylation in response to external stimuli. In plants, members of the MKK gene family are commonly involved in various abiotic stress responses, such as cold stress, salt stress, and drought stress. The MKK gene family also participates in plant growth and development, and plays an important role in plant hormone signal transduction.

[0004] The maize ZmMKK gene belongs to the MAPK kinase family. Current research on the maize ZmMKK gene family mainly focuses on its potential functions in abiotic stress response and plant growth and development. Regarding the regulation of maize kernel development, Jin Jiao et al. obtained a gene related to maize kernel development, ZmMKK4, through map-based cloning. This is currently the only ZmMKK gene discovered related to maize kernel development. The main characteristics of this gene deletion mutant compared to the wild type are smaller kernels, wrinkled tops, a powdery and opaque endosperm, extremely low seedling rate, weak growth, and stunted plants (Jin Jiao, Map-based cloning of maize kernel size gene ZmMKK4, Henan Agricultural University, 2021, No. 02). However, empirical evidence is still lacking regarding whether other maize MAPKK homologs participate in kernel development. Summary of the Invention

[0005] To address the shortcomings of existing research, the purpose of this invention is to provide an application of the maize ZmMKK5 gene in regulating maize kernel development. The maize ZmMKK5 gene has a stronger regulatory ability on maize kernel development, providing more theoretical basis for breeding maize varieties with higher quality kernels.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] This invention provides an application of the maize ZmMKK5 gene in regulating maize kernel development. The nucleotide sequence of the ZmMKK5 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the ZmMKK5 gene is shown in SEQ ID NO.2.

[0008] As a further optimization of the present invention, the expression of the ZmMKK5 gene promotes maize kernel development, and the loss of function of the ZmMKK5 gene inhibits maize kernel development.

[0009] As a further optimization of the present invention, the development of corn kernels includes the development of the length, width, and weight of 100 kernels of corn kernels, as well as the development of corn kernel starch granules.

[0010] As a further optimization of the present invention, the corn is the corn inbred line KN5585.

[0011] This invention also provides a method for obtaining new maize germplasm. The method involves editing the ZmMKK5 gene in maize using CRISPR-Cas9 gene editing technology to suppress its expression. The nucleotide sequence of the ZmMKK5 gene is shown in SEQ ID NO.1. After constructing a ZmMKK5-CRISPR-Cas9 gene editing vector, maize genetic transformation is performed. The resulting positive seedlings are new maize germplasm with smaller kernel length, width, 100-kernel weight, and smaller starch granule diameter. Alternatively, a ZmMKK5 gene overexpression vector is constructed using genetic engineering technology and maize genetic transformation is performed. The resulting positive seedlings are new maize germplasm with larger kernel length, width, 100-kernel weight, and larger starch granule diameter.

[0012] As a further optimization of the present invention, in the CRISPR-Cas9 gene editing technology, two sgRNA target sites were designed using the nucleotide sequence of the ZmMKK5 gene as a template to construct the ZmMKK5-CRISPR-Cas9 gene editing vector. The sgRNA target sequence is as follows:

[0013] SEQ ID NO.3: Nucleotide sequence containing target site one: CCGCGGCACGGGGCGCCCCTACG;

[0014] SEQ ID NO.4: Nucleotide sequence containing target site 2: CGGCATGTACGAGCGCGCGGCGGGG.

[0015] As a further optimization of the present invention, editing of the ZmMKK5 gene includes inserting a 1 bp base and mutating a 1 bp base at target site one, and deleting a 23 bp base at target site two, or deleting a 139 bp base between target site one and target site two.

[0016] The present invention has the following beneficial effects:

[0017] This invention is the first to discover that by inhibiting the expression of the gene encoding the ZmMKK5 protein, the length, width, 100-kernel weight, and diameter of starch granules in maize kernels can be significantly reduced. This provides a scientific basis for further fully tapping the production potential of maize and increasing its yield. The application prospects are promising, and the rational use of the ZmMKK5 gene has important guiding significance for improving maize yield and breeding high- and stable-yield varieties. Attached Figure Description

[0018] Figure 1 The images show the identification of the knockout mutants zmmkk5-KO1 and 2. WT is wild-type KN5585.

[0019] Figure 2 The images show the identification of the knockout mutants zmmkk4-KO1 and 2. WT is wild-type KN5585.

[0020] Figure 3 Phenotypic analysis of the grains of the knockout mutants zmmkk5-KO1, 2, zmmkk4-KO1, 2 and wild-type KN5585;

[0021] Figure 4 Scanning electron microscopy analysis of the knockout mutants zmmkk5-KO1, 2, zmmkk4-KO1, 2 and wild-type KN5585. Detailed Implementation

[0022] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] 1. Materials

[0024] The full-length CDS sequence (SEQ ID NO.1) and protein sequence (SEQ ID NO.2) of the maize ZmMKK5 gene were obtained from the Plant Genome Database website (https: / / phytozome.jgi.doe.gov / pz / portal.html).

[0025] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0026] 2. Method

[0027] 2.1 Identification of the knockout mutant zmmkk5

[0028] Based on the full-length CDS sequence of the ZmMKK5 gene, a ZmMKK5-CRISPR-Cas9 gene editing vector was constructed and genetically transformed to obtain maize mutant seeds. To ensure the accuracy of gene editing, specific dual-target sequences were designed: target site 1 was located on the antisense strand, and target site 2 was located on the sense strand. The sequence containing target site 1 is (SEQ ID NO.3: 5'-CCGCGGCACGGGGCGCCCCTACG-3'); the sequence containing target site 2 is (SEQ ID NO.4: 5'-CGGCATGTACGAGCGCGGCGGG-3'). Maize leaf genome was extracted and fragments containing gene knockout target sequences were amplified and sequence alignment was performed.

[0029] The results are as follows Figure 1 As shown, two homozygous maize lines, zmmkk5-KO1 and zmmkk5-KO2, were successfully obtained through gene editing. zmmkk5-KO1 had a 1bp insertion and a 1bp mutation at the first target site, and a 23bp deletion at the second target site. zmmkk5-KO2 had a deletion between the first and second target sites, totaling 139bp, which resulted in a frameshift in the ZmMKK5 gene nucleotide sequence, preventing it from encoding the normal ZmMKK5 protein.

[0030] 2.2 Identification of the knockout mutant zmmkk4

[0031] Based on the full-length CDS sequence of the ZmMKK4 gene (Zm00001d018326) (as shown in SEQ ID NO.7), a ZmMKK4-CRISPR-Cas9 gene editing vector was constructed and genetically transformed to obtain maize mutant seeds. To ensure the accuracy of gene editing, specific dual-target sequences were designed, with target sites 1 and 2 both located on the antisense strand. The sequence containing target site 1 is (SEQ ID NO.3: 5'-CCGCCGGGCACGCCGGGGCGGTC-3'); the sequence containing target site 2 is (SEQ ID NO.4: 5'-CCGCAGCGGGATCTGACCTCCCT-3'). Maize leaf genome was extracted and fragments containing gene knockout target sequences were amplified and sequence alignment was performed.

[0032] The results are as follows Figure 2 As shown, two homozygous maize lines, zmmkk4-KO1 and zmmkk4-KO2, were successfully obtained through gene editing. In zmmkk4-KO1, a deletion of 55 bp occurred between the first and second target sites. In zmmkk4-KO2, a 2 bp insertion occurred at the first target site, and a 1 bp deletion occurred at the second target site, resulting in a frameshift in the ZmMKK4 gene nucleotide sequence that prevented it from encoding the normal ZmMKK4 protein.

[0033] 2.3 Phenotypic identification of knockout mutants zmmkk5 and zmmkk4

[0034] The identified knockout lines zmmkk5-KO1-2, zmmkk4-KO1-2, and wild-type KN5585 were planted in the Nongcuiyuan experimental field of Anhui Agricultural University. After maturity, they were harvested, and grain phenotypes were observed, with grain length, grain width, and 100-grain weight recorded. Figure 3 ).like Figure 3 As shown, compared with the wild type, the kernels of zmmkk5-KO1-2 and zmmkk4-KO1-2 are smaller in size than those of the wild type (e.g., ...). Figure 3 A); the kernel length and width of zmmkk5-KO1-2 and zmmkk4-KO1-2 were also significantly smaller than those of the wild type (e.g., ...). Figure 3 AC); the 100-kernel weight of zmmkk5-KO1-2 and zmmkk4-KO1-2 seeds was significantly smaller than that of the wild type (e.g., AC); Figure 3 D), among which the length, width and 100-grain weight of the zmmkk5-KO1-2 grains were significantly greater than those of the mutant zmmkk4-KO1-2. This result indicates that reducing the expression of the ZmMKK5 gene significantly inhibits the development of grain size and greatly reduces crop yield.

[0035] 2.4 Effects of knockout mutants zmmkk5 and zmmkk4 on grain starch development

[0036] Starch is the most important storage substance in maize kernels, and starch development is closely related to kernel size. To clearly observe the morphology of starch granules in maize kernels, this study used scanning electron microscopy to observe longitudinal sections of knockout mutants zmmkk5-KO1 and zmmkk4-KO1, as well as wild-type KN5585(WT) maize kernels; the results are as follows. Figure 4 As shown in Figure A, the granules of the zmmkk5-KO1 and zmmkk4-KO1 mutants were significantly smaller than those of the wild type. Using ImageJ software, 100 starch granules were randomly selected from the zmmkk5-KO1, zmmkk4-KO1 mutants, and wild type for diameter analysis. The results are shown below. Figure 4 As shown in Figure B, compared with the wild type, the diameter of starch granules in both the zmmkk5-KO1 and zmmkk4-KO1 mutants was shorter. Among them, the diameter of starch granules in the zmmkk5-KO1 mutant was shortened more significantly. This result indicates that reducing the expression of the ZmMKK5 gene will significantly inhibit the development of starch granules, thereby affecting the development of grain size.

[0037] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A maize ZmMKK5 gene in regulating maize kernel development, characterized in that, The ZmMKK5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ZmMKK5 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2. The corn kernel development includes the development of the length, width, and 100-kernel weight of the corn kernel, as well as the development of the corn kernel starch granules. ZmMKK5 Loss of gene function inhibits corn kernel development.

2. Use according to claim 1, characterized in that, The corn is corn inbred line KN5585.

3. A method of obtaining a new maize germplasm ， characterized in that: Using CRISPR-Cas9 gene editing technology to manipulate maize ZmMKK5 Genes are edited to suppress the aforementioned ZmMKK5 Gene expression, the ZmMKK5 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and it was constructed to obtain... ZmMKK5-CRISPR-Cas9 Genetic transformation of maize using gene-edited vectors resulted in positive seedlings, which were new maize germplasm with smaller kernel length, width, 100-kernel weight, and starch granule diameter.

4. The method for obtaining new maize germplasm according to claim 3 ， characterized in that, in the CRISPR-Cas9 gene editing technology, two sgRNA target sites are designed with the nucleotide sequence of the gene as a template to construct ZmMKK5 ZmMKK5- CRISPR-Cas9 gene editing vectors, and the sgRNA sequences are as follows:​ The nucleotide sequence comprising target site one is SEQ ID NO. 3: CCGCGGCACGGGGCGCCCCTACG. The nucleotide sequence comprising target site two is SEQ ID NO. 4: CGGCATGTACGAGCGCGGCGGGG.

5. The method of obtaining novel maize germplasm according to claim 4 ， characterized in that, to ZmMKK5 the editing of the gene comprises an insertion of 1 bp base at target site one and a mutation of 1 bp base at target site two or a deletion of 139 bp bases between target site one and target site two.

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